Measuring the susceptibility and adhesion of microorganisms to light-activated antimicrobial

Measuringthesusceptibilityand

adhesionofmicroorganismstolight‐

activatedantimicrobialsurfaces

AthesispresentedtoUniversityCollegeLondoninpartialfulfilmentofthe

requirementsforthedegreeofDoctorofPhilosophy

ZoieAlexandraAiken

DivisionofMicrobialDiseasesUCLEastmanDentalInstitute

Supervisedby

DoctorJonathanPratten


ProfessorMichaelWilson


2012

2

Declaration

I Zoie Alexandra Aiken confirm that the work presented in this thesis is my own

WhereinformationhasbeenderivedfromothersourcesIconfirmthatthishasbeen

indicatedinthethesis

3

AbstractThe prevention of healthcare‐associated infections (HCAIs) is a major challenge

currently being faced by hospitals in both the UK and worldwide The hospital

environment acts as a reservoir for nosocomial organisms contributing towards the

transmissionofbacteriaand thus thecolonisationand infection ratesof the patient

populationThereforeitisdesirabletoimplementmeasurestodecreasethemicrobial

load within the hospital environment as a whole and particularly on frequently

touchedsurfacesAntimicrobialcoatingscouldbeappliedtothesesurfacesandused

asanadjuncttootherinfectioncontrolpoliciestoreducetheincidenceofHCAIs

Novelnitrogen‐dopedsulfur‐dopedandsilver‐coatedtitaniumdioxidephotocatalytic

thin films were generated by sol‐gel or chemical vapour deposition The materials

exhibitedantibacterialpropertiesafterexposuretoawhitelightcommonlyusedinUK

hospitalsHoweveritwasdifficulttosynthesisereproduciblethinfilmsusingtheCVD

method of deposition An additional antibacterial material was generated with the

potential tobeused inendotracheal tubesto reducethe incidenceofHCAIssuchas

ventilator‐associated pneumonia The novel polymer was impregnated with a

photosensitiserusingaswellencapsulationmethodandactivatedwithlaserlightthe

antibacterialandanti‐adhesivepropertieswerethenassessed

Sampling the test surfaces by swabbing and subsequently performing viable counts

was shown to provide an adequate estimate of concentration of bacteria on a test

surfaceThenitrogen‐andsulfur‐dopedtitaniumdioxidecoatingsdisplayedsignificant

photocatalyticactivityagainstEscherichia coliafterexposure toawhite light source

4

whichdemonstratedashiftinthebandgapfromtheUVtothevisibleregionofthe

electromagnetic spectrum Visible light photocatalysis was confirmed on the silver‐

coated titania thin films when a UV filter was used to block out the minimal UV

componentofthewhitelightsourceintheformofphoto‐oxidationofstearicacida

reduction in thewater contactangleandphotocatalyticactivityagainstanepidemic

strain of meticillin resistant Staphylococcus aureus (EMRSA‐16) This is the first

example of unambiguous visible light photocatalysis and photo‐induced

superhydrophilicity alongside a titanium dioxide control that shows no activation A

reduction in the viability of EMRSA‐16 adhered onto the surface of the irradiated

silver‐coatedtitaniathinfilmswasalsodemonstrated

AsignificantreductionintherecoveryofPseudomonasaeruginosaStenotrophomonas

maltophilia Acinetobacter baumannii and Candida albicans was observed on TBO‐

impregnated polymers after irradiation with a HeNe laser light A recently isolated

clinicalstrainofPaeruginosashoweddecreasedsusceptibilitytothephoto‐activityof

the TBO‐impregnated polymers compared with a laboratory type strain Finally a

significant reduction in the adhesion of P aeruginosa on the TBO‐impregnated

polymers was demonstrated after a 3‐step irradiation schedule A photo‐bleaching

effect was noted after light exposure that reduced the antibacterial activity of the

polymerswhichdemonstratestherequirementforfurthermodificationtoretainthe

photosensitiserwithinthepolyurethanematrix

These novel materials have the potential to be used as anti‐microbial surfaces in

healthcareenvironments

5

AcknowledgementsIwould liketothankmysupervisorsDrJonathanPrattenandProfessorMikeWilson

fortheirsupportoverthelastfouryearsIthasbeenaneventfuljourneyandIthank

youforalltheknowledgeandwisdomyouhavesharedwithmeThankstoDrCharlie

DunnillandDrGeoffHyett forsynthesisingtheCVDthinfilmsandtoCharlie forthe

assistancewith theoretical concepts especially duringmywriting up period ndash it has

beeninvaluableThankstoProfessorIvanParkinDrKristopherPageandDrStefano

PerniforteachingmaterialschemistrytoamicrobiologistndashitcanrsquothavebeeneasyI

wouldliketoacknowledgetheEngineeringandPhysicalSciencesResearchCouncilfor

financial support Dr Aviva Petrie for providing statistical assistance and Dr Nicky

Mordan forpreparing samples forSEManalysis andhelp inanalysing thegenerated

images

IwouldliketothankthestafffromtheDivisionofMicrobialDiseasesatTheEastman

Dental Institute past and present who made the experience more enjoyable

especially Mike Brouwer (for motivating tea breaks Body Combat Stroopwafels

Bastongne amp beer) Dr Sarah Tubby Linda Dekker Dr Katherine McCurrie Salim

IsmalDrLenaCiricDrRachaelWhealanDrFlorentChangPiDrJohnWrightandDr

GilShalomThankstoDrTomMorganandDrWillKoningforchallengingmyviewson

statisticalanalysis

ThankyoutoKerryWilliamsRebeccaGortonMichelleCairnsandDrCassiePopefor

yourfriendshipsupportloveandscientificadvicendashmy(other)LondonfamilyThanks

to Samantha KaiserHelen Castle CatrionaWright AliceOrsquoSullivan andBeccaOwen

6

for your continued friendship and patience during the tough times Thanks to Emiel

Aiken Dad Mike Nelson and also to the Derbyshire family for your laughter and

continuedsupport

IrsquomindebtedtoDrTimMcHughforhelpingmetobelievethatIcoulddoaPhDandto

Dr Clare Ling and Simon Rattenbury for hiring me as a trainee Clinical Scientist all

those years ago supporting me since and allowing me to pursue a career in

MicrobiologyThankstoDrMathewDiggleandDrKatrinaLeviforyourflexibilityand

understandingduringmywrite‐upperiod

Finally thank you tomyMum for being a constant support inmy life Irsquove enjoyed

sharingmypositiveresultswithyouandyourwordsofencouragementhavekeptme

goingthroughthebadtimesIcouldnothavedonethiswithoutyouThisisdedicated

toyouandtoNannyGrandadandAuntieAnnwhowouldhavelovedtobearoundto

readthis

7

TableofcontentsDeclaration 2

Abstract 3

Acknowledgements 5

Tableofcontents 7

Listoffigures 13

Listoftables 19

1 Introduction 20

11 Healthcare‐associatedinfections 20

111 OrganismscausingHCAIs 22

12 RelevanceoftheenvironmentinHCAIs 26

121 Bacterialsurvivalofdesiccation 31

122 Cleaningfrequencyandstandards 32

123 Levelofsurfacecontamination 34

124 Frequencyofsurfacere‐contaminationpost‐cleaning 36

125 Frequencyofcontactwiththehand‐touchsurface 37

126 Hygienepracticesofstaffpatientsandvisitors 39

13 Antimicrobialcoatings 40

131 Silverasanantimicrobialagent 41

132 Copperasanantimicrobialagent 48

133 Titaniumdioxidephotocatalyticthinfilms 49

14 Relevanceofsurfacesinventilator‐associatedpneumonia 63

141 Photodynamictherapy 66

15 Methodsofproducinglight‐activatedantimicrobialmaterials 70

151 Chemicalvapourdeposition 72

152 Sol‐gel 71

153 Swellencapsulation 72

16 Measuringenvironmentalcontamination 73

161 Swabbing 73

8

162 Dipslides 73

163 Airsampling 74

164 ATPbioluminescence 75

165 Stainingtechniques 77

166 Summaryofenvironmentalsamplingtechniques 78

17 Methods of characterising and assessing the functionality of light‐activatedantimicrobialmaterials 79

171 UV‐visible‐IRspectroscopy 79

172 Photooxidationofstearicacid 79

173 Contactanglemeasurements 81

174 Standardmethodsofassessment 82

18 Overviewandprojectaims 84

2 Materialsandmethods 86

21 Targetorganisms 86

22 Growthconditions 87

23 Preparationofthebacterialinoculum 87

24 Lightsources 87

241 Whitelightsource 87

242 Ultraviolet(UV)lightsources 88

243 Laserlightsource 89

25 Generalsamplingmethodology 89


261 Luminometer‐specificmethodologies 91

27 DirectvisualisationofbacteriandashLiveDeadstaining 93

28 Effectofwhitelightonbacterialsurvival 93

29 Optimisationofthesamplingtechnique 94

210 Preparationoflight‐activatedantibacterialmaterials 95

2101 Thinfilmsgeneratedbychemicalvapourdeposition 95

2102 Thinfilmsgeneratedbysol‐geldeposition 99

2103 Toluidine Blue O‐containing polymers generated by swell encapsulation 101

211 Characterisation and functional assessment of light‐activated antibacterial materials 102

9




212 Microbiologicalassessmentoflight‐activatedantimicrobialmaterials 105

2121 Decontaminationofthethinfilms 105

2122 Measuringtheeffectof lightonthethinfilmsgeneratedbyAPCVDor sol‐gel 105

2123 Measuring the effect of light on Toluidine Blue O‐impregnated polymersgeneratedbyswellencapsulation 107

213 Statisticalanalysis 108

3 Development of protocols used to assess the activity of thephotocatalyticthinfilms 110

31 Introduction 110




323 Measuringtheeffectofwhitelightonbacterialsurvival 114

33 Results 115




34 Discussion 130



343 Theeffectofwhitelightonbacterialsurvival 135

35 Conclusions 138

4 AssessmentofnovelCVD‐synthesisedlight‐activatedantibacterialmaterialsforuseinthehospitalenvironment 139

41 Introduction 139


421 Synthesisofthethinfilms 140

10

422 Measuringtheantibacterialeffectofthethinfilms 140

423 Assessmentofthedecontaminationregimen 141

424 Effectofthecoveringmaterialonthinfilmactivity 141

43 Results 142

431 Photocatalyticactivityoftitaniumdioxidethinfilms 142

432 Photocatalytic antibacterial activity of nitrogen‐containing titanium dioxidethinfilmsTiON‐1andTiON‐2 144

433 Photocatalytic antibacterial activity of nitrogen‐doped titanium dioxidethinfilmsN1N2andN3 149

434 EffectofchangingthedecontaminationregimenonthinfilmN1 153

435 Effectofcoveringmaterialonthinfilmactivity 154

436 Photocatalytic antibacterial activity of sulfur‐based titanium dioxide thinfilms 157

44 Discussion 161

441 UVlight‐inducedphotocatalyticactivity 161

442 Whitelight‐inducedphotocatalyticactivity 162

443 Limitationsoftheexperimentalwork 166

45 Conclusions 168

5 Assessment of novel sol‐gel synthesised light‐activatedantibacterialmaterialsforuseinthehospitalenvironment 170

51 Introduction 171


521 Thinfilmsynthesis 171

522 Characterisationandfunctionalassessmentofthethinfilms 171

523 Antibacterialassessmentofthethinfilms 172

53 Results 173


532 AntibacterialactivityagainstEcoliATCC25922 184

533 AntibacterialactivityagainstEMRSA‐16 189

54 Discussion 195

541 Synthesisofthesilver‐dopedtitaniathinfilms 196

542 Characterisation and functional assessment of the silver‐doped titania thinfilms 197

11

543 Antibacterialactivityofthesilver‐dopedtitaniathinfilms 200

55 Conclusion 203

6 Assessment of a novel antibacterial material for use inendotrachealtubesinintubatedpatients 204

61 Introduction 204


621 Materialsynthesis 206

622 Measuring the antibacterial photo‐activity of the TBO‐impregnated polymers 206

63 Results 207

631 Assessmentoftheantibacterialphoto‐activityoftheTBO‐ impregnated polymersagainstPaeruginosaPAO1atypestrain 207

632 Assessmentoftheantibacterialphoto‐activityoftheTBO‐ impregnated polymersagainstaclinicalstrainofPaeruginosa 213

633 Assessmentoftheantibacterialphoto‐activityoftheTBO‐ impregnated polymersagainstaclinicalstrainofAbaumannii 217

634 Assessmentoftheantibacterialphoto‐activityoftheTBO‐ impregnated polymersagainstaclinicalstrainofSmaltophilia 220

635 Assessmentoftheantibacterialphoto‐activityoftheTBO‐ impregnated polymersagainstaclinicalstrainofCalbicans 223

64 Discussion 226

641 TBO‐mediatedphotodynamicbacterialinactivation 226


643 Novelmaterials for potential use as antimicrobial endotracheal tubes 232

65 Conclusions 234

7 Assessment of the disruptive and anti‐adhesive properties ofnovellight‐activatedmaterials 235

71 Introduction 235


721 Silver‐dopedtitaniumdioxidethinfilms 236

722 TBO‐impregnatedpolymers 240

73 Results 243


12


74 Discussion 256

741 AssessmentofinitialattachmentofEMRSA‐16 256

742 DisruptionofanimmaturebiofilmofEMRSA‐16 258

743 PreventionofinitialPaeruginosaPAO1attachment 260


75 Conclusions 263

8 Concludingremarksandfuturework 265

9 Publicationsarisingfromthiswork 270

91 Peer‐reviewedPublications 270

92 Posterpresentations 271

93 Otherpublications 271

10 References 272

13

ListoffiguresFigure11TheWHOFiveMomentsforHandHygiene 27

Figure12Transmissionroutesofpathogenswithinahospitalenvironment 28

Figure13Schematicofaconductionbandinaconductor 49

Figure14Freemovementofelectronswithinaconductor 50

Figure15Schematicofaconductionbandinaninsulator 50

Figure16Schematicdisplayingthebandgapwithinasolidstatematerial 51

Figure 17 Promotion of an electron from the valence band (VB) to the conductionband(CB)inasemiconductorafterlightabsorption 52

Figure18n‐typesemiconductors 53

Figure19p‐typesemiconductors 53

Figure110Electronicexcitationofasemiconductormolecule 55

Figure111Generationofsingletoxygen 68

Figure112SchematicrepresentationofaCVDapparatus 71

Figure113Chemicalstructureofstearicacid 80

Figure21Spectralpowerdistributiongraphforthewhitelightsource 88

Figure22Experimentalsetupofthemoisturechamber 94

Figure23Thesol‐geldippingapparatus 100

Figure24Whitelightirradiationofnitrogen‐dopedthinfilms 106

Figure31ComparisonofdifferentswabtypestoincreasetherecoveryofEcoliandEfaecalis 115

Figure32ComparisonofdifferentsamplingmethodsusedtoincreasetherecoveryofEcoli 116

Figure33ComparisonofSaureusdetectionmethods 118

Figure34ComparisonofEcolidetectionmethods 120

Figure35EffectofthewhitelightsourceonthesurvivalofSaureusNCTC6571 123

Figure36EffectofthewhitelightsourceonthesurvivalofEcoliATCC25922 124

14

Figure37EffectofthewhitelightsourceonthesurvivalofEfaecalis 125

Figure38EffectofthewhitelightsourceonthesurvivalofSpyogenesATCC12202 126

Figure39EffectofthewhitelightsourceonthesurvivalofEMRSA‐16 127


Figure311EffectofthewhitelightsourceonthesurvivalofMRSA43300 128

Figure312Effectofthewhite lightsourceonthesurvivalofSaureusNCTC8325‐4 129

Figure41Photo‐activityoftheTiO2thinfilms 142

Figure42PhotocatalyticactivityofPilkingtonActivTMonEcoli 143

Figure43EffectofthethinfilmTiON‐2againstEcoliafterexposureto1hour254nmlightand4hours365nmlight 145


Figure 45 Effect of the thin film TiON‐2 on the survival of E coli Thin films wereexposedtowhitelightfor24hoursthebacterialdropletwasaddedthenthesamplewasexposedasecondlightexposureperiodofeither618or24hours 148


Figure47EffectofthethinfilmN1onthesurvivalofEcoliThinfilmswereexposedto white light for 24 hours the bacterial droplet was added then the sample wasexposedasecondlightexposureperiodof24hours 150



Figure 410 Light‐activated antimicrobial killing of E coli on thin film N1 and afterinactivation 154

Figure411ConcentrationofEcoliremainingonthethinfilmTiON‐2usingaclingfilmcovering 155

15

Figure 412 UV‐visible light transmission trace of the petri dish lid and the clingfilmcovers 157

Figure413EffectofthethinfilmS2onthesurvivalofEcoliThinfilmswereexposedto white light for 72 hours the bacterial droplet was added then the sample wasexposedasecondlightexposureperiodof24hours 158



Figure51PhotographoftheAg‐TiO2thinfilms 174

Figure52TransmissiondataoftheAg‐TiO2andTiO2thinfilmsdepositedontoaquartzsubstrateobtainedbyUV‐visible‐IRspectrometry 176

Figure53 Tauc plotsof theUV‐visible‐IRdata taken for the (a)Ag‐TiO2and (b) TiO2thinfilmspreparedonquartzsubstrates 177

Figure54UV‐VisspectrumfortheOptivextradeUVfiltershowingthecut‐offforradiationbelow400nminwavelength 179

Figure55IRabsorptiondatashowingthephoto‐oxidationofstearicacidmoleculesonthesurfaceofthethreematerialsover72hoursusinga254nmlightsource 181

Figure56IRabsorptiondatashowingthephoto‐oxidationofstearicacidmoleculesonthesurfaceofthethreematerialsover96hoursusingawhitelightsource 182

Figure 57 Raw data showing the photo‐oxidation of stearic acid molecules on thesurface of the three samples over 500 hours using a white light source and theOptivextradeUVfilter 183

Figure 58 Effect of the thin film Ag‐TiO2 on the survival of E coli Thin films wereirradiatedwithwhitelightorincubatedinthedarkfor2hours 185


Figure 510 Effect of the thin filmAg‐TiO2 on the survival ofE coli Thin filmswereirradiatedwithwhitelightorincubatedinthedarkfor12hours 187

Figure 511 Effect of the thin filmAg‐TiO2 on the survival ofE coli Thin filmswereirradiatedwithwhitelightfilteredwiththeOptivextradeglassorincubatedinthedarkfor12hours 187

16


Figure513EffectofthethinfilmAg‐TiO2onthesurvivalofEMRSA‐16Thinfilmswereirradiatedwithwhitelightorincubatedinthedarkfor6hours 190


Figure515EffectofthethinfilmAg‐TiO2onthesurvivalofEMRSA‐16ThinfilmswereirradiatedwithwhitelightfilteredwiththeOptivextradeglassorincubatedinthedarkfor12hours 192



Figure 61 A catheter tube impregnated with the photosensitising agent methyleneblue 205

Figure 62 Antibacterial activity of TBO‐impregnated polymer against P aeruginosaPAO1after30seconds 208








Figure610AntibacterialactivityofTBO‐impregnatedpolymeragainstaclinicalstrainofPaeruginosaafter90seconds 214

17



Figure613AntibacterialactivityofTBO‐impregnatedpolymeragainstaclinicalstrainofAbaumanniiafter90seconds 218



Figure616AntibacterialactivityofTBO‐impregnatedpolymeragainstaclinicalstrainofSmaltophiliaafter90seconds 221



Figure619AntimicrobialactivityofTBO‐impregnatedpolymeragainstaclinicalstrainofCalbicansafter90seconds 223



Figure71Theflowcellchamberusedtoassessbacterialattachment 237

Figure72Microtitreplatelayoutforthebiofilmdisruptionassays 241

Figure73AttachmentofEMRSA‐16toeitheran(a)uncoatedslideor(b)Ag‐TiO2thinfilmafter0hexposuretothewhitelightsource 244



Figure76ConfocalmicrographofEMRSA‐16inPBSontheAg‐TiO2thinfilmafter24hoursgrowthat37degCinthedarkand24hoursexposuretowhitelightat22degC 246

18

Figure77ConfocalmicrographofEMRSA‐16inPBSontheAg‐TiO2thinfilmafter24hoursgrowthat37degCinthedarkand24hoursincubationat22degCinthedark 247

Figure78ConfocalmicrographofEMRSA‐16 inBHIontheAg‐TiO2thinfilmafter24hoursgrowthat37degCinthedarkand24hoursexposuretowhitelightat22degC 249

Figure79ConfocalmicrographofEMRSA‐16 inBHIontheAg‐TiO2thinfilmafter24hoursgrowthat37degCinthedarkand24hoursincubationat22degCinthedark 250

Figure710AbilityoftheTBO‐impregnatedpolymerstopreventtheinitialattachmentofPaeruginosaPAO1 252

Figure711 SEM imageofPaeruginosaPAO1on the surfaceofaTBO‐impregnatedpolymerafter3hoursirradiationwiththelaserlight 253

Figure712 SEM imageofPaeruginosaPAO1on the surfaceofaTBO‐impregnatedpolymerafter3hoursincubationintheabsenceoflaserlight 254

Figure 713 Effect of photo‐bleaching on the anti‐P aeruginosa activity of the TBO‐impregnatedpolymers 256

19

Listoftables

Table21Bacterialandfungalstrainsusedinthesestudies 86

Table22Nomenclatureusedduringmicrobiologicalassessmentofthethinfilms107

Table 31 Definitions of the terms used to compare the luminometer‐specificmethodologies 110

Table32ReproducibilityoftheATPbioluminescenceassay‐Saureus 118

Table33ReproducibilityoftheATPbioluminescenceassay‐Ecoli 121

Table34Effectofwhitelightonbacterialsurvival 130

Table41Summaryofthephotocatalyticactivityofthenitrogenandsulfurdopedthinfilms 161

Table51WatercontactanglesoftheAg‐TiO2thinfilmsandthecontrolsamples 178

Table 52 Photo‐oxidisation of stearic acid during irradiation by the different lightsources 184

Table 61 Nomenclature used during microbiological assessment of the TBO‐impregnatedpolymers 207

Table62SummaryofPaeruginosaPAO1experiments 212

Table63ComparisonofthetwoPaeruginosaexperiments 217

Table64TBO‐impregnatedpolymers‐Summaryofresults 226

Table71Confocalscanninglasermicroscope‐samplesdescriptions 240

Table72Resultsofthebacterialattachmentassays 251

20

1 Introduction

11 Healthcare‐associatedinfections

Healthcare‐associated infections (HCAIs)aredefinedbytheDepartmentofHealthas

ldquoany infection by any infectious agent acquired as a consequence of a personrsquos

treatmentinhealthcarerdquo(DepartmentofHealth2008)andtheyareamongthemost

commonadverseevents inhospitalisedpatients (Leapeetal 1991)Organisms that

cause HCAIs are able to cause disease in the susceptible host and survive in the

hospital environment for long periods of time (Dancer 2011) The prevention and

control of HCAIs within healthcare institutions both in the UK and worldwide is a

majorpriorityandtherecentlyreviseddocumentfromtheDepartmentofHealthlsquoThe

Health Act 2006 Code of Practice for the Prevention and Control of Healthcare‐

AssociatedInfectionsrsquodetailsstandardsrequiredtoachievetheseaims(Departmentof

Health 2008) Mandatory surveillance of certain infections such as orthopaedic

surgical site infections and those caused by specific bacteria such as meticillin‐

resistantStaphylococcusaureus(MRSA)andClostridiumdifficilehavebeenintroduced

becauseofthemorbidityandmortalityassociatedwiththoseinfections(Reportbythe

Comptroller and Auditor General ‐ HC Session 2003‐2004) Surveillance data are

updatedfortnightlyandareavailableatwwwdatagovukThemandatorysurveillance

schemewasextendedinJune2011to includeratesofEscherichiacoliandmeticillin‐

sensitive S aureus bacteraemia (Health Protection Agency 2011a) Government

targetsarealsoinplacetoreducetheincidenceofinfectionscausedbySaureusand

CdifficileBothoftheseorganismscanresideinharmonywithinhealthyhumanhosts

but cause serious problemswhen growth is uncontrolled or permitted outside their

usualniches

21

Approximately 17 million HCAIs are acquired annually in the American healthcare

environment resulting in nearly 99000 deaths a year greater than the number of

casesofanynotifiablediseasewithanassociatedcostperpatientofbetween$16359

and $19430 (Scott II 2009)When this figure is scaled up it amounts to a cost of

between$284to338billiondollarsperannum(Klevensetal2007ScottII2009)In

responsetotherisingcostof in‐patientcaretheCentersforMedicareandMedicaid

Services which provide health insurance for certain sections of the American

populationhavediscontinuedpaymenttohospitalsifthepatientisafflictedbyoneof

eight lsquopreventable complicationsrsquo during their stay (Rosenthal 2007) The HCAIs

included in this list are catheter associated urinary tract infections and vascular

catheter‐associated infections An estimated 13000 deaths were caused by urinary

tractinfectionalonein2002(Klevensetal2007)

InEnglandapproximately1 in10patientshaveanHCAIatanyonetimeaccounting

for100000casesand5000deathsperannum(ReportbytheComptrollerandAuditor

General‐HC230Session1999‐2000ReportbytheComptrollerandAuditorGeneral‐

HCSession2003‐2004)PatientsthatacquireanHCAIarerequiredtostayinhospital

for an average of eleven additional days and incur treatment costs of nearly three

timesthatofanuninfectedpatienttheyarealsoseventimesmorelikelytodiethan

patientsthatdidnotacquireanHCAI(Plowmanetal2000ReportbytheComptroller

and Auditor General ‐ HC Session 2003‐2004 2004) The financial cost of HCAIs in

Englandhasbeencalculatedtobeapproximatelypound1billionperannumandupto30

oftheseinfectionscanbeprevented(Plowmanetal2000)Introducingpreventative

measurescostslessthantreatingtheinfectionitselfsointensiveeffortsareinplaceto

22

reduce infection rates (Report by the Comptroller andAuditorGeneral ‐ HC Session

2003‐20042004)

111 OrganismscausingHCAIs

1111 Meticillin‐resistantSaureus(MRSA)

S aureus is found in the anterior nares of 20 of the population (Report by the

Comptroller andAuditorGeneral ‐ HC Session 2003‐2004 2004 Alekshun and Levy

2006)butcausesinfectioninwoundswhichcanleadtoosteomyelitisifitreachesthe

boneabscessesif itpenetratesdeepintothetissuesbacteraemiaandsepticaemiaif

itgetsintothebloodstreamandfromthispointitcouldseedintoanyorganandcause

disseminateddiseaseMeticillin‐resistantSaureus(MRSA)isresistanttotheβ‐lactam

group of antibiotics which was the first line therapy before the widespread

development of resistance This resistance decreases the number of available

treatmentoptionsrequiringtheuseofantibioticswithgreatersideeffectswhichcan

prolongthedurationoftreatmentandthetimespentinhospital

MRSA ismost commonly transmittedbetweenpatientsvia contaminatedhandsbut

thepersistenceoftheorganismintheenvironmentalsoprovidesanimportantsource

AdditionallythepresenceofMRSAinthenasalpassagesofcolonisedpatientsenables

spreadviarespiratorydropletnucleiForthesereasonsthenearpatientenvironment

is often contaminatedwith bacteria and themost likely sources ofMRSAmeticillin‐

sensitive (MSSA) contamination in colonised patients are the floor and bedframe

followedbythepatientlockerandtheoverbedtable(Mulveyetal2011)

23

1112 Glycopeptide‐resistantenterococci

Glycopeptide‐resistant enterococci (GRE) predominantly cause infections of the

bloodstreamabdomenpelvisoropenwoundsinimmunocompromisedpatientsThis

patient group is likely to have had previous antibiotic treatment and a prolonged

hospital stay due to significant co‐morbidities such as liver or renal disease

haematologicalmalignanciesordiabetes(Hanetal2009)usuallyinaspecialistward

such as intensive care or a renal unit (Health Protection Agency 2011b) GRE are

resistant to the glycopeptide group of antibiotics which includes vancomycin and

teicoplaninInfectionsareusuallyeithernosocomialorduetoendogenousinoculation

andaredifficulttotreatduetothelackoftreatmentoptionsandthevulnerabilityof

theaffectedpatient

The first reportsofglycopeptide resistantenterococciweredocumented in themid‐

1980s(Uttleyetal1988)andtherehasbeenasignificantincreaseintheincidenceof

bothGREcolonisationand infectionsincebetween1989and1995theproportionof

glycopeptide‐resistant strains of enterococci isolated in the United States rose from

03to104(Gaynesetal1996)TheemergenceofGREcoincidedwithanincrease

in the use of vancomycin (Ena et al 1993) and it is possible that sub‐inhibitory

concentrationsofvancomycinweregeneratedinthetissuesofthesepatientssothat

vancomycin‐resistance was selected alongside an overgrowth of the resistant

Enterococcusfaecalis(Uttleyetal1988)Arecent10‐yearstudycalculatedthe60‐day

mortalityofpatientswithGREbacteraemiaat57andasstandardempiricaltherapy

oftendoesnot includecover forGREsuitableantimicrobial therapy isoftendelayed

whichfurtherincreasesmortality(Hanetal2009)

24

GRE have increased tolerance to environmental conditions and therefore have an

improved survival rate compared withMRSA However transmission of GRE is less

frequent because the colonisation site is usually the gastrointestinal tract whereas

MRSAcommonlycolonisesthenasalpassagesallowingfortransmissionviarespiratory

droplets (Dancer 2002) Unwashed hands remain an important fomite in the

transmissionofGRE

1113 Cdifficile

C difficile can be found in small numbers in the large intestines of some healthy

humansHoweverwhenthenormalmicrobiotaofthegut iscompromisedeitherby

theuseofbroadspectrumantibioticssuchascephalosporinsduetoco‐morbiditiesor

oldagethecolonisationresistanceeffectofthegutisdepletedwhichallowsCdifficile

to proliferate (Wilcox 1996) The clinical presentation ranges from asymptomatic

carriage through to profuse diarrhoea and in serious cases toxic megacolon and

pseudomembranous colitis which carries a significant mortality rate (Alekshun and

Levy2006)Cdifficile produces toxinsduringgrowthwhichdamage the integrityof

thecolonandthisdamagecontributestotheclinicalsymptomsCdifficile iscapable

of entering a dormant phase during which the bacterial cells sporulate and these

spores have increased resistance to harsh environmental conditions such as

desiccationextremesintemperatureanddisinfectantsSporesareoftenfoundinhigh

numbers in the areas surrounding C difficile positive patients (Dancer 1999) and

elimination of this environmental source has been cited as a contributing factor in

haltingtheonwardtransmissionofinfection(Samoreetal1996)

25

1114 Organismscausingventilator‐associatedpneumonia

Ventilator‐associatedpneumonia(VAP)isanosocomialbacterialinfectionofthelungs

withamultifactorialetiologyAnendotrachealtube(ETT)isplacedalongthetrachea

andisconnectedtoaventilatortoallowmechanicallyassistedbreathingThephysical

presenceofthetube interfereswiththenormalclearingofsecretionssuchasmucus

from the upper airways and allows micro‐aspiration of contaminated subglottic

secretionsintothelungsThesesecretionsarecontaminatedwithcommensalbacteria

which provide a source for a pulmonary infection The lumen of the ETT itself can

become colonised with bacteria providing an additional source of infection The

organisms most commonly implicated are S aureus Pseudomonas aeruginosa

Acinetobacter species and Stenotrophomonas maltophilia (Johanson et al 1972

Weberetal2007Bouadmaetal2010)theseorganismsarenotusualcommensals

of the upper respiratory tract but the normal flora of hospitalised patients tends to

containagreaterproportionofGram‐negativebacilliwhicharealso likelytodisplay

multidrugresistancephenotypesVAPisthemostcommonHCAIintheintensivecare

unitaccountingfor30‐50ofinfectionsandisassociatedwithincreaseddurationof

intubationand increased lengthofhospital stay (Kollefetal 2008Bouadmaetal

2010)

TheestimatednumberofinfectionscausedbyVAPintheUnitedStatesis52543with

anattributablecostofbetween$14806and$27520perpatient(Klevensetal2007)

Whenallnosocomialpneumoniaswereconsideredtherewerenearly36000deaths

intheUnitedStatesandofthepatientsthatsurvivedtheextra lengthofstay inthe

hospitalwas9days(Wenzel1995)

26

12 RelevanceoftheenvironmentinHCAIs

Dr Ignac Semmelweis dubbed the lsquoFather of Infection Controlrsquo first described the

importanceofcleanhandsinthepreventionofinfectionin1861(Semmelweis1861)

Henoticedanincreasedrateofpuerperalfeverinalabourwardattendedexclusively

by clinicians compared toaneighbouringwardattendedexclusivelybynursing staff

Thecliniciansperformedautopsiesoncadaversbeforeattendingtoparturientpatients

butdidnotwashtheirhandsaftertheinvestigationsthusallowingthetransferofthe

lsquocadavericparticlesrsquotothewomeninlabourSemmelweisproposedthatallexaminers

should wash their hands in a solution of chlorinated lime to destroy the cadaveric

materialadheringtothehandsByintroducingthismeasurehereducedtheratesof

childhoodmortalityfrom114in1846to18in1848(Semmelweis1861)

MorerecentlytheNHSNationalPatientSafetyAgencylaunchedthelsquocleanyourhandsrsquo

campaignwiththeaimtoimprovethehandhygieneofhealthcareworkersinorderto

reduce the incidence of HCAIs (NHS National Patient Safety Agency 2004) Hand

hygiene plays an essential role in preventing the transmission of microorganisms

(CasewellandPhillips1977Haydenetal2006Dancer2010)anditisrecommended

both in the scientific literature and by the World Health Organisation that hands

should be decontaminated before and after touching a patient before any aseptic

procedureandafterexposuretobodyfluidsasdetailedinFigure11

27

Figure 11 The World Health Organisation Five Moments for Hand Hygienerecommend hand decontamination after touching the near patient environment(Pittetetal2009)

The guidelines also recommend that hands should be decontaminated after contact

withtheenvironmentsurroundingapatientasevidenceshowsthatsitesclosetothe

patientcanbeheavilycontaminatedwithbacteriaorbacterialspores(Samoreetal

1996WeberandRutala1997Devineetal2001BoyceandPittet2002Oieetal

2007 Dancer et al 2008 Pittet et al 2009) The role of the environment in the

transmission of HCAIs has been demonstrated in the scientific literature and is

illustratedinFigure12

28

Figure 12 Transmission routes of pathogens within a hospital environment Boldarrows indicate potential routes of pathogen transfer and red crosses denote adisruptionintransmission

Two independent routes have been described (Talon 1999 Boyce and Pittet 2002

Boyce2007Dancer2008)

1 A healthcare worker (HCW) contaminates their hands by touching the

environmentthentouchesapatientleadingtomicrobialtransferor

2 Asusceptiblepatienttouchesacontaminatedsurfaceandthemicroorganisms

aretransferreddirectlyfromtheenvironmenttothesamepatient

Surfaces that are frequently touched by people in the hospital environment are

termedlsquohand‐touchsurfacesrsquoandthosethathavebeenstudiedinthemostdetailto

determine levels of microbial contamination include the bed‐frame bedside tables

doorhandlestoiletrailsandtoiletseats(Dancer2004Dentonetal2004Boyceet

29

al2008Danceretal2008Huslageetal2010)Hand‐touchsurfacesinthehospital

environment are being increasingly implicated in the transmission of nosocomial

pathogenspatientcolonisationbytheseorganismsandoutbreaksofHCAIs(Boyceet

al1994WeberandRutala1997Bartleyetal2001DepartmentofHealth2001

Ramplingetal2001Frenchetal2004Johnstonetal2006Dancer2010Dancer

and Carling 2010) In reality adherence to hand washing practices has remained

substandard but even exemplary hand hygiene cannot stop transmission if the

environment has a high bacterial load (Dharan et al 1999 Boyce and Pittet 2002

Dancer20042010Erasmusetal2010)

The risk of acquiring MRSA GRE or C difficile has been demonstrated to be

significantlyhigherinpatientsadmittedtoaroomwhosepreviousoccupanthadbeen

MRSAGREorCdifficilepositive(McFarlandetal1989Huangetal2006Dancer

2009CarlingandBartley2010Shaughnessyetal2011)Dreesetal(2008)showed

patientswhoacquiredGREduringtheirhospitalstayweremorelikelytobeinaroom

inwhichaGRE‐positivepatienthadpreviouslyoccupiedandGREwas isolated from

the near‐patient environment in 25 of cases Bacteria are frequently found to

contaminate hand‐touch surfaces even after cleaning and organisms commonly

foundincludeMRSAGREandothercausesofHCAIssuchasMSSAandAcinetobacter

baumannii (Dentonetal 2004 Lewisetal 2008Boyceetal 2009Mulveyetal

2011)

Theenvironmenthasalsobeenshowntoplaya role inthetransmissionof infection

outsideahospitalsettingAnAmericanstudyshowedanincreasedrateofdiarrhoeal

diseaseinchildrenattendingdaycarecentreswheretheenvironmentwasfoundtobe

30

contaminatedwithfaecalcoliforms(Labordeetal1993)Theenvironmentalsources

implicatedweremoistsitessuchassinksandtapsandatwo‐foldincreaseintherate

of diarrhoea was found in children attending these facitilites compared to centres

withanuncontaminatedenvironmentInaseparatestudyofhouseholdcasesofinfant

salmonellosistheserotypeofSalmonellaexcretedbytheinfectedindividualwasalso

isolated from the environment (van Schothorst et al 1978) Chopping boards have

beencommonly implicated inthespreadofgastroenteritisForexample inadequate

cleaning of a chopping board contaminated with juices from raw turkeys led to an

outbreak of gastroenteritis when the chopping board was later used to prepare

sandwiches Additionally an individual investigating the outbreak also developed

symptomsaftertouchingthechoppingboardbeforesmoking(Sanborn1963)

31

The riskofacquiringan infection fromacontaminatedenvironment ismultifactorial

anddifficulttodirectlyassess(Boyce2007Lewisetal2008)Howeveritislikelyto

belinkedto

bull theabilityoftheorganismtosurvivedesiccation

bull thefrequencyandlevelofcleaning

bull thelevelofsurfacecontamination

bull thefrequencyofrecontaminationaftercleaning

bull the frequencyof contactwith thehand‐touch surfacebyhealthcareworkers

patientsandvisitors

bull thehygienepracticesofthehealthcareworkerspatientsandvisitors

121 Bacterialsurvivalofdesiccation

Somebacterialstrainsaremoreresilienttodesiccationbecauseoftheecologicalniche

theyoccupyForexamplestaphylococcalspeciesarewelladaptedforsurvivalonthe

arid environment of the skin and on environmental surfaces which is likely to be

linkedtomatricand ionicstressresistance(ChaibenjawongandFoster2011)MRSA

has been shown to survive for over 2months on a cotton‐blanket (Duckworth and

Jordens 1990) GRE has been shown to survive for up to 4 months on a polyvinyl

chloride surface (PVC) (Wendt et al 1998) andA baumanniiwas recovered froma

patients room 6months after discharge (Zanetti et al 2007) ConverselyNeisseria

gonorrhoeaethrivesinthemoisture‐richenvironmentofthegenitalandbuccaltracts

but is not so well adapted for survival on the predominantly dry hospital surfaces

32

(Griffith et al 2000) Furthermore some epidemic strains of MRSA (EMRSA) have

beenshowntohaveanincreasedsurvivalrateandcansurviveintheenvironmentat

higherconcentrationsthansporadicstrains(Farringtonetal1992Wagenvoortetal

2000)Thisprovidesaselectiveadvantageandcontributestowardsitspersistenceand

endemicityinthehospitalenvironment(Talon1999)Cdifficilesporescansurvivein

the environment formany years and spores are resistant to hand decontamination

products such as alcohol hand gels which further contributes to the persistence of

theseorganismsintheenvironment(BAPS1994)

122 Cleaningfrequencyandstandards

Thepurposeofcleaningistwofoldthemicrobiologicalpurposeistoreduceboththe

microbial load and any nutrientswhich support bacterial growth or substances that

inhibittheactivityofdisinfectantsthenon‐microbiologicalpurposeisaestheticandis

torestoretheappearanceofthematerialandpreventdeterioration(Collins1988)As

thoroughcleaningcanreducethemicrobialloadthenitcanassistinbreakingthecycle

of transmissionof infectionwithin thehospitalenvironment (Dancer2002 Lewiset

al 2008) Indeed regular disinfection of surfaces has been shown to reduce the

transmission of hospital pathogens by 40 and enhanced cleaning of the patient

environment reduces acquisition of bacteria known to cause HCAIs (Hayden et al

2006 Boyce 2007 Carling and Bartley 2010) Despite this the frequency and

standard of cleaning has decreased in recent years due to out‐sourcing of contracts

andlimitationsoncleaningbudgets(Dancer1999Carlingetal2008Dancer2008)

33

Cleaningwithadetergentsolutionisusuallysufficientbuttheuseofdetergentalone

hasbeenshowntoleadtoanincreaseinbacterialcontaminationofhospitalsurfaces

(Dharanetal1999Dancer2011)Asporicidalagentsuchasachlorinecontaining

formulationisrequiredwhentheenvironmentiscontaminatedwithCdifficile(Weber

andRutala2011)

UsingATPtoassessthecleaningprocessisaneffectivetoolasthetotalorganicsoiling

ofasurfacecanbedetermined(HawronskyjandHolah1997)Asurfacecouldbefree

from microbial contamination but could still contain a high level of organic soil

originating from food residues which would provide nutrients to support microbial

growth(Whiteheadetal2008)Deadbacteriaandviablebutnon‐cultivable(VBNC)

organismscanalsobedetectedusingATPbioluminescenceandwouldbemissedby

traditional culturing methods (Poulis et al 1993) ATP bioluminescence has been

shown to be a good indicator of the cleanliness of a surface and of likely bacterial

contamination(Griffithetal2000Maliketal2003Andersonetal2011)

The Department of Health has drawn up a set of lsquoStandard Principles for the

PreventionofHealthcare‐AssociatedInfectionsrsquoforhospitalstoadhereto(Department

ofHealth 2001NHS Estates2004)The first guidelinecovers themaintenanceofa

clean hospital environment and describes the potential link between inadequate

environmentalhygieneandthespreadofmicroorganismscapableofcausingHCAIsIt

recommends that the hospital environment should be visibly clean and free from

soilageanddustbutnomicrobiologicalguidance isprovided(DepartmentofHealth

2001)Morerecentguidancestatesthathospitalsalsohavetoprovideandmaintaina

clean and appropriate environment for healthcare (Department of Health 2008)

34

althoughnospecificrecommendationonthecleanlinessoftheenvironmentisgiven

TheAmericanbasedCenters forDiseaseControlandPreventionhaveacknowledged

this association in a set of guidelines which recommend cleaning or disinfection of

environmentalsurfacesonaregularbasisinadditiontowhenvisiblysoiled(Rutalaet

al 2008) and more frequent cleaning and disinfection of high‐touch surfaces than

minimaltouchsurfaces(Sehulsteretal2003)Theserecommendationsareallbased

onvisualassessmenttodeterminethecleanlinessoftheenvironmentwhichisapoor

indicationoftheefficiencyofthecleaningprocess(Maliketal2003)

However proposed cleaning standards are not always adhered to This is

demonstratedbyanenvironmentalauditofarenalunitinanAustralianhospitalthat

showed just 43 of theminimum standardswere beingmet during an outbreak of

GRE (Bartley et al 2001) The epidemic was terminated with a combination of

measures including enhanced environmental cleaning and isolation of colonised

patientstopreventonwardtransmission

123 Levelofsurfacecontamination

Thelevelofenvironmentalcontaminationispartlydependentonthepatientsrsquositeof

colonisationorinfectionpatientswithMRSAintheurinestoolsorinawounddisplay

higher levelsofenvironmentalcontaminationthanpatientswithMRSA isolatedfrom

other body sites (Rutala et al 1983 Boyce et al 1997 2007 2007 2008) The

environment surrounding a GRE‐positive patient was seven times more likely to be

contaminatedwithGREthananun‐colonisedpatient(Haydenetal2006)andwhen

the routine environmental cleaning regimen was improved a decrease in

35

environmentalcontaminationwasobservedCertainlycontaminatedroomsarearisk

factor for the acquisition of nosocomial pathogens (Hota 2004) and a positive

correlationhasbeendemonstratedbetweenthelevelofAbaumanniienvironmental

contamination and the number of patients colonised or infectedwithA baumannii

(Dentonetal2004)

Theminimumlevelofcontaminationonasurfaceneededtoinitiatecolonisationofa

patientwhich could lead to an infection has not been quantified and is difficult to

measureMicrobiological standards have been proposed for hand‐touch surfaces in

hospitalsinanattempttodeterminewhetherthemicrobialcontaminationofagiven

surface presents a risk of infection for any patients in that vicinity (Dancer 2004

Mulvey et al 2011) It was proposed that an integrated and risk based approach

should be used encompassing visual assessment rapid assays to detect organic soil

and microbiological testing The standards for the microbiological assessment were

splitintotwosections(i)thepresenceofindicatororganismsand(ii)thetotalaerobic

colonycount

Indicator organisms are pathogens that pose a significant threat to patients and

include MSSA MRSA C difficile Salmonella species multi‐drug resistant Gram‐

negativebacilliGREanda numberofotherorganisms thatare important in certain

clinical situations such as Aspergillus species in a ward for severely

immunocompromised patients This standard was set at less than 1 cfu cm2 The

secondstandardwassettoprovideanindicationofthecompletemicrobialloadona

given surfaceasahighmicrobial loadonahand‐touch surface is likelyto represent

poorenvironmentalcleaningandtheheavygrowthofotherorganismsmayshieldthe

36

presenceofanindicatororganism(Dancer2004)Thisstandardwassetatlessthan5

cfucm2Thesestandardshavesincebeentestedandadapted indifferenthospitals

using various detection systems to validate the set benchmarks and are still under

review(Griffithetal2000Maliketal2003Ayciceketal2006Griffithetal2007

Oieetal 2007Danceretal 2008 Lewisetal 2008Dancer2011Mulveyetal

2011)

124 Frequencyofsurfacere‐contaminationpost‐cleaning

Thehospitalenvironmentisrapidlyre‐contaminatedaftercleaning(WeberandRutala

1997)andhospitalfloorscanbecomere‐contaminatedtothesamelevelasbeforethe

cleaning event within 2 hours (Collins 1988 Dettenkofer and Spencer 2007)

Benchmarkscouldbeusedtoestablishhowlongittakesforasurfacetobecomere‐

contaminated after cleaning so that the frequency of cleaning could be optimised

(Lewis et al 2008) Bed occupancy rates also have an effect on the microbial

contaminationofthehospitalenvironmentandtheriskof infectionwithMRSAOne

studydemonstratedgreaterbacterialcontaminationofsampledhand‐touchsurfaces

whenbed occupancy rateswere above 95 comparedwith bed occupancy rates of

below80anda separate study showed the riskof cross‐infectionwithMRSAwas

increasedforpatientsinafive‐beddedbaycomparedwiththoseinafour‐beddedbay

(Kibbleretal1998Danceretal2008)Bedmakinghasalsobeenshowntoincrease

airborne levelsofSaureuswhich thenhave thepotential to settleonnear‐patient

surfacesand further contaminate theenvironment (Shiomorietal 2002Hansenet

al 2010) Re‐contamination of the patient environment is not surprising given that

viable skin colonising microorganisms are carried on skin squames one million of

37

which are shed from healthy skin each day efficiently transferring bacteria into the

immediatesurroundings(Noble1975)

Bacterial contamination of the environment is not necessarily detrimental to a

patientrsquos health Bacterial contamination of the hospital environment is ubiquitous

even though the environment is dry and free from substances that encourage

microbial growth (Collins 1988 Dettenkofer et al 2011) Gram‐positive cocci are

most commonly found and more than 99 are likely to be coagulase negative

commensals and thus unlikely to cause serious disease To create an environment

completely free from bacteria would require sterilisation which is both impractical

andunnecessaryItwouldhoweverbeadvantageoustocreateanenvironmentwhere

thebacterialpopulationpresentdoesnotcontainpathogensand isunlikelytocause

infection(Collins1988)

125 Frequencyofcontactwiththehand‐touchsurface

Bydefinitionhandcontactuponhand‐touchsurfaces is frequentsothenumbersof

occasions for thepotential transferof pathogens fromcontaminatedhands to these

surfaces or vice versa is high The near‐patient environment contains numerous

hand‐touchsurfacesonanintensivecareunitforexampletherearevariousitemsof

instrumentationsuchasventilatorsandmonitorsthatcouldbepotentialreservoirsof

infection (Dancer 2008) Nursing staff rather than domestic staff are usually

responsible forcleaningthesesurfacesand it isoftena lowprioritytask in factonly

40 of these surfaces were shown to be cleaned adequately (Dancer et al 2008

Dancer 2009 Carling and Bartley 2010) Ten hand‐touch surfaceswere sampled in

38

two surgical units over a one year period and itwas found that near‐patient hand‐

touch sites cleaned by trained nursing staff were most likely to fail microbiological

hygiene standards as opposed to surfaces cleaned by domestic staff (Dancer et al

2008)Dentonetal(2004)clearlydefinedtheresponsibilityforcleaningthesehand‐

touch surfaces to thedifferent staff groupsduringanoutbreak ofAbaumannii and

this measure along with a number of others assisted in terminating the outbreak

Andersonetal(2011)demonstratedmorerecentlythatsurfacescleanedbydomestic

staff are more likely to pass defined hygiene standards than surfaces which are

cleanedbyotherstaffsuchasnursesandclinicalsupportworkers

Hands are an important fomite implicated in the transfer of pathogens between

patientsandimprovementsinroutinecleaningregimenshavebeenassociatedwitha

decrease in the contamination on the hands of healthcare workers (Hayden et al

2006) An association has been demonstrated between positive cultures from the

hands of healthcare workers and C difficile environmental contamination which

impliesthattheenvironmentcanplayarole in contaminatingthehandsofthestaff

(Samore et al 1996 Weber and Rutala 2011) Bhalla et al (2004) showed the

transfer of pathogens from the near‐patient environment to the hands of the

investigatorsinoverhalfofthesamplingoccasionsandsurprisinglypathogentransfer

occurred inoccupiedpatientroomsregardlessofthecolonisationor infectionstatus

of the patient These examples demonstrate the importance of adhering to defined

cleaningstandardswithdefinedrolesandresponsibilitiesforstaffmembers

39

126 Hygienepracticesofstaffpatientsandvisitors

There isa largevariation in thehandhygienepracticesofhealthcareworkersanda

recent systematic review of 96 studies reported hand hygiene compliance rates

ranging from 4 ndash 100with an overall average rate of 40 (Erasmus et al 2010)

Compliancewaslowerintheintensivecareunitsettingamongstcliniciansandbefore

patient contact even though this is the first of TheWorld Health Organisation Five

Moments for Hand Hygiene (Pittet et al 2009 Erasmus et al 2010) Intervention

campaigns to improve hand‐washing compliance are often effective during and

immediatelyafterthecampaign(Chengetal2011)butcomplianceratesoftendrop

inthemonthsaftertheintervention

Educating staff about the importance of cleaning the hospital environment has

resulted in improvements in the quality of cleaning as assessed by a number of

methodsUVpowdersandgelshavebeenappliedtosurfacestoassesstheefficiency

ofthecleaningregimenandanincreaseincleaningrateswasachievedafterfeedback

of surveillance results (Carling et al 2008 Munoz‐Price et al 2011) ATP

bioluminescencehasalsobeenusedtoassesscontaminationonhand‐touchsurfaces

and a reduction in the relative light unit (RLU) values was observed after a similar

education programmes (Poulis et al 1993 Griffith et al 2007 Boyce et al 2009

Mulvey et al 2011) Patient and visitor involvement in hand decontamination also

decreasesbacterialcontaminationofthehealthcareenvironment

40

13 Antimicrobialcoatings

Antibacterial materials could be used to supplement cleaning of the hospital

environmentandTheCentresforDiseaseControlandPreventionrecommendfurther

evaluating implementation of antimicrobial materials for use in the hospital

environment(Rutalaetal2008)Ithasbeenshownthatbacteriacanbespreadfrom

acontaminatedareatoanon‐contaminatedareaduringthecleaningprocess(Dharan

et al 1999) Recontamination of the hospital environment also occurs readily after

cleaning events (Collins 1988) and cleaning has often been found to be inadequate

with studies showing only 34 compliancewith policies (Carling and Bartley 2010

Carlingetal2010)

Ifhospitalsurfaceswerecoatedwithanantibacterialmaterialthenthecontaminated

areaswouldbesusceptibletothekillingeffectofthecoatinganddecontaminationof

theaffectedareascouldoccur inbetweencleaningeventsContinuousprotectionof

thehospitalenvironmentinthiswayhasbeenproposedbyanumberofauthorsasan

adjunct to other infection control procedures (Casey et al 2010) Reducing the

bacterial load in the environment can help to prevent person‐to‐person spread of

bacteriaandthedevelopmentofinfection

MRSAhasbeen isolatedfromcomputerkeyboardswithinahospitalward (Devineet

al 2001) howeverwhen self‐cleaning keyboardswere used in a surgicalward in a

Scottish hospital sampled surfaces were consistently below the defined ATP

benchmarks and passed the hygiene standards in the cleanliness audit (Anderson et

al2011)

41

131 Silverasanantimicrobialagent

Silver has a broad spectrum of activity and is active against Gram‐negative and ‐

positive bacteria fungi viruses and protozoa (Davies and Etris 1997 Martinez‐

Gutierrezetal2010)Theantibacterialeffectofsilverhasbeenknownforcenturies

andwas used by the ancient Egyptians and Greeks to treat infectious ailments For

exampleHippocratesdescribedtheuseofasilverpowdertotreatulcers(Hippocrates

400 BC) and at around the same time Alexander the Great kept his drinkingwater

clean by the use of silver water vessels (White 2002) Silver was re‐introduced for

topical applications in the 1960s in the forms of silver nitrate or silver sulfadiazine

especiallyinthepreventionofwoundinfections(Moyeretal1965Foxetal1969)

Inmore recent times silver has been coated ontomany substrates or impregnated

throughoutsubstancestoprovideantibacterialprotection(MelaiyeandYoungs2005)

Theuseofsilvernanoparticlesisincreasingduetotheirhighantibacterialactivityand

smallsizewhichprovidesalargesurfaceareatovolumeratio(Rupareliaetal2008

Lvetal2010)

1311 Mechanismofaction

Themechanismbehindtheantibacterialactivityofsilverandothermetalionsisdueto

theoligodynamiceffectfirstdescribedbyKarlWilhelmvonNaumlgeliasthelethaleffect

thatsmallmetalionsexertonlivingcells(Kraemer1905)Silverbindstothiolgroups

on the bacterial proteins including the ribosome and NADH dehydrogenase which

inhibitstheexpressionofenzymesrequired inATPproductionandpreventselectron

transfer and respiration respectively (Davies and Etris 1997 Plowman et al 2000

Percivaletal2005Yamanakaetal2005Kimetal2008Liuetal2010)Oxidation

42

ofkeycomponentsoftherespiratorypathwayinhibitsbacterialrespiration(Braggand

Rainnie1974)andsilveralsoreactswithmicrobialDNAtocausethefreeDNAtoform

a condensedAg‐DNA complex in the centre of the cellwhich results in a loss in its

replicative function (Feng et al 2000Melaiye and Youngs 2005) Externally silver

targetsthebacterialcellmembraneandonceboundcausespittingand interference

of membrane function which has been visualised by electron microscopy (Clement

andJarrett1994Linetal1996Percivaletal2005Kimetal2007)Interactions

withthecellmembranealsocauseacollapseintheprotonmotiveforceleadingtothe

leakageofH+de‐energisationof themembraneandcelldeath (Dibrovetal 2002)

Silver nanoparticles have also been shown to form silver‐sulfur aggregates on the

surfaceofbacterialcellswhich interfereswiththegenerationof freeradicalswhich

cancausedamagetobacterialcellmembranes(Kimetal2007)

Serious adverse effects of silver in humans is limited to neurotoxicitywhich is only

experienced if theblood‐brainbarrier isbreechedand invitro toxicitytomammalian

cells has not been replicated in the treatment of wound infections (Melaiye and

Youngs2005Tayloretal2009)

Zone of inhibition or agar pour plate tests were used to demonstrate the diffusible

antibacterialactivityofsilver‐basedcompoundsagainstarangeofbacteriaincludingE

coli Klebsiella pneumoniae P aeruginosa Streptococcus mutans S epidermidis S

aureusBacillusanthracisAcinetobacterbaylyiMycobacteriumfortuitumandCandida

albicans(Furnoetal2004Ebyetal2009DurucanandAkkopru2010Gerasimchuk

etal2010Pollinietal2011Riveroetal2011)Thisdiffusibleantibacterialactivity

wouldbeadvantageousforimplantsorsurgicalinstrumentstogiveaninitialhighdose

43

of silver to the surrounding environment which would decrease the likelihood of

resistancedeveloping(Stobieetal2008)Thereleaseofsilver fromthesurfacecan

be further controlled bymodifying the composition of the coating (Liu et al 2010)

Combiningsilverwithanantibioticagentcanfurtherenhancetheantibacterialactivity

(Fox1968Shahverdietal2007Kimetal2008)

1312 Resistancetosilver

Silver isabiocideandassuchhasmultiplemodesofactionunlikeanantibioticthat

tendstotargetaspecificsite(Percivaletal2005)Biocidesthereforehaveabroader

spectrum of activity and resistance is less likely to occur Silver resistance was not

detectedinanybacterialstrainscausingurinarytractinfectionsinpatientswithsilver‐

coated catheters in situ over a 12‐month period (Rupp et al 2004) However

resistance has been identified inmany species of bacteriamainly from burns units

where silver‐based dressings are used to prevent bacterial infection (Clement and

Jarrett1994Silver2003)

A strain of silver‐resistantSalmonellawas isolated froma hospital inMassachusetts

andtheresistancedeterminantwasfoundtobea180kbplasmidpMG101(McHughet

al 1975) Much work has since been performed on this plasmid to elucidate the

molecular basis for resistance and the sequenced region is available on Genbank

(Gupta et al 1999) The gene cluster includes a periplasmic silver‐specific binding

protein(SilE)andtwoparalleleffluxpumps(SilPandSilCBA) (Guptaetal1999)and

amplification of these genes provides a rapidmethodof identifying resistant strains

(Percival et al 2008) Genotypic resistance does not typically translate directly into

phenotypic resistance three strains of Enterobacter cloacae isolated from burn

44

woundswerefoundtocarrytheseresistantgenesbutstilldemonstratedsusceptibility

to therapeutic levels of silver in vitro (Percival et al 2005) The widespread

developmentofresistancetosilver isunlikelyasbacteriahavebeenexposedtosub‐

inhibitory concentrations of this metal ion for centuries however greater use will

increasethelikelihoodofresistancedeveloping(Percivaletal2008)

1313 Applicationsofsilverasanantimicrobialmaterial

13131 Centralvenouscatheters

Silver‐coatedcathetershavebeendevelopedwiththeaimtoreducetheprobabilityof

developingline‐associatedinfectionswhichareacommoncauseofHCAIs(Noimarket

al2009Syedetal2009)Experimentallysilver‐coatedpolyurethanecatheterswere

inserted intoaratmodelandbacteriacouldnotbe isolatedfromthesurfaceofthe

linesafter6weeksimplantationintheinternaljugularvein(Bambaueretal1997)A

significant reduction in E coli adhesion on silver‐coated polyurethane catheterswas

demonstrated in vitroandofthosebacteriathatdidadhereagreaterproportionof

cells found on the silver‐containing polymer were non‐viable compared to the

uncoatedcontrols(Grayetal2003)

13132 Urinarycatheters

The American‐based Healthcare Infection Control Practices Advisory Committee

publishedguidelinesdetailingbestpractices inthepreventionofcatheter‐associated

urinarytractinfectionandtheuseofantimicrobialcathetersweretobeconsideredif

othermethodsofdecreasingratesofinfectionwerefailing(Gouldetal2010)Inthe

USAa trialon theuseof silverhydrogel coatedcatheterswas conductedcompared

45

with standard siliconehydrogel urinary catheters and the incidence of catheter‐

associated urinary‐tract infections fell from63 infections per 1000 catheter days to

26infectionsper1000catheterdaysachievinga57reductionoverall(Ruppetal

2004) In a separate study a 60 reduction in catheter‐associated urinary‐tract

infectionswasachievedfollowingintroductionofsilvercoatedcathetersachievingan

annual saving estimated to be in the region of pound38000 and the release of 192 bed

days(ReportbytheComptrollerandAuditorGeneral‐HCSession2003‐2004)

1314 Endotrachealtubes

An endotracheal tube (ETT) containing silver nitrate and sodium hydroxide reduced

adhesionofPaeruginosa(Monteiroetal2009)andanumberofotherstudieshave

demonstrated clinical efficency of silver coated ETTs this is further discussed in

Section 14 Silver coated endotracheal tubes have been approved for clinical use in

the USA but the increased cost and risk of breakthrough events of VAP have

preventeditsrsquowidespreaduse(Raadetal2011)

1315 Environmentalsurfaces

Silver‐based compounds can also be employed on inanimate surfaces which could

potentiallybeaddedtohand‐touchsurfacessol‐geldepositionwasusedtosynthesise

silver‐doped phenyltriethoxysilane films that prevented S epidermidis adhesion and

biofilm formation over a 10‐day period (Stobie et al 2008) Silver‐doped TiO2 and

titaniumnitridethinfilmscausedsignificantdecreases intheviabilityofSaureusE

coliStreptococcuspyogenesandAbaumannii(Kellyetal2009Wongetal2010)P

aeruginosa appeared more sensitive to the titanium nitride films and growth was

46

inhibitedforupto7dayssupportingthehypothesisthatGram‐positivebacteriaare

more resistant to the antibacterial effects of silver This could be due to the larger

amount of negatively‐charged peptidoglycan in the thicker Gram‐positive cell wall

whichcouldbind silver thus reducing the silveravailable toactupon the interiorof

thecell to causedamage (Schierholzetal 1998Kawaharaetal 2000Grayetal

2003Monteiroetal 2009)Howeverothergroupshave shown thatGram‐positive

and ‐negative strains possess similar susceptibility to silver (Ruparelia et al 2008

Wongetal2010)Inarecenthospitalstudyarangeofsilver‐coatedproductswere

placed in ward areas to monitor the effect on bacterial contamination of the

environment and up to 98 fewer bacteria were recovered from the environment

compared with a control ward which contained uncoated products (Taylor et al

2009)Theantimicrobial activity lasted for thedurationof the12‐month testperiod

andadverseeffectstosilverwerenotreported

1316 Otherapplications

Surgicalmaskshavebeenimpregnatedexperimentallywithtitaniumdioxide(TiO2)and

silvernanoparticlesandnoviableSaureusorEcoliwasdetectedafter48hoursNo

adversereactionswereobservedinhumanvolunteers(Lietal2006)Silverhasbeen

incorporated intodental composite resinsanda slowand sustained releaseof silver

intothesurroundingenvironmenthasbeendemonstratedwitha6‐logreductioninS

mutans growth after 12 hours (Kawashita et al 2000) These composites could

potentiallyreduceinfectivecausesofsurgicalimplantfailure(Floresetal2010)Silver

nanoparticleshavebeen incorporatedwith lysozymeandcoatedonto stainless steel

surgical blades and needles and significant antibacterial activity against a panel of

47

Gram‐positiveandGram‐negativebacteriawasobserved(Ebyetal2009)Silverwas

added to an ethanol‐based disinfectant to generate additional residual antibacterial

activitypost‐application(Bradyetal2003)Silvernanoparticleshavealsobeenused

inenvironmentalsettingssuchasinwastewatertreatment(Linetal1996Daviesand

Etris1997)

132 Copperasanantimicrobialagent

TheantibacterialactivityofcopperhasalsobeenknownforcenturiesandHippocrates

describeditasacureforulcers(Hippocrates400BC)Awiderangeofmicroorganisms

aresusceptibletocopperincludingSaureusEcoliCdifficileEfaecalisEfaecium

Mycobacterium tuberculosisAspergillus fumigatusCalbicansand influenzaAH1H1

(Grassetal2010)Copper‐dopedTiO2coatingswereappliedtoatitaniumalloyasa

model formetal implants used for total joint arthroplasty and a 6‐log reduction in

MRSAgrowthwasobservedafter24hourscomparedwiththeTiO2coatingswithout

the copper ions (Haenle et al 2010) Noyce et al (2006) inoculated MRSA onto

coppersurfacesandwereunabletorecoverviablebacteriafromthesurfacesafter45

minutesincubationatroomtemperatureSignificantreductionswerealsoachievedat

4degC and frombrasswhich contains 80 copper although extended exposure times

wererequired

Coppersurfaceshavebeenassessedfortheiruseinthehealthcareenvironmentinthe

UKUSAChileandJapan(Pradoetal2010Schmidtetal2011KeevilandWarnes

2011)Copper‐containingtapsdoorpushplatesandtoiletseatswere installed inan

acute medical ward in the UK and compared with non‐copper containing control

48

surfaces and the level of bacterial contamination found on the copper‐containing

surfaceswassignificantly lowerthanthatfoundonthecontrolsurfaces(Caseyetal

2010)Thetoiletseatandtaphandlesurfacespassedthebenchmarkmicrobiological

standards proposed by Dancer (2004) for hand‐touch surfaces whereas 50 of the

controlsurfacesfailedHoweverthecleanlinessofthesurfaceaffectscopperactivity

and cumulative soiling and cleaning of copper surfaces was shown to inhibit

antibacterial activity this decrease in antibacterial activity was not observed on

stainlesssteelcontrolsurfaces(AireyandVerran2007)

The mechanism of activity of copper has been shown to be predominantly due to

disruption of cellular respirationDNAdamage by the generation of reactive oxygen

and ionic copper species which cause damage to bacterial enzymes and proteins

(Yoshidaetal1993Noyceetal2006Weaveretal2010)Thecellmembranemay

also be damaged during exposure to copper which leads to rupture and loss of

membranepotential (Grassetal2010)althoughthis isnotthemainmechanismof

celldeath(WarnesandKeevil2011)

133 Titaniumdioxidephotocatalyticthinfilms

Titanium dioxide has inherent light‐activated antibacterial activity and its

functionalitieshavealreadybeencommerciallyexploitedTiO2 coatingsareavailable

as self‐cleaning glasses with Pilkington Activtrade and Saint Gobain BIOCLEANtrade as the

marketleadersTheglasscanbeusedinwindowsconservatoriesandglassroofsand

requires less frequent cleaning because of the dual photocatalytic and

superhydrophilic activities of TiO2 Modified TiO2 has the potential for use in

49

healthcare institutions to reducebacterial contamination of theenvironmentbut to

understand how the TiO2 thin films are activated by light to exert an antibacterial

effect it is firstnecessarytogainabasicunderstandingofbandtheoryofsolidstate

materials

1331 Bandtheoryofsolids

Solid state materials can be split into three categories conductors insulators and

semiconductors (West1999)Their characterisationwithinthesegroupsdependson

theband structurewhich in turn dependson thepositioningof theelectronswithin

theatomsandmoleculesastheycometogethertomakeasolidmaterialElectronsare

arrangedintobandsthatcontainspaceorlsquoholesrsquofortheelectronstoexistinNotwo

electronscanoccupythesamespaceanditispreferentialfortheelectronstoexistin

pairsThecategoryofthesoliddependsuponthenumberofspacesavailableandhow

manyelectronstherearetofillthesespaces

13311 Conductors

Materialscharacterisedasconductorshaveanlsquounfilledconductionbandrsquo(Figure13)

Figure13Schematicofaconductionbandinaconductor

Electronhole

Electronlyingwithinahole

50

Theelectronsinconductorsarefreetomovefromoneholetoanotherwithnoenergy

inputandahole isleftinthespacefromwhichtheelectronhasmoved(Figure14)

The electrons are able to transport charge because of this free movement and

therefore the material is an electronic conductor Metallic materials fall into this

category

Figure14Freemovementofelectronswithinaconductor

13312 Insulators

If theconductionbandofamaterial is full (Figure15) theelectronsarenotableto

moveandsoconductionofelectricitywillnotbepossibleThismaterialisclassifiedas

aninsulator

Figure15Schematicofaconductionbandinaninsulator

Electronhole


51

13313 Semi‐conductors

Inadditiontothepreviouslydescribedbandsanadditionalsetofelectronholesalso

exists above the conduction band and there is a further set found above that

However an input of energy is required in order to promote an electron from the

valence band (highest band occupied by electrons) to the conduction band (lowest

bandwithspacesforelectrons(Figure16))Thisenergyinputiscalledthebandgap

Figure16SchematicdisplayingthebandgapwithinasolidstatematerialwhereCB=conductionbandandVB=valenceband

The band gap of insulators like rubber is very high and a large input of energy is

required to promote the electron to the conduction band Semiconductors however

have an accessible band gap (Figure 17) a small amount of energy is required to

promoteanelectron to theconductionbandand thus createa conductoroutofan

insulator (Carp et al 2004) Once the electron has been promoted conduction can

occurviatwopossiblerouteseitherwithinthevalencebandusingthepositiveholes

createdorwithintheconductionbandsthroughthemovementofelectrons

Electronhole


Bandgap

CB

VB

52

Figure17Promotionofanelectron fromthevalenceband (VB) to theconductionband(CB) inasemiconductorafterabsorptionof lightwithawavelengthmatchingthebandgapenergyofthematerial

Theexcitedelectroncansubsequentlyfallfromtheconductionbandintoaholeinthe

valencebandwhichresultsintheemissionoflightenergyofthesamewavelengthas

theabsorbedincidentrayAlternativelysemi‐conductormaterialssuchasTiO2canbe

dopedwithelementssothattheseparationoftheholeandelectroncanbestabilised

andtheabsorbedenergycanbeutilised

13314 DopedSemiconductors

Doped semiconductors can be classified into one of two groups depending on the

chemical properties of the dopant material n‐type semiconductors or p‐type

semiconductorsInann‐typesemiconductorthedopantmaterialhasavalenceband

which isslightly lower inenergythantheconductionbandofthesemiconductorbut

higherinenergythanthevalencebandofthesemiconductor(Figure18)(Carpetal

2004)Conductionoccurswhenanelectronispromotedfromthevalencebandofthe

dopanttotheconductionbandofthesemiconductorwhichrequireslessenergythan

thenormalelectronictransition

Electronhole


Lightin

CB

VB

53

Figure18n‐typesemiconductors‐positioningofthedopantvalencebandinrelationtothesemiconductorconductionband(CB)andvalenceband(VB)

Alternativelyinap‐typeconductorthedopantmaterialhasaconductionbandwhich

isslightlylowerinenergythantheconductionbandofthesemiconductor(Figure19)

Electronsaretrapped inthedopantconductionbandandconductionoccursthrough

the positive holes The number of electrons should always equal the number of

positiveholesbecausetheproductionofasinglefreeelectronresultsinthecreation

ofasinglepositivehole

Figure 19 p‐type semiconductors ‐ positioning of the dopant conduction band inrelationtothesemiconductorconductionband(CB)andvalenceband(VB)

Anumberofprocessescanoccuronthesemiconductorafterelectronicexcitationand

thesearesummarised inFigure110(MillsandLeHunte1997)Anelectron(‐)anda

positivehole(+)aregeneratedandasmentionedpreviouslyTheelectroncouldreturn

Normaltransition

Dopantmaterialwithlower

conductionband

CB

VB

Normaltransition

Dopantmaterialwithhighervalenceband

CB

VB

54

to the valence band of the semiconductor which is termed electron‐hole

recombinationThisprocesscouldoccuronthesurfaceofthesemiconductor (Figure

110 i) or within the bulk of the semiconductor (Figure 110 ii) Alternatively the

electroncouldreduceanelectronacceptor ina redoxreactiononthesurfaceofthe

semiconductor(Figure110iii)orthepositiveholecouldoxidiseanelectrondonoron

thesurfaceofthesemiconductor(Figure110iv)

55

Figure110Diagramtoillustratethemainreactionstakingplaceonasemiconductormoleculeafterexposure toa light sourcecausingelectronicexcitation (i)electronholerecombinationatthesurface (ii)electron‐holerecombination inthebulk (iii)reductionofanelectronbyanelectronacceptorat the surface (iv)oxidationofapositive hole by an electron donor at the surface Figure amended from thesemiconductorreviewbyMillsandLeHunt(MillsandLeHunte1997)

1332 Titaniumdioxideasasemiconductor

Titanium dioxide (TiO2) is commonly used as a semiconductor as it is inexpensive

chemically stable non‐toxic possesses a high refractive index and has excellent

transmission inthe infraredandvisibleregions(DoboszandSobczynski2003Parkin

andPalgrave2005Dunnilletal2011)TiO2existsinmanypolymorphsandthemost

abundant are anatase and rutile (Parkin and Palgrave 2005) Pure anatase tends to

display greater photocatalytic properties than rutile due to the faster electron‐hole

recombinationrateofrutiletitania(MillsandLeHunte1997Allenetal2005Brook

56

etal2007b)WhenTiO2intheanatasecrystallineformisexposedtowavelengthsof

lightbelow385nmitbehavesasann‐typesemiconductor(Carpetal2004)andfree

electronsandpositiveholesarecreatedinthefollowingreaction

TiO2 h+vb+e‐cb

The positive holes react with water present on the surface of the thin films in the

followingreactionstogeneratehydroxylfreeradicals

h+vb+H2Oadsorbed OH+H+

h+vb+‐OHsurface OH

Thefreeelectronsparticipateinthefollowingreactionstogeneratethesuperoxideion

andsubsequentlyhydroxylfreeradicals

e‐cb+O2 O2‐

2O2‐+2H2O 2HO+2OH‐+O2

Thegeneratedreactiveoxygenspeciescanreactwithorganicmaterialonthesurface

ofthesemiconductorwhichundergooxidationorreductionreactionsPhotoreactions

occurring on the surface of a catalyst such as TiO2 are termed heterogeneous

photocatalysis(MillsandLeHunte1997)

ThegenerationoffreeelectronsandpositiveholesinTiO2wasfirstdescribedin1972

whenwaterwasdecomposedafterexposuretoUVlight(FujishimaandHonda1972)

λlt385nm

57

Thiswasfollowed in1979byresearchdemonstratingthegenerationofthehydroxyl

radical by electron spin resonance after irradiation of TiO2 by UV light (Jaeger and

Bard1979)Theheterogeneousphotocatalyticprocessisdependentonthepresence

ofwateronthesurfaceofthecatalystandoxygenasanelectronacceptor(Figure110

iii)

1333 Titaniumdioxide‐basedantibacterialphotoactivity

The bactericidal activity of the TiO2 photocatalyst increases proportionately as the

concentration of oxygen is increased from 0 to 100 (Wei et al 1994) Near UV

lightwithwavelengthsbetween300and400nmisthe lightsourcemostcommonly

used for bacterial photoinactivation experiments becauseUV lightwithwavelengths

under300nmareabsorbedbynucleicacidsandcancausemajordamagetoorganisms

(Saitoetal1992)NearUVlightisnotabsorbedbynucleicacidsandsoanyobserved

damagecanbeattributedtothephotoactivityofthecatalystandnottheincidentlight

source

13331 Demonstratingthelossofcellviability

Theseminalpaperinthefieldofphotocatalysisdescribedthephotoinactivationofthe

Gram‐positive bacterium Lactobacillus acidophilus the Gram‐negative bacterium E

coli the yeast Saccharomyces cerevisiae and the green alga Chlorella vulgaris

(Matsunagaetal1985)Asuspensionofplatinum‐loadedtitaniumoxidewasadded

toeachmicrobialsuspensionbeforeaUVlightsourcewasappliedareductioninthe

viability of all organisms was observed The concentration of coenzyme A (CoA)

generatedthroughoutthecourseoftheexperimentwasmonitoredandadecreasein

58

CoAconcentrationwasassociatedwithalossofcellviabilityTheypostulatedthatthe

mechanismofactionwasthephotoelectrochemicaloxidationofCoAwhichresulted

inadecreaseinthemetabolicactivityofthecellsandsubsequentcelldeath

Thegroup followeduptheseexperimentsby immobilisingtheTiO2particleswithina

membraneinacontinuousflowsystemwhichwasusedtosterilisewaterspikedwith

Ecoli(Matsunagaetal1988)AdecreaseinCoAconcentrationwasagainobserved

and reactive oxygen specieswere implicated in the photoinactivation ofE coli The

electrondonorCoAwasoxidisedbythepositively‐chargedholesinthevalenceband

A similarexperimental rigwasusedby Irelandetal (1993) to furtherelucidate the

mechanism of the photocatalytic bactericidal activity of TiO2 E coli in an aqueous

suspension was photoinactivated and after a 9 minute exposure time a 9 log10

reductionwasobservedWhenhydrogenperoxide(H2O2)wasaddedtothesystemit

actedasanirreversibleelectronacceptorandparticipatedinthefollowingreactions

H2O2+e‐cb OH+OH‐

H2O2+O2‐ OH+OH‐+O2

Thegenerationofhydroxylradicalswaspromotedwhich inturnreducedtherateof

electron‐holerecombinationwhichwasaccompaniedbyanincreaseinphotocatalytic

activity Photoinactivation of Streptococcus sobrinus was also demonstrated after

exposureto21nmdiameterparticlesofTiO2andUVlighta5log10decreaseinviable

bacteria was seen after just 1 minute at a bacterial concentration of 105 cfu mL

Photocatalytic activity was reduced when the bacterial inoculum was higher and it

59

took 60minutes to achieve a 5 log10 decrease in S sorbrinus when a 109 cfu mL

inoculumwasused(Saitoetal1992)

A combination of reactive oxygen species is necessary to exert a photocatalytic

bactericidaleffectwith thehydroxyl radical as theprimary radical actingdirectlyon

the cell (Yan et al 2009) Hydrogen peroxide has also been postulated to directly

contribute towards the bactericidal activity as an increase in the concentration of

catalase which degrades hydrogen peroxide to water and oxygen increased the

survival rate of E coli (Kikuchi et al 1997) Therefore hydrogen peroxide could

provide a source of hydroxyl radicals and act as a direct attacking agent (Yan et al

2009)

Viruses have also been shown to be susceptible to the photocatalytic effect of

irradiated TiO2 The non‐enveloped polio virus was spiked intowastewater samples

containingastocksolutionofanataseTiO2andarapid inactivationofthepoliovirus

wasobserved(Wattsetal1995)A2log10decreaseinviablepolioviruswasdetected

after30minutes comparedwitha150minutesexposure time toachieve the same

reductionofEcoliTheincreasedsusceptibilityofthepoliovirustophotoinactivation

waspostulatedtobeduetothelowsurfacetovolumeratiocomparedwithbacteria

whichprovidedahigherrateofhydroxylradicalreactionwiththeextracellularprotein

capsidofthevirus(Wattsetal1995)

60

13332 Detectingchangesinthebacterialcellarchitecture

The activity of the hydroxyl radical is limited by diffusion through the outer and

cytoplasmic membranes (Watts et al 1995 Sunada et al 1998) therefore

compromiseofthesebarrierswillallowgreateractivityofthereactiveoxygenspecies

Potassium ion (K+) leakage was used to demonstrate increased cell membrane

permeability as an indicator of damage to the integrity of the cell membrane An

increaseintheextracellularK+concentrationwasdetectedafterlightirradiationwith

TiO2presentasapowderwhichoccurredinparallelwiththelossincellviability(Saito

etal1992Luetal2003)TheleakageoflargermoleculessuchasRNAandprotein

hasalsobeendetectedaccompaniedbyalossincellviability(Saitoetal1992)

Using transmission electronmicroscopy (TEM) the internal changes associatedwith

photocatalysis couldbevisualisedand thedestructionof thecytoplasmicmembrane

andintracellularcontentswasobservedafter60ndash120minuteslightirradiation(Saito

et al 1992) The reactive oxygen species generated initially damaged the bacterial

peptidoglycan layerbeforeattacking thecytoplasmicmembrane causing irreversible

damageChangesintheoutermembranestructureofEcoliinoculatedontoTiO2thin

films has been demonstrated by atomic force microscopy (AFM) (Lu et al 2003

Sunadaetal2003)After10minutescellviabilityhaddecreasedandacompleteloss

inintegritywasseenafter60minutesWhenbacterialspheroplasts(which lackacell

wall)wereinoculatedontoTiO2thinfilmstherateofbactericidalactivitywasgreater

than thatobserved for the intact cells suggesting that thecellwall hasaprotective

effect on E coli and is the initial site of photocatalytic attack (Sunada et al 2003)

Quantumdots(QD)havealsobeenusedasamarkerofchangesinthepermeabilityof

61

thecellmembraneQDarelightemittingcolloidalnanocrystallinesemiconductorsand

after 20minutes irradiation QDwere shown to enter E coli cells demonstrating a

changeincellmembranepermeability(Luetal2003)

Lipid peroxidation has been demonstrated to occur at the surface of E coli during

photoinactivation inthepresenceofTiO2 (Manessetal1999Soumlkmenetal 2001)

Lipidperoxidationisaprocessinwhichfreeradicalsremoveelectronsfromlipidssuch

as those within the bacterial cell membranes which results in a reduction in the

integrityofthemembraneandthuscellviabilityMalondialdehyde(MDA)aproduct

oflipidperoxidationwasusedasamarkerandanaccumulationofMDAwasdetected

withanaccompanyingdecrease incellularrespiratoryactivityTheauthorsproposed

that reactive oxygen species were generated on the TiO2 surface and attacked the

polyunsaturatedphospholipidspresentintheoutermembrane(Manessetal1999)

TiO2particlesalsointeractwiththeoutermembranecausingreversibledamagewhich

doesnotaffecttheviabilityofthecells(Huangetal2000)Oxidativedamagefollows

whichincreasesthepermeabilityofthecellcausingeffluxofintracellularcomponents

Once thecytoplasmicmembranehasbeen severely compromisedTiO2particles can

enter the cell and directly attack intracellular components Intracellular components

arethenabletoleakoutofthecellandtheo‐nitrophenol(ONP)assaycanbeusedto

detectthisAnincreaseinONPlevelswasobservedinEcoliwhichsignifiedincreased

permeability of the cellmembranes (Huang et al 2000) Bacterial endotoxin is also

degraded in the photocatalytic process and occurs simultaneously with E coli cell

death(Sunadaetal1998)

62

13333 Photoinducedoxidativebacterialdecomposition

InterestinglybacteriacanundergooxidativedecompositionuponthesurfaceofTiO2

thinfilmsuponexposureto356nmlight(Jacobyetal1998)AsuspensionofEcoli

was inoculatedonto irradiatedTiO2thinfilmsandSEMandcarbondioxideevolution

was used tomonitor photocatalytic oxidation After 75 hours exposure to UV light

decompositionofthebacterialcellswasevidentinstarkcontrasttotheuncoatedglass

slidesusedascontrolsAconcomitantincreaseintheconcentrationofcarbondioxide

(CO2)wasalsodetectedPhotocatalyticoxidationofBacillussubtilisvegetativecellsB

subtilissporesandAspergillusnigersporeswasalsodemonstratedandincreasedCO2

concentrations were used as markers of microbial decomposition (Wolfrum et al

2002) The rate of oxidationwas slower forA niger cells comparedwith the other

testedorganismsThishasimportanttranslationalimplicationsasitprovidesevidence

that the coatings are self‐cleaning and do not require a physical removal step after

photoinactivation organic matter present on the surface of the catalyst can be

mineralisedifexposedtothelightsourceforanadequatetimeperiodprovidingmore

spaceforphotocatalyticreactionstotakeplace

1334 Enhancingthepropertiesoftitaniumdioxidethinfilms

AdditionalelementscanbeaddedtoTiO2toalterthechemistryofthematerialTiO2

can be dopedwith substances such as nitrogen or sulfur to cause a batho‐chromic

shiftwhichalters thebandonsetenergy (Section13314) so thatphotonsof light

withalowerfrequencyareabsorbedandareabletoexcitetheelectronstoahigher

energystate(Asahietal2001Carpetal2004)Transitionmetalionssuchasiron

leadandcoppercanalsobeusedasdopantstoenhancethephotocatalyticproperties

63

ofTiO2(ThompsonandYates2006)Theaimofthisdopingistogenerateamaterial

that can be activated by visible light such as indoor lighting conditions which

broadens the commercial applications of the material A ten‐fold increase in the

numberofphotonsavailable forphotocatalysiswouldbegeneratedbyashift inthe

TiO2bandonsetofjust40ndash50nm(DunnillandParkin2009)

The exact mechanisms governing visible light photocatalysis are poorly understood

althoughitisgenerallyagreedthatnitrogendopingcausesincreasedphotocatalysisat

lower photon energies and localised nitrogen 2p states above the valence band are

generatedbytheadditionofnitrogen(ThompsonandYates2006)Itisnotyetagreed

whether substitutional or interstitial nitrogen binding provides the most favourable

visiblelightdrivenphotocatalyticproperties

14 Relevanceofsurfacesinventilator‐associatedpneumonia

Ventilator‐associated pneumonia (VAP) is a serious healthcare‐associated infection

that affects patients on ventilators predominantly in the intensive care unit The

intubatedpatientusuallyhasseriousco‐morbiditiessuchthattheyrequireassistance

with theirbreathingand thephysicalpresence of theendotracheal tube (ETT)both

compromisesthenormalactionoftherespiratorytractandallowsmicro‐aspirationof

contaminatedsubglotticsecretions

AnumberofclinicalmeasurescanbeappliedtopreventVAPaspreventionrequiresa

multifactorial approachand research into the subject includes theuseofalternative

ETTmaterials (Balk2002Pneumatikosetal 2009Torresetal 2009Bouadmaet

al 2010 Berra et al 2011 Blot et al 2011 Coppadoro et al 2011 Rewa and

64

Muscedere 2011) Bacteria originating from the oropharynx colonise the ETT and

produceabiofilmonthelumenofthetubewhichisdifficulttoremoveandprovidesa

potentialsourceofcolonisationandinfectionofthelowerairways(Sottileetal1986)

Therefore the prevention of bacterial adhesion to the surface of the ETT and the

destructionandremovalofboundorganismsisofclinicalinterest(Berraetal2003)

Polyurethane cuffed ETTs are being used in preference to the traditional

polyvinylchlorideETTsas theyaremore flexibleandabetter seal isproducedat the

base of the tube which prevents leakage of oropharngeal contents into the lower

airways (Berra et al 2008b Miller et al 2010) An alternative novel way to

decontaminate theETT isbyusing theMucusShaverwhichphysically removesboth

mucus and bacterial biofilms from the inner lumen of the tubing (Kolobow et al

2005)

ETTs can also be impregnated with antibiotics or other antibacterial compounds to

preventtheinitialbiofilmformationstageortokilltheadherentorganismsSilverions

have been added to polyurethane ETTs and a series of in vitro studies have

demonstrated reduced adherence of MRSA P aeruginosa Enterobacter aerogenes

andAbaumanniitothesilver‐coatedmaterials(Berraetal2008aRelloetal2010)

Colonisationof silver‐coated ETTsbyPaeruginosawas shown tobe lowerand take

longerthanonuncoatedcontrolETTswithlowerlevelsoflungcolonisationobserved

inventilateddogsasa consequence (Olsonetal 2002Relloetal 2010)A similar

study used silver‐sulfadiazine and chlorhexidine coated ETTs in ventilated dogs and

demonstratedareductionintrachealcolonisationandanabsenceoflungcolonisation

(Berraetal2004)

65

Whensilver‐coatedETTswereusedinastudyinvolvingninepatientsnoneoftheETTs

werecolonisedwithpathogens therewas lesscolonisationofcommensalorganisms

andtherewasadecreaseinbiofilmformationcomparedwiththenon‐coatedcontrol

ETTs(Relloetal2010)AdelayedETTcolonisationtimeandpositivetrachealaspirate

culture time was demonstrated in an earlier study using the same coated material

(Relloetal 2006)andnobacterial growthorbiofilmproductionwasdetectedona

silversulfadiazinecoatedpolyurethaneETTused inacohortof46 intubatedpatients

(Berra et al 2008b) A reduced incidence of VAPwithin 10 days of intubationwas

observedintheNASCENTtrialwhichrecruitedover2000patientssilver‐coatedETTs

were used in the test group and were compared with non‐coated equivalents that

wereusedinthecontrolgroup(Kollefetal2008)

A number of silver‐coated ETTs are now commercially available butwidespread use

has been hindered by the pricewhich is up to 45 timesmore than uncoated ETTs

however a theoretical cost‐analysismodel showed silver‐coated ETTswere actually

associatedwithfinancialsavingsofover$12000peravertedcaseofVAP(Shorretal

2009Torresetal2009)

Chlorhexidinehasbeencombinedwiththedyebrilliantgreenorgentianviolettoform

the novel compounds gardine and gendine respectively These compounds have

displayedsignificantantibacterialactivity invitroand inanelegantbiofilmdisruption

assaydemonstratedsuperioritytosilvercoatedETTsThesecompoundsarerelatively

cheap to produce and the authors propose clinical use after thorough in vivo

assessment(Chaibanetal2005Hannaetal2006Hachemetal2009Reitzelet

al2009Raadetal2011)Thesestudies illustratethebenefitsofantibacterialand

66

novel ETTmaterials and to further improve the incidence of VAP and other device‐

relatedinfectionsfurtherresearchshouldbeconducted

141 Photodynamictherapy

AdifferentmethodofgeneratinganantibacterialeffectonthesurfaceoftheETTsis

viaaprocess calledphotodynamic inactivation (PDI)Phototherapywas firstusedby

theNobelPrizewinnerNielsFinsentotreatatuberculosisskinconditioncalled lupus

vulgaris in the 1890rsquos by applying light directly onto the lesions (Bonnett 1995

Dolmansetal2003)Photodynamictherapy(PDT)evolvedfromthisinitialworkand

involves the use of a photosensitising agent and a light source to generate toxic

reactive oxygen species (Wainwright 1998) The procedure can be used in the

targetedtreatmentofcanceroustumours(MarcusandMcIntyre2002Dolmansetal

2003) in ophthalmology to treat age‐related macular degeneration (Bressler and

Bressler2000)atherosclerosis(Rocksonetal2000)andinthelocalisedtreatmentof

bacterial infectionsparticularlyindentistry(Wainwright2003)WhenPDTisusedto

killbacteriaitistermedphotodynamicinactivation(PDI)(HamblinandHasan2004)

There are two types of photosensitisation reactions type I and type II and the

pathwaysinvolvedingeneratingthesereactionsareillustratedinFigure111Whena

photosensitisermolecule is irradiatedwith lightofanappropriatewavelength itcan

undergoanelectronictransitiontoformthesingletexcitedstatewithpairedelectron

spinsThemoleculetheneitherundergoeselectronicdecayandreturnstotheground

stateortheenergycanbetransferredsothatthemoleculeundergoesanelectronic

transitiontothetripletexcitedstateTheelectronspinsatthispointareunpairedThe

67

molecule could once again lose the energy depending on the environmental

conditions and the structure of the molecule itself and return to the ground state

Alternatively ifoxygen ispresent theenergycouldbetransferredandusedtodrive

redoxreactionsandgenerateradicalions(typeI)ortogeneratesingletoxygen(typeII

reaction) Themajor pathway involved in generating the bactericidal effect in PDI is

the production of singlet oxygen (Wakayama et al 1980) To be an efficient

photosensitiseramoleculemustbeefficientatproducingsingletoxygenandthat in

turn isdependentonthegenerationofa largepopulationof long‐livedmolecules in

thetripletstate(Wainwright1998)

68

Figure111FlowdiagramtodemonstratethegenerationofsingletoxygenTheboldarrows indicate the pathway to the Type II reaction (Bonnett 1995 Wainwright1998)

The reactiveoxygen species‐drivenbactericidaleffect is similar to thatgeneratedby

TiO2 thin films upon irradiation with suitable wavelengths of light Singlet oxygen

speciesexertadirecteffectonmicrobialcellsbyoxidisingcellconstituentssuchasthe

cellwall cellmembrane or intracellular components such as nucleic acidswith the

cytoplasmicmembraneastheprimarytargetPDIcausesalossofmembraneintegrity

suchthattheintracellularcontentsleakoutofthecellcontrolledtransportofsolutes

across themembrane is compromised and the cell loses viability due to the lack of

essential constituentsneeded foranabolicandcatabolicpathways (Jorietal 2006)

69

The reactiveoxygen speciesare thenable toaccess the intracellularDNAandcause

further damage (Dunipace et al 1992 Salmon‐Divon et al 2004 Chi et al 2010)

Singlet oxygen has a diffusion distance of approximately 20 nm therefore if the

bacterial species are in contactwith the light‐activatedmaterial then the generated

singlet oxygen should be active against both the bacterial cell wall and underlying

membrane

Anadvantageous featureofPDI is thatmulti‐drug resistantstrainsofbacteriawhich

are resistant to a number of different antibiotic classes do not show enhanced

resistancetoPDIcomparedwiththeequivalentantibioticsensitivestrains(Maliketal

1990) The susceptibility of 60 multi‐drug resistant strains of P aeruginosa to the

photosensitiser toluidine blue and red laser light were comparedwith 19 antibiotic

sensitivestrainsandnodifference insusceptibilitywasobserved(Tsengetal2009)

InadditionthegrowthphaseofPaeruginosadoesnotimpactonitssusceptibilityto

TBO‐mediatedphotosensitisation(KomerikandWilson2002)unlikesomeclassesof

antibioticswhichhaveselectiveactivityforbacteriaintheexponentialphaseofgrowth

(Tuomanenetal 1986)Duetothemulti‐siteactivityofthereactiveoxygenspecies

generated during light irradiation it is unlikely that resistant phenotypes will be

selected(HamblinandHasan2004)

1411 Typesofphotosensitisers

There are a number of different aromatic compounds which can act as

photosensitiserswhenirradiatedbyspecificwavelengthsoflightThecompoundsare

usually coloured as they reflect light in the visible part of the electromagnetic

spectrum An ideal photosensitiser would contain an overall cationic charge as

70

bacterial cells carry an overall anionic charge because of the presence of the

cytoplasmic membrane (Hamblin and Hasan 2004) Examples of photosensitisers

whichhavebeenusedforPDIarethephenothiazinestoluidineblue(Wakayamaetal

1980Paardekooperetal1992Wainwrightetal1997Pernietal2009bRagaset

al 2010) and methylene blue (Decraene et al 2009 Perni et al 2009a) the

halogenated xanthene rose bengal (Decraene et al 2006) and acridines such as

acridineorange(Wainwrightetal1997)

Photosensitiserscanbeusedinsolutionandappliedtothetreatmentareaorcanbe

impregnatedintoapolymerwhichcanbeusedinavarietyofsettingsForexamplea

solution of photosensitiser can be injected into a periodontal pocket before the

applicationof laserlighttoexertPDIonthepathogenspresent(Wilson19931996)

Alternatively the photosensitiser could be immobilised in a polymer used in as a

cathetermaterialsothatanybacteriapresentinthelumenorexteriorofthetubing

would be exposed to the reactive oxygen species generated during PDI upon

applicationofthelightsource(Pernietal2011)

15 Methodsofproducinglight‐activatedantimicrobialmaterials

151 Chemicalvapourdeposition

Thin films of TiO2 are commonly synthesised using the chemical vapour deposition

(CVD)technique indeed itisthemethodusedindustriallybyPilkingtontosynthesise

theirPilkingtonActivtradeself‐cleaningglasses(Millsetal2003)Thedepositionprocess

requiresheatingtoahightemperature(gt500degC)thereforethechoiceofsubstrateis

limited as the substrate has to withstand the rise in temperature this constraint

71

makesglassan idealchoicePrecursormoleculescontainingtitaniumandoxygenare

heated into a gaseous phase and transported via the nitrogen carrier gas into the

reaction chamber The precursormolecules are adsorbed onto the heated substrate

anddecompose theelementsof choice remainadhered to the substrateandwaste

productsareremovedfromthesystembythenitrogencarriergas(West1999Carp

etal2004Page2009)AschematicofatypicalCVDrigisdisplayedinFigure112

Figure112Schematic representationofaCVDapparatusThe setupshown in thisdiagram was used to deposit thin films of titanium oxynitride as discussed inChapter4(Aikenetal2010)

152 Sol‐gel

The sol‐gel technique is considered to be more reproducible than CVD and the

production of a uniform film is possible on a small scale (Carp et al 2004) To

synthesiseTiO2 thin filmsby the sol‐gelmethodahomogenous solution isprepared

containing thecationic reactants required for the synthesis analkoxide isusedasa

72

sourceofTiO2waterisrequiredtohydrolysethealkoxideandanalcoholisaddedto

catalyse the reaction (West 1999 Rampaul et al 2003 Page 2009) A viscous gel

develops containing colloidal particleswhich grows further as the solution is left to

age During this time the water and alcohol trapped in the matrix of the polymer

evaporate and so the resultant aged sol is transparent and homogenous with no

crystallinephasesorprecipitatesTheglasssubstratecanthenbedippedintothesol

andthesoladherestothesurfaceoftheglassitisremovedataconstantratesothat

thethinfilmproducedisofaconsistentthicknessalongthelengthofthematerialThe

sol dries readily but is mechanically weak so is sintered at a high temperature to

removeanyorganicmatterandadensecrystallineoxidecoatingisproduced

153 Swellencapsulation

Swell encapsulation is a chemical method used to impregnate polymers with an

organic compound and can be modified to add a photosensitiser molecule to a

polymer in order to generate a light‐activated antibacterial material When an

elastomer is immersed in an organic solution containing a photosensitiser the

photosensitiserisabletopenetratethepolymerastheelastomericmatrixswellsThe

elastomer is removed from the photosensitiser‐containing solution after a defined

periodandthepolymerrevertsbacktoitsoriginalsizeasthesolventevaporatesThe

photosensitiserremainsembeddedintheelastomericmatrixduringevaporationand

thefinalconcentrationofphotosensitisercanbeadjustedbyvaryingtheconcentration

intheorganicsolution(Pernietal2009aPernietal2011)

73

16 Measuringenvironmentalcontamination

Accuratemethodsarerequiredtomonitormicrobialcontaminationofenvironmental

surfacestoassesscleaningregimensandtodetectanybacteriapresent (Manheimer

andYbanez1917SaloandWirtanen1999MooreandGriffith2002Verranetal

2002Hedinetal2010Verranetal2010a)

161 Swabbing

Bacterial culture is a widely used method as any viable bacteria present can be

detected quantified and identified at a relatively low cost The test surface can be

sampled using a swab or spatula which can be made from a variety of materials

includingcotton viscosenylon orman‐madesubstances suchas thebrush‐textured

nylon flock Samples can then either be streaked directly onto an agar plate or re‐

suspendedintoagrowthenhancingbrothbeforesubcultureontosolidmedia(Moore

andGriffith2007) If thebacterial inoculum ishigh thesamplecanbeserialdiluted

before plating out to allow enumeration of the single colonies on the culture plate

ensuring a more accurate estimation of the original bacterial inoculum Pathogenic

yeastsandfungicanalsobedetectedinthiswayHoweverthetechniquereliesupon

theabilityof the swab to collectallmicrobial contaminationon the surfaceand the

releaseoftheorganismsfromtheswabheadduringprocessing(Faveroetal1968)

162 Dipslides

Environmental surfaces can alternatively be directly sampled by placing a section of

agar directly onto the surface by use of a RODAC (replicate organismdetection and

counting)plateorasimilarsamplingdeviceandenumerationofthecoloniesafteran

74

incubation period Dipslides have a greater sensitivity and reproducibility compared

with swabbingwithout enrichment culturewhen sampling surfaces especially if the

surface isdry (Mooreetal2001MooreandGriffith2002FoodStandardsAgency

2004Obeeetal2007)Howeverquantificationcanbedifficultifthesurfacelevelof

contamination is too high as the microbial load on the surface cannot be diluted

resulting in confluentgrowth on theagarwhichmakes colonycounting impractical

Growth is instead classified instead as moderate or heavy based on the surface

coverageoftheslideandcomparisonwithvisualimagesofcontrols

163 Airsampling

Air sampling devices are used to sample the microbial contamination of the

surroundingairAdefinedvolumeofairisdrawnintothedeviceandispassedoveran

agar plate so that microorganisms found in the air are inoculated onto the plate

surfaceAirbornesporesarealso inoculatedontotheplatesandgrowthoccursafter

germination These units have been employed in the healthcare environment to

monitor efficiency of cleaning schedules and terminal decontamination regimens

(Jeanesetal2005Wongetal2011)thefungalcontaminationofairduringbuilding

work(Goodleyetal1994)andthequalityofairinoperatingtheatres(Whyteetal

1982Hambraeus1988Landrinetal2005)Ariskfactorforsurgicalsiteinfectionsis

microbial contamination of the air in operating theatres so knowledge of the air

quality isessential toensureairhandlingunitsare functioningcorrectlyandprevent

theseinfectionsoccurring(Whyteetal1982Hambraeus1988Whyteetal1992)

Microbialcontaminationoftheaircanalsobemonitoredusingsettleplateswhichare

large agar plates that are placed in the test environment Airbornemicro‐organisms

75

which fall onto the plates are then detected by colony counting after incubation

However droplet nuclei stay suspended in the air so cannot be detected using this

methodandtheplatesrequire longerperiodsofsampling(circa24hours)compared

withamechanicaldevicethattakesminutestoobtainasample

164 ATPbioluminescence

All of themethodsdescribedabovehave thedisadvantage that theyaredependent

upontheabilityoforganismstogrowonsolidmediasobacteriaintheviablebutnon‐

cultivable (VBNC) state would not be cultured Alternative sampling methods that

overcometheselimitationswouldthereforebeuseful(MooreandGriffith2007)ATP

bioluminescence is a process based upon a naturally occurring light‐generating

reactionfoundintheNorthAmericanfireflyPhotinuspyralis(HawronskyjandHolah

1997) Both themale and female fireflies use the generation of light to locate one

anotherandasmatingsignals(EncyclopediaBritannica2011)Theluciferaseenzyme

isolated from P pyralis can be used in the laboratory to catalyse the oxidation of

luciferinusingATPastheenergysourceandthereactionisasfollows

ATP+D‐luciferin+O2 AMP+PPi+oxyluciferin+CO2+light

The light produced during the reaction can be quantified by a luminometer and the

output is given in relative light units (RLU) (Lundin 2000) The generated light is

directlyproportionaltotheamountofATPpresentintheinitialsampleasonephoton

oflightisemittedpermoleculeofATP

luciferase

76

ATP is found inall living organismsand isalsopresentas freeATP (Hawronskyjand

Holah1997)Luminometerscanbeusedtoprovidedataontheleveloforganicdebris

andmicrobialcontaminationonasurface(Davidsonetal1999)EukaryoticATPand

ATPfromextracellularsourcescanbedegradedpriortothelysisofthebacterialcells

withcertainmodels (HawronskyjandHolah1997)enablingthenumberofbacterial

cellstobecalculatedfromtheamountoflightemittedResultscanbeavailablefrom

fivetothirtyminuteseliminatingthetime‐consumingovernightincubationofculture

plates

ATP bioluminescence has been used for the last decade in the food industry and is

especiallyusefulincomplyingwithspecificfoodregulationswhichservetoreducethe

riskoffoodspoilageandcontamination(HawronskyjandHolah1997Davidsonetal

1999Wagenvoortetal2000)Qualitativemeasurementsareusuallytakensothata

surfacewill eitherpass if anacceptablenumberofbacteriaarepresentor fail if the

numberofbacteria is aboveapredetermined level (Cooperetal 2007)Theuseof

ATPbioluminescenceinthesesituationsisadvantageousastheresultsareavailablein

minutes so if the surface contaminationwas deemed too high then it could be re‐

cleanedre‐testedandfoodproductioncouldcontinueifitsubsequentlypassed

ThereareanumberofcommerciallyavailableluminometersincludingtheClean‐Trace

(BioTraceBridgendUK)aportableluminometerwhichdetectsATPbioluminescence

ofbothmicrobialandnon‐microbialoriginThissystemiscommonlyusedtoassessthe

effectiveness of cleaning regimens as organic debris is also detected The easily

transportable BioProbe (Hughes Whitlock Gwent UK) and the Junior (Berthold

TechnologiesGmbHBadBadwildGermany)luminometersrequireadditionalreagents

77

to generate RLU readings as does the Lumat luminometer (Berthold Technologies

GmbH) The Microbial ATP Kit (BioThema AB Sweden) can be used to degrade

exogenousATPbefore thebacterial cells are lysed soamoreaccurate indication of

theactualnumberofbacteriapresentonthetestsurfacecanbeobtained(BioThema

AB2006)Thesemethodologiesarenot commonlyused in thehealthcareora food

environmentastheyrequireasamplepreparationstepandtakeslightlylonger(upto

30 minutes) These methodologies can be used for molecular experiments such as

reporter gene assays where a higher sensitivity is required (Dyer et al 2000

McKeatingetal2004BioThemaAB2006)

165 Stainingtechniques

Staining techniques could alternatively be used to estimate the level of bacterial

contaminationonasurfaceAcridineorangeisacommonlyuseddyeusedtoperform

direct counts on test surfaces although no indication of bacterial viability is given

Fluorescentprobessuchascyanoditolyltetrazoliumchloride(CTC)andrhodamine123

canbeusedasindicatorsofcellviabilityCTCisreducedtocrystallineCTC‐formazan

present as red crystals within bacterial cells and rhodamine 123 is concentrated in

functioningmitochondriaandcellsfluorescegreen(YuandMcFeters1994Pyleetal

1995)Visualisationrequirestheuseofappropriateexcitationandemissionfiltersona

fluorescentmicroscope(YuandMcFeters1994)TheLiveDeadBacLighttradeBacterial

Viability stain (Molecular Probes Inc) is a fluorescent dye which can differentiate

betweenviableandnon‐viablebacterialcellsThekitcontainstwodyesSYTO9and

propidiumiodideSYTO9emitsat500nmandstainsallcellsgreenwhereaspropidium

iodide is a red stain that emits at 635 nmand penetrates cellswith a damaged cell

78

membrane(Boulosetal1999AireyandVerran2007)Allgeneratedimagescanbe

capturedonacameraattachedtoafluorescentmicroscopetoenableenumerationof

the organisms present using computer software such as ImageJ

(httprsbwebnihgovijindexhtml) Direct visualisation techniques can also detect

thepresenceofnon‐microbialcontaminationsuchasorganicsoil thatcouldprovide

sustenanceforbacterialgrowth(Verranetal2002)

166 Summaryofenvironmentalsamplingtechniques

Thereiscurrentlynostandardisedtechniqueforsamplingenvironmentalsurfacesina

hospital environment so a variety of methods are used (Hedin et al 2010) ATP

bioluminescence provides a snapshot of bacterial contamination and can detect the

presence of organic soil Viable bacteria can be enumerated by performing viable

counts which is cheap and easy to perform and improvements in the swab head

material and sampling diluent have been shown to increase sampling efficiency

althoughtheimprovementsobservedwereminimal(Hedinetal2010)Visualisation

techniquesrequiremorespecialisedequipmentandstainsbutintactbiofilmscanbe

observedwithoutdisruptionandnon‐viablebacteria included in thebacterial count

Thesetechniquesallpossessinherentadvantagesanddisadvantagessoarebestused

with clear knowledge of these limitations especially when interpreting any data

generated(Verranetal2010a)

79

17 Methodsof characterisingandassessing the functionalityof light‐

activatedantimicrobialmaterials

171 UV‐visible‐IRspectroscopy

UV‐visible‐IRspectroscopycanbeusedtopredictthelikelyphotocatalyticactivityofa

potentialantibacterialmaterialbycalculatingthebandonset(Section13313)When

incidentlightwithawavelengthbetween200nmand700nmisappliedtoacandidate

materialthreereadingscanbetaken(i)thetransmissionoflightthroughthesample

(ii) the absorption of light by the sample and (iii) the reflectance of light from the

sampleThesereadingscanbeusedtoestimatethebandgapAplotof(αhv)12against

hv isthengeneratedwherehvequalstheincidentlightandaequalstheabsorbance

coefficient(a=‐logTT0whereTequalsthetransmissionreadingofthesampleandT0

equals the transmission of the substrate)When thecurve isextrapolatedalong the

linearportionofthecurvethebandgapcanbereadfromthexaxis(Tauc19681970

Sharmaetal2009)ThisiscalledaTaucplotThetransmissiondatacanalsobeused

tocalculatethethicknessofthethin filmsusingtheSwanepoelmethod (Swanepoel

1983)

172 Photooxidationofstearicacid

Thephotodegradationoftheorganicmoleculestearicacid(Figure113)canbeusedto

quantify the photocatalytic self‐cleaning ability of candidate antibacterial materials

andisbasedonthefollowingequation(Millsetal2002)

CH3(CH2)16CO2H+26O2 18CO2+18H2Ohvgebandgapenergyofsemiconductor

80

Carbondioxideandwater isgeneratedfromorganicmolecules inacoldcombustion

reaction(ParkinandPalgrave2005)Theprocessisrelativelysimpletoperformandso

a large number of thin films can be screened for potential photocatalytic activity

Infrared (IR) spectroscopy is used to monitor the degradation of the stearic acid

molecules The thin films that show the greatest activity by this method can then

selectedforantibacterialtesting

Figure113ChemicalstructureofstearicacidC18H36O2

Infraredspectroscopyisananalyticalmethodusedtoobservethevibrationalenergies

of molecular bonds Photons of light from the IR portion of the electromagnetic

spectrum interact withmolecular bondswithin the sample The incident light has a

lower frequency than UV or visible light and causes molecular bonds to bend and

stretchastheyabsorblightAbsorptionofthephotonofIRlightcausesanincreasein

thevibrationalenergyofthebondraising it toahighervibrationalenergy levelThe

modeofvibrationvariesdependingupontheconstituentatomsinthebondandthese

chemicalstretchesandbendsareidentifiableontheIRspectragenerated(McCarthy

1997)

TheIRmeasurementsareplottedonagraphofwavenumberagainsttransmittanceor

absorption The changes in the vibrational energies of the molecular bonds are

detected as inverted peaks on the resultant IR spectra as the transmittance of the

incident light decreases because of the absorbance of the light by the molecular

81

bondsTheseinvertedpeaksaretermedabsorptionbandsandarecharacteristicofthe

IR vibrations of specific molecular bonds Stearic acid has three modes which are

visibleintheIRspectrumthesymmetricCndashHstretch(CH2)hasanabsorbanceband

of2923 cm‐1 theCndashH stretch (CH3)hasanabsorbancebandof 2958cm‐1and the

asymmetric C ndash H stretch (CH2) has an absorbance band of 2853 cm‐1 The

concentrationofstearicacidcanbeapproximatedbyintegratingtheareaofthelatter

twopeaks the firstpeak isof low intensityand is generallynotusedAn integrated

areaof1cm‐1equatestoapproximately97x1015molecules(MillsandWang2006)

andsothedestructionofstearicacidcanbemonitoredovertimebynormalisingthe

concentrationofstearicacidmoleculesonthetestsurfaceasCxC0readingswhere

C0istheinitialconcentrationandCxistheconcentrationofstearicacidatagiventime

point

173 Contactanglemeasurements

Photo‐inducedsuperhydrophilicitycanbeinducedonphotocatalyticthinfilmssuchas

TiO2 after irradiationwith light possessing band gap energy (Mills et al 2002) The

hydrophilicity or indeed hydrophobicity of a substrate can be calculated by

determiningthecontactangleofadropletofwaterinoculatedontothesurfaceofthe

materialAhydrophilicmaterialwillpossessalowwatercontactangleasthedroplet

will spread flat on the lsquowater‐lovingrsquo hydroxylated surface with an accompanying

increase in the diameter of the droplet Conversely a hydrophobicmaterialwill not

have an affinity for the droplet of water so the diameter of the droplet will be

reduced resulting in a highwater contact angle (Page 2009)Hydrophobic surfaces

82

havewatercontactanglesabove90deghydrophobicsurfaceshavewatercontactangles

below90degandsuperhydrophilicsurfaceshavewatercontactanglesapproaching0deg

During photo‐induced superhydrophilicity on a TiO2 semiconductor light exposure

causes the trapping of holes at lattice sites near the surface of thematerial and a

concomitant reduction of Ti4+ to Ti3+ (Carp et al 2004) The bonds between the

titanium and oxygen within the lattice are weakened by the trapped holes which

enable the release of oxygen atomswhich in turn creates oxygenvacancies and an

increaseinthehydroxylationstateofthesurfaceHydroxylgroupsareadsorbedonto

thesurfacewhichbindwiththewaterinoculatedontothesurfaceduetoanincrease

inthevanderWaalsforcesandhydrogenbonding(Carpetal2004)

174 Standardmethodsofassessment

International standards have been developed to assess the activity of novel

antimicrobial products such as the Japanese Industrial Standard JIS Z 2801 which

measuresantibacterialactivityandefficiencyandnumerousISOstandardsdeveloped

by the International Organisation for Standardisation (International Organisation for

Standardisation 2011) Antibacterial activity can be calculated using the following

formula R = log(BC) where R is a measure of the antibacterial activity B is the

averagenumberofviablecellsofbacteriaonanuntreatedsampleafter24hoursand

Cistheaveragenumberofviablebacteriaontheantibacterialsampleafter24hours

If a test sample has a value of greater than 20 then it is denoted an antibacterial

materialaccordingtoJISZ28012006

83

The methylene blue reduction test can also be used for the assessment of

photocatalytic surfaces and has recently been adopted as an ISO standard (ISO

106782010)Whenmethyleneblueisinoculatedontoatestsurfacephotogenerated

electronsreduceatmosphericoxygentoproducesuperoxidewhichdegradesthedye

or photogenerated holes either directly oxidisemethylene blue or generate reactive

oxygenspeciesthatdirectlyattackthedye(AthertonandNewlander1977Zitaetal

2009)These reactions result inadecrease in the intensityof thecolouration of the

dye and this colour change can be monitored on a spectrophotometer over time

comparedwithanuntreatedcontrolsampletodeterminetheabilityofUV‐activated

surfaces to photodegrade dissolved organic molecules Therefore this would be a

useful tool toscreena largenumberofdifferentphotocatalystsbeforefocusingona

smallernumberofsamplestotestagainstbacterialsuspensionsHowevertheassayis

notvalidatedtouseonsurfacesactivatedbyvisible lightoragainstbacterial targets

AcidOrange7isanotherdyethatisoxidisedduringphotocatalysisanddegradationof

themoleculecanbemonitoredasamethodofdeterminingphotocatalyticactivityA

morerecentdevelopmentistheuseofaninkResazurinwhichisdescribedasafaster

and simpler method (Mills andMcGrady 2008) During photocatalysis the positive

holes generated are trapped by glucose which is containedwithin the preparation

and thephotogeneratedelectrons reduceResazurin (Zitaetal 2009)Thecolourof

theinkchangesfrombluetopinkwhichoccursinsecondscomparedwiththehours

requiredfortheformermethodsandthecolourchangecanbedetectedbyeyewhich

providesaninexpensivesemi‐quantitativemeasureofphotocatalyticactivity

84

18 Overviewandprojectaims

A multi‐disciplinary approach is required to prevent HCAIs as the acquisition and

transmissionofinfectionisrarelycausedbyanisolatedeventbutasaconsequenceof

anumberoffailuresinprocedure(Dettenkoferetal2011)Handhygieneisviewedas

themost important and effectivemethod for preventing the transmission ofHCAIs

Adequate isolation facilities need to be available and high‐risk patients need to be

transferred into these areas promptly This requires sensitive specific and rapid

detection of the infective organisms so that these scarce resources are used

appropriately (Cheng et al 2011) Prudent antibiotic prescribing is important to

preventtheemergenceofresistantorganismsandhasbeenshowntoreducetherates

ofCdifficile infection (Mearsetal 2009)The patientenvironment shouldbekept

free of pathogens by methods as basic as regular scheduled cleaning and hand

decontamination after each patient contact This has been shown to significantly

reduce the transmission of microorganisms and prevents the transfer of organisms

from patient‐to‐patient and from the environment‐to‐patient (Devine et al 2001

Rampling et al 2001 Dancer 2004 Johnston et al 2006 Department of Health

2008Danceretal2009)Novel technologiescouldalsobeemployedaspartofthe

armoury of interventions used to prevent the transmission of infectious

microorganismswithinhospitalsascurrentlyemployedmethodssuchascleaningand

handhygienealonearenotprovingtobesufficient(Ramplingetal2001Frenchet

al 2004) Recontamination of surfaces occurs readily after disinfection of areas

surrounding an infected patientwhich allows further transmission of the organisms

(Collins1988WeberandRutala1997Bradyetal2003)Self‐cleaningsurfacescould

potentially lower the bacterial load in the near‐patient environment and reduce re‐

85

colonisation rates as organisms shed in‐between cleaning events would be killed

breakingthecycleofre‐colonisationAntimicrobialpolymerscouldbeusedtoproduce

ETTsandcatheters to reduce theadherenceof bacteriawithin the lumenof tubing

andpotentiallydecreasetheincidenceofdevice‐relatedHCAIs

Thepurposeofthisprojectwastogenerateandassesstheantibacterialactivityofa

rangeoflight‐activatedmaterialswiththepotentialtobeusedinahealthcaresetting

toreducethetransmissionandacquisitionofHCAIs

86

2 Materialsandmethods

21 Targetorganisms

Bacterial typestrainsused inthesestudiesare listed inTable21Allof thebacterial

strainswerestoredat‐80degCinbrainheartinfusionbroth(BHI)containing10glycerol

andmaintainedbyweeklysubcultureonto5Columbiabloodagarplates (allmedia

fromOxoidLtdBasingstokeUK)AclinicalisolateofCalbicanswasalsoused(Table

21)andwas stored onaSabourauddextroseagar slopeat22degCandmaintainedby

weeklysubcultureontoSabourauddextroseagarplates

Table21Bacterialandfungalstrainsusedinthesestudies

Bacterialfungalstrain Referencenumber

Escherichiacoli ATCC25922

Staphylococcusaureus NCTC6571

Staphylococcusaureus ATCC8325‐4

Epidemicmeticillinresistant‐Staphylococcusaureus16 Clinicalisolate


Meticillinresistant‐Staphylococcusaureus ATCC43300

Streptococcuspyogenes ATCC12202

Enterococcusfaecalis Clinicalisolate

Pseudomonasaeruginosa PAO1

Pseudomonasaeruginosa Clinicalisolate

Acinetobacterbaumannii Clinicalisolate

Stenotrophomonasmaltophilia Clinicalisolate

Candidaalbicans Clinicalisolate

87

22 Growthconditions

Bacteria were grown aerobically in either nutrient broth (P aeruginosa E coli S

maltophiliaandAbaumannii)orBHIbroth(SaureusSpyogenesSepidermidisand

E faecalis) and incubated for 18 hours at 37degC in an orbital incubator (Sanyo BV

Loughborough UK) at a speed of 200 rpm C albicans was grown aerobically in

Sabourauddextroseliquidmediafor18hoursat37degCinanorbitalincubator

23 Preparationofthebacterialinoculum

A1mLaliquotoftheovernightculturewascentrifugedat12000rpmandthepellet

was re‐suspended in 1 mL phosphate buffered saline (PBS) (Oxoid Ltd) An optical

densityof005Aatawavelengthof600nmwasachievedbyaddinganaliquotofthe

re‐suspendedsolutionto10mLPBSwhichequatestoapproximately107cfumLFor

C albicansexperiments the entire re‐suspendedpelletwas added to 10mL PBS to

achieveanopticaldensityof1100Awhichcorrespondedtoapproximately107cfu

mL

ForexperimentsinvolvinganalginateswabthePBSwassubstitutedwith3mLCalgon

ringerrsquos solutionand for thoseusing LiveDead stains1mLbufferedpeptonewater

(BPW)wasused

24 Lightsources

241 Whitelightsource

Forwhite light photocatalysis experiments aGeneral Electric 28WBiax 2D compact

fluorescentlamp(GELightingLtdEnfieldUK)wasusedThislampiscommonlyfound

88

inUK hospitals and emits light across the visible spectrum the spectral distribution

chartisshowninFigure21Forexperimentalpurposesthelampwasaffixedinsidea

cooled incubator tomaintain a constant temperature of 22degC (LMS Series 1 Cooled

Incubator Model 303 LMS Ltd Sevenoaks Kent) The intensity of the light was

measured using a luxmeter (LX101 Luxmeter Lutron Electronic Enterprise Co Ltd

Taiwan) and readings were recorded in lux units The term visible light indicates

wavelengths of light in the visible portion of the electromagnetic spectrum namely

between 400 ndash 700nm however the terms white light and visible light are used

interchangeablyinthisthesisandindicateuseofthisfluorescentlightsource

Figure21Spectralpowerdistributiongraphforthelightsourceused inthevisiblelightphotocatalysisexperiments(Technicalpublicationforthe2Dserieslamp2005)

242 Ultraviolet(UV)lightsources

2421 365nmlightsource

For theUV light photocatalysis experiments aUV fluorescent lampwas used (Vilber

LourmatVL‐208BLB LeicestershireUK)The light sourceemitted lightprimarilyata

89

wavelength of 365 nmand the intensity of the lightwasmeasured using aUV light

meterSolarmeterModel50(SolartechIncHarrisonTownshipMichiganUSA)with

the readings recorded inmWcm‐2 Experimentswereconducted ina cabinet (Philip

HarrisLtdShenstoneUK)fittedwithaUVsafetyscreen


AsecondUVlightsourcewasused(VilberLourmatVL‐208GVWRLtdLeicestershire

UK)eitherasamethodfordecontaminatingtheusedsamplesortoactivatetheTiO2

slidesbeforeexposuretothe365nmlightsourceThisgermicidalUVfluorescentlamp

emitted lightprimarilyatawavelengthof254nmExperimentswereconducted ina

cabinet(PhilipHarrisLtdShenstoneUK)fittedwithaUVsafetyscreen

243 Laserlightsource

AHeNelaserlightsource(ChangchunNewIndustriesOptoelectronicsTechCoLtd

Changchun China) was used for the photodynamic therapy experiments The light

sourceemitted lightprimarilyatawavelengthof660nmanda light intensityof230

mW

25 Generalsamplingmethodology

Asuspensionofbacteriacontaining107cfumLbacteriaasdescribedinSection23

wasdilutedtenfoldinPBStoproduceaseriesofbacterialconcentrationsrangingfrom

107 ‐ 104 cfu mL The standard volume of bacterial suspension used in these

experimentswas25microLwhichoccupiedanareaofapproximately1cm2uponthetest

samplesthereforethefinalbacterialpopulationrangedfrom25x105ndash25x102cfucm‐

90

2 A standard volume (25 microL) of bacterial suspension was inoculated onto a clean

microscope slide of dimensions 76 x 26 x 08 ndash10mm (length xwidth x thickness)

(VWR International Ltd Lutterworth UK) and was sampled using a cotton‐tipped

swab The surface was swabbed for 20 seconds in three directions with continual

rotation of the swabhead ina standardisedmanner before inoculation intoabijou

containing1mLofPBSThebijouwasvortexedfor2minutestoremovetheadherent

bacterialcellsandpriortopreparationoftenfoldserialdilutionsTwentymicrolitresof

eachdilutionwasplatedoutontoeitherMacConkeyagar forE coli ormannitol salt

agar for S aureus and the plates were incubated at 37degC for up to 48 hours The

aerobic colony count (ACC) was calculated by counting the resultant colonies to

determinethenumberofcolonyformingunitspersquarecentimetre(cfucm2)


AseriesofluminometerswereusedtomeasureATPbioluminescenceasanalternative

methodofdetectingandquantifyingbacteriafromthetestsurfacesAllluminometers

were programmed to capture luminescence readings every 1 second and themean

reading in relative light units (RLU)was reported after 10 seconds Test tubeswere

requiredforthedetectionofATPusingcertainmodelsofluminometerandtodestroy

any exogenous ATP before use theywere placed under the 254 nm germicidal UV

lamp(Section2422)for30minuteswithinsealedplasticbagsThebagwasinverted

atthehalfwaypointtoprovideevenexposuretothelightsource

91

261 Luminometer‐specificmethodologies

2611 Juniorluminometer

The cotton‐tipped swabwas added to a test tube containing 50 microL ATP Eliminating

Reagent from theMicrobialATPKit (BioThemaABHandenSweden)post sampling

Thetubewasincubatedfor10minutesatroomtemperatureaccordingtothereagent

kit instructions before 50 microL Extractant BS was added and the covered tube was

vortexedfor5secondstothoroughlymixthesolutionFourhundredmicrolitresofATP

ReagentHSwas finally added and the light generatedwas quantified by placing the

tubeintotheJuniorLB9509luminometer(BertholdTechnologiesGmbHampCoKGBad

WildbadGermany)AnATPstandardwasusedoneachrunand10microLofthepremixed

100nmolLATPstandardwasaddedtothefinalsolutionsothattheequivalentof1

pmolATPwasaddedtothetestsolutionTheATPbioluminescenceofthetestsample

plustheATPstandardwasthenquantifiedbytheJuniorluminometer

Foreachbacterialconcentrationonasurfacethreeindependentswabswereusedto

generate an ATP bioluminescence reading and one swab was used for ACC

measurements with each dilution plated out in duplicate Each experiment was

performedatleastintriplicatetodemonstratereproducibility

2612 Lumatluminometer

The Lumat LB9507 luminometer (Berthold Technologies GmbH amp Co KG) is a more

sensitivebutlessportablemodelthantheJuniorluminometerThemethodologyused

tomeasureATPbioluminescenceemittedfromtestsamples incombinationwiththe

Lumat luminometerwas as described for the Junior luminometer in Section 2611

92

with the exception that the test tubewas placed in the Lumat luminometer for the

bioluminescencereadings

2613 BioProbeluminometer

TheBioProbeluminometer(HughesWhitlockLtdGwentUK)wasusedincombination

withtheMicrobialATPKitasinthepreviouslydescribedmethodologiesHoweverthe

ATP bioluminescence generated from the bacterial suspension could be measured

directlyfromthetestsurfacesothereagentswereapplieddirectlytothetestsurfaces

andtheunnecessaryswabbingstagewasomittedInsteadtheBioProbeluminometer

wasplacedabovethetestsurfacecreatingasealbetweenthe inoculated laboratory

benchandtheluminometerTheluminescencegeneratedwasthenquantifiedbythe

BioProbeluminometer

2614 Clean‐TraceNGluminometer

TheMicrobialATPKitwasnotrequiredforthedetectionassayutilisingtheClean‐Trace

NG luminometer (3M Bracknell UK) This luminometer was designed for use with

custom‐made pre‐moistened swabs which after sampling in the standard manner

were returned to thecasingand immersed ina reagent solution locatedat itsbase

The entire swab casing was placed in the luminometer for quantification after

vortexingfor5secondsApositivecontrolwasusedoneveryrunThiswasa freeze‐

driedpowdercontaining5pmolATPwhichwassampledwiththepre‐moistenedswab

andhandledusingthesamemethodologyasthetestsamples

93

27 DirectvisualisationofbacteriandashLiveDeadstaining

Slideswereexaminedunderthefluorescentlightmicroscopepost‐samplingusingthe

LiveDeadBacLightBacterialViabilityKit (InvitrogenLtdPaisleyUK)tovisualiseany

remaining bacterial cells and to determine their viability The kit consisted of two

stains SYTO9tradewhichpenetrated themembranesofall cells andpropidium iodide

which penetrated bacterial cells with damaged membranes (Boulos et al 1999)

Viable cells appeared green under the fluorescent microscope whereas non‐viable

cellsgeneratedaredfluorescenceImageswerecapturedonacameraattachedtothe

microscopeandbacterialcellswereenumeratedandtheproportionofviableandnon‐

viable cells was noted The final bacterial populationwas compared to the starting

inoculumvaluetoevaluatetheefficiencyofthesamplingprocess

28 Effectofwhitelightonbacterialsurvival

Glass microscope slides were placed in a moisture chamber which was custom‐

designed topreventevaporationof thebacterial inoculumduringexposure towhite

light (Figure 22) Filter paper 150 mm in diameter (Whatman plc Maidstone UK)

soakedinsteriledistilledwaterwasusedtolinethebaseofasquare24cmx24cm

petridishWoodenstickswereplacedontopofthefilterpapertoresttheslideson

Anadditionalmoisturechamberwascovered infoiltopreventlightpenetrationand

slideswhichweretobeincubatedintheabsenceoflightwereplacedinthismoisture

chamber for the exposure period as a dark control The moisture chambers were

placedinthecooledincubatorandtheuncoveredchamberwasplacedonashelf20

cmfromthelightsourcewiththefoilcoveredchamberontheshelfdirectlybelow

94

Figure 22 Experimental set up of the moisture chamber used during white lightexperimentswhereA=woodenswabsB=glassslidesC=moistenedfilterpaperD=bacterialinoculum

Theeffectof thewhite light source on theviabilityofanumberofbacterial species

was investigated A suspension of bacteria was inoculated onto a microscope slide

priortoincubationunderthewhitelightsourcefor24hoursAnydecreaseintheACC

aftertheirradiationperiodwascalculatedasapercentageandlogreduction

29 Optimisationofthesamplingtechnique

To increasetheproportionofbacteriathatwererecovered fromthetestsurfacesa

seriesofexperimentswereperformedandasinglevariablewaschangedUncoated

cleanmicroscopeslideswereinoculatedwithasuspensionof25microLofaGram‐negative

bacterium(Ecoli)oraGram‐positivebacterium(Efaecalis)andtheneither

(i)sampledusingarangeofdifferentswabtypes

(ii) sampledwith a cotton swab and either vortexed or sonicated to remove

adherentbacteria

A

B

C

D

95

(iii)sampledwithuptothreedifferentcottonswabswhichwerere‐suspended

intothesamebijou

(iv)sampledwithuptothreedifferentcottonswabswhichwerere‐suspended

intoseparatebijoux

Total bacterial numberswere calculated by serially diluting the bacterial suspension

within thebijouand inoculatingduplicate20microLaliquotsonto 5bloodagar plates

TheACCwascalculatedafterupto48hoursgrowthat37degCtodeterminethecfumL

andthisvaluewascomparedwiththeACCrecoveredfromthestartinginoculum

210 Preparationoflight‐activatedantibacterialmaterials

2101 Thinfilmsgeneratedbychemicalvapourdeposition

Novelantibacterialthinfilmsweregeneratedbyoneoftwopost‐doctoralresearchers

based at the UCL Department of Chemistry The thin films were prepared by

atmospheric pressure chemical vapour deposition (APCVD) (Section 151) The

depositionswerecarriedoutontheSiO2surfaceofslidesofstandardfloatglassfrom

Pilkingtonofdimensions220x85x4mm(lengthxwidthxthickness)coatedonone

sidewithabarrierlayerofSiO2topreventiondiffusionfromtheglasstothefilmThe

glasswaswashedpriortoinsertionintotheAPCVDreactorusingsequentialwashings

ofwateracetonepetroleumether(60‐80)andpropan‐2‐olgivingacleanandsmear

freefinish

96

21011 Nitrogen‐containingtitaniathinfilmsTiON‐1andTiON‐2

The nitrogen containing thin films TiON‐1 and TiON‐2 were prepared by Dr Geoff

Hyett with anhydrous ammonia (BOC Ltd) as the nitrogen source titanium (IV)

chloride (TiCl4 999 Sigma‐Aldrich Ltd) as the titanium source and ethylacetate

(EtAc990BOCLtdGuildfordUK)astheoxygensource(Hyettetal2007Aiken

etal2010)Depositionswerecarriedoutat550degCfor60secondsandtheresulting

filmswerecutintosevenequallysizedsectionsof32mmx89mmoncecooled

AnitrogencarriergaswasusedfortheTiCl4andEtAcataflowrateof2LminThe

TiCl4bubblerwasheatedto61degCandtheEtAcbubblerto44degCataflowrateof05L

minwhichproducedamolarmassflowratioof12TheTiCl4andEtAcwerecarriedto

a singlemixing chamber through gas delivery lineswhichweremaintained at 200degC

andheatedto250degCwithanadditionalflowofnitrogencarriergasatarateof12L

min The glass substratewas dopedwith nitrogen by flowing ammoniawithout the

carrier gas through the reservoir at a flow rate of 026 L min The TiCl4 and EtAc

mixture and the ammonia gas were introduced just before contact with the glass

substrateandtheTiCl4EtAcammoniamassflowratiooftheresultantthinfilmwas

28541TheresultantthinfilmwasTiON‐1thetitaniumoxynitrideThinfilmTiON‐

2waspreparedusingthesamemethodologyexceptthedepositionwascarriedoutat

450degCinsteadof550degC

21012 Nitrogen‐dopedtitaniumdioxidethinfilmsN1N2andN3

ThenitrogencontainingthinfilmsN1N2andN3werepreparedbyDrCharlesDunnill

witht‐butylamine(995FisherScientificUKLtdLoughboroughUK)asthenitrogen

97

sourcetitanium(IV)chloride(TiCl4999Sigma‐AldrichLtd)asthetitaniumsource

andethylacetate(EtAc990BOCLtdGuildfordUK)astheoxygensource(Dunnill

et al 2009bDunnill et al 2009cDunnill and Parkin 2009) The resultant coatings

werenitrogen‐dopedtitaniumdioxide(N‐dopedTiO2)thinfilmsanddepositionswere

carriedoutat500degCfor30seconds

AnitrogencarriergaswasusedfortheTiCl4andEtAcwhichwaspreheatedto150degC

ataflowrateof05LminTheTiCl4bubblerwasheatedto70degCandtheEtAcbubbler

to 40degC which produced a molar mass flow ratio of 12 The TiCl4 and EtAc were

carried to a singlemixing chamber and heated to 250degC with an additional flow of

nitrogencarriergaspreheatedto150degCatarateof6LminTheglasssubstratewas

doped with nitrogen by flowing the carrier gas preheated to 60degC through the t‐

butylamine reservoir set at 5degC the temperature of the t‐butylamine reservoirwas

controlledusingawaterbathcontainingwaterandethyleneglycolinequalpartsThe

TiCl4andEtAcmixtureandthet‐butylaminegaswere introduced justbeforecontact

withtheglasssubstrateat100degCwithanadditional flowofcarriergasat1Lmin

TheTiCl4 EtAc t‐butylaminemassflowratiooftheresultantthin filmwas1 25

03Sectionsofthesamesheetofthegeneratedfilmweredivided into25x25cm

samplesoncecooledanddividedintothreegroupsrepresentingthinfilmsN1N2and

N3

21013 Sulfur‐dopedtitaniumdioxidethinfilms

Threesetsofsulfurcontainingthinfilms(S‐dopedTiO2)werepreparedbyDrCharles

Dunnillusingtitaniumtetrachloride(TiCl4Sigma‐AldrichLtd)asthetitaniumsource

ethylacetate(EtAc990BOCLtd)astheoxygensourceandcarbondisulfide(CS2

98

999AlfaAesarHeyshamUK)asthesulfursource(Dunnilletal2009a)Anitrogen

carriergaswasused for theTiCl4 andEtAcwhichwaspreheated to150degCata flow

rateof05LminTheTiCl4bubblerwasheatedto70degCandtheEtAcbubblerto40degC

whichproducedamolarmassflowratioof12TheTiCl4andEtAcwerecarriedtoa

singlemixingchamberandheatedto250degCwithanadditionalflowofnitrogencarrier

gas preheated to 150degC at a rate of 6 L min The glass substratewas dopedwith

sulfurbyflowingthecarriergaspreheatedto60degCthroughtheCS2reservoirsetata

temperaturebetween0and10degCthetemperatureoftheCS2reservoirwascontrolled

usingawaterbathcontainingwaterandethyleneglycol inequalpartsTheTiCl4and

EtAc mixture and the CS2 gas were introduced just before contact with the glass

substrate at 100degC with an additional flow of carrier gas at 1 L min Depositions

were carried out at 500degC for 30 seconds and three thin filmswere producedwith

different TiCl4 EtAc CS2 mass flow ratios which varied dependent upon the

temperatureoftheCS2reservoirduringsynthesis

(i) during synthesis of sample S1 the reservoir was set at 0degC generating a

massflowratioof12509

(ii) during synthesis of sample S2 the reservoir was set at 5degC generating a


(iii)during synthesisof sampleS3 the reservoirwas setat10degCgeneratinga


Theresultingfilmswerecutintosevenequallysizedsectionsof32mmx89mmonce

cooled

99

21014 Controlthinfilms

ThinfilmsofTiO2weresynthesisedusingAPCVDwiththesamesyntheticconditionsas

that described above but omitting the addition of the dopant (ie ammonia t‐

butylamine or carbon disulfide) Uncoated glass of the same size was used as an

additionalcontrol

2102 Thinfilmsgeneratedbysol‐geldeposition

Thesilver‐titaniathinfilmsweregeneratedinatwo‐stepprocess(Dunnilletal2011)

glassslideswereinitiallycoatedwithtitaniumdioxideandannealedbeforeacoating

ofsilvernitratewasadded

21021 Titaniumdioxidesolpreparationandthinfilmsynthesis

TheTiO2 solwaspreparedbyadding25246gofacetylacetone (002526mol99+

Sigma‐AldrichLtd) toa250mLglassbeakercontaining32cm3butan‐1‐ol (035mol

994 Sigma‐Aldrich Ltd) This produced a clear and colourless solution to which

1750 g titanium n‐butoxide (005 mol 970 Fluka) was added The solution was

vigorously stirred for 1 hour before 364 mL distilled water dissolved in 905 g

isopropanol (015 mol analytical grade Fisher Scientific) was added to the stirring

titanium n‐butoxide solution The yellow colouration of the sol deepened but

remained clear and itwas stirred for a further hour Lastly 166 g acetonitrile (004

mol99minFisonsScientificUKLtd)wasaddedtothesolutionanditwasstirredfor

an hour The deep yellow coloured sol was covered with parafilm and left to age

overnightinthedark

100

21022 Titaniumdioxidethinfilmsynthesis

On the following day clean single cavity ground glass slides (Jencons Scientific Ltd

EastGrinsteadUK)ofdimensions76x26x1mm (lengthxwidthxthickness)were

attachedtothedipcoatingapparatusinbatchesof4(Figure23)

Figure23ThedippingapparatususedtoproduceaxerogelonthemicroscopeslidesPhotographreproducedwithpermissionfromDrKristopherPage

Thecavityslideswereloweredintoaglassbeakercontainingtheagedsolandafter20

secondsthecavityslideswerewithdrawnbytheapparatusatasteadyrateof120cm

min The first coat was allowed to dry before the process was repeated The

deposited xerogel films required calcination in order to adhere the coating to the

cavityslideandtobecomecrystallineThereforethecoatedcavityslideswereplaced

insideamufflefurnaceandfiredat500degCfor1hourwithaheatingrateof10degCmin

101

andacoolingrateof60degCminThethinfilmswerethenleftinthefurnaceovernight

to cool and stored in a dark container until required The resultant coatings are

referredtoasTiO2thinfilms

21023 Silver‐titaniumdioxidethinfilmsynthesis

Asolutionofsilvernitratewaspreparedbyadding042gsilvernitrateto500mLof

methanol(bothFisherScientificUKLtd)toproduceafinalconcentrationof5x10‐3mol

dm3TheTiO2thin filmswereattachedtothedipcoatingapparatusdipped inthe

silvernitrate solutionandwithdrawnata rateof120cm minThe thin filmswere

thenexposedtothe254nmUVlampfor5hourswithinacustommadelightboxand

were stored in the dark for at least 72 hours before bacteriological testing

Photodepositionoccursquickly (lt30min)butanexcessoftimewasusedtoremove

the time of irradiation as a variable and ensure that the filmswere fully clean and

activatedpriortoinitialcharacterizationTheresultantcoatingsarereferredtoAg‐TiO2

thinfilms

2103 ToluidineBlueO‐containingpolymersgeneratedbyswellencapsulation

Toluidine Blue O (TBO) was incorporated into polyurethane polymers by swell

encapsulation To achieve this 125mg of TBOwas added to 25mL distilledwater

beforetheadditionof225mLacetoneforminga91ratioofacetonetodistilledwater

(H2O10vv)Thesolutionwasplaced inasonicatingwaterbathfor15minutesto

ensure the TBO was evenly distributed throughout the suspension To prevent

interaction of the solution with light the container was covered in foil during

sonicationTenmillilitrealiquotsof theTBOsolutionwasdispensed intoglass screw

102

capped bottles and a 1 cm2 square of polyurethane was added The bottles were

stored horizontally in the dark for 24 hours The polyurethane squares were then

removedandlaidtodryonapapertowelandcoveredfor1hourAfterthistimethey

wererinsedwithsteriledistilledwateruntiltheexcessTBOadheredtothesurfaceof

the polymers had detached and thewater remained clear The polymerswere then

driedandstoredinthedarkforafurther24hoursbeforeuseBatchesof24polymers

were made and control polymers were also prepared without the addition of TBO

(Pernietal2009b)

211 Characterisation and functional assessment of light‐activated

antibacterialmaterials


UV‐visible‐IR spectroscopy was employed to determine the band onset of the thin

filmsandassessthe likelyphotocatalyticactivityofthematerialThethinfilmswere

decontaminated by exposure to the 254 nm germicidal UV lamp for 12 hours and

storedinthedarkfor72hoursThethinfilmwasthenplacedinsidetheUV‐Visible‐IR

spectrophotometer (Perkin Elmer λ950 Massachusetts USA) and percentage

transmission readings were measured which were converted to absorption and

absorbanceusingthereflectancetogaugethethicknessofthefilmsbytheSwanepoel

method(Swanepoel1983)DataweretransformedandaTaucPlotwasgeneratedto

determinetheopticalbandgapofthethinfilmsbyextrapolatingthe linearcurveto

theabscissaATaucplotcanbecalculatedusingtheformula(axhv)12againstenergy

whereadenotes the absorbance of thematerial andhvdenotes the energy of the

103

photon of light (Tauc 1968 1970) Measurements were also taken of the titanium

dioxidethinfilmanduncoatedglassslidesothatthereadingscouldbecompared


Water droplet contact angles were measured to determine the potential photo‐

induced hydrophilicity of the thin films The thin films were decontaminated by

exposuretothe254nmgermicidalUVlampfor12hoursandstoredinthedarkfor72

hours A FTA 1000 droplet analyserwas used tomeasure the diameter of a 86 microL

dropletofdeionisedwaterinoculatedontothethinfilmusingasidemountedcamera

The dropwas formed and dispensed by gravity from the tip of a gauge 27 needle

Readings were taken before and after irradiation with UV light (Section 2421) or

filteredwhitelight(Section241)between200and2500nmAnuncoatedglassslide

and titanium dioxide thin film were used as controls Results were entered into a

computer programme to calculate the contact angles based upon the volume‐

diameterdataAnaverageof5readingsweretakenateachexposuretimesothatthe

resultsobtainedwerereproducible


Thestearicacidtestwasusedtoquantifythephotocatalyticactivityofthethinfilmsas

a preliminary indicator of their potential antibacterial activity The destruction of

stearicacidwasmeasuredbyFourierTransform InfraredSpectroscopy (FTIR)usinga

PerkinElmerSpectrumRX1FTIRspectrometer

The thin filmsweredecontaminatedbyexposure to the254nmgermicidalUV lamp

for12hoursandstoredinthedarkfor72hoursThethinfilmswerethenattachedto

104

an IR sample holder comprised of a sheet of aluminiumwith a circular hole in the

centre beforea 10 microL dropofa001Msolutionof stearicacid inmethanol (Fisher

ScientificUKLtd)wasappliedtotheexposedportionofthethinfilmAcharacteristic

white smearwas observed once the droplet had evaporated and the sampleswere

thenstoredonceagain inthedarkforat72hourspriortothebaselinereadingat0

hours FTIR spectrawereobtained for the stearicacid layerbetween2800and3000

cm‐1 andanuncoatedglass slidewasusedasacontrol for thebackground readings

Baselinereadings(C0)weretakenofthethinfilmsandblankcontrolsthenallsamples

were placed in the custom‐made light box and were exposed to the light source

Readings (Cx)were takenat24hour intervalsand the sampleswere returned to the

lightboxaftereachreadingForeachtimepointtheareaofthepeakswereintegrated

andthevaluescombinedtogiveanapproximateconcentrationofstearicacidonthe

surfacewhere1cmminus1intheintegratedareabetween2700and3000cmminus1corresponds

to approximately 97times1015 molecules cm2 (Mills and Wang 2006) A graph was

plottedofthenormalisedconcentrationofstearicaciddetectedonthesurface(CxC0)

againsttimewhichallowedthedestructionofstearicacidtobeobserved

The light sources were attached to the lids of the custom‐made light boxes which

were suspended 25 cm from the surface of the thin films Three lighting conditions

wereexaminedaUVlightsource(Section2422)awhitelightsource(Section241)

andthewhitelightsourcefittedwithaUVfilterTheUVfilterusedwasa3mmthick

sheet of Optivextrade glass which is described to cut off all radiation below 400nm

(InstrumentGlasses2000)Thefilterwaspositioned1cmabovethesamplesandwas

setupsuchthatalllightarrivingatthesampleshadpassedthroughthefilter

105

212 Microbiological assessment of light‐activated antimicrobial

materials

2121 Decontaminationofthethinfilms

Priortomicrobiologicalassessmentcoatedsamplesweresoakedin70isopropanol

for 30 minutes to kill and remove any adherent contaminants rinsed with fresh

isopropanolandair‐driedThesampleswerethen incubated inahotairoven(Weiss

Gellenkamp oven BS Leicestershire UK) for 1 hour at 160degC to kill any residual

organisms and stored in the dark until required This process was repeated after

microbiologicalassessmentinpreparationforfurthertesting

The decontamination procedure was later amended and after microbiological

assessment the slides were rinsed with sterile distilled water and air‐dried before

exposuretothe254nmgermicidalUV lamp(Section2422) for18hourstokillany

remainingadherentorganismsTheslideswerethenplacedinthedarktoreversethe

activating effect of theUV light Sampleswere then ready for re‐use after 72 hours

dark storage Thin films were re‐used due to the lack in reproducibility of the

depositionmethod

2122 Measuringtheeffectof lightonthethinfilmsgeneratedbyAPCVDor

sol‐gel

Thethinfilmswereplacedina24x24cmpetridishlid20cmfromthelightsourcefor

theactivationstep(designatedA+)forthedesiredtimeperiodThethinfilmswerenot

coveredduringthislightexposureperiodAsacontrolduplicatethinfilmswerealso

106

placed inthecabinetbutwithina foil‐encased24x24cmpetridishtopreventlight

penetration(designatedA‐)

Thethinfilmswerethenpositionedwithinthemoisturechamber(Figure24)beforea

25microLdropletofbacterialsuspensionwasaddedThelidwasaddedtopreventdroplet

evaporation and the moisture chamber was placed under the light source at a

distanceof20cmfortheirradiationstep(designatedL+)andexposedforthedesired

periodoftimebeforesamplingControlduplicatethin filmswere incubatedwithina

foil‐encasedmoisturechamberduringthewhitelightexposureperiod(designatedL‐)

ThenomenclatureusedforthelightexposureexperimentsissummarisedinTable22

Figure 24 Irradiation of the nitrogen‐doped thin films to white light with thesamplesplacedwithinthecustomdesignedmoisturechamber

107

Table22Nomenclatureusedduringmicrobiologicalassessmentofthethinfilms

Nomenclature Description

A+L+Sample exposed to first light dose bacterial droplet addedthensampleexposedtosecondlightdose

A‐L+Sample stored in the dark bacterial droplet added thensampleexposedtosecondlightdose

A+L‐Sample exposed to first light dose bacterial droplet addedthensamplestoredinthedark

A‐L‐Sample stored in the dark bacterial droplet added thensamplestoredinthedark

Bacteria were recovered by sampling the thin films as described in Section 25

Experiments were performed in at least duplicate and repeated on a minimum of

threeseparateoccasionsforeachtypeofthinfilmandexposuretime

2123 Measuring the effect of light on Toluidine Blue O‐impregnated

polymersgeneratedbyswellencapsulation

Newly synthesised polymers (described in Section 2103) were used for each

experimentandwerediscardedaftereachuseApolymerwasplacedinawellwithina

6‐wellmicrotitreplatebeforea25microLdropletofthemicrobialsuspensionwasadded

Aglasscoverslipwascarefullyplacedontoptospreadthedropletevenlyacrossthe

surfaceofthepolymerandtheplatewastransferredtoaraisedplatform24cmfrom

thelaserlightsourceThelightemittedfromthelaserpassedthroughabeamdiffuser

tospreadthelightbeamsothattheentirepolymerwasexposedtothelaserlightand

thepolymerwasexposedtothelaserlightfortherequiredperiodoftime

108

Oncetheexposuretimehadendedthecoverslipwasasepticallyremovedandplaced

insidea50mLtubecontaining135microLPBSA10microLaliquotofthemicrobialdroplet

wasremovedfromthepolymerandinoculateddirectlyontoanappropriateagarplate

andspreadusinganL‐shapedspreaderTheremaining15microLofmicrobialsuspension

was recovered placed in the 50mL tube and briefly vortexed before tenfold serial

dilutions were prepared Twenty microlitres of each dilution was inoculated and

spread onto an appropriate agar plate in duplicate As controls TBO‐containing

polymerswereinoculatedwiththemicrobialsuspensionforthesamelengthoftimein

the absence of laser light (L‐S+) or polymers preparedwithout the addition of TBO

were inoculated with the microbial suspension and exposed to identical periods of

laser light (L+S‐) or not exposed to the laser light (L‐S‐) The sampling process was

repeated three times for each polymer type and exposure time and the entire

experimentwasrepeatedonatleastthreeseparateoccasionsforeachorganismand

exposuretime(Pernietal2009b)

213 Statisticalanalysis

Inordertodeterminethesignificanceofanydecreases inthecfuobservedbetween

the light‐activated antibacterialmaterials exposed to different conditions theMann

WhitneyUtestwasusedThenumberofsurvivorsrecoveredfromthetestgroup(ie

thelight‐activatedmaterialexposedtolight)wascomparedtothenumberofsurvivors

fromthecontrolgroups(ie the light‐activatedmaterialsnotexposedto lightorthe

uncoated samples)Median valueswere taken because the datawere not normally

distributedand thevalueswere transformed to log10 fornormalisationAp valueof

less than 005 was considered statistically significant Statistical significance is

109

diagrammaticallyrepresentedontheboxandwhiskerplots intheresultssectionsas

asterisksoneasteriskdenotesapvaluelt005twoasterisksdenotesapvaluelt001

andthreeasterisksdenotesapvaluelt0001Allstatisticalanalyseswereperformed

usingtheSPSSstatisticalpackage(version160SPSSIncChicagoILUSA)

110

3 Developmentofprotocolsusedtoassesstheactivityofthephotocatalyticthinfilms

31 Introduction

The purpose of the work described in this chapter was to develop a reproducible

method of testing the antibacterial photocatalytic activity of thin films Initially the

sampling technique was examined to determine the sampling efficiency and an

optimised regimen was developed Researchers from our laboratory had previously

used swabs (Page et al 2007) to remove bacteria from the test surface in order to

detectchangesinthebacterialconcentrationpost‐exposuretoantibacterialcoatings

Othergroupshaveuseddipslidesasadirectdetectionmethodbutthisisunsuitable

for accurately quantifying high concentrations of bacteria as it results in confluent

growth which only generates an estimate of the bacterial load The recovery of

bacteriafromglasssurfaceswasinitiallycomparedusingarangeofswabswithswab

headscomprisedofdifferentmaterialsusingadifferingnumberofswabspersample

and using sonication as a method of releasing bacterial cells from the swab head

There are however inherent problems with swabbing as bacteria are either left

behindonthesurfaceafterswabbingorgetcaughtwithinthemeshoftheswabhead

andarenotreleasedintothediluentaftersampling(Davidsonetal1999)

Antimicrobial coatings are generally assessed using the viable count technique and

bacterialsurvivalisdeterminedbycountingcoloniesoriginatingfrom(i)serialdilutions

ofthebacterialsuspensiononthecoating(Wilson2003Decraeneetal2006Page

et al 2007) (ii) those grown on an agar overlay applied to the entire coating

(Decraeneetal2008b)(iii)serialdilutionsofthebacterialsuspensionaftertheentire

111

coating has been immersed in a sterile fluid and agitated to remove adherent

organisms(Decraeneetal2008a)or(iv)acombinationofthese(Pernietal2009a)

These techniques have proven to be effective at determining the activity of novel

antimicrobial coatingsbut the turnaround time for results is around48hours soan

alternativefastermethodisstilldesirable

ATPbioluminescencehasbeenusedasarapiddiagnostictesttodetectbacteriafrom

urinesamples(Selanetal1992)andmorerecentlyhasbeenappliedinthehospital

environment to rapidly assess the efficiency of cleaning regimens in hospitals as

described in Section 164 (Griffith et al 2000 Malik et al 2003 Dancer 2004

Ayciceketal2006Griffithetal2007Willisetal2007Lewisetal2008Boyceet

al2009Mulveyetal2011)followingonfromthesuccessfuluseofthismethodin

the food industry for the monitoring of surface cleanliness (Poulis et al 1993

HawronskyjandHolah1997Ayciceketal2006)Thecleanlinessofasurfacecanbe

rapidlyassessedand if the levelofATP isaboveanacceptable level thenthesurface

canbere‐cleanedandretested

ATPbioluminescenceutilisesthefirefly luciferaseenzymetocatalysetheconversion

ofATPintoAMPresultingintheemissionoflight(Lundin2000)Theamountoflight

emitted is quantified by a luminometer and is directly proportional to the initial

amountofATPinthesampleIftheeukaryoticATPisremovedfromthesurfacebefore

sampling then this value is in turn proportional to the amount of bacteria in the

startingsampleasonephotonoflightisgeneratedpermoleculeofATPForthisstudy

themethodwasevaluatedforitspotentialuseasatooltoassesstheeffectivenessof

novel antibacterial coatings by quantifying bacteria present on a surface before and

112

after light exposure The generation of quantitative data especially at low bacterial

concentrationswouldbeusefulanditwaspostulatedthatATPbioluminescencecould

supersede swabbing as the first choice for bacterial detection from surfaces in this

project

Alsoassessedinthischapterwastheeffectoftheincidentlightsourceonthesurvival

ofbacteriaCertainspecificwavelengthsofwhite lightareknownto inactivatesome

Gram‐positivestrainsofbacteria(Macleanetal20082009)soitwasimportanttobe

aware of the effect of the light source used to activate the novel thin films Any

decreaseinthebacterialconcentrationcouldthenbeattributedtotheactivityofthe

thinfilmsandnottoincidentlightsource



BacterialstrainsweremaintainedasdescribedinSection21andbacterialsuspensions

ofEcoliandEfaecaliswerepreparedasdetailedinSection23resultinginastarting

inoculumofapproximately107cfumlAnumberofstrategieswereemployed inan

attempttoimprovebacterialrecoveryfromthesurfaceofuncoatedmicroscopeslides

as described in Section 29 Three different cotton swabs were used (all Fisher

ScientificUKLtd)woodstickcottontippedswabs‐CottonAcottonswabssterilisedby

ethyleneoxide‐CottonBandcottonswabssterilisedbyUVlight‐CottonCAlginate

andviscoseswabswerealsousedinthecomparison

113



ofEcoliandSaureuswerepreparedasdetailedinSection23resultinginastarting

inoculum of approximately 107 cfu ml ATP bioluminescence was used to detect

bacteria inoculated onto the surface of uncoated microscope slides as described in

Section261Anumberofcommercialluminometerswereusedwithoutputgivenin

relativelightunits(RLU)andtheamountofATPpresentinthesampleswascalculated

usingthefollowingformula(HughesWhitlockLtd1995)

ATPsample=RLUsample(RLUsample+standardndashRLUsample)

The number of bacteria present in each sample was then calculated based on

previously documented studies which estimate that each bacterial cell contains

approximately2x10‐18molATP(Lundin2000BioThemaAB2006)Itwasimportantto

determinetheinitialamountsofATPpresentasotherwisetheRLUreadingsobtained

fromdifferent luminometerscouldnotbedirectlycompared(HawronskyjandHolah

1997)Toassessthesensitivityoftheassayusingeach instrumentone‐tailedt‐tests

were performed where the sensitivity was the lowest concentration that was

significantlydifferentfromthenegativecontrolwith95confidenceThecoefficient

ofvariation(CV)wascalculatedasapercentageforeachdilutiontodemonstratethe

reproducibilityofeach luminometerwheregreater reproducibility is representedby

lower CV values particularly below 100 (Griffith et al 1994) The luminometer‐

specific methodologies were assessed to determine the precision accuracy and

sensitivityofeachassayusingthedefinitionsdescribedinTable31

114

Table 31 Definitions of the terms used to compare the luminometer‐specificmethodologies

Parameter Definition

PrecisionA measure of the reproducibility of the luminometer‐specificmethodAssessedbycalculatingthecoefficientofvariation(CV)

SensitivityThe lowest concentrationofbacteria that is significantlydifferenttothenegativecontrolAssessedbyperformingone‐tailedt‐tests

AccuracyHow close the value generated by the luminometer‐specificmethod is to the true value Assessed by comparison with theinoculumlevelestimatedbyviablecolonycount

323 Measuringtheeffectofwhitelightonbacterialsurvival


of S aureus NCTC 6571 E coli ATCC 25922 E faecalis S pyogenes ATCC 12202

EMRSA‐16 EMRSA‐15 MRSA 43300 S aureus NCTC 8325‐4 and S epidermidis 01

were prepared as detailed in Section 23 resulting in a starting inoculum of

approximately 107 cfu ml equating to approximately 25 x 105 cfu sample The

effectofthewhitelightontheviabilityofbacterialstrainswasdeterminedusingthe

methodologydescribedinSection28andFigure22TheMannWhitneytestwasused

to determine the statistical significance of any differences observed as described in

Section213

115

33 Results


The use of different swabs during sampling did not result in a notable increase in

bacterial recovery (Figure 31) the greatest recovery of E coli and E faecaliswas

observedusing thealginate swabbut there remaineda973and 992 respective

loss compared with the starting inoculum Recovery of E coli and E faecalis using

cottonswabCresultedina989and996lossofbacteriarespectivelyandtheuse

ofcottonswabAresultedina989and997lossofbacteriarespectivelyOverall

recoveryofEcoliwasbetterthanrecoveryofEfaecalis

Figure31ComparisonofdifferentswabtypestoincreasetherecoveryofEcoliandEfaecalisTheuseofanyoftheswabtypesresultedinalossofmorethan97ofbacteriaduringtheswabbingprocessBarsindicatemeanvalues(n=8)anderrorbarsrepresentstandarddeviations

116

ThereforeEcoliwasusedtoassessfurtherimprovementsinthesamplingtechnique

withcottonswabASonicatingtheswabsaftersamplingthesurfacedidnotresultina

greater recoveryofE colinor did theuseofmore than one swab (Figure32) The

methodwhichresultedinthegreatestrecoveryofbacteriawasthe2‐swabin1bijou

methodbuttherewasstilla98differencebetweenthestartingconcentrationofE

coli and the concentration recovered All nine methods tested resulted in losses of

morethan98ofEcoliThereforethe1‐swabtechniquewithcottonswabAanda

120secondvortexwasusedforallsubsequentexperimentsThedifferenceinrecovery

betweenthevarioustechniqueswasnotsubstantialandthechosenmethodwasthe

leastlabourintensiveandmostcosteffective

Figure32ComparisonofdifferentsamplingmethodsusedtoincreasetherecoveryofEcoliAllsamplingmethodstrialledresultedinlossesofmorethan98ofEcoliBarsindicatemeanvalues(n=8)anderrorbarsrepresentstandarddeviations

117


3321 Saureus

Themost accurate prediction of the concentration ofS aureuswas producedwhen

the BioProbe luminometer was used to detect ATP bioluminescence a starting

inoculumof 25x105 cm2was reportedas67x105 cm2 (Figure 33)However the

highest dilutions of bacteriawere not always detected andwere falsely reported as

negativewhichresultedinlargestandarddeviationsandacoefficientofvariation(CV)

of over 100 for the lowest concentration of bacteria (Table 32) Furthermore the

methodology was not the most sensitive the calculated sensitivity of the BioProbe

assaywas 25x104 cm2 (plt005)whichmeant that lower bacterial concentrations

couldnotbedifferentiatedfromthenegativecontrolAnaccurateestimateoftheS

aureus concentrationwas also producedwhen the Junior luminometerwas used to

detectATPbioluminescenceHoweveratthelowesttestconcentrationthevariance

ofthedatawasverylargewhichsimilarlyresultedinaCVvalueabove100

118

Figure33Comparisonofthefivedifferentmethodsemployedforthedetectionofsurface‐associated S aureus Data points represent mean values and error barsrepresentstandarddeviations(Aikenetal2011)

Table 32 Reproducibility of the ATP bioluminescence assay using the fourluminometerstodetectSaureusdisplayedascoefficientsofvariation(CV)wherealower CV represents a greater reproducibility All values are expressed aspercentagesThesensitivityofeachassayismarkedwithanasterisk

cfucm2

SaureusLumat Junior BioProbe

Clean‐Trace

25x105 16 62 52 21

25x104 20 64 70 29

25x103 27 51 62 35

25x102 44 158 137 133

The most precise estimate of the bacterial load on the test surface was generated

when the Lumat luminometer was used to detect ATP bioluminescence (p lt001)

whereprecisionisanindicationofthereproducibilityofthemethodThepresenceof

119

25x102cm2(thelowestdilutionfactortested)ofSaureuswasconsistentlydetected

(Figure33)and low levelsofbacteriawerenotmisreportedasnegativewhichwas

confirmedbythelowCVvaluesobtained(Table32)foralldilutionfactorsHowever

theaccuracyofthedevicewaspoorasthedetectedconcentrationofbacteriawasat

leastafactorof10lowerthantheinoculumaddedtothetestsurface

When the Clean‐Trace luminometer was used to detect ATP bioluminescence an

inaccurate result was always generated although the data produced was always

reproducibleTheconcentrationofSaureuswasunderestimatedbyalmostafactorof

10 at each dilution factor At low bacterial concentrations an absence of ATP was

commonlyreportedresultinginlargestandarddeviationsandaCVvalueover100at

thelowestbacterialconcentration

Reproducible estimateswere obtained using the viable countmethod however the

bacterial loadwasunderestimatedbyuptoa factorof10andwas lowerthanthose

values generated by the ATP bioluminescence assays using the BioProbe or Junior

luminometersA largevariation in thevaluesobtainedathigher concentrationswas

alsoseenalthoughthepresenceofbacteriawasnevermisreported

3322 Ecoli

ThemostaccuratepredictionoftheconcentrationofEcoliwasproducedwhenthe

BioProbe luminometer was used to detect ATP bioluminescence and a starting



negativewhich resulted in large standarddeviationsandCVvaluesofover100A

120

lessaccuratepredictionoftheconcentrationofEcolipresentonthetestsurfacewas

providedwhentheJunior luminometerwasusedtodetectATPbioluminescenceFor

examplewhen the starting inoculumwas 25x105 cm2 the bacterial concentration

was underestimated by a factor of 10 and at the lowest bacterial concentration no

bacteria were detected on any of the six replicates performed (Figure 34) The

reproducibilityoftheassaywaspoorwhichwasreflectedbythehighCVvaluesaCV

valueof0wasobtainedwhenthestarting inoculumwas25x102 cm2butthiswas

onlybecauseoftheinabilityoftheassaytodetectthepresenceofEcoli

Figure34Comparisonofthefivedifferentmethodsemployedforthedetectionofsurface‐associated E coli Data points represent mean values and error barsrepresentstandarddeviations(Aikenetal2011)

121

Table 33 Reproducibility of the ATP bioluminescence assay using the fourluminometers to detect E coli displayed as coefficients of variation (CV)where alower CV represents a greater reproducibility All values are expressed aspercentagesThesensitivityofeachassayismarkedwithanasterisk

cfucm2Ecoli

Lumat Junior BioProbeClean‐Trace

25x105 14 85 52 32

25x104 23 67 32 36

25x103 15 254 58 54

25x102 13 0 98 104

ThemostsensitiveandreproducibleestimateofthenumberofEcolipresentonthe

test surface was generated when the Lumat luminometer was used to detect ATP

bioluminescence (Figure 34) Low levels of bacteria were always detected and not

misreportedasnegativeand therewasvery little variationobserved in the readings

generatedwhichwasconfirmedbythe lowCVvaluesobtainedforallconcentrations

ofbacteriatested(Table33)Howevertheaccuracyoftheestimatewaspooraswas

alsoseenintheSaureusassayandthedetectedconcentrationofbacteriawasatleast

afactorof10lowerthantheinoculumlevelForexamplejust74x103cm2ofEcoli

wasdetectedbythismethodwhenthestartinginoculumwas25x105cm2


accuratepredictionoftheconcentrationofEcoliatthelowestdilutionswasprovided

(Figure 34) However there was little differentiation between the highest two

dilutionsofbacteriatestedForexampleastartingconcentrationofEcoliof25x103

cm2 was reported as 34x102 cm2 and a starting concentration of 25x102 cm2

122

reported as 17x102 cm2 and this problem was compounded by the fact that the

highestdilutionsofeitherbacteriawerenotalwaysdetectedandthusfalselyreported

asnegativeresultinginlargestandarddeviationsandCVvaluesofover100

Theviable countmethodwassuperiortoallothermethodsforEcolidetectionFor

examplewhenthestarting inoculumofEcoliwaseither25x105 cm2or25x102

cm2 respective concentrations of 11x105 cm2 and 14x102 cm2 were obtained

(Figure 34) The presence of bacteria was always reported even at low

concentrationswhichwasnotshownforalltheluminometer‐basedmethods


3331 Comparisonof4bacterialstrainsonaglasssubstrate

White lightwasobservedtohaveanantibacterialeffecton the survivalofSaureus

NCTC6571 onaglass surface (Figure 35)After24hoursexposure towhite light a

statisticallysignificantreductioninviableorganismswasseen(56log10cfusample)

comparedwiththecontrolconditionswithoutwhitelightexposureThemediancount

wasbelowthedetectionlimitoftheassaybuttherewasawiderangeincountsand

valuesbetween0and47log10cfusamplewereobtained(plt0001)

White light did not have an effect on the survival of E coliATCC 25922 on a glass

surface (Figure36)After24hoursexposure towhite light anegligible reduction in

viableorganismswasseen(02log10cfusample)comparedwiththecontrolsample

which was not exposed to white light Although when the data were statistically

analysedahighly significantdifference in countswasobserved thiswasdue to the

123

very smallerrorbars in this seriesofexperimentsattributed to the little variation in

counts obtained on each experimental repeat Such small differenceswould not be

consideredmicrobiologicallydifferent

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions

Figure35EffectofthewhitelightsourceonthesurvivalofSaureusNCTC6571onaglasssurfaceA25microlbacterialsuspensionwas inoculatedontoaglassslidebeforeexposetowhite lightfor24hours(L+n=29)Asacontrol inoculatedglassslideswerealsoincubatedinthedarkfor24hours(L‐)Thethickhorizontallinesindicatemedianvaluesthebaseandtopofeachboxrepresentsthe25and75quartilesrespectivelyandtheerrorbars the10and90percentilesandthesmallcirclesareoutliersThedottedhorizontallineindicatesthedetectionlimitofthesamplingmethod14log10cfusample

124

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions

Figure36EffectofthewhitelightsourceonthesurvivalofEcoliATCC25922onaglasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=10)

TheeffectofwhitelightonthesurvivalofEfaecalisonaglasssurfacecanbeseenin

Figure37After24hoursexposure towhite light a smallbut statistically significant

reduction in viable organismswas seen (01 log10 cfu sample) comparedwith the

controlsamplethatwasnotexposedtowhite light(plt005)Awiderange incounts

was obtained with values between 22 and 54 log10 cfu sample observed on the

surfaceexposedtolight

125

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions

Figure 37 Effect of thewhite light source on the survival ofE faecalis on a glasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=6)

White lightwasalsoobservedtohaveaneffectonthesurvivalofSpyogenesATCC

12202 inoculatedontoaglasssurface(Figure38)After24hoursexposuretowhite

lighta13 log10cfusamplereduction inviableorganismswasseencomparedwith

thecontrolconditionswithoutwhitelightexposurewhichwasstatisticallysignificant

(plt005)Therewasawiderangeincountsandvaluesbetween0and45log10cfu

samplewereobtained

126

Figure38EffectofthewhitelightsourceonthesurvivalofSpyogenesATCC12202onaglasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=4)

3332 ComparisonofSaureusstrainsonaglasssubstrate

Thedata collected in the previous sections suggested thatSaureusNCTC6571was

particularlysusceptibletothewhitelightusedforthisseriesofexperimentssoitwas

decided toexamineotherSaureus strains to seewhether theyshare this increased

sensitivity towhite light inactivation This was particularly important as it would be

usefultoassesstheactivityofthelight‐activatedantimicrobialcoatingsagainststrains

ofSaureus especially theepidemic strainsEMRSA‐15and EMRSA‐16because they

areacommoncauseofHCAIstheyhavebeenthepredominantcirculatingstrainsof

MRSAintheUKandarecitedasthecauseofmorethan95ofMRSAbacteraemias

(Johnsonetal2001Ellingtonetal2010)

AreductionintherecoveryofbothEMRSA‐16(Figure39)andEMRSA‐15(Figure310)

wasseenfromtheglasssurfacesexposedtothewhitelightsourcecomparedtothat

127

recoveredfromthesurfacesnotexposedtowhitelightTheobservedreductionswere

statistically significantandwere09 log10 cfu sampleand15 log10 cfu sample for

EMRSA‐16 (p lt001) and EMRSA‐15 (plt001) respectively indicating that EMRSA‐16

waslesssusceptibletothewhitelightcomparedwithEMRSA‐15

WhitelightwasobservedtohaveamuchgreatereffectonthesurvivalofMRSA43300

inoculatedontoaglasssubstrate(Figure311)After24hoursexposuretowhitelight

a statistically significant reduction in viable organisms was seen (46 log10 cfu

sample) compared with the control conditions without white light exposure The

mediancountwasbelowthedetectionlimitoftheassaybuttherewasawiderangein

countsandvaluesbetween0and46log10cfusamplewereobtainedTheseresults

were similar to thoseobservedafterSaureusNCTC6571wasexposed to the same

lightconditions(Figure35)

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample

Figure 39 Effect of thewhite light source on the survival of EMRSA‐16on a glasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=8)

128

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample

Figure310Effectof thewhite light sourceon the survivalofEMRSA‐15onaglasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=12)

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample

Figure311EffectofthewhitelightsourceonthesurvivalofMRSA43300onaglasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(L‐n=10L+n=12)

129

Theeffectofwhite lightonthesurvivalofSaureusNCTC8325‐4 isshown inFigure

312A33log10cfusamplereductioninbacterialcountwasobservedcomparedwith

thecontrolgroupwhichwasnotexposedtowhitelightandthisreductionwashighly

statistically significant The survival of S aureus NCTC 8325‐4 also appeared to be

affectedbytheexperimentalsetupasareductionintherecoveryofbacteriafromthe

control groupwas seen whichwas also statistically significant at the 01 level S

aureusNCTC8325‐4appearedtobeslightlymoretoleranttotheeffectsofthewhite

lightcomparedwithSaureusNCTC6571(Figure35)

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample

Figure312EffectofthewhitelightsourceonthesurvivalofSaureusNCTC8325‐4onaglasssurfaceSampleswereexposedtowhitelightfor24hoursafteradditionof25microlofabacterialinoculum(n=8)

130

Table34SummaryofresultsfromtheseriesofexperimentsexaminingtheeffectofwhitelightonbacterialsurvivalDataareexpressedasmedianvalues

BacterialstrainReductioninbacterialrecovery

(log10cfusample)

SaureusNCTC6571 56

EcoliATCC25922 02

Efaecalis 01

SpyogenesATCC12202 13

EMRSA‐16 09

EMRSA‐15 15

MRSA43300 46

SaureusNCTC8325‐4 33

34 Discussion


Accurateassessmentoftheactivityofthelightactivatedcoatingsisdependentupona

reliable and reproduciblemethod of detecting bacteria found on the surface of the

coatings both before and after light exposure (Verran et al 2010a) Therefore the

sampling technique used previously in this laboratory was examined to determine

whetheritcouldbefurtherimprovedDifferenttechniqueswereusedtomeasurethe

levelofmicrobialcontaminationonuncoatedsurfacesSwabsarethemostcommonly

used technique for measuring surface contamination but it has been well reported

that the rate of bacterial recovery using thismethod is poor (Davidson et al 1999

MooreandGriffith2007)Cotton‐tippedswabsareoftenusedbecausetheyabsorba

large volumeof the bacterial suspension on the surface so the surface appears dry

after sampling However bacteria become entangled within the meshwork of the

131

cellulose fibres of the swab head and are not readily released during vortexing

resulting in a low count during enumeration (Favero et al 1968) Viscose is a

derivativeof cottonsowouldbe likelytoabsorb liquidtothesamedegreeAlginate

swabshavebeenreportedtoimprovetherecoveryofbacteriafromsurfaces(Pageet

al2007)butthesedatashowthatthisimprovementwasnotsubstantialandthatthe

bacterial recovery was comparable to the other swab head materials Swab heads

comprisedofman‐madefibressuchasnylondonotretainliquidtothesamedegree

and so any organisms taken up by the swab are readily released However fewer

bacteria are taken up by the initial sampling event so a similarly low count is

generated(Davidsonetal1999)Detergentbasedsamplingsolutionshavebeenused

to increase sampling efficiency and could have been used instead of PBS in these

studiestoimprovebacterialrecovery(SaloandWirtanen1999)

Other factors to consider when interpreting data generated from viable counts are

thateachcolonyformingunitcountedonaplatedoesnotnecessarilycorrespondto

one bacterial cell as a clump of numerous cells will form one colony as will one

bacterial cell Light exposure causes bacterial stress which in turn causes bacterial

clumping and a concomitant reduction in the number of organisms recovered

Furthermore both the swabbing and vortexing processes used to remove adherent

organismsfromthesurfaceandswabheadrespectivelycandamagethe integrityof

thebacterialcellwallwhichwouldalsoresultinasmallernumberofviablecellsanda

lower viable count (Obee et al 2007) To detect the presence of residual

microorganisms remaining on the surface post‐sampling microscopic examination

132

could be employed and any remaining bacteria could be stainedwith a differential

viabilitystain(Verran2010Verranetal2010a)


Samplingasurfacewithaswabcangiveagoodindicationofthepresenceofbacteria

but does not provide an exact concentration of the bacteria present on the surface

(MooreandGriffith2007Verranetal2010a)Luminometersareusedfrequentlyin

thefoodindustry(Davidsonetal1999Storgardsetal1999)andincreasinglyinthe

healthcareprofession(Griffithetal2000Dancer2004Lewisetal2008)todetect

thepresenceofmicrobialcontaminationandorganicsoilFourdifferentluminometers

were tested as alternative sampling methods to swabbing and performing viable

counts

Previousstudieshaveshownthatitisnotpossibletodetectlownumbersofbacteria

fromatestsurfaceusingATPbioluminescence(Saloetal1999)specificallylt103cfu

cm2(Davidsonetal1999Mooreetal 2001MooreandGriffith2002) Improved

more sensitive luminometers such as the Lumat and the Junior were used in this

chapter inaddition toan improveddetection reagent thateliminatednon‐microbial

ATPandclaimedtobeabletodetectasfewasfivebacterialcells(BioThemaAB2006)

soanincreasedsensitivitywasexpected

However this study supports previous findings and has demonstrated that ATP

bioluminescencewasnotsuitableforaccuratelydetectingthenumberofbacteriaona

test surface over a range of concentrations (Aiken et al 2011) The methodology

utilising the BioProbewas able to detect higher concentrations of both E coli or S

133

aureus but no one method was able to reproducibly detect both organisms at all

bacterial concentrations At lower concentrations of bacteria the BioProbe‐based

assayeitherdidnotdetectthepresenceofbacteriaormadenodistinctionbetween

the suspensions containing 25x103 cm2 and 25x102 cm2 The BioProbe

methodologywaslikelytohaveproducedthebestresultsbecausetheinstrumentwas

specificallydesigned fordetectingbacteriadirectly froma flat surfaceHowever the

BioProbe is no longer commercially available so the use of this instrument was

unsuitable for future studies The methods employing the Junior Clean‐Trace and

LumatluminometersandindeedviablecountsallincorporateaswabbingstepForthe

organisms to be detected by these methods they therefore needed to be both

capturedbytheswabfromthetestsurfaceandreleasedfromtheswabheadintothe

diluentpriortoquantification(MooreandGriffith2002)whichlimitstherecoveryof

bacteriafromthesurface

TheLumat luminometerwasstatisticallythemostsensitivemodeltested(plt001at

25x102 cm2 for both E coliandS aureus)andproduced consistent data at every

dilution tested However the estimate although reproducible was not always

accurateandwasuptotenfoldlowerthanboththeknownconcentrationofbacteria

inoculated onto the test surface and the estimates made using alternative

luminometersThiswasdisappointingasunderoptimumconditionstheinstrumentis

abletodetect1amolATPwhichcorrespondstolessthanonebacterialcell(BioThema

AB2006BertholdTechnologiesGmbHampCoKG2007)The instrument isdesigned

forexperimentssuchasgenereporterassaysandluminescentimmunoassays(Dyeret

134

al2000McKeatingetal2004)andthisworksuggeststhatthepublishedsensitivity

cannotbetransferredtothequantificationofbacteriafromsurfaces

Inthepresent laboratorystudyacorrelationbetweencolonyformingunitsandRLU

wasmadebutithaspreviouslybeendifficulttodemonstrateahighdirectcorrelation

between these parameters outside of laboratory conditions because ATP

bioluminescence detects all ATP present on the sampled surface including organic

material of bacterial origin food residues human secretions and dirt (Poulis et al

1993)GenerallyofthetotalATPisolatedfromahandtouchsurface33ismicrobial

in origin therefore it is likely that theRLUvaluesobtainedwillbehigher than that

expectedifonlymicrobialATPwasdetected(Griffithetal2000)Howeveranumber

ofgroupshavedemonstratedacorrelationbetweentheseparameters

Selanetal(1992)usedATPbioluminescencetodetecturinarypathogensfromeither

bacterial culture or patient samples and employed the NRB Lumit PM kit At high

bacterialconcentrations(gt105cfuml)acorrelationbetweencfumlandRLUwas

observedwhere105cfumlEcolicorrespondedto10ndash500RLUand109cfumlE

coli corresponded to an RLU of around 87000 A statistically significant but low

correlationbetweencfumlandRLUvalueswasdemonstratedwhenthe3MClean‐

Trace ATP system was used to monitor the effectiveness of cleaning in a hospital

(Boyceetal2009)Othergroupshavedemonstratedaweakcorrelationbetweenthe

ATPscoreandmicrobialgrowthwhendifferentATPsystemswereusedtoassessthe

cleanlinessofhospitalwards(Ayciceketal2006Mulveyetal2011)Inaseparate

cleaning study sites which were considered unsatisfactory by ATP bioluminescence

werealsoshowntobeunsatisfactorybymicrobiologicalswabbing(Willisetal2007)

135

Articles in the literaturehavequestioned thevalue in correlating theaerobic colony

count and ATP bioluminescence RLU values because they measure different

parameterstheformermeasuresthenumberofviablemicroorganismsandthelatter

measurestheresidualorganicsoilwhichcouldbeofmicrobialornon‐microbialorigin

(Lewisetal2008) Inthischaptera relationshipbetweentheviablecountandATP

bioluminescence readings was sought and this was valid because the test surfaces

weredecontaminatedbeforeuse so itwasassumed thatno residualATP remained

Additionallythereagentkitthatwasusedcontainedan initialstepwhicheliminated

non‐microbialATPwhichfurtherincreasesthelikelihoodthatanyATPdetectedonthe

surfaceswasofbacterialoriginandnotfromanotherexogenoussourceHoweverthis

questionisperhapsinvalidwithinthecontextofassessingthecleanlinessofahospital

environment

An important limitation of ATP bioluminescence is that no information about the

bacterialspeciesisgiven(HawronskyjandHolah1997)Withinahospitalenvironment

itwouldbeadvantageoustodifferentiatebetweenbacterialspeciesforexamplethe

presenceofMRSAonapatientrsquosbed‐railwouldbeofmuchgreater interestclinically

thanthepresenceofcoagulase‐negativestaphylococcionthesamesurfaceMolecular

techniques such as the polymerase chain reaction (PCR) or culture‐based methods

wouldberequiredtospeciatethebacteriapresent

343 Theeffectofwhitelightonbacterialsurvival

Finally the effect of white light on the viability of a range of microorganisms was

investigated to ensure that any reduction in bacterial counts observed on the novel

136

lightactivatedthinfilmstobetestedwasattributeddirectlytotheintrinsicactivityof

thecoatingsandnotduetothelightexposureitselfWhenEcoliandEfaecaliswere

inoculated onto uncoated glass surfaces and then exposed to white light an

insubstantialreductionincellnumberwasobservedAreductionintherecoveryofE

coli has previously been observed after irradiation with 458 and 488 nm light

(Vermeulenetal2008)althoughazenonarclampwasusedwhichgenerateslightof

amuchgreaterintensityInterestinglythiswasnotthecasewithSaureusNCTC6571

An average reduction of 56 log10 cfu sample was observed on an uncoated glass

surfaceThiseffectwasalsoseentoa lesserextent inadifferentstrainofSaureus

ATCC 8325‐4 and an average reduction of 33 log10 cfu sample was observed S

aureusNCTC6571haspreviouslybeenshowntobeunaffectedby6hoursexposureto

the samewhite light source (Decraene et al 2006 2008b) implying that the killing

occursafteraprolongedirradiationtimeIndeedMacleanetal(2009)demonstrated

that longer exposure times were required for photoinactivation of certain bacterial

species suchasE coliandE faecalis Thisgroupandothers haveused lightwitha

wavelengthofbetween400ndash420nmtophotoinactivatearangeofbacterialspecies

(GuffeyandWilborn2006Macleanetal200820092010)

Themechanism of action is proposed to be due to photo‐excitation of endogenous

intracellularporphyrinsresultinginthegenerationofcytotoxicsingletoxygenspecies

(Hamblin and Hasan 2004 Lipovsky et al 2009) It is proposed that the observed

reductionsinbacterialviabilitydescribedinthesestudiesarelikelytobecausedbythe

samemechanismbutthishasnotbeeninvestigatedfurtherThevariationinbacterial

countsobserved in someof theexperiments couldalsobedue todifferences in the

137

intracellular concentration of porphyrins but the reason for this variation is unclear

(Hamblinetal2005)

InterestinglytheepidemicstrainsofMRSAdidnotshowthesamelevelofsensitivity

to the effect of the white light source EMRSA‐16 appears to show an increased

tolerancetotheinhibitoryeffectofthewhitelightsourcecomparedtoothertestedS

aureusstrainsasa09 log10cfusampledecreaseintherecoveryofEMRSA‐16was

seenafter24hoursexposuretothewhitelightcomparedwitha15log10cfusample

decrease when EMRSA‐15 was used and much greater reductions for meticillin‐

sensitivestrainsVariations inthesensitivityofSaureustotheeffectsofwhite light

hasbeendescribedpreviouslyandwasproposedthatthedifferencesinsusceptibility

were due to increased production of porphryns increased generation of reactive

oxygenspeciesanddecreasedproductionofcarotenoidsinthelight‐sensitivestrains

(Lipovskyetal2009)Amutationcouldbepresentinepidemicstrainswhichconfers

increasedtolerancetowhite lightbyoverproductionofthecarotenoidsantioxidants

ordecreasedproductionofporphyrinsAmplificationofthegenesflankingeitherthe

S aureus‐specific porphyrin coproporphyrin or golden pigment carotenoid and

sequencingofthePCRproductcouldconfirmthishypothesis

The observed decreased susceptibility to white light could contribute towards the

persistence of epidemic strains such as EMRSA‐16 in the hospital environment

ThereforewhenchoosinganepidemicMRSAstraintouseforassessmentofthelight‐

activatedantimicrobialcoatingsitwouldbelogicaltoselectthestrainthatislesslight

sensitiveandthesestudiesshowthistobeEMRSA‐16

138

35 Conclusions

Samplingthetestsurfacesbyswabbingandsubsequentlyperformingviablecountshas

been shown toprovideanadequateestimateof concentrationofbacteriaona test

surfaceDatageneratedinthischaptersuggestthatamethodincorporatingtheuseof

ATP bioluminescence for testing novel antimicrobial coatings would not be

appropriateThesuperiorityoftheviablecounttechniquewasespeciallyapparentat

lowbacterial concentrationswhen theATPbioluminescencebased techniqueswere

unable to consistently confirm the presence of small numbers of bacteria Two

meticillin‐sensitive strains of S aureus were shown to be susceptible to

photoinactivation by white light alone whereas the meticillin‐resistant strains of S

aureustestedshowedincreasedtoleranceindicatingapossiblevirulencefactorfound

inEMRSA‐16EcoliandEfaecalisalsodisplayedtolerancetotheinhibitoryeffectsof

thewhitelightsourcesoEcoliwillbeinitiallyusedtoassesstheantibacterialactivity

ofthelight‐activatedcoatings

139

4 Assessment of novel APCVD‐synthesised light‐activatedantibacterialmaterialsforuseinthehospitalenvironment

41 Introduction

Presentedinthischapterarethefindingsfromaseriesofnovelantimicrobialcoatings

thatwereactivatedbyeithervisibleorultravioletlightThefilmsweregeneratedusing

aprocesscalledAPCVD(Section151)wheredopantswereaddedduringthesynthesis

of the TiO2 thin films in order to alter the photochemical properties TiO2 is awell‐

described photocatalyst both as a powder and when immobilised within thin films

(Matsunagaetal1985)andisnormallyactivatedbyultraviolet(UV)lightTheaimof

thecurrentworkwastoshiftthebandwidthofnovelTiO2filmssothatlightofalower

frequencywas able to initiate photocatalysis (Section 133)E coliwas used as the

test organism for the initial screening as it has been demonstrated that it is not

affected by the white light used for activation unlike some of the staphylococcal

speciestested(Section333)whichhavepreviouslybeenshowntohaveanincreased

resistance to theactivityofphotocatalysis (Decraeneetal 2006Pageetal 2007)

Pure TiO2 thin films were also tested to demonstrate the difference between the

dopedandun‐dopedmaterialsTheantibacterialactivityofthematerialswasassessed

usingaswab‐basedmethodologyandnotanATPbioluminescencebasedtechniqueas

viablecountsproducedthemostreproducibleresultsinChapter3thepresenceofE

coliwasalwaysreportedevenatlowconcentrations

140


421 Synthesisofthethinfilms

Thetitanium(IV)oxynitridefilms(Ti285O4N) (TiON‐1)wereproducedbyAPCVDusing

ammoniaas thenitrogen sourceasdescribed inSection21011Anitrogen‐doped

thin film (TiON‐2) was also synthesised using ammonia as the nitrogen source as

described in Section 21011 The nitrogen‐doped TiO2 films N1 N2 and N3 were

producedbyAPCVDusingt‐butylamineasthenitrogensourceasdescribedinSection

21012andwerecutfromdifferentareasofasinglesheetofcoatedglassThesulfur

containingthinfilmsS1S2andS3werepreparedwithcarbondisulfideasthesulfur

sourceandtitaniumtetrachloride(TiCl4)asthetitaniumsourceasdescribedinSection

21013TiO2thinfilmswerepreparedascontrolsasdescribedinSection21014

Theconditions chosen forall experimentsallowed for the rapid deposition ofa thin

filmwhichremaineddefect‐andpinhole‐freebyeyeThefilmswereallwelladhered

tothesubstrateandresistanttoabrasionThethinfilmswerecharacterisedandthe

functionalactivityassessedasdescribedpreviously(Dunnilletal2009a2009bAiken

etal2010)

422 Measuringtheantibacterialeffectofthethinfilms


ofEcoliATCC25922werepreparedasdetailed inSection23resultinginastarting

inoculum of approximately 107 cfu ml equating to approximately 25 x 105 cfu

sampleTheeffectofthephotocatalyticthinfilmsontheviabilityofbacterialstrains

was determined using the swab‐basedmethodology described in Section 2122 and

141

Figure22SamplesweredenotedCforthenitrogenorsulfur‐containingsamplesTfor

theTiO2thinfilmsandGfortheuncoatedglassTheMannWhitneytestwasusedto

determine the statistical significance of any differences observed as described in

Section213

423 Assessmentofthedecontaminationregimen

Priortomicrobiologicalassessmentthethinfilmsweredecontaminatedasdescribed

in Section 2121 The decontamination procedurewas later amended and stored in

thedarktodeactivateandusedonlyafteraperiodof72hours

424 Effectofthecoveringmaterialonthinfilmactivity

To prevent dehydration of the bacterial inocula the effect of thematerials used to

coverthemoisturechamberwasinvestigatedThethinfilmswereincubatedunderthe

whitelightfor24hourswitharangeofcoveringswhichstillallowedlightpenetration

ontothebacterialsuspensioninoculatedontothethinfilmThefollowingcoverswere

used(i)glasscoverslips(ii)quartzcoverslips(iii)thepetridishlid(iv)clingfilmAUV‐

visiblelighttracewasalsogeneratedtomeasurethetransmissionoflightthroughthe

petri dish lid and the clingfilm The intensity of light generated by the lamp was

quantifiedusinga lightmeter (LX101LuxmeterLutronElectronicEnterpriseCoLtd

Taiwan)

142

43 Results

431 Photocatalyticactivityoftitaniumdioxidethinfilms

The activity of the TiO2 films was initially examined to check whether any

photocatalyticactivitywasobservedusingwhite lightasthesourceof incident light

TiO2thinfilmspreparedin‐housewereassessedalongsidecommerciallyproducedthin

filmsWhentheTiO2thinfilmswereassessedforphotocatalyticantibacterialactivity

againstEcoli(Figure41)nostatisticaldifferenceinbacterialrecoverywasobserved

from the thin films after a 24 hour exposure period compared with the bacterial

recoveryfromtheglassslides (pgt005) thereforetheseTiO2thinfilmswereusedas

controlsfortheremainingexperimentswherenecessary

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions

Figure41ActivityoftheTiO2thinfilmspreparedin‐houseAnaliquotofEcoliwasaddedtothethinfilmsbeforeexposuretothewhite lightsourcefor24hours(L+)Alternativelythinfilmswereincubatedinthedarkthroughout(L‐)UncoatedglasssidesandTiO2thinfilmsaredenotedbyGandTirespectivelyThethickhorizontallinesindicatemedianvaluesthebaseandtopofeachboxrepresentsthe25and75quartilesrespectivelyandtheerrorbarsthe10and90percentilesandthesmallcirclesareoutliersThedottedhorizontal line indicates thedetection limitofthesamplingmethod14log10cfusample

143

ThecommerciallyproducedTiO2thinfilmPilkingtonActivTMwasalsoassessedforany

photocatalytic activity using the white light source and a 03 log10 cfu sample

reduction in the recovery of E coli was observed compared with the thin film

incubated in the absence of light (Figure 42) This small decrease was statistically

significant (plt 0001)which is likely to be due to the small level of variance in the

viable count recovered from the thin films in the control group rather than to a

differencefromthenumberofbacterialcoloniesobservedinthetestgroupandsuch

smalldifferenceswouldnotbeconsideredmicrobiologicallydifferent

$ amp$$()$$+-$(-

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Figure42Effectof thecommerciallyproducedTiO2 thin filmPilkingtonActivTMonthesurvivalofEcoliThinfilmswereexposedtowhite lightfor24hours(A+) thebacterialdropletwasaddedthen thesamplewasexposedasecond lightexposureperiodof24hours(L+)Alternativelythinfilmswereexposedtojustthelatterlightdose(A‐L+)thefirstlightdoseonly(A+L‐)orincubatedinthedarkthroughout(A‐L‐)The asterisk denotes statistical significance compared with an uncoated controlincubatedunderthesamelightingconditionsasdescribedinSection213

144

432 Photocatalytic antibacterial activity of nitrogen‐containing titanium

dioxidethinfilmsTiON‐1andTiON‐2

4321 Photocatalyticactivityafterexposuretoultravioletlight

Theactivityofthenitrogen‐dopedthinfilmsTiON‐2wereassessedinitiallyusingtwo

UVlamps(254nm365nm)asthelightsourcesWhenthethinfilmTiON‐2waspre‐

exposed to 1 hour of 254 nm light inoculated with E coli and then subjected to 4

hoursof365nmlight(CA+L+)a14log10cfusample(955)reductioninbacteria

was observed compared with the uncoated control exposed to the same light

conditions (GA+L+)Thisdifference is statistically significant (plt001)and is shown

graphically alongwith the bacterial counts for a number of the other conditions in

Figure43

Exposingtheuncoatedslidestobothlightincubationsteps(GA+L+)orjustthelatter

light incubation step (GA‐L+) resulted ina05 log10 cfu sample reductionofE coli

comparedwiththeslidesincubatedintheabsenceoflight(GA‐L‐)asthisdifference

wasstatisticallysignificant(plt001)theGA+L+slidewasusedasthenegativecontrol

throughout

The pre‐inoculation activation step did not substantially enhance the activity of the

thin films when they were subsequently exposed to the 365 nm light A similar

decreaseinbacterialrecoverywasobservedwhetherthethinfilmswerepre‐activated

(14 log10cfu samplereduction)ornot (11 log10cfusamplereduction)andthese

valueswerenot statisticallydifferent (pgt005) Therewasno significantdecrease in

the number of bacteria recovered from thin films which were exposed to just the

activationstep(CA+L‐)andnosignificantdecreaseinthenumberofrecoverableEcoli

145

was observed from the thin films which were incubated in the absence of light

throughout (CA‐L‐) in fact the bacterial recoverywasgreater from these thin films

thanfromthenegativecontrol

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Figure43ConcentrationofEcoliremainingonthethinfilmTiON‐2afterexposureto1hour254nmlightand4hours365nmlight(CA+L+)orjustthelatterlightdose(C A‐L+) Thin films were also exposed to the activation step only (C A+L‐) orincubated in the dark throughout (C A‐L‐) Uncoated glass slideswere exposed tobothlightconditions(GA+L+)orneither(GA‐L‐)

Whenthetitanium(IV)oxynitridefilmTiON‐1waspre‐exposedto1hourof254nm

lightinoculatedwithEcoliandthenexposedto4hoursof365nmlight(CA+L+)a

41 log10cfusample(9999)reduction inbacterialcountwasobservedcompared

withtheuncoatedcontrolexposedtothesamelightconditions(GA+L+)(Figure44)

Thisdifferencewashighlystatisticallysignificant(plt001)

Thepre‐inoculationactivationstepwasfoundtoenhancetheactivityofthethinfilms

TherecoveryofEcoli fromtheoxynitridethinfilmswhichwereexposedtothe365

nmlightforfourhourswithoutprioractivationwasnotsignificantlydifferentfromthe

146

recoveryfromtheuncoatedcontrolslides(pgt005)Similarlynosignificantdecrease

inthenumberofbacteriarecoveredfromthethinfilmswasobservedwhentheywere

justactivated(CA+L‐)orwhenthethinfilmswereincubatedintheabsenceoflight(C

A‐L‐)

IncomparisonwhentheTiO2thinfilmswereexposedto365nmlightwitha254nm

activationsteptherewasa41 log10cfusamplereduction inbacterialcount Itwas

converselyfoundthatfortheTiO2thinfilmstheactivationstepwasunnecessaryand

exposure to 365 nm light alone led to a 41 log10 cfu sample reduction after four

hoursoflightexposure(datanotpresented)

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147

4322 Photocatalyticactivityafterexposuretowhitelight

Thephotoactivityofthesethinfilmswassubsequentlyassessedusingvisible lightas

theactivatinglightsourceAswhitelighthasalowerfrequencythanultravioletlight

the sampleshad tobeexposed to thewhite light fora longer timeperiodThe thin

films were exposed to the white light for 24 hours as an lsquoactivatingrsquo step then

inoculatedwithEcoliandexposedtothewhitelightforeither618or24hoursThe

thin film TiON‐2 did not display any significant photoactivity after 6 18 or 24 hours

exposure to thewhite light (Figure 45) The greatest decrease in bacterial recovery

was exhibited after 24 hours where just a 05 log10 cfu sample reduction was

observedcomparedwith the thin films incubated in theabsenceof light throughout

the duration of the experiment (A‐L‐) However the effect of the light source alone

should be incorporated into this reduction to show that any reduction in bacterial

recoverywasduetothephotoactivityofthethinfilmsandnotanartefactcausedby

thelightsource

Itwasdemonstrated inSection3331andFigure36that24hoursexposuretothe

whitelightresultedina02log10cfusampledecreaseintherecoveryofEcoliThis

figurewassubtractedfromthereductionsseeninthissectionandthisvaluewasused

astheoverallnegativecontrol(GA+L+)Thereforethegreatestdecreaseinbacterial

recoveryforthenitrogen‐dopedthinfilmwasjust02log10cfusampleafterexposure

toboth24hourlightincubationstepswhichwasnotstatisticallysignificant

148

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions

Figure 45 Effect of the thin film TiON‐2 on the survival ofE coli Thin filmswereexposedtowhite lightfor24hours(A+) thebacterialdropletwasaddedthenthesamplewasexposedasecondlightexposureperiodofeither618or24hours(L+)Alternatively thin filmswere exposed to just the latter light dose (A‐L+) the firstlightdoseonly(A+L‐)orincubatedinthedarkthroughout(A‐L‐)

Whenthetitanium(IV)oxynitridefilmTiON‐1wasexposedtothewhitelightforeither

6or18hours therewasno significant reduction in the recoveryofE coliHowever

after24hours irradiationareductiveeffectwasseenandtheaveragerecoveryofE

colifromthethinfilm(A+L+)was06log10cfusamplelowerthantherecoveryfrom

theuncoatedglassslidesexposedtothesamelightconditions(GA+L+)asdisplayedin

Figure 46 This result was statistically significant (p lt 001) However the observed

effect was not consistent demonstrated by the variability of the A+L+ 24h data

showninFigure46Evenafterfiveexperimentalrepeatsaconsistentresultcouldnot

beachievedandreductionsinthebacterialcountrangedfrom49log10cfusampleto

05log10cfusamplewithanaveragereductionofjust06log10cfusample

149

$ amp$$()$$+-$(-

0123)45$6-+-3


Theanti‐Ecolieffectoftitanium(IV)oxynitridethinfilmTiON‐1wasgreaterthanthe

nitrogen‐doped thin filmTiON‐2 underboth lighting conditionswhichdemonstrates

thattheformerthinfilmwasamoreeffectivephotocatalystunderthetestconditions

433 Photocatalyticantibacterialactivityofnitrogen‐dopedtitaniumdioxide

thinfilmsN1N2andN3


Theactivityofasecondsetofnovelnitrogen‐containingthinfilmswasassessedusing

whitelightastheactivatingsourceof irradiationThethinfilmswereexposedtothe

whitelightfor24hourstheninoculatedwithEcoliandre‐exposedtothewhitelight

for24hoursThegreatestreduction inbacterial recoverywasseenwhenEcoliwas

150

inoculated onto thin film N1 and a 28 log10 cfu sample (999) reduction was

observed(Figure47)comparedwiththethinfilms incubated intheabsenceof light

throughout the duration of the experiment (A‐L‐)When the uncoated glass sample

exposedtobothlightconditionswasusedasacontrol(GA+L+)theoverallreduction

inEcolicauseddirectlybytheactivityoftheN‐dopedthinfilmN1wasapproximately

25log10cfusample(997)whichwashighlystatisticallysignificant(plt0001)

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions

Figure47EffectofthethinfilmN1onthesurvivalofEcoliThinfilmswereexposedtowhitelightfor24hours(A+)thebacterialdropletwasaddedthenthesamplewasexposed a second light exposure period of 24 hours (L+) Alternatively thin filmswereexposed to just the latter lightdose (A‐L+) the first lightdoseonly (A+L‐)orincubatedinthedarkthroughout(A‐L‐)

Exposingthethinfilmstojustthesecondlightcondition(A‐L+)resulted ina09log10

cfu sample reduction in the recovery of E coli (p lt 005) compared with the

uncoated control incubated under the same conditions (G A+L+) Exposing the thin

filmstotheinitialactivatinglightdoseonly(A+L‐)didnothaveasignificanteffecton

151

therecoveryofEcolinordidexposuretothethinfilmsintheabsenceoflightinfact

a higher recovery of E coli was observed in this control group Hence an additive

effectwasobservedwherebyexposure toeither the second lightdoseor both light

doses resulted in a significant reduction in bacterial recovery with the greatest

decreaseobservedafterbothlightexposureperiods

WhenthethinfilmN2wasexposedtowhitelightforboth24hourperiodsa16log10

cfu sample reduction was observed (Figure 48) compared with the thin films

incubatedinthedarkthroughoutWhentheuncoatedglassslideexposedtothesame

lightconditionswasusedasthecontrolthentherecoveryofEcoliwasreducedto11

log10cfusampleNostatisticalsignificantdifferencewasseenbetweenthetestand

control groups as the data sets were small No decrease in bacterial recovery was

observedwhen the thin filmswere exposed to thewhite light for 24 hourswithout

pre‐activation(A‐L+)whenthethinfilmswere justpre‐activated(A+L‐)orwhenthe

thinfilmswere incubated intheabsenceof light (A‐L‐)comparedwiththeuncoated

controlexposedtobothlightdoses

152

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


AlargevariationintherecoveryofEcoliwasobservedfromthesetofthinfilms(N3)

displayedinFigure49Onaveragethereductioninbacterialrecoveryfromthepre‐

activatedthinfilmsincubatedunderwhitelightfor24hourswas09log10cfusample

whencomparedwiththethinfilmsincubated inthedarkthroughoutthedurationof

the experiment The reduction drops to a 05 log10 cfu sample reduction when

compared with the uncoated control incubated exposed to both light doses These

reductions were not statistically different The recovery of E coli from these films

rangedfrom58log10cfusampletobelowthelimitofdetectiondemonstratingthe

wide spectrum of activity that these thin films displayed under the experimental

conditionsWhetherthethinfilmN3wasexposedtojustthesecondlightdosewhilst

inoculatedwithEcolijustthepre‐activatingwhitelightdoseorneithertherewasno

153

significant reduction in bacterial recovery compared with the uncoated control

exposedtobothperiodsoflight

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


434 EffectofchangingthedecontaminationregimenonthinfilmN1

The effect of themodified decontamination regimewas evaluated by repeating the

white lightexposureexperimentson the thin filmdesignatedN1However the thin

films could not be reproduced to the samespecifications and had therefore already

been exposed to the original decontamination regime before the newmethod was

usedTheactivityofthethinfilmwasmaintainedforthefirstfourreplicateswhenthe

new decontamination regimen was used (Figure 410a) a statistically significant

reduction in bacterial recovery was observed (p lt 001) and the new regime was

thought to be successful However the photocatalytic activity of the thin filmswas

154

thenlostwhentheexperimentwasrepeatedonasubsequentthreeoccasions(Figure

410b)andnostatisticallysignificantreductionintherecoveryofEcoliwasobserved

WhenthethinfilmswerestainedusingtheLiveDeaddifferentialstainafluorescent

greensmearwasseenonsurfaceofthefilmsbutnoviableornon‐viablebacterialcells

werepresent

(a) (b)

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

Figure410Light‐activatedantimicrobialkillingofEcolionthinfilmN1(a)andafterinactivation (b) The thin film was exposed to first light dose (A+) the bacterialdropletwas added and then the thin filmwas exposed to second light dose (L+)Alternatively thin filmswere exposed to just the latter light dose (A‐L+) the firstlightdoseonly(A+L‐)orincubatedinthedarkthroughout(A‐L‐)

435 Effectofcoveringmaterialonthinfilmactivity

Theeffectofthematerialusedtocoverthemoisturechamberwasinvestigatedwith

regardtobacterialviabilityGlassorquartzcoverslipswereusedtocoverthebacterial

inoculumduringexposuretothewhitelightsourcebutafter24hoursincubationthe

dropletshadevaporateditwasnotpossibletoculturetheorganismsontosolidagar

using the viable count technique and the cells had become non‐viable This was

confirmedbyvisualisationusingtheLiveDeadstain(datanotincluded)whichshowed

100ofcellsweredeadAbathofwaterwasplacedatthebaseofthe incubatorto

155

saturate the environment with moisture to prevent evaporation but the bacterial

inoculumhadonceagaindriedoutafterthe24hourincubationperiod

Whenthemoisturechamberwascoveredwithaplasticpetridishlidorclingfilmthe

bacterialdropletsdidnotdryoutthereforetheeffectivenessofthesecoveringswas

assessedE coli inoculated onto thin film TiON‐2 showed a greater susceptibility to

killingbyUVlightwhenthemoisturechamberwascoveredwithclingfilm(Figure411)

comparedtowhenitwascoveredwiththepetridishlid(Figure43)A49log10cfu

samplereductioninviableorganismswasseenwiththeclingfilmcoveringcompared

witha14log10cfusamplereductionwhentheplasticpetridishcoverwasused

$ amp$$()$$+-$(-

0123)45$6-+-3

Figure 411 Concentration of E coli remaining on the thin film TiON‐1 using aclingfilmcoveringThethinfilmswereexposedto1hour254nmlightand4hours365 nm light (C A+L+) or just the latter light dose (C A‐L+) Thin films were alsoexposedtotheactivationsteponly(CA+L‐)orincubatedinthedarkthroughout(CA‐L‐)Uncoatedglassslideswereexposedtobothlightconditions(GA+L+)orneither(GA‐L‐)

156

AUV‐visible lighttransmissiontracewasproducedtohighlightanydifferencesinthe

transmissionoflightthroughandthereflectancefromthetwocoveringmaterialsThe

UV‐visiblelighttransmissiontrace(Figure412)showedthataround90oflightfrom

the visible portion of spectrum (with a wavelength between 400 and 700 nm)

penetrated through both the petri dish and the clingfilm coverings Less than 2of

lightwithawavelengthbelow280nmwasabletopenetratethroughthepetridishlid

However more than 80 of light of this wavelength could penetrate through the

clingfilm covering This finding indicates that this coveringwould not be suitable for

the series of experiments evaluating the effect of the light activated antimicrobial

coatingsasbacteriaareinactivatedbylightofthiswavelengthandbelow(Saitoetal

1992)Thegreaterreductioninbacterialrecoveryshownwhentheclingfilmwasused

to cover the moisture chamber suggests that wavelengths of light with a higher

frequencywereabletopassthroughtheclingfilmresultinginthegreatersusceptibility

ofE coliobservedwhen inoculatedonto the thin filmTiON‐2which suggests there

couldbe some leakageof sub‐365nmUV light from the light source that caused the

observedincreaseinphotoactivityThereforethepetridishlidwasusedtocoverthe

moisturechamberinalllight‐activationexperiments

157

$

amp

(

)

amp $ $amp amp amp ampamp amp (

+-012345406

78096

990454lt

=284gt934-8

01A6

)06

06

Figure412UV‐visible lighttransmissiontraceofthepetridish lidandtheclingfilmcoversThewavelengths280nmand400nmareindicatedbyverticaldottedlines

436 Photocatalytic antibacterial activity of sulfur‐based titanium dioxide

thinfilms

The photocatalytic activity of a series of novel sulfur‐doped thin filmswas assessed

Thethinfilmswereexposedtowhitelightfor72hoursbeforeasuspensionofEcoli

wasaddedThethinfilmswerethenre‐incubatedunderthewhite light fora further

24hoursbeforesamplingThephotocatalyticactivityofthinfilmS2isshowninFigure

413whereasignificantdecreaseinbacterialrecoverywasobserved(plt001)A25

log10 cfu sample decreasewas observed comparedwith the sulfur‐doped thin film

incubatedinthedarkthroughoutthedurationoftheexperimentTheoveralldecrease

in bacterial recovery when compared to a TiO2 thin film exposed to the same light

conditionswas22log10cfusamplewhichremainsstatisticallysignificant(p=001)

158

AlargevariationinbacterialrecoverywasobservedwhenthethinfilmS2wasexposed

to thewhite light for 24 hourswithout prior activation ranging from62 log10 cfu

sample to below the limit of detection with an average recovery of 41 log10 cfu

sample indicating that the activation step did not have a significant effect on the

photoactivity of the S‐doped thin film No statistically significant decrease in the

recovery of E coli was observed under these conditions when the thin film was

exposedtotheactivating lightdosealoneorwhen incubated intheabsenceof light

entirely

$ amp$$()$$+-$(-

0123)45$6-+-3

Figure 413 Effect of the thin film S2 on the survival of E coli Thin films wereexposedtowhite lightfor72hours(A+) thebacterialdropletwasaddedthenthesamplewasexposedasecond lightexposureperiodof24hours(L+)Alternativelythinfilmswereexposedto justthe latter lightdose(A‐L+) thefirst lightdoseonly(A+L‐) or incubated in the dark throughout (A‐L‐) TiO2 controls were exposed toeitherbothlightdoses(TiA+L+)orneither(TiA‐L‐)

ThethinfilmsS1andS3werelesseffectiveatreducingtheEcolibacterialloadafter

exposuretothewhitelightTherewasnosignificantdecreaseintherecoveryofEcoli

fromthesurfaceofpre‐activatedthinfilmS1afterthe24hourexposureperiod(Figure

159

414)comparedwitheithertheTiO2controlexposedtothesamelightingconditions

or the sulfur‐doped thin film incubated in the absence of light Similarly the pre‐

activatedthinfilmS3didnotproduceasignificantreductiveeffectintherecoveryofE

coli from the surface of the thin films after the 24 hour exposure period when

comparedwitheithertheTiO2controlexposedtobothlightdosesorthesulfur‐doped

thinfilmnotexposedtowhitelight(Figure415)Howeveraninconsistenteffectwas

seenontheS3thinfilmswhichwerenotpre‐exposedtothewhitelightfor72hours

but incubated under the white light for 24 hours after addition of the bacterial

suspension This result was not reproducible demonstrated in the box andwhisker

plotbythelargesizeofboththeboxanderrorbarsA09log10cfusamplereduction

was seen comparedwith the thin film incubated in the absence of light (p lt 005)

HoweverthemedianreductionwaslowerwhencomparedwiththeTiO2thinfilm(06

log10 cfu sample) or the uncoated glass control (01 log10 cfu sample) and these

reductionswerenotstatisticallysignificant

160

log 1

0 cfu

t

hin

film

Exposure conditions


Exposure conditions

log 1

0 cfu

t

hin

film


161

Table41SummaryofthephotocatalyticactivityofthenitrogenandsulfurdopedthinfilmsassessedinthischapterThinfilmswereexposedtowhitelightfor24or72hoursforN‐dopedandS‐dopedsamplesrespectivelyThebacterialdropletwasaddedbeforethesamplewasexposedasecondwhitelightexposureperiodof24hoursBacterialcountsobtainedwerecomparedwithuncoatedglassslidesexposedtothesamelightingconditions

SamplenameWhitelight‐induced

photocatalyticactivitylog10cfupersample

Statisticalsignificance

TiON1 06 plt001

TiON‐2 02 Nil(pgt005)

N1 25 plt0001

N2 11 Nil(pgt005)

N3 05 Nil(pgt005)

S1 Nodecrease Nil(pgt005)

S2 17 pgt0001


44 Discussion

441 UVlight‐inducedphotocatalyticactivity

Thedatapresentedinthischapterhasdemonstratedtheantibacterialphotoactivityof

anumberofnoveldopedTiO2thinfilmsgeneratedbyAPCVDThethinfilmsthatwere

initially assessed were doped with nitrogen and exposed to UV light in order to

demonstrateequivalencewithpuretitaniaThetitanium(IV)oxynitridethinfilmTiON‐

1 demonstrated greater photoactivity than theN‐doped thin film TiON‐2 and a 41

log10cfusamplereductionwasachievedonthepre‐activatedtitanium(IV)oxynitride

sampleafterjust4hoursexposuretothelightsourceTheseresultsalsoshowthatthe

162

titanium(IV)oxynitridethinfilmsdemonstratednoanti‐bacterialactivitywithoutUV

exposure after the inoculation of the bacterial suspension therefore the mode of

actionisunlikelytoberelatedtothediffusionofionsontothesurfaceandisgenuinely

photo‐activated

442 Whitelight‐inducedphotocatalyticactivity

Thephotocatalyticactivityofthethinfilmswasthenassessedusingwhitelightasthe

activatinglightsourceWhitelightwasusedasanactivatingsourcelightsourceasUV

light is known to have a bactericidal effect (Vermeulen et al 2008) and the

applications of the resultant thin film would be wider using a lower energy light

source Any reduction in bacterial count observed under these conditions would

indicate a shift in the band gap of the material caused by the doping process

demonstratingthatactivationby lightofa lowerwavelength ispossible (Dunnilland

Parkin2009)A reductionofup to49 log10 cfu sampleofE coliwasobservedon

thinfilmTiON‐1(Ti285O4N)butthiswasnotconsistentandtheaveragereductionwas

just06 log10 cfu sampleHowever thisdoes provideapromisingbasis for further

dopingexperiments

The photocatalytic activity of the N‐doped thin films N1 N2 and N3were assessed

next using white light as the activating light source Thin film N1 displayed the

greatestphotocatalyticactivityanda25log10cfusampledecreaseintherecoveryof

E coli was observed after exposure to both light incubation steps These findings

confirm the chemical characterisation tests performed on these samples such as

photooxidation of stearic acid and contact anglemeasurements and these data are

163

published elsewhere (Dunnill et al 2009b 2009c 2010) A 09 log10 cfu sample

decrease was observed when the 24 hour activating step was omitted which

demonstrated that the activation stepwas required to increase the photoactivity of

the thin films This increase in activity is attributed to the pre‐cleaning effect of the

treatmentThelackofactivityonthethinfilmsthathadbeenactivatedbutthennot

exposedtothesecondlightstepindicatedtheshortlifetimeofthereactivespecieson

the surface of the thin films that are responsible for killing the bacterial cells It is

unlikely that the oxygen radicals generated in the presence of light survive long

enoughtokillthebacteriathatwereappliedaftertheactivationstephasendedgiven

that singletoxygenhasahalf lifeof just1 micros (Pernietal 2009a)Thevariability in

photocatalytic activity observed on the N1 N2 and N3 thin films which were

synthesised on the same sheet of float glass demonstrates the inherent lack of

reproducibility in the composition of coatings produced using this deposition

technique

TheactivityoftheN‐dopedthinfilmN1wasgreaterthanthatseenforthetitanium

(IV) oxynitride thin film TiON‐1 The two thin films were synthesised with different

precursors the N‐doped thin films were synthesised using t‐butylamine as the

nitrogensourceandammoniawasusedforthetitanium(IV)oxynitridethinfilmsThe

chosen nitrogen precursor was introduced into the titanium (IV) chloride and ethyl

acetatevapoursatthepointofentrytothedepositionchamberresultinginthermal

decompositionofthenitrogenprecursoronthesurfaceoftheglasssubstrateduring

formationofTiO2(DunnillandParkin2009)Pre‐reactioncomplexesweremorelikely

toformwhenammoniawasusedasthenitrogensourceratherthant‐butylamineand

164

thesecomplexescancausecontrollineblockageswhichcanaffecttheconcentration

of nitrogen deposited onto the surface of the glass The activity of the thin films is

dependentupontheconcentrationofnitrogen intheTiO2thinfilm(Irieetal2003)

so perhaps the greater control of nitrogen deposition displayed when t‐butylamine

was used as the nitrogen precursor conferred the increased photocatalytic activity

observed

The S‐doped thin film S2 also displayed significantwhite light driven photocatalytic

activityanda22 log10 cfu sampledecrease in the recoveryofE coliwasobserved

after a 24 hour exposure periodOnce again themicrobiological findings confirmed

the initialchemicalcharacterisationscreeningtestsandthethinfilmwiththefastest

rateofstearicacidphotodegradationdemonstratedthemostsignificantantibacterial

activity (Dunnill et al 2009a 2010) However the N‐doped thin films displayed

greaterphotocatalyticactivitythantheS‐dopedthinfilmsevenwhentheinitialwhite

lightactivationtimewasextendedfrom24to72hours

Reports in the literature have described the antibacterial properties of white light

activated N‐ and S‐doped thin films but direct comparison is difficult due to

differences in the method of synthesis used (Asahi et al 2001 Mills et al 2002

Diwaldetal2004ThompsonandYates2006)Indeedthethinfilmsdescribedinthis

chapterarethefirstpublishedthinfilmswith interstitialnitrogen‐orsulphur‐doping

possessingwhitelightactivatedantibacterialpropertiesN‐dopedthinfilmshavebeen

shown to generate a greater photocatalytic effect against E coli compared with

carbon‐doped thin films (Wong et al 2006) However the reduction in bacterial

recovery was minimal (less than a 1 log10 reduction) and when these films were

165

characterised the nitrogen doping was shown by XPS to be substitutional with an

ionisation peak at 396 eV (Yang et al 2004) in contrast to the interstitial‐doped

nitrogen described in this chapterwith an ionisation peak at 400 eV (Dunnill et al

2009c)Thisdoeshoweverdemonstratethatnitrogenisabetterchoiceofdopantthan

carbon if photocatalytic properties are desired Titanium oxide doped with both

nitrogen and carbon was shown to exhibit enhanced photocatalytic properties and

reductionsofmorethan3log10cfumLwereobserved(Lietal2007)butahalogen

bulbwas used as the light sourcewhich has a higher intensity than thewhite light

sourceusedinthischapterandsoagreaterphotocatalyticeffectwouldbeexpected

Additionallypowdershaveagreatersurfaceareapervolumeratiothansolidsfurther

boostingthepredictedlevelofphotocatalysis

Thequantityofnitrogenpresentinthethinfilmisofparamountimportanceandsome

groups show high levels of nitrogen doping can result in the production of poor

photocatalysts (Irie et al 2003) whereas other groups show increased levels of

photocatalysis when the nitrogen concentration is higher (Li et al 2007) When

nitrogen concentrations are higher less TiO2 reduction occurs and there are more

oxygenvacanciesthatactasrecombinationsitesforpositiveholesandelectronsthus

reducing the overall photocatalytic activity The concentration of nitrogen in the N‐

doped thin film N1 was 013 at and reports in the literature surmise that

concentrationsaround1ndash2atisfavourablealthoughtheoptimallevelisstillunder

debate(Irieetal2003Dunnilletal2011)ConverselywhenTiO2powderwasdoped

withsulfurincreasedlevelsofthedopantledtoahigherlevelofphotocatalysisandan

166

increasedbactericidaleffectwasobservedagainstMicrococcuslylae(Yuetal2005)

Theoptimallevelofdopingisthereforedebatable

443 Limitationsoftheexperimentalwork

Problemswere experienced in synthesising reproducible thin films using the APCVD

apparatusTheprecursorgasesusednamelytitanium(IV)chlorideandethylacetate

werechosenastheyareusedindustriallyintheproductionofTiO2‐basedself‐cleaning

glassbutthesetupofthedepositionchambersusedinthisprojectweredifferentIn

an industrial setting general mass flow controllers would be used to deliver the

reactantsandthegasoutletswouldbestablewiththeglasssheetsmovingunderneath

the float at 500 ‐ 600degC (Dunnill et al 2009b) These conditions result in a more

consistentreactiononthesurfaceoftheglassandamorehomogenouscoatingwhich

is essential for a commercial product The flow rate of the precursor gases are also

more tightly regulated which was more difficult to control using the in‐house

apparatus overall this meant that the resultant thin films varied in their chemical

composition with differences observed between batches of samples samples

synthesised during the same run and even on different areas on the same piece of

floatglass Forexample theN‐dopedsamplesN1N2andN3wereall cut from the

samepieceof floatglassandyetdisplayeda largevariation inphotocatalyticactivity

against E coli This inconsistency is an inherent disadvantage of the APCVD

methodologyandmadeitverydifficulttoassessthethinfilmsmicrobiologicallyasfor

accurate assessment the samples should at least be identical and tested at least in

triplicate for each light exposure condition on three separate occasions for each

bacterialspecies

167

Asaresultthethinfilmsweredecontaminatedaftereachmicrobiologicalassessment

toenablere‐useItwaspostulatedthatbacterialcellsremainingonthesurfaceofthe

thinfilmswouldbeinactivatedbytheisopropanolandheattreatmentswhichwould

restorethethinfilmstotheirnativestateIthasbeenshownpreviouslythattherewas

no residual antimicrobial effect when isopropanol treatment was used to

decontaminate thin films so any activity observed after decontamination can be

attributed to the activity of the coatings alone (Page 2009) However the

photoactivityofthethinfilmsdecreasedaftereachroundofmicrobiologicaltestingso

thedecontaminationregimenwasamendedsothatastageincludingexposuretoUV

light was incorporated Any remaining bacterial cells were postulated to undergo

photoinduced oxidative decomposition (Section 13333) and non‐bacterial debris

wouldalsobedegradedaftertheextendedlightexposureperiodThethinfilmswere

thenincubatedinthedarkforatleast48hourssotoallowoxygenintheairtoreact

withthehydroxylspeciestonegatetheactivatingeffectoftheUVlight(ONeilletal

2003)

Amendment of the decontamination regimen did not prevent the decrease in

antibacterial activity observed on the thin films after sequential use and the exact

mechanismforthis loss inphotoactivitywasnotestablishedBacterialcellswerenot

presentonthethinfilmafterdecontaminationbutafluorescentsmearwasobserved

whichwasnotseenontheunusedthinfilmsIn‐depthmicrobiologicalassessmentof

thesethinfilmswasthereforenotpossibleandanalternativereproduciblemethodof

synthesiswassoughtwhichwillbeexploredinthefollowingchapterHoweverre‐use

168

ofthethinfilmsdiddemonstratethedurabilityofthecoatingsandtheintegrityofthe

coatingwasnotcompromisedafterrepeateduseanddecontaminationcycles

Another limitation of the testmethodwas the choice ofmedia used to recover the

bacterialstrains fromthetestsurfacesTheselectivemediumMacConkeywasused

to culture E coli because round discrete colonies were formed which made

enumeration easier to perform than when the counts were performed on a non‐

selectivesolidmediumsuchasbloodagarHoweverbacteriarecoveredwerelikelyto

besubletallydamagedbyexposuretothephotocatalyticeffectsofthethinfilmsand

cultivationonselectivemediahasbeenshowntoinhibittherepairofthesedamaged

strains (Sandel and McKillip 2004) A non‐selective agar overlay could have been

poured over the selective medium after inoculation to increase the recovery of

damagedcells(SandelandMcKillip2004)

45 Conclusions

Twosetsofnitrogenbasedthinfilmsweresynthesisedbychemicalvapourdeposition

namely N‐doped TiO2 and titanium oxynitride These coatings displayed significant

photocatalyticactivityagainstEcoliafterexposuretoUVlightandimportantlyawhite

light sourcewhich demonstrates a shift in the band gap from theUV to the visible

region of the electromagnetic spectrum TheN‐doped thin films displayed a greater

photocatalyticactivitycomparedwiththetitanium(IV)oxynitridethinfilmsAseriesof

sulfur‐doped thin films were synthesised using the same apparatus which also

displayed significant photocatalytic activity against E coli after exposure to awhite

light source The N‐doped thin film N1 displayed the greatest photoactivity The

169

reproducibilityofthethinfilmssynthesisedusingAPCVDwaspoorandadecrease in

the photocatalytic activity of the thin films was observed after repeated use An

alternativemethodofdepositionwillbeexploredinthenextchapter

170

5 Assessment of novel sol‐gel synthesised light‐activatedantibacterialmaterialsforuseinthehospitalenvironment

51 Introduction

InthepreviouschapteraseriesofTiO2basedthinfilmsweresynthesisedbychemical

vapourdeposition(APCVD)whichdisplayedphotocatalyticpropertieswhenexposed

tovisiblelightThethinfilmsweredopedwitheithernitrogenorsulfurwhichcaused

a shift in the band gap energy of the coating so that lower energy photons of light

could cause excitation of electrons from the valence band to the conduction band

resultingintheproductionofreactiveoxygenspeciesthataretoxictobacteriaThere

were however issueswith the reproducibility of the thin filmswhichmeant itwas

difficulttosynthesisealargenumberoffilmswithidenticalcompositionsInaddition

theactivityofthethinfilmsdecreasedovertimesomicrobiologicalassessmentofthe

usedthinfilmsgeneratedresultswithalargevariation

Analternativemethodofsynthesiswasthereforesoughtandsol‐geldepositionwas

chosenAlargenumberofsamplescouldbesynthesisedfromthesamehomogenous

solandthereislittlevariationintheconstitutionofdifferentbatchesofpreparedsols

so the composition of the resultant films are easier to control However sol‐gel

synthesisedfilmsaregenerallythickerlessmechanicallyrobustandrequiredsintering

aftercoatingtoannealthefilmtothesubstratecomparedwithAPCVDgeneratedthin

films (Brook et al 2007b) Therefore the synthesis methodology included a post‐

coating annealing step and the thickness and robustness of the thin films was be

examinedtodeterminewhetherthiswasdetrimentaltothephotocatalyticactivity

171

Silver ions were added to the titania base layer to improve the photocatalytic and

photo‐activatedantibacterialpropertiesoftitaniaSilverhasbeenusedextensivelyin

antibacterialmaterialsbecauseof itsintrinsicactivity(Silver2003Silveretal2006

Noimark et al 2009) silver ions can move from the surface of the antibacterial

materialthroughthecellmembraneofbacteriawheretheyareabletoelicitapotent

toxiceffect(Kawashitaetal2000Page2009Pageetal2009)


521 Thinfilmsynthesis

The thin films were synthesised using sol‐gel deposition in a two‐step process

describedinSection2102ThesilvercoatedTiO2thinfilmsweredenotedAg‐TiO2and

TiO2 thin films and uncoated glass microscope slides were used as controls The

adherence of the TiO2 and Ag‐TiO2 thin films to the glass substrates was tested by

scratchingwith(i)fingernails(ii)aHBpencil(iii)a2Hpencil(iv)asteelscalpel(v)a

diamondtippencilandapplicationandremovalofscotchtapeThestabilityofthethin

filmswereassessedbyimmersioninthefollowingliquidsfor2hours(i)methanol(ii)

acetone(iii)distilledwater(iv)2MHCl(v)2MNaOH

522 Characterisationandfunctionalassessmentofthethinfilms

Thin films of TiO2 and Ag‐TiO2 were prepared on both glass and quartz substrates

beforecharacterisationusingUV‐visiblespectroscopyasdescribed inSection2111

The reflectance datawas used to calculate the thickness of the thin films using the

SwanepoelmethodandtoestimatethebandonsetofthethinfilmsusingaTaucplot

172

Further methods employed to characterise the thin films included XRD Raman

spectroscopyAFMandXPSasdescribedinDunnilletal(2011)


Waterdropletcontactanglemeasurementsweretakenofadropletofdeionisedwater

inoculated onto both the Ag‐TiO2 and TiO2 thin films and uncoated glass control as

describedinSection2112Measurementsweretakenafter(i)incubationinthedark

for72hours(ii) irradiationwiththeUVlightsourcefor30minutes(Section2421)

(iii) irradiation with the filtered white light source for 30 minutes (Section 241)

(InstrumentGlasses2000)

5222 Photo‐oxidationofstearicacid

A solution of stearic acidwas inoculated onto both the thin films and the uncoated

glass control slides to assess the rate of photo‐oxidisation as described in Section

2113 The rate of photo‐activity was determined after exposure to three lighting

conditions (i)254nmUV light source forup to 72hours (Section2422) (ii)white

lightsourcefor96hours(Section241)(iii)thesamewhitelightsourcewithafilter

attachedthatabsorbedvirtuallyallsub‐400nmradiation(InstrumentGlasses2000)

523 Antibacterialassessmentofthethinfilms


ofEcoliATCC25922andEMRSA‐16werepreparedasdetailedinSection23excepta

50 microL bacterial droplet was inoculated onto the surface resulting in a starting

inoculumofapproximately5x105cfusampleTheeffectofthephotocatalyticthin

films on the viability of bacterial strains was determined using the methodology

173

described in Section 2122 and Figure 22 except the activation stepwas omitted

WhenrequiredaUV light filterwaspositioned25cmabovethemoisturechamber

The Mann Whitney test was used to determine the statistical significance of any

differencesobservedasdescribedinSection213

53 Results

ThinfilmsofAg‐TiO2andTiO2weresuccessfullysynthesisedusingthesol‐gelmethod

ofdeposition(Figure51)Controlthinfilmsconsistingofjustsilvernanoparticleswere

alsoproducedbutthesecoatingswereunstabledemonstratingtheessential roleof

theTiO2under‐layer foradherenceof the silvernanoparticles to theglass substrate

The TiO2 and Ag‐TiO2 thin films were well adhered to the glass substrates after

applicationandremovalofscotchtapeandwereresistanttoscratchingbyfingernails

aHBpencila2HpencilandasteelscalpelBoththinfilmswereeasilyscratchedwitha

diamondtippencilThethinfilmswerestableafterimmersioninmethanolacetone

distilledwateror2MHClfor2hoursbutweredissolvedin2MNaOH

174

Figure51PhotographoftheAg‐TiO2thinfilmsThepurplecolouredthinfilm(left)wasstoredinthedarkandtheorangecolouredthinfilm(right)wasirradiatedwithUVlighttoinducethecolourchange

Thethinfilmswereuniformlyadheredtotheglassmicroscopeslidesandwereorange

incolourandtransparentwhensynthesisedAfterstorage inthedarkforat least72

hoursthethinfilmsturnedpurplereversiontotheorangecolourcouldbeinducedby

irradiationwith UV light for 10minutes or standard indoor lighting conditions for 1

hourThereversiblephoto‐inducedcolourchangecanbedescribedusingthefollowing

formula

Silveroxide(purple) silver(orange)+oxygen

To confirm this orange and purple thin films were placed inside separate Schlenk

flasksandtheairwasevacuatedThepurplesamplewasirradiatedwithUVlightinthe

createdvacuumandturnedorangeHoweverwhentheorangethinfilmswerestored

in the dark for 72 hours the orange colour remained indicating that oxygen was

hv+TiO2

air

175

required for the backward reaction and light exposurewas needed for the forward

reaction


5311 UV‐visiblespectroscopy

ThinfilmsofAg‐TiO2andTiO2werepreparedusingquartzastheunderlyingsubstrate

inplaceofglassasitallowedbettermeasurementofthebandonsetusingaTaucplot

withouttheinterferenceoftheunderlyingglassbandonsetexpectedatabout33eV

TheUV‐visible‐IRspectroscopyresultsaredisplayedinFigure52andtheAg‐TiO2and

TiO2arevery similar TheAg‐TiO2 thin filmshoweda smalldecrease in transmission

due to silver ions on the surface and a minimal red shift compared with TiO2 The

uncoatedquartzslideshowednofeaturesabove300nm

176

0

10

20

30

40

50

60

70

80

90

100

200 700 1200 1700 2200

Wavelength

T

Qaurtz

TiO2

Ag-TiO2

Figure 52 Transmission data of the Ag‐TiO2 and TiO2 thin films deposited onto aquartzsubstrateobtainedbyUV‐visible‐IRspectrometry

ThethicknessoftheAg‐TiO2andTiO2thinfilmswereestimatedat211nmand196nm

respectivelyusingtheSwanpoelmethodwhich indicatedthatadditionofsilverhad

littleeffectonthethicknessofthethinfilmsThethicknessofthinfilmssynthesised

from the same sol can vary by 10 nm suggesting that the difference observed

betweentheAg‐TiO2andTiO2thinfilmswasunsubstantial

ThebandonsetoftheAg‐TiO2andTiO2thinfilmswereestimatedusingtheUV‐visible‐

IRdatatoproduceTaucplots(Figure53)Theincorporationofsilverontothesurface

of the TiO2 caused a shift in the bandonset towards lower energy radiationwith a

shift from 32 eV for titania to 29 eV for the silver‐doped titania This indicates an

interactionbetweensilverandthetitaniasubstratecausingashifttowardsactivation

inthevisibleregionofthespectrum

177

0

20

40

60

80

100

120

140

160

180

200

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2320 eV29 eV

0

50

100

150

200

250

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2

320 eV

Figure53TaucplotsoftheUV‐visible‐IRdatatakenforthe(a)Ag‐TiO2and(b)TiO2thinfilmspreparedonquartzsubstrates


When the Ag‐TiO2 thin film was exposed to UV light the water contact angle

decreasedfrom60degto8degasthesurfacebecamesuperhydrophilic(Table51)Asimilar

decreaseinwatercontactanglewasobservedontheTiO2thinfilmafterexposureto

UVlight(64degto8deg)Thewatercontactangleontheuncoatedglassslidedidnotchange

afterirradiationwithUVlightalthoughtheinitialreadingwascomparativelylow

ThesamplesweresubsequentlyexposedtowhitelightusingtheOptivexUVfilterto

eliminate any higher energy photons of light and the UV‐visible IR spectrum of this

178

filter isdisplayed inFigure54which showsalmost zero transmissionof lightbelow

400nmThedecreaseinwatercontactangleontheAg‐TiO2thinfilmwasthesameas

thatobservedafterUV irradiation(Table51)Thefilteredwhite lightsourcedidnot

haveaneffectontheTiO2thinfilmandtherewasnosubstantialchangeinthewater

contactangleTheseresultsclearlydemonstratethevisiblelight‐inducedhydrophilicity

oftheAg‐TiO2thinfilms

Table51ThewatercontactanglesoftheAg‐TiO2thinfilmsandthecontrolsamplesMeasurementsareaccuratetoplusmn2deg

Samplename Lightsource Watercontactangle

Uncoatedglassslide None 25(2)deg

UV 24(2)deg

TiO2 None 64(2)deg

UV 8(2)deg

Filteredwhitelight 60(2)deg

Ag‐TiO2 None 60(2)deg

UV 8(2)deg


179

0

10

20

30

40

50

60

70

80

90

100

200 300 400 500 600 700 800 900 1000 1100

Wavelength nm

T

Figure 54 UV‐Vis spectrum for the Optivextrade UV filter showing the cut‐off forradiationbelow400nminwavelength


Theeffectofthe lightsourcesontheconcentrationofstearicacidonthesurfaceof

theuncoatedglassslide is illustrated inFigure55aFigure56aandFigure57aThe

heightsofthelinesonthegraphrepresenttimewiththehighestpeakscorresponding

to the shortest irradiation timeTheuncoatedglass slidesdidnot showany signsof

photo‐activityafterexposuretoanyofthethreelightingconditionsandtherewasno

appreciabledecrease in the concentrationof stearicacid detectedon the surfaceof

the samples after the exposure periods Significant destruction of stearic acid was

demonstratedontheTiO2andAg‐TiO2thinfilmsafterexposuretothe254nmUVlight

source(Figure55bandFigure55c)andafter29hoursthepeakshaddisappearedThe

rateofstearicaciddestructionforboththeTiO2andAg‐TiO2thinfilmswascalculated

tobeapproximately11x1014moleculescm2perhourbasedupontheassumption

that1unitofintegrationbetween2700and3000cmequatedtoapproximately97x

180

1015moleculescm2(MillsandWang2006)Thereforesilverdopingdidnothavean

effectonthephoto‐oxidisationofstearicacidafterirradiationwithUVlight

Whenthewhitelightwasusedastheirradiationsourceasignificantdecreaseinthe

stearicacid concentrationwasdemonstratedon theAg‐TiO2 thin films (Figure56c)

whereasaminimal reductionwasobservedon theTiO2 thin films (Figure56b)The

rateofstearicaciddestructionfortheTiO2andAg‐TiO2thinfilmswerecalculatedto

be approximately 16 x 1014 and 42 x 1014 respectively (Table 52) However TiO2

shouldnotdisplayanyphoto‐activityafterirradiationwiththewhite lightsourceand

activationshouldonlyoccurafterexposuretowavelengthsoflightbelow385nmas

thebandonsetofTiO2 is32eVTherefore theOptivextradeUVfilterwasfittedtothe

light box to eliminate any higher energy photons of light The photo‐oxidation of

stearic acid on the TiO2 thin film was seriously compromised and only a negligible

changeintheconcentrationofthecompoundwasobserved(Figure57b)Incontrast

thephotocatalyticactivitywasretainedontheAg‐TiO2thinfilms(Figure57c)which

was shown to be 200 timesmore effective at destroying stearic acid than the TiO2

control(Table52)Thisisthefirstunequivocalevidenceofvisiblelightphotocatalytic

destructionofstearicacid(Dunnilletal2011)

181

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

48

53

72

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

Figure55IRabsorptiondatadisplayingthephoto‐oxidationofstearicacidmoleculeson the surface of the threematerials over 72 hours using a 254 nm light sourcewherea)uncoatedglassslideb)TiO2andc)Ag‐TiO2Linetimesareshowninorderof height on the graph and in all cases the area under the curve indicates theamountofstearicacidremainingonthesurface

a

b

c

182

-002

000

002

004

006

008

010

012

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

016

018

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

Figure56IRabsorptiondatashowingthephoto‐oxidationofstearicacidmoleculesonthesurfaceofthethreematerialsover96hoursusingawhitelightsourcewherea) uncoated glass slide b) TiO2 and c) Ag‐TiO2 Line times are shown in order ofheightonthegraphandinallcasestheareaunderthecurveindicatestheamountofstearicacidremainingonthesurface

a

b

c

183

Figure 57 Rawdata showing the photo‐oxidationof stearic acidmolecules on thesurface of the three samples over 500 hours using a white light source and theOptivextrade UV filter where (a) uncoated glass slide (b) TiO2 and (c) Ag‐TiO2 Linetimes are shown in order of height and in all cases the area under the curveindicatestheamountofstearicacidremainingonthesurface

a

b

c

184

Table52Thenumberofmoleculesofstearicacidphoto‐oxidisedduringirradiationbythedifferentlightsourcesRatesaregivenasmoleculescm2perhourExposuretimestotheUVwhitelightandfilteredwhitelightwere29hours96hoursand500hoursrespectively

TiO2 Ag‐TiO2

Lightsource Moleculesoxidised

RateMoleculesoxidised

Rate

UVndash254nm 332x1016 114x1015 330x1016 114x1015

Whitelight 149x1016 155x1014 405x1016 422x1014

Filteredwhitelight 149x1016 299x1011 312x1016 625x1013

532 AntibacterialactivityagainstEcoliATCC25922

Theantibacterial activityof the thin filmswasassessedagainstEcoliAfter2hours

irradiationwithwhitelighta09log10cfusampledecreasewasobservedcompared

withboth the uncoated controlsand theTiO2 controlsexposed to the same lighting

conditions (Figure58) Thedecrease inbacterial recoverywasmuchgreaterafter6

hours irradiationwith thewhite light sourceE coliwasnot recovered from theAg‐

TiO2thinfilmsafterthe6hourexposureperiodonanyoftheexperimentalrepeats

Thisreductioncorrespondstoa48 log10cfusampledecreaseinbacterialrecovery

comparedwiththeglasscontrolsexposedtothesamelightingconditions(plt0001)

ThedecreaseinrecoverywasslightlylesswhencomparedtotheTiO2thinfilmsbuta

statistically significant 44 log10 cfu sample decreasewas still achieved (p lt 0001)

However E coli could not be recovered from the Ag‐TiO2 thin films which were

incubated in thedark for the6 hour incubation period indicating that theobserved

antibacterialactivityobservedwasnotlight‐dependent

185

Figure58 Effectof the thin filmAg‐TiO2on the survivalofE coli Thin filmswereirradiatedwithwhitelight(L+)orincubatedinthedarkfor2hours(L‐)TheuncoatedglassslidesTiO2andAg‐TiO2arerepresentedbylsquoUnrsquolsquoTirsquoandlsquoAgrsquorespectively


186

TheantibacterialactivityoftheAg‐TiO2thinfilmswasfurtherassessedtheexposure

periodwasextendedto12hoursandonceagainitwasnotpossibletorecoverEcoli

fromtheAg‐TiO2thinfilmsaftertheincubationtimeandthiseffectwasindependent

of light exposure (Figure 510) Interestingly the activity of the TiO2 thin films

increasedwithextendedexposuretowhitelightanda24log10cfusampledecrease

inbacterial recoverywasobserved comparedwith theglass controlexposed to the

samelightingconditionsThisfindingsupportstheresultsfromthefunctionaltesting

whichdemonstratedphoto‐oxidationofstearicacidafterexposuretothiswhitelight

sourceThereforetheOptivextradeUVfilterwasplacedabovethemoisturechamberto

eliminatetheUVcomponentofthewhitelightsourceTheantibacterialactivityofthe

TiO2thinfilmswaseliminated(Figure511) the reductionobservedontheTiO2thin

filmswasnegligible (002 log10cfusampledecrease)The light‐independentactivity

of the Ag‐TiO2 thin films was retained and the decrease in bacterial recovery was

maintained at 49 log10 cfu sample on the Ag‐TiO2 thin films in the presence and

absenceoffilteredlight

187

Figure510EffectofthethinfilmAg‐TiO2onthesurvivalofEcoliThinfilmswereirradiated with white light (L+) or incubated in the dark for 12 hours (L‐) Theuncoated glass slide TiO2 and Ag‐TiO2 are represented by lsquoUnrsquo lsquoTirsquo and lsquoAgrsquorespectively

Figure511EffectofthethinfilmAg‐TiO2onthesurvivalofEcoliThinfilmswereirradiatedwithwhitelightfilteredwiththeOptivextradeglass(L+)orincubatedinthedarkfor12hours(L‐)TheuncoatedglassslideTiO2andAg‐TiO2arerepresentedbylsquoUnrsquolsquoTirsquoandlsquoAgrsquorespectively

188

The antibacterial activity of theAg‐TiO2 thin filmswere further determined after 18

hours exposure to thewhite light source The light‐independent activity of the thin

filmswasmaintainedanda46 log10cfu sampledecrease intherecoveryofEcoli

was observed compared with the glass controls exposed to the same lighting

conditions (p lt0001)No re‐growthofE coliwasobservedoneither the thin films

incubated in the presence or absence of light indicating a sustained antibacterial

effect Aminimal decrease in the recovery ofE coliwas observed on the TiO2 thin

filmsafterthe18hourincubationperiod(03log10cfusample)whichparadoxically

wasmuchlessthanthatseenafter12hoursThisdifferencewashoweverstatistically

significant(plt001)ThewhitelightalonedidnothaveaneffectonthesurvivalofE

coliontheuncoatedcontrolslidesandnosignificantdifferenceinbacterialrecovery

wasobservedonthesesamplesafterincubationinthepresenceorabsenceofwhite

lightwhichimpliesthatthephoto‐activityobservedontheTiO2thinfilmswasnotdue

totheeffectofthewhitelightsourcealone

189


533 AntibacterialactivityagainstEMRSA16

TheantibacterialactivityofthethinfilmswasassessedagainstEMRSA‐16A03log10

cfu sample decrease in the recovery of EMRSA‐16 was observed after 6 hours

irradiation with white light (Figure 513) compared with the uncoated glass slides

exposedtothesamelightingconditionswhichdidnotreachstatisticalsignificance

190

Figure 513 Effect of the thin filmAg‐TiO2 on the survival of EMRSA‐16 Thin filmswere irradiatedwithwhite light (L+)or incubated in thedark for6hours (L‐) Theuncoated glass slide TiO2 and Ag‐TiO2 are represented by lsquoUnrsquo lsquoTirsquo and lsquoAgrsquorespectively

TheAg‐TiO2thinfilmsweresubsequentlyexposedto12hourswhite lightanda26

log10 cfu sample decrease in the recovery of EMRSA‐16 was observed (p lt001)

comparedwith the uncoated glass slides (Figure 514)Negligible photo‐activitywas

observedontheTiO2thinfilmsandtherewasan insignificantdifferenceobserved in

the recovery from the irradiated TiO2 thin films compared to those incubated in the

dark (02 log10 cfu sampledecrease) Theantibacterialeffectappeared to be light‐

dependentandtherewasa23log10cfusampledifferenceintherecoveryofEMRSA‐

16 from the irradiated Ag‐TiO2 thin films comparedwith the non‐irradiated Ag‐TiO2

thinfilms(plt001)anda26log10cfusampledifferenceintherecoveryofEMRSA‐16

fromtheuncoatedirradiatedsamples(plt0001)

191

Figure 514 Effect of the thin filmAg‐TiO2 on the survival of EMRSA‐16 Thin filmswere irradiatedwithwhite light (L+)or incubated inthedarkfor12hours(L‐)Theuncoated glass slide TiO2 and Ag‐TiO2 are represented by lsquoUnrsquo lsquoTirsquo and lsquoAgrsquorespectively

TheexperimentwasrepeatedwiththeOptivextradeUVfilterinsitutoeliminateanystray

photons of sub 400 nm light and the antibacterial activity of theAg‐TiO2 thin films

decreased(Figure515)A11log10cfusamplereductionintherecoveryofEMRSA‐

16 was observed compared with the uncoated sample irradiated with the same

filteredlightsource(plt0001)Theminimalphoto‐activityobservedontheTiO2thin

films in the presence of unfilteredwhite light wasmaintained and a 02 log10 cfu

sampledecreasewasdetectedcomparedwiththeuncoatedsamples irradiatedwith

filteredwhitelightThisdifferencewasnotstatisticallysignificant(pgt005)

192

Figure 515 Effect of the thin filmAg‐TiO2 on the survival of EMRSA‐16 Thin filmswereirradiatedwithwhitelightfilteredwiththeOptivextradeglass(L+)orincubatedinthe dark for 12 hours (L‐) The uncoated glass slides TiO2 and Ag‐TiO2 arerepresentedbylsquoUnrsquolsquoTirsquoandlsquoAgrsquorespectively

TheAg‐TiO2thinfilmsweresubsequentlyirradiatedwithwhitelightfor18hoursand

theresultsareshowninFigure516A34log10cfusamplereductionintherecovery

of EMRSA‐16was observed comparedwith the glass controls exposed to the same

lighting conditions (p lt 0001) The light‐dependent activity of the thin films was

sustainedanda29log10cfusampledecreaseinbacterialrecoverywasobservedon

the irradiated Ag‐TiO2 thin films compared with those incubated in the dark (p lt

0001) However significant photo‐activity was detected on the TiO2 thin films

althoughthiseffectwasextremelyinconsistentasindicatedonthegraphbythelarge

errorbarsandwasalsolessstatisticallysignificant(plt005)A34log10cfusample

decrease in the recovery of EMRSA‐16was observed comparedwith the uncoated

glasscontrolsexposedtothesamelightingconditionsNoactivitywasdetectedonthe

TiO2thinfilms incubated inthedark indicatingthattheactivitywas lightdependent

andcouldonceagainbeduetotheUVcomponentofthewhitelightsource

193

Figure 516 Effect of the thin filmAg‐TiO2 on the survival of EMRSA‐16 Thin filmswere irradiatedwithwhite light (L+)or incubated inthedarkfor18hours(L‐)Theuncoated glass slides TiO2 and Ag‐TiO2 are represented by lsquoUnrsquo lsquoTirsquo and lsquoAgrsquorespectively

Therefore the Optivextrade filter added and the samples were irradiated with filtered

white light (Figure 517) The antibacterial activity of the Ag‐TiO2 thin films was

retained but at a reduced rate the average decrease in bacterial recovery dropped

from34 log10cfusampleto23 log10cfusampleusingtheunfilteredandfiltered

whitelightsourcesrespectivelyThisresultmirrorsthatseenafter12hoursirradiation

with the filtered light sourceand remainedhighly statistically significant (plt0001)

ThelightdependentactivityoftheAg‐TiO2thinfilmswasalsoreplicatedand14log10

cfu sample decrease in bacteriawas observed on the irradiatedAg‐TiO2 thin films

comparedwiththoseincubatedinthedark(plt005)butagainthisreductionwasless

thanthatobservedwhentheunfilteredlightsourcewasused

194

Figure 517 Effect of the thin filmAg‐TiO2 on the survival of EMRSA‐16 Thin filmswereirradiatedwithwhitelightfilteredwiththeOptivextradeglass(L+)orincubatedinthedarkfor18hours(L‐)TheuncoatedglassslideTiO2andAg‐TiO2arerepresentedbylsquoUnrsquolsquoTirsquoandlsquoAgrsquorespectively

Themostsurprisingresultwastheretainedphoto‐activityoftheTiO2thinfilms(Figure

517) the photo‐activity was reduced when filtered white light was used as the

irradiationsourcebutastatisticallysignificant31log10cfusampledecreaseinviable

bacteriawasstillobserved(plt001)whichwasagreaterdecreasethanthatseenon

theAg‐TiO2thinfilmsAwiderangeofbacterial recoverywasobservedindicatedby

the large box on the graph on occasion no bacteria were recovered at all and on

otherexperimentalreplicatesthenumberofcoloniespresentequalledthatobserved

from the control samples incubated in the dark The bacterial recovery from the

control samples Ag‐TiO2 and TiO2 which were incubated in the dark was also

significantly lower than theuncoatedglass samples incubated in thedark (plt001)

Furthermore the values obtained from the TiO2 thin film incubated in the darkwas

significantly lower than that obtained in the previous 18 hour experiment (Figure

516)

195

54 Discussion

Silverhasbeenshownboth inthischapterand inthe literature to improvetitanium

dioxide photo‐activity and this is achieved through three mechanisms The first

involvesreductionofsilverionstosilverbyphoto‐excitedelectronsTheelectronsare

furtherattractedtosilverparticlesinthefollowingreactionwherethesilverparticles

actaselectrontraps(Herrmannetal1997Heetal2002Brooketal2007b)

(Ag)+e‐ e‐Ag

The electrons move to the interior of the thin film and the holes move to the

interfacial region which enhances their separation and inhibits electron‐hole

recombination The photo‐generated holes then react with surface hydroxyl groups

and water to form hydroxyl radicals and other reactive species which possess

antibacterial activity (Sclafani et al 1991 Herrmann et al 1997 Stathatos et al

2001 He et al 2002) Secondly the electric field around the silver particles is

increased by surface plasmon resonance effects which further enhance photo‐

excitationoftheelectronsandelectron‐holeseparation(Zhaoetal1996)Finallythe

surface roughness of the titaniumdioxide thin film changes upon silver addition so

that the titanium dioxide particle size in the resultant thin films is smaller which

exposes a greater surface area available for photo‐reactionwhich further increases

photo‐activity(Herrmannetal1997Heetal2002Martinez‐Gutierrezetal2010)

Therefore thepropertiesofaphotocatalyst can beadaptedby reducing theparticle

sizetocoupletheintrinsicbandonsetpropertiestoallowlowerenergyphotocatalysis

(Herrmannetal1997Heetal2002Dunnilletal2011)

196

541 Synthesisofthesilver‐dopedtitaniathinfilms

Analogoustonitrogenandsulfurdopingoftitaniathesilverconcentration iscritical

and a decrease in the photo‐activity of the thin films will occur if the silver

concentrationexceedsanoptimumlevel(Sclafanietal1991DoboszandSobczynski

2003 Brook et al 2007b) This is due to the lsquoscreening effectrsquo where the silver

depositedonthesurfaceofthethinfilmmasksthephoto‐reactivesitessothatthey

are inaccessible for interaction with photons (Dobosz and Sobczynski 2003) In

additionthenegativelychargedsilverparticlesonthethinfilmcouldattracttheholes

beforeanyinteractionwithwaterwhichwoulddecreasetheconcentrationofreactive

oxygenspeciesgeneratedandtheobservedphoto‐activity(Heetal2002)

Sol‐geldepositionwasusedtosynthesisethethinfilms inthischapter incontrastto

APCVDwhichwasused togenerate the thin filmsassessed in theprevious chapter

APCVD was initially chosen as a deposition method as the resultant coatings are

transparentrobustandstronglyadheredtothe substrateSol‐gel filmsaregenerally

thicker less mechanically robust and require sintering after coating to anneal the

coating to the substrate (Brook et al 2007b) A post‐coating annealing step was

includedinthesol‐gelmethodofsynthesissothethinfilmsgeneratedinthischapter

were well adhered to the substrate and as mechanically stable as the APCVD

generatedthinfilms

197

542 Characterisationand functionalassessmentof thesilver‐dopedtitania

thinfilms

The silver‐coated titania thin films exhibited photo‐chromic behaviour which was

causedbyachangeintheoxidationstateofthesilvernanoparticlesfromsilveroxide

tometallicsilver(Ohkoetal2003Paramasivametal2007Gunawanetal2009)

BothUVandvisiblelightwereabletoinducethemorecolouredorangemetallicstate

and the less coloured purple oxide state occurred after storage in the dark Excited

electronsgeneratedduring lightexposurephoto‐reactedwith the silver ionspresent

withinthepurplefilmandthefilmsturnedorangeasthesilveroxidewasreducedto

silvermetal(Ohtanietal1987)Whenthefilmsweresubsequentlystoredinthedark

inthepresenceofairthephoto‐reducedsilverwasoxidisedformingsilveroxideand

the films reverted to the purple colour due to a decrease in light absorbance

(Paramasivametal2007)Thesechangesarecausedbysurfaceplasmonresonance

effects which in turn are influenced by the nanoparticle size shape and the local

refractiveindex(Jinetal2001Mocketal2002Ohkoetal2003Gunawanetal

2009)

Thebandonsetofthesilver‐coatedtitaniathinfilmshadshiftedto29eVtowardsthe

visible regionof theelectromagnetic spectrumwhich in theabsenceofparticle size

modification indicated doping of silver nanoparticles within the titanium dioxide

structureWehadpreviouslyshownthatdopingtitaniathinfilmswitheithernitrogen

orsulfurcausedashiftinthebandonsetto29eVand30eVrespectivelyindicating

thatthesethinfilmswouldmakebetterwhitelightphotocatalyststhantitaniaaloneA

lowerbandonsetfromsilver‐dopedtitaniasampleshasbeenreportedabandonset

198

of 26 eV was estimated by Medina‐Ramirez et al (2011) although these were

nanoparticulatecompositesandnotthinfilmsTheobservedshifttowardsthevisible

spectrum could also be partly due to mixing of the band onsets silver oxide at

approximately1eVforAgOand14eVforAg2O(Idaetal2008Rajuetal2009)

Thewatercontactangleofthethin filmswasmeasuredtodetermineanychange in

the hydrophilicity of the surface after irradiation with the different light sources

Superhydrophilicity occurs after photo‐oxidation of hydrocarbons adsorbed onto the

substrate which results in the production of a hydroxylated surface (Zubkov et al

2005) Predictably thewater contact angle of the titania thin films decreased after

irradiation with the UV light source (Mills and LeHunte 1997 Parkin and Palgrave

2005)andthewatercontactangleofthesilvercoatedtitaniathinfilmsalsodecreased

byasimilaramountTheadditionofsilvernanoparticlestothesurfaceofthetitania

thinfilmwaspredictedtoresult inanalterationofthehydrophilicityofthethinfilm

prior to light exposure as the surface roughness of the thin film had changed and

largercontactanglesareusuallyfoundonroughersurfaces(Wenzel1936Cassieand

Baxter 1944) but these data show this effect is insignificant even though silver

coverageofthesurfacereached64(Dunnilletal2011)IrradiationwithUVlightdid

nothaveaneffecton thewater contactangleon theuncoatedglass slidealthough

thewatercontactangleontheslidewasinitiallylowTheexpectedcontactangleona

glasssurfaceisapproximately70degandthelowreadingobservedintheseexperiments

indicatedthattheglasssubstratewasinaverycleancondition(Zubkovetal2005)

Thevisiblelight‐inducedhydrophilicityofthethinfilmswasdeterminedbyirradiation

withwhitelightfilteredwithasheetofOptivexglasstoeliminateanystrayhigher

199

energy photons of light with awavelength of less than 400 nm Thewater contact

angle on the silver‐coated titania thin film decreased to the same degree as that

observed after UV irradiation In contrast no change in water contact angle was

observedonthetitaniathinfilmsThis clearlydemonstratesthevisible‐light induced

natureofthesilvercoatedtitaniathinfilms

The photo‐oxidisation of stearic acid has been used extensively in the literature to

indicate the photocatalytic activity of novel thin films and estimate their potential

antibacterial activity (Mills et al 2002 Mills andWang 2006 Brook et al 2007a

2007bPageetal2007)TherateofstearicaciddegradationwascalculatedfortheN‐

dopedandS‐dopedthinfilmsassessed inthepreviouschapterafterexposuretothe

white light source The N‐doped sample (N1) displayed a rate of destruction of

approximately 14 x 1014 molecules cm2 per hour and the S‐doped sample (S2)

demonstrated a similar rate of 11 x 1014 molecules cm2 per hour (Dunnill et al

2010)Thesilver‐coatedtitaniathinfilmsgeneratedinthischapterdemonstratedrate

of destruction of approximately 42 x 1014molecules cm2 per hourwhich is three

timesmoreefficientthantheN‐dopedandS‐dopedthinfilmsandtwiceasefficientas

thetitaniumdioxidethinfilmsThisimpliesthatsurfacesilverdopingdoesnotinduce

asmuch electron‐hole recombination as that observed in theN‐doped and S‐doped

titaniawhichresultsinimprovedphotocatalysis

The anatase titanium dioxide thin film should not exhibit any photo‐activity after

irradiationwiththewhitelightsourceandactivationshouldonlyoccurafterexposure

towavelengthsoflightbelow385nmasthebandonsetoftitaniumdioxideis32eV

The photo‐activity observed suggests that therewas light of an increased frequency

200

emitted from the white light source The emission spectrum for the light source is

shown in Figure 21 and no emission is detectable below 410 nm however the

spectrumstartsat380nm so theprofileat lowerwavelengths isnotknownWhite

light sources suchas the fluorescent lampused in theseexperiments can leakvery

small amounts of higher energy photons of light as they age due to the release of

phosphor from the inside of the fluorescent tubing which could explain the photo‐

activitygeneratedonthetitaniumdioxidethinfilm

TheOptivexUVfilterwasemployedoncemoreandthephoto‐activityofthesilver‐

coatedtitaniathin filmswasretainedandthephoto‐activityofthetitaniathinfilms

was terminated This demonstrated the true visible light driven photo‐oxidation of

stearicacidonthesilver‐coatedtitaniathinfilmsTherateofstearicaciddegradation

wasslowerwhentheUVfilterwasemployedpartlybecausetheintensityofthewhite

lightwasreducedasonlyaround80ofemitted lightwasabletotransmitthrough

the glass shield and partly due to the loss of the UV part of the electromagnetic

spectrum

543 Antibacterialactivityofthesilver‐dopedtitaniathinfilms

Theantibacterialpropertiesofthesilver‐coatedtitaniathinfilmswereassessedusing

E coliand EMRSA‐16as representative strainsGram‐negative strains suchasE coli

havebeendemonstratedtobemoredifficulttokillusinglight‐activatedantimicrobial

coatingsthanGram‐positivestrainssuchasMRSA(Decraeneetal2006Pageetal

2009) However in these experiments E coli was eradicated from the silver‐coated

titaniathinfilmsataquickerratethanEMRSA‐16AreductionintherecoveryofEcoli

201

fromthesilver‐coatedtitaniathinfilmswasobservedafterjust2hoursandnoviable

bacteriacouldberecoveredfromthesamplesafter6hoursincubationHoweverthe

observedantibacterialeffectwasindependentoflightexposureasasimilarreduction

in bacterial recovery was observed on the silver‐coated titania incubated in the

absenceoflightwhichillustratestheactivitywasduetothetoxicityofthesilverions

ratherthanalightinducedeffectwhichhasbeendemonstratedintheliterature(Feng

etal2000Kimetal2007Jungetal2008)TheincreasedsusceptibilityofGram‐

negative bacteria to the silver containing thin filmwas postulated to be due to the

thinnerpeptidoglycanlayerinthecellmembranewhichallowsincreaseduptakeinto

the interior of the bacterial cell (Schierholz et al 1998) Conversely Kowal et al

(2011) showed a greater susceptibility of MSSA and MRSA to silver‐doped titania

nanopowderscomparedwithEcoli

EMRSA‐16 has been responsible for a significant proportion of the healthcare‐

associatedcasesofMRSAbacteraemiaoverthelastdecadeandwasshowninChapter

3tobealighttolerantstrainofMRSA(Johnsonetal2001Ellingtonetal2010)The

antibacterial activity of the silver‐coated titania thin films increasedwith prolonged

exposuretowhitelightwiththelargestreductioninbacterialrecoveryobservedafter

18 hours irradiation Enhancement of the photocatalytic properties of the light‐

activatedsurfacebythesilverparticlesandtheenhancementofthetoxicpropertiesof

thesilverbytitaniawasobservedonthesilver‐coatedtitaniawhichdemonstrateda

synergisticrelationshipbetweenthetwocomponentsofthethinfilmThiseffectwas

muchgreaterthanthatobservedwhenthesilver‐coatedtitania filmswereincubated

intheabsenceoflightorwheneitherthetitaniaoruncoatedsampleswereirradiated

202

with white light The silver ions alone appeared to have an effect on EMRSA‐16

especially after a prolonged incubation time but this was less significant than the

effect seenafter lightexposureThe lack ofactivityobservedon theuncoatedglass

slidesdemonstratedthatthewhitelightsourcedidnothaveaninhibitoryeffectonthe

viability of EMRSA‐16 The lack of activity observed on the titania thin film in the

presenceof6or12hourswhite light indicatedthattheUVcomponentofthewhite

lightsourcewasnotsufficienttophoto‐activatethetitaniafilmsHoweverthispattern

wasnotmaintainedandasignificantdifferenceintherecoveryofEMRSA‐16fromthe

irradiatedTiO2thinfilmswasobservedcomparedwiththeuncoatedglassslidesafter

18hoursThiseffectwasnoteliminatedwhentheOptivextradeUVfilterwasappliedThe

significantdecreaseinrecoveryofEMRSA‐16observedontheTiO2thinfilmincubated

inthedarksuggeststhatalight‐independentmechanismofactionwasinvolved

It is possible to conclude that the photo‐induced destruction was due to reactive

oxygenproducedbytitaniadrivenbywhitelightphotocatalysisinducedbythesilver

These effects did not occur in the absence of white light or silver An alternative

explanationcould involvephoto‐assisted releaseof silver ions from the silver‐coated

titaniawhichinturncausedtheantibacterialeffect

Amajor limitation of the experimentswas that the test conditionswere laboratory‐

controlledanddidnottakeintoaccountfactorssuchasorganicsoilwhichwouldbe

presentonhand‐touch surfaces Substancessuchas sebaceousoilsbloodandother

humansecretionswouldbe likelytocontaminatethethinfilms if theywereusedas

antibacterial coatings in a patient environment and the effect of these substances

should be investigated as they are likely to cause an inhibition in the photocatalytic

203

activity of the thin films (Furno et al 2004)Organic soiling of a surface is likely to

precedebacterialcontamination(Verranetal2002)soifthethinfilmswereableto

photo‐degrade any organic soil present it would keep the surface hygienically clean

andeliminateapotentialnutrientsourceofanycolonisingbacteria

55 Conclusion

Thischapterhasdemonstratedthattheantibacterialactivityoftitaniathinfilmscan

be significantly enhanced by the addition of surface‐bound silversilver oxide

nanoparticles The thin films displayed photochromic behaviour and were found as

either silver oxide or pure silver depending on the storage conditions oxidation of

silvertosilveroxideoccurredafterstorageinthedarkandapurplecolourationwhilst

exposuretoindoorlightingconditionscausedphoto‐reductionofthesilveroxideback

to silver and an orange coloured film White light induced photocatalysis was

generatedbyashiftinthebandonsetofthethinfilmscausedbytheadditionofsilver

nanoparticlesVisiblelightphotocatalysiswasdemonstratedwhenaUVfilterwasused

to block out the minimal UV component of the white light source and this was

observed in the form of photo‐oxidation of stearic acid a reduction in the water

contactangleandphotocatalyticactivityagainstEMRSA‐16Thisisthefirstexampleof

unambiguous visible light photocatalysis and photo‐induced superhydrophilicity

alongsideatitaniumdioxidecontrolthatshowsnoactivation

204

6 Assessment of a novel antibacterial material for use inendotrachealtubesinintubatedpatients

61 Introduction

Ventilator‐associatedpneumonia(VAP)isaHCAIassociatedwithsignificantmorbidity

and mortality Intubated patients have an endotracheal tube (ETT) in situ to allow

mechanicallyassistedbreathingwhichcompromises thenormal clearanceofmucus

and other upper airway secretions and allows micro‐aspiration of contaminated

subglotticsecretionsintothelungsThesesecretionscontaincommensalbacteriathat

provide a source for pulmonary infection In addition the lumen of the ETT itself

becomes colonised with bacteria which provides a secondary source of infective

organisms (Deem and Treggiari 2010) A number of studies investigating the

microbiology of VAP have shown that Gram‐negative bacilli are isolated more

commonly in patients with VAP compared with patients with hospital‐acquired

pneumonia (ie pneumonia acquired in hospital in the absence of mechanical

ventilation) P aeruginosa Acinetobacter species and S maltophilia are the most

commonly observed Gram‐negative pathogens causing VAP (Johanson et al 1972

Richards et al 1999 Weber et al 2007 Bouadma et al 2010) Both meticillin‐

sensitive and resistant S aureus have also been isolated but were observed more

frequentlyinnon‐intubatedpatients(Weberetal2007)

It is advantageous to reduce microbial load and decrease biofilm formation in the

lumenoftheETTasthiswouldeliminatethebacterialreservoirand lowertheriskof

developing VAP The use of antimicrobial silver ETTs has been recommended in

combinationwithadditionalclinicalmeasures inthepreventionofVAP(Torresetal

205

2009 Coppadoro et al 2011) and it would be desirable to expand on the pool of

antimicrobialETTsavailablePhotodynamicinactivation(PDI)ofbacteriahasprovento

beaneffectivemethodofreducingthebacterialloadonsurfacesandthistechnology

has the potential to be applied to an ETT A laser light could be inserted along the

length of the ETT and switched on periodically to activate the surface and kill any

bacteriapresentFigure61showshowthismaybeachievedinacathetertube

Figure61Acathetertube impregnatedwiththephotosensitisingagentmethyleneblueItissuggestedthatlightfromalasercouldbeprojectedthroughthetubewiththeuseoffibreopticsPhotographcourtesyofProfWilson(UCL)

This chapter describes the development of a polyurethane polymer which was

impregnatedwiththephotosensitisingagenttoluidineblueO(TBO)Theantibacterial

effect of the impregnated polymers after irradiation with laser light was observed

206

againstaseriesofpathogensknowntocauseVAPBothclinicalandtypestrainswere

tested to assess any difference in susceptibility to PDI The published literature

describedabovewasusedtoguidethechoiceofbacteriaandmaterialtypeassessed

inthischapter


621 Materialsynthesis

Thepolyurethanepolymersrequiredforthisseriesofexperimentsweresynthesisedas

described inSection2103PolymerswerepreparedcontainingTBO(S+)andcontrol

polymerswerepreparedinparallelwithouttheadditionofTBO(S‐)

622 Measuring the antibacterial photo‐activity of the TBO‐impregnated

polymers


of P aeruginosa PAO1 and clinical strains of P aeruginosa A baumannii and S

maltophiliawerepreparedasdetailedinSection23resultinginastartinginoculumof

approximately107cfumlwhichequatedtoaconcentrationofapproximately106cfu

polymerasdescribedinSection2123AsuspensionofCalbicans(107cfuml)was

alsopreparedasdescribed inSection23TheMannWhitneyUtestwasusedforall

statistical analyses to determine the statistical significance of any differences

observed as described in Section 213 The nomenclature used during this series of

experimentsisdetailedinTable61

207

Table 61 Nomenclature used during microbiological assessment of the TBO‐impregnatedpolymers

63 Results

631 Assessmentoftheantibacterialphoto‐activityoftheTBO‐impregnated

polymersagainstPaeruginosaPAO1atypestrain

TheactivityoftheTBO‐impregnatedpolyurethanepolymerswasfirstassessedagainst

atypestrainofPaeruginosaPAO1Thepolymerswereexposedtothelaserlightfor

timeperiodsofbetween30secondsand240secondsandtheresultsareillustratedin

Figure62throughtoFigure610


L+S+ TBO‐impregnatedsampleexposedtolaserlight

L+S‐ TBO‐impregnatedsampleNOTexposedtolaserlight

L‐S+ NonTBO‐impregnatedsampleexposedtolaserlight

L‐S‐ NonTBO‐impregnatedsampleNOTexposedtolaserlight

208

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure62AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainstPaeruginosaPAO1after30secondsThedottedhorizontallineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


209

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure65AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainstPaeruginosa PAO1 after 120 seconds The dotted horizontal line indicates thedetectionlimitofthesamplingmethod080log10cfupolymer

210

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


211

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Highly statistically significant reductions in the numberof viablePaeruginosa PAO1

recoveredfromtheTBO‐impregnatedpolymerswasobservedatalltimepointstested

212

(allplt0001)Thereductioninbacterialcountfollowedadose‐dependentresponse

whereby as the dose of laser light was increased the antibacterial activity of the

impregnatedpolymers increasedwhich resulted ina lower recoveryofbacteria For

examplea141log10cfupolymerdecreasewasobservedafter90secondsexposure

to the laser light (Figure65) rising toa294 log10 cfu polymerdecreaseafter180

seconds(Figure67)anda333log10cfupolymerdecreaseafter240seconds(Figure

69)TheresultsfromalloftheexperimentsaresummarisedinTable62

Table62SummaryofthedataobtainedfromthePaeruginosaPAO1experimentsThestatedreductions inbacteriaarecalculatedbycomparingthemedianbacterialrecoveryfromtheL‐S‐samplewiththeL+S+sample

ExposuretimesecondsLogreductioncfuper

polymerPercentagereduction

cfuperpolymer

30 044 639

60 049 679

90 141 961

120 209 992

150 282 9985

180 294 9989

210 305 9991

240 333 9995

Theobservedreductions inbacterial recoverywerehighlystatisticallysignificant (plt

0001) at all time points (L‐S‐ comparedwith L+S+)which demonstrates the potent

light‐dependent antibacterial activity of the TBO‐impregnated polymers When the

twogroupsofTBO‐impregnatedpolymerswerecomparedandtheeffectofthe laser

213

lightwas investigated (L‐S+ and L+S+) the recovery ofP aeruginosa from the TBO‐

impregnatedpolymersexposedtolightwassignificantlylowerthanrecoveryfromthe

TBO‐impregnated polymers incubated in the dark This difference was highly

statisticallysignificant(plt0001)foralltimepointsabove60secondsthedifference

wasalsostatisticallysignificantafter30secondswithapvalueofplt001Thesedata

further confirm the photocatalytic nature of the TBO‐impregnated polymers There

wasno statisticaldifference in thebacterial recoveryobtained from the twosetsof

polymers incubated in the dark (L‐S‐ compared with L‐S+) which demonstrates the

intrinsic lackofantibacterialactivityofTBO intheabsenceof lightofanappropriate

wavelength


polymersagainstaclinicalstrainofPaeruginosa

The photo‐activity of the TBO‐impregnated polyurethane polymers was assessed

againstaclinicalstrainofPaeruginosatoassesswhethertherewereanydifferences

in the susceptibility of the laboratory type strain compared with a strain recently

isolatedfromapatientwithclinicallyconfirmedVAPThepolymerswereexposedto

thelaserlightfortimeperiodsof90seconds180secondsand240secondsusingthe

sameinitialbacterialinoculumofapproximately106cfubacteriaperpolymer

214

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure610AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainsta clinical strain of P aeruginosa after 90 seconds The dotted horizontal lineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


215

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Ahighlysignificantreduction intherecoveryoftheclinicalstrainPaeruginosa from

theTBO‐impregnatedpolymersafterexposuretothelaserlightwasachievedafter90

seconds (Figure610)180 seconds (Figure611)and240 seconds (Figure 612) This

reductionwas highly statistically significant for all time points tested (p lt 0001) A

highly statistically significant decrease (p lt 0001) was observed on the TBO‐

impregnatedpolymersexposedtothelaserlightcomparedwiththosenotexposedto

thelaserlightAlackofantibacterialactivitywasdemonstratedintheabsenceoflaser

lighttherewasnostatisticaldifferenceintherecoveryofPaeruginosafromthetwo

sets of polymers which were not exposed to the laser at any light exposure time

Combining these data illustrates the laser light‐induced antibacterial nature of the

polymers

216

ThedirecteffectofthelaserlightontheviabilityofPaeruginosawasdeterminedby

comparingthebacterialcountsfromthenon‐impregnatedpolymerswiththebacterial

counts from the TBO‐impregnated polymers irradiated with laser light A small

decreasecanbeobservedontheboxplotswhichwasstatisticallysignificant(plt0001

at90sand240splt005at180s)howeverthisreductionwasnotsubstantial(lt05

logcfupolymerreduction)anditismorelikelythatthisisduetothesmallvariation

in the bacterial count rather than a genuine effect of the laser To reinforce this

statement the bacterial count of P aeruginosa from the non TBO‐impregnated

polymersexposedtothelaserlight(L+S‐)wascomparedwiththatobtainedfromthe

TBO‐impregnated polymers exposed to the laser light (L+S+) large reductions in

bacterial countswere observed for all three timepoints tested (088 151 and 129

log10cfupolymerdecreasesafter90180and240secondsrespectively)whichwere

allhighlystatisticallysignificant(plt0001)

Thedifference in the susceptibilityof the twoPaeruginosa strainswas investigated

and summarised in Table 63 It was immediately evident that the laboratory type

strainofPaeruginosaPAO1wasmoresusceptibletothephotodynamiceffectofthe

TBO‐impregnatedpolymerscomparedwiththeclinical isolateAgreaterrecoveryof

bacteriawas obtained during the experimentswith the clinicalP aeruginosa isolate

compared with the type strain and this was demonstrated after 90 180 and 240

seconds

217

Table 63 Comparison of the data obtained from the two sets of P aeruginosaexperiments The stated reductions in bacteria are calculated by comparing themedianbacterialrecoveryfromtheL‐S‐samplewiththeL+S+sample

ClinicalstrainofPaeruginosa PaeruginosaPAO1

Exposuretimeseconds

Logreductioncfuperpolymer

Percentagereductioncfuperpolymer



90 106 913 141 961

180 170 980 294 9989

240 155 972 333 9995


polymersagainstaclinicalstrainofAbaumannii

The activity of the TBO‐impregnated polyurethane polymers was subsequently

assessedagainstarecentlyisolatedclinicalstrainofAbaumanniiandtheresultsare

displayedinthefollowingthreefiguresThepolymerswereexposedtothelaserlight

for time periods of 90 seconds 180 seconds and 240 seconds using the same

concentrationofapproximately106cfubacteriaperpolymer

218

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure613AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainstaclinicalstrainofAbaumanniiafter90secondsThedottedhorizontallineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

$amp())+-

01+2()amp3456532

Figure614AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainsta clinical strain of A baumannii after 180 seconds The dotted horizontal lineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

219

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


AreductionintherecoveryofAbaumanniifromtheTBO‐impregnatedpolymerswas

achieved after 90 seconds (Figure 613) 180 seconds (Figure 614) and 240 seconds

(Figure615) irradiationwiththe laserlightdemonstratingthephotocatalyticactivity

of the TBO‐impregnated polymers These reductions were all highly statistically

significant (p lt 0001) There was no statistical difference in the recovery of A

baumanniifromthetwosetsofpolymerswhichwerenotexposedtothelaserlight(L‐

S‐ and L‐S+) confirming the light dependent properties of the TBO‐impregnated

materialWhen theeffect of the laser lightalonewas investigated (L‐S‐andL+S‐) a

statistically significant differencewas observed at 180 seconds (p lt 0001) and 240

seconds(plt005)andnotat90secondsbutthefiguresshowthatthisreduction is

minimal and this is likely to be a consequence of the small amount of variation in

bacterialcountsseeninthesetwogroupsFurthermorehighlystatisticallysignificant

220

reductions (plt0001)wereachievedwhen the recovery from the TBO‐impregnated

polymers exposed to the laser light were compared with the irradiated non‐

impregnated polymers further emphasising the requirement for both the laser light

andthephotosensitisertoexertahighlysignificantconsistentantibacterialeffect


polymersagainstaclinicalstrainofSmaltophilia

The activity of the TBO‐impregnated polyurethane polymerswas assessed against a

newly isolated clinical strain of S maltophilia and the results are displayed in the

followingfiguresThepolymerswereexposedtothelaserlightfortimeperiodsof90

seconds 180 seconds and 240 seconds using the same concentration of

approximately106cfubacteriaperpolymer

221

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure616AntibacterialactivityofTBO‐impregnatedpolyurethanepolymeragainsta clinical strain of S maltophilia after 90 seconds The dotted horizontal lineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer


222

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


The TBO‐impregnated polymers exerted a significant antibacterial effect on S

maltophiliaafterexposuretothelaserlightfor90seconds(Figure616)180seconds

(Figure 617) and 240 seconds (Figure 618) This reduction was highly statistically

significant (p lt 0001) for all of the three exposure times Comparison of the two

groupsofTBO‐impregnatedpolymersshowedastatisticallysignificantdecreaseinthe

recoveryofSmaltophilia fromthepolymersexposedtothe laser lightcomparedto

that recovered from those polymers not exposed to the laser light There was no

statisticaldifference in the recoveryofSmaltophilia from the twosetsofpolymers

incubated in the absence of laser light (L‐S‐ and L‐S+) demonstrating the light

dependent activity of the polymers A small but statistically significant reduction in

bacterialcountswasobservedwhenthedirecteffectofthelaserlightwasinvestigated

bycomparingvaluesobtainedfromrecoveryfromthetwogroupsofnon‐impregnated

223

polymers but the effect of the laser light in combination with the impregnated

photosensitiserwasmuchlargerThisfindingmirrorsthedataobtainedintheprevious

experimentalsectionsassessingtheactivityoftheTBO‐impregnatedpolymersagainst

Abaumannii(Section633)andPaeruginosa(Sections0and632)


polymersagainstaclinicalstrainofCalbicans


recently isolated clinical strain of C albicans and the results are displayed in the




log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure619AntimicrobialactivityofTBO‐impregnatedpolyurethanepolymeragainstaclinicalstrainofCalbicansafter90secondsThedottedhorizontal line indicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

224

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions

Figure620AntimicrobialactivityofTBO‐impregnatedpolyurethanepolymeragainstaclinicalstrainofCalbicansafter180secondsThedottedhorizontallineindicatesthedetectionlimitofthesamplingmethod080log10cfupolymer

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


225

A decrease in the recovery ofC albicans from the TBO‐impregnated polymerswas

noted after exposure to the laser light for 90 seconds (Figure 619) 180 seconds

(Figure 620) and 240 seconds (Figure 621) The observed reduction was highly

statisticallysignificant(plt0001)forallofthethreeexposuretimesThefindingswere

similar to thoseobtained from theexperiments involvingbacterial causesofVAP in

that a decrease in the recovery of C albicans was not detected from the TBO‐

impregnatedpolymerswhenincubatedinthedark(L‐S‐comparedwithL‐S+pgt005)

MoreoverthelaserlighthadnoeffectontherecoveryofCalbicansafter90seconds

or 180 seconds irradiation and although a statistically significant decrease was

observedafter240secondsthedifferenceisrathersmallinabsoluteterms(031log10

cfu polymer) When the effect of the laser light in combination with TBO was

comparedwith theTBOaloneahighly statistically significantdecrease in countwas

observeddemonstratingthelight‐activatednatureoftheTBO‐impregnatedpolymers

The data from this chapter are summarised below in Table 64 It is immediately

evidentthattheTBO‐impregnatedpolymers incombinationwiththe laser lightexert

anantimicrobialeffectagainstalltheorganismstestedafter90seconds180seconds

and 240 seconds The TBO‐impregnated polymers were most effective against A

baumannii where a reduction of over 4 log10 cfu polymerwas achieved after 240

seconds and was least effective against C albicans but a significant reduction

approaching2log10cfupolymerwasstillobservedafter240secondsAsmentioned

previouslytheclinicalisolateofPaeruginosawaslesssusceptibletothephoto‐active

nature of the TBO‐impregnated polymers and a smaller reduction was observed

comparedwiththelaboratorytypestrain

226

Table 64 Summary of the data obtained from the experiments investigating theactivity of the TBO‐impregnated polymers The stated reductions in bacteria arecalculatedbycomparing thebacterial recoveryfromtheL‐S‐ samplewith theL+S+sample


Exposuretimeseconds

Paeruginosa

PAO1

Paeruginosa

clinicalisolate

Abaumanniiclinicalisolate

Smaltophilia

clinicalisolate

Calbicansclinicalisolate

90 141 106 172 096 054

180 294 170 190 282 148

240 333 155 416 312 179

64 Discussion

641 TBO‐mediatedphotodynamicbacterialinactivation

The assessment of novel antimicrobial materials for use in endotracheal tubes is a

timely and pertinent task Therefore in this chapter polyurethane polymers were

impregnatedwiththephotosensitiserTBOandexposedtowavelengthsoflightknown

tocausephotoactivityPolyurethaneisamaterialcommonlyusedinETTs(Berraetal

2008a2008bRelloetal2010)andthepolymerswereimpregnatedwithTBOrather

thancoatedastheprocessallowsapplicationoftheantibacterialagentonboththe

inner and outer surfaces of the catheter which can increase overall antibacterial

activity (Furnoetal 2004)TheTBO‐impregnatedpolymerswereassessedagainsta

rangeofbacterialspeciescommonlyisolatedfrompatientswithVAPandtheyeastC

albicans which has also been cultured from this patient group (Weber et al 2007

Bouadma et al 2010) Previous work in our laboratory has shown that the TBO‐

impregnated polymers produced photodynamic inactivation (PDI) of a meticillin‐

227

resistant strainofSaureus (EMRSA‐16)andE coli (Pernietal 2009b)Thecurrent

studyexpandedonthesedatatoinvestigatethephotoactivityofthepolymersagainst

themostcommoncausesofVAP

These experiments have shown that the TBO‐impregnated polymers exerted a

significantantimicrobialeffectonallorganismstestedafterirradiationwithlaserlight

Thereductionsfollowedadose‐dependentresponsesothatthegreatestreductionsin

bacterial (or yeast) numbers were observed after the longest irradiation time A

baumanniiwasshowntobemostsusceptibletophotodynamic inactivationwiththe

TBO‐impregnated polymers and a reduction of over 4 log10 cfu polymer was

achieved after a 4minute irradiation time Reductions of over 3 log10 cfu polymer

werealsoachievedintherecoveryofPaeruginosaPAO1andSmaltophiliaafterthe

sameirradiationtime

Many groups have reported photodynamic inactivation of a range of planktonic

bacteriaandyeasts inthepresenceofanaqueoussolutionofTBOand laser lightE

coliwasfirstshowntobesusceptibletoa25microMsolutionofTBOinthepresenceofa

tungstenlampatalightintensityof5400luxThegenerationofsingletoxygenduring

irradiationwasconfirmedastheadditionofthesingletoxygenquencherα‐tocopherol

reduced thephotoactivityof thedye (Wakayamaetal 1980)A2 ‐ 3 log10 cfu ml

decreaseintherecoveryofAbaumanniiwasdescribedafterexposureto635nmlight

at a concentration of 2 microM and 225 J cm2 energy (Ragas et al 2010) but a pre‐

sensitisation step of 30 minutes was required to achieve this level of

photoinactivationMRSAwas shown to be susceptible to a suspension of TBO after

exposuretoaHeNe laser light for just30seconds(WilsonandYianni1995)andthe

228

susceptibilityofE faecalisB cereusandPaeruginosawasdemonstratedagainsta

variety of phenothiazinium dyes including TBO after 60 minutes light exposure

(Wainwrightetal1997)

Gram‐negative bacteria have been shown to be less susceptible than Gram‐positive

bacteria to the photoactivity of the TBO‐impregnated polymers (Perni et al 2009b)

andtophotodynamictherapyusingotherphotosensitiserssuchasmethyleneblueand

rose bengal (Phoenix et al 2003 Decraene et al 2006 Perni et al 2009a) The

cytoplasmicmembrane is the primary target of the singlet oxygen generated during

irradiationwith the laser light (Wakayama et al 1980 Jori et al 2006) which has

been demonstrated in E coli and S cerevisiae (Ito 1977 Ito and Kobayashi 1977)

Gram‐negative bacteria have a reduced rate of uptake of singlet oxygen due to the

presenceoftheoutermembrane(Jorietal2006)whichpreventsdirect interaction

of the singlet oxygen with the underlying cytoplasmic membrane It also acts as a

permeabilitybarrierpreventingthediffusionofsmallmoleculesintothecytoplasmof

thecellConverselyGram‐positivebacteriaaresurroundedbyarelativelyporouslayer

of peptidoglycan and aremore likely to be susceptible to the action of the reactive

oxygen species generated on the surface of the polymers DNA damage occurs in

Gram‐positiveandGram‐negativebacteriaandinyeastcellsoncethepermeabilityof

the externalmembrane has been compromised and the reactive oxygen species are

abletopenetratetheinteriorofthecells(Dunipaceetal1992Chietal2010)The

susceptibility of Gram‐negative bacteria to the effect of the TBO‐impregnated

polymerssuggeststhatthemechanismofactivityistheTypeIIpathway(Figure111)

The photosensitiser was immobilised in the polymer and was not able to interact

229

directly with the bacterial cell wall and so the phototoxic effect occurred via the

generationofsingletoxygenwhichoxidisedmoleculesintheoutermembraneItwas

hypothesised that reactive oxygen species generated by the Type I pathway

wereunabletocauselethaldamagetotheoutermembraneandrequiredpenetration

ofthemembraneinordertoexertlethalPDI(Jorietal2006)

It was hypothesised that the reductions observed for the Gram‐negative organisms

usedintheseexperimentswouldbelessthanthatobservedforSaureus(Pernietal

2009b)Although these results support the hypothesis the data cannot be directly

comparedwiththepublishedworkasalargerstartinginoculumwasusedinthisseries

ofexperimentsandcellsaremoresusceptibletoPDIwhenalowerinoculumisused

(Soetal2010)TheinitialbacterialconcentrationusedinthePernistudyequatedto

approximately4x104cfupolymerandinpreliminaryexperimentsa354log10cfu

polymerreductioninPaeruginosaPAO1wasdetectedwhichwasbelowthedetection

limitof theexperiment(datanotshown)Thereforeahigher initialbacterial loadof

106 cfu polymerwas selected so that colonieswere always detectable on the test

(L+S+) plates and the values obtained were within the detectable limits of the

experimental design Alternatively the exposure time to the laser could have been

decreased to ensure the recovered bacteria werewithin the detection limits of the

assay For reference the Perni et al (2009a) study showed a gt4 log10 cfu ml

reduction in EMRSA16 after a 1 minute irradiation time and a gt4 log10 cfu ml

reductioninEcoliATCC25922aftera2minuteirradiation

These data also show that C albicans was less susceptible to TBO‐mediated

photodynamic inactivation than the Gram‐negative bacteria S maltophilia A

230

baumanniiandPaeruginosaPAO1IthaspreviouslybeenshownthatCalbicanswas

susceptible toPDIusinga solutionof TBOand irradiationwith red light (Wilsonand

Mia 1993) and an increased tolerance to these conditionswas displayed compared

with the Gram‐negative oral bacteria Fusobacterium nucleatum Actinobacillus

actinomycetemeomitans and Porphyromonas gingivalis (Wilson et al 1993 Wilson

andMia1994)Yeastcellsaremuchlargerinsizethanbacterialcellsthediameterof

aCalbicanscellisapproximately3to4microm(MerzandRoberts1999)comparedwith

Abaumanniiwhichisapproximately1to15by15to25microminsize(Schreckenberger

and von Graevenitz 1999) and S aureus which is approximately 05 to 15 microm in

diameter (Kloos and Bannerman 1999 Sandel and McKillip 2004) Therefore the

yeastcellislikelytorequirealargerdoseofreactiveoxygenspeciestoexertasimilar

photodynamiceffect (Jorietal2006)Thestructureoftheyeastcellwallcouldalso

contributetowardsincreasedtolerancetoPDT(BowmanandFree2006)


The clinical strain of P aeruginosa was shown to be the least susceptible to the

photoactivityoftheTBO‐impregnatedpolymersaftera4minuteirradiationtimeand

the reduction in bacteria observed was substantially less than that seen in for the

laboratory strain ofP aeruginosa PAO1P aeruginosaPAO1was originally isolated

fromawoundinMelbourneAustraliain1955(Holloway1955)Sincethenithasbeen

serially passaged for many years and shared with laboratories around the world

where further passages have taken place (Fux et al 2005) The PAO1 strain was

selectedbecauseitrsquosubiquitoususeallowsthedatageneratedintheseexperimentsto

becomparedwithresultsgeneratedbygroupsaroundtheworldonthesensitivityof

231

P aeruginosa to the TBO‐laser combination However itrsquos limitations should be

acknowledged and it is probable that the PAO1 strain in use today has lost

characteristicsfoundintheoriginalstrainasaresultofserialpassage(Fuxetal2005)

Theconditionsthatbacteriaareexposedtoduringlaboratoryculturearesubstantially

differentfromthoseexperiencedwithinthehostileenvironmentofthehumanbody

An abundance of nutrients are present in laboratory media to encourage bacterial

growth and incubation conditions are optimal for rapid replication Therefore the

genesthatarerequiredforcolonisationandsurvivalwithinthehumanhostaresurplus

to requirement For example in E coli genes required for flagella production are

inactivatedafterserialpassagersquos(Edwardsetal2002)whichbenefitsthelaboratory‐

adaptedstrainasflagellaproductionisanenergy‐richprocessthatrequireshighlevels

ofaminoacidproduction If thesegenesare inactivated the replication timewillbe

shorterwhichwillgivethelaboratory‐adaptedstrainafitnessadvantageoverthewild

typestrain

Theabilityofthe laboratoryadaptedcells toadhereandformbiofilmscouldalsobe

reduced(Fuxetal2005)MucoidstrainsofPaeruginosaarecommonlyisolatedfrom

patientswithcysticfibrosisandthisphenotypeisoftenlostduringlaboratoryculture

due to a series of point mutations and a non‐mucoid rough colony morphology

predominates(Govan1975DrenkardandAusubel2002)Mucoidstrainsproducea

greater quantity of alginate (Simpson et al 1989) a known scavenger of reactive

oxygen species such as singlet oxygen which is produced in abundance during the

photodynamicreactionontheTBO‐impregnatedpolymers(Wakayamaetal1980)A

possible reason for the decreased susceptibility of the clinical isolate to the

232

photoactivityofTBO‐impregnatedpolymerscouldthereforeberelatedtoanincreased

production of alginate which is a defencemechanism against the respiratory burst

released by macrophages within the human hostWong et al (2006) showed that

clinical isolates exposed to the visible‐light driven photocatalytic effect of N‐doped

TiO2 thin films displayed increased tolerance to killing compared with a laboratory

strainofEcoliOP50and itwassuggestedthatthemechanismbehindthiswasalso

linkedtoresistancetoreactiveoxygenspecies

Thebacterial isolatesused in this seriesofexperimentswerecultured inbrainheart

infusion (BHI) liquid media and subsequently re‐suspended in PBS which is a low

protein saline solution It has been shown that the PDI effect is reduced by the

presence of proteins in the medium and so it is possible that the inhibitory effect

observed in these experiments would be reduced under in vivo conditions as the

trachealsecretionscontainhighlevelsofproteins(WilsonandPratten1995Nitzanet

al 1998) These proteins could absorb light which would reduce the number of

photonsavailablewhichwouldinturndecreasetheconcentrationofreactiveoxygen

species generated (Komerik and Wilson 2002) The proteins may also be used as

alternativetargetsbythesingletoxygenspeciesandshieldbacteriafromthecytotoxic

effectsgenerated

643 Novelmaterialsforpotentialuseasantimicrobialendotrachealtubes

Numerous invitrostudieshavebeenconductedonmaterialswhichcouldbeusedas

novel antibacterial ETTs Methylene blue was incorporated into silicone and the

photodynamic effect with and without the addition of gold nanoparticles was

233

investigated (Perni et al 2009a) A significant level of photoactivity was observed

againstEcoliandMRSAafter5minutes irradiationwithared laser lightwhichwas

enhanced with the addition of gold nanoparticles Berra et al (2008a) coated

polyurethaneETTswithsilversulfadiazineandchallengedthetubeswithPaeruginosa

PAO1 The silver coated ETT was examined by both scanning electron microscopy

(SEM)andconfocal laser scanningmicroscopy (CLSM)and sectionsof the tubewere

culturedafteraperiodof72hoursadhesionofPaeruginosaPAO1tothesubstrate

hadbeenpreventedandthegrowthratewasalsoreducedThesilvercoatedETTwas

subsequentlyused inaventilated sheepmodelNobacteriawerecultured from the

coatedETTsafter24hoursandathinnerlayerofmucuswaspresentonthelumenof

the tube compared with the uncoated control where bacterial colonisation was

present(Berraetal2008a)

Rello et al (2010) coated a proprietary hydrophilic polymer with silver ions and

investigated the adherence of 18 organisms after an exposure time of 4 hours A

reducedlevelofbacterialattachmentwasobservedforrespiratorystrainsofMRSAP

aeruginosaandEaerogenesbuttheattachmentofanumberofotherorganismssuch

asCalbicansandKpneumoniaewasnotpreventedTheantibacterialactivityofthe

silverion‐coatedETTwasthenassessedinarabbitmodelwhichwaschallengedwitha

respiratoryisolateofPaeruginosaAfter16hoursareducedlevelofETTcolonisation

wasobservedonthesilverion‐coatedtubesandPaeruginosawasnotisolatedfrom

thelungsoftherabbitsIncomparisonPaeruginosawasculturedfromallnon‐coated

ETTsand from the lungsofall rabbits intubatedwith the control tubes (Relloetal

2010)

234

A large‐scale randomised trial published in 2008 aimed to ascertain whether silver

coatedETTscouldreducetheincidenceofVAPinhumans(Kollefetal2008)Nearly

10000patientswerescreenedfortheireligibilityintothestudyandsuitablepatients

wereassignedasilver‐coatedETToranon‐coatedtubeAreduction inthe incidence

of VAP was observed in patients with silver‐coated tubes These findings were

extremelypromisingastheyshowedthatbysimplyusingadifferentventilatortube

theincidenceofVAPcouldbereducedanditrequirednoadditionalinvolvementfrom

themedical team treating the patientHowever some authors have questioned the

meritofreducingbacterialloadontheETT(Balk2002Spronketal2006)asthereis

no direct evidence to demonstrate that antibacterial ETTs can reduce length of

hospital stay ormortality rates and the silver coated ETTs cost over $100 per tube

compared with less than $1 for a traditional uncoated tube (Deem and Treggiari

2010)

65 Conclusions

The antibacterial photodynamic inactivation of P aeruginosa S maltophilia and A

baumanniiwasassessedonTBO‐impregnatedpolymersafter irradiationwithaHeNe

laser light A significant reduction in the recovery of all bacterial strains testedwas

observed after 90 180 and 240 seconds A recently isolated clinical strain of P

aeruginosa showed decreased susceptibility to the photo‐activity of the TBO‐

impregnated polymers compared with a laboratory type strain Significant

photodynamicinactivationofCalbicanswasalsoobservedafterexposuretothesame

lightsourcedemonstratingthatthelight‐inducedeffectisnotrestrictedtobacteria

235

7 Assessment of the disruptive and anti‐adhesive propertiesofnovellight‐activatedmaterials

71 Introduction

Theanti‐adhesivepropertiesoftwoofthenovellight‐activatedantibacterialmaterials

generatedinthisthesiswasexploredinthischapterusingarangeoftechniquesThe

silver‐doped titanium dioxide thin films were examined to determine whether in

addition to the photo‐activated bactericidal effects already demonstrated initial

bacterialadhesiontothesurfacecouldbepreventedandwhethertheformationofan

immaturebacterialbiofilmcouldbedisruptedTheinitialattachmentofbacteriatothe

TBO‐impregnated polyurethane polymers was assessed after irradiation with the

HeNe laser which prompted the examination of the photo‐bleaching effect of the

laserontheantibacterialactivityoftheTBO‐impregnatedpolymers

Demonstratingareductionintherecoveryofviablebacteriainoculatedontothenovel

surfacesafterlightexposureisausefulinitialmethodofestablishingtheantibacterial

activityofthenovelmaterialsHoweveritwouldalsobeadvantageoustopreventthe

initialattachmentofbacteriatothesurfaceDuringthe initialadhesioneventsthere

willbea lowerbacterial loadsophotoinactivationmayoccuratafasterrateAlsoin

the clinical environment the risk of onward transmission of bacteria from a hand‐

touch surface via the hands of patients or healthcare workers would be further

reduced due to the smaller inoculum present An additional measure which would

provebeneficialintheclinicalenvironmentwouldbethedetachmentandinactivation

ofbacteriaalreadyboundtothesurfacebeforelightexposure

236


721 Silver‐dopedtitaniumdioxidethinfilms

7211 AssessmentofinitialattachmentofEMRSA‐16

BacterialattachmenttotheAg‐TiO2thinfilmswasmeasuredusingtwosinglechannel

transmissionFC81‐PCflowcells(BioSurfaceTechnologiesCorporationMontanaUSA)

Two flowcell chambers (50x13x235mm)were joined togetherwith tapebefore

autoclavingandrinsingwithwaterTheflaskwaspreparedbyconstituting500mLPBS

ina1000mLconicalflaskwithamagneticstirreraddedarubberstopperwasloosely

placedonandcoveredwith foil The two female connectorswerewrappedwith foil

andsealedwithautoclavetapeClampswereattachedtotheendsofbothtubesby

the male connectors and on either side of the air filters and the entire unit was

autoclavedfor15minutesat121degC

237

Figure 71 The flow cell chamber used to assess bacterial attachment TheAg‐TiO2thin film was placed within the chamber and adhesion was assessed by lightmicroscopyasabacterialsuspensionflowedacrossthematerial

Theflowcellchamberwasassembledandasealantwasappliedbetweeneachlayerto

preventthe leakageof liquidAcoverslipwasplacedontheclearplastic lidandthe

entry and exit points in the flow cell chamber were cleaned with an isopropanol‐

containingwipetoensuretherewasnoobstructioncausedbysealantTheuncoated

glassslidedenotedS‐wasplacedintheridgeontheclearplasticlidandscrewswere

addedtothetopandnottightenedTheAg‐TiO2thinfilmscouldnotbeautoclavedso

thesewere not added at this point The screwswere loosely positioned on top and

coveredwithtapeFoilwasaddedtothetopofthebubbletrapandtheendsofthe

twomale connectors Clamps were affixed to the ends of both tubes by the male

connectorsTheflowcell chamberwasthen laid flat inanautoclavebagandsealed

thenplacedintoasecondautoclavebagsealedandlabelledThebagwassterilisedby

autoclavingat121degCfor12minutes

238

Afterautoclavingtherubberstopperonthetopoftheconicalflaskwassecuredand

theclamps fromeither side of theair filterwere removedThe flaskofPBSand the

flow cell chamberswere allowed to cool before the Ag‐TiO2 slide denoted S+ was

placed into the flow cell chamber and all screws on the flow cell chamber were

tightened to prevent any leakages The clamps from the end of each tube were

removedand the flowcell chamberwas joined to the flaskbyplacing themaleand

female connectors together Finally a 045 nm filter (Nalgenereg Labware Roskilde

Denmark) was added to the top of the bubble trap A culture of EMRSA‐16 was

preparedinBHIasdescribedinSection22

After24hoursgrowth5mLoftheovernightculturewasdispenseddirectly intothe

flaskcontaining500mLPBSprovidingadilutionofapproximately1in100Theflow

cell chamber and bubble trap was placed into a large white tray and the narrow

section of tubing was passed through the peristaltic pump (Watson‐Marlow Pumps

GroupFalmouthUK)toachievealowflowrateThewholesystem(peristalticpump

flask and tubing)was transferred into the 22degC incubator containing thewhite light

sourcealongwithamagneticstirrerTheperistalticpumpwasthenswitchedonand

thespeedsetto30equatingtoashearrateof40s‐1Thevalveonthebubbletrap

waskeptopenuntiltheliquidhadreachedthehalfwaymarkatwhichpointthevalve

wasclosedandtheliquidcouldpassthroughthesystembacktotheconicalflask

After06and18hourstheflowcellsystemwasmovedtothelightmicroscopesothat

theattachmentofbacteriaonthesurfaceofthethinfilmscouldbevisualisedThex40

objectivelens(OlympusULWDCDPlan40)wasusedandatleasttenrandomfieldsof

viewwereexaminedpersampleandrepresentativeimageswerecaptured

239

7212 DisruptionofanimmaturebiofilmofEMRSA‐16


ofEMRSA‐16werepreparedinPBSasdetailedinSection23Alternativelyanaliquot

of the re‐suspendedpelletofbacteriawasadded toa10mL ofBHIand the optical

densitywasmeasuredonthespectrophotometerInbothcasestheresultingbacterial

suspensioncontainedapproximately107 cfu mL Silver‐doped titaniumdioxide thin

filmsoruncoatedcontrolswereplacedinthemoisturechambersdescribed inFigure

22before50microLofthebacterialsuspensionwasaddedandthemoisturechambers

wereincubatedinthedarkfor24hourstoallowanimmaturebiofilmtodevelop

Themoisture chamberswere subsequently transferred to the cooled incubator and

incubated at 22degC for 24 hours under thewhite light source The Live Dead stain

(Molecular Probes)was prepared by adding 20 microL of both SYTO9trade and propidium

iodidetoafoil‐covereduniversalcontaining40mLPBSandwasincubatedinthedark

for 30minutes before use The Live Dead stainwas poured into a petri dish the

sampleswere immersed inthepetridishand incubated inthedark for5minutesto

allow the stain to penetrate the bacterial cells before viewing Two slides were

examinedforeachexposureconditionasdetailedinTable71andatleasttenfieldsof

view were examined per sample and representative images were captured The

sampleswereexaminedontheconfocal laserscanningmicroscope(CLSM)usingthe

x40 lenswithabluefilterand lateranalysedusingthe ImageJcomputerprogramme

which can be accessed for free from httprsbwebnihgovij The experimentwas

repeatedtodemonstratereproducibility

240

Table 71 Description of the samples examined under the confocal scanning lasermicroscope

Samplereference Sampletype Exposureconditions Inoculum

K2K3 Ag‐TiO2 light EMRSAinPBS

K4K5 Ag‐TiO2 dark EMRSAinPBS

K6K7 Ag‐TiO2 light EMRSAinBHI

K8K9 Ag‐TiO2 dark EMRSAinBHI

K10K13 Ag‐TiO2 light Nobacteria

K14K17 Ag‐TiO2 dark Nobacteria

B1B2 Uncoatedslide light Nobacteria

B3B4 Uncoatedslide dark Nobacteria

722 TBO‐impregnatedpolymers

7221 PreventionofinitialPaeruginosaPAO1attachment


ofPaeruginosaPAO1weregrownandpreparedinPBSasdetailedinSection22and

Section23resultinginabacterialsuspensioncontainingapproximately107cfumL

Thedescribedmethodwasadapted fromapaperbyChrzanowskietal (2010)The

testsampleswerepreparedandplaced ina24wellmicrotitreplateas illustrated in

Figure 72 Empty wells were filled with foil to prevent laser light penetrating into

adjacent wells One millilitre of bacterial suspension was added to the test well

ensuring the polymer did not float to the surface and the remaining wells were

covered with a sheet of black paper The well was irradiated with the HeNe laser

source described in Section 243 for the designated exposure time and theemitted

light was passed through a beam diffuser to ensure that the entire polymer was

241

exposed to the laser light The process was repeated for each appropriate sample

beforestaticincubationat37degCforthedesignatedtimeperiodbeforere‐exposureto

the laser source After three hours each sample was placed into a separate bijou

containing3mLPBSandincubatedat22degCfor5minorpreparedforscanningelectron

microscopyThepolymerwassubsequentlytransferredtoabijoucontaining1mLPBS

and 5 glass beads each with a diameter of 3 mm and vortexed for 1 min Twenty

microlitresofthebacterialsuspensionwasthenremovedseriallydilutedandspread

ontoMacConkey agar plates before incubation at 37degC for 48 hours The resultant

colonieswerecountedandcomparedwiththecontrolstocalculatethelevelofbiofilm

disruption

Figure 72 The layout of themicrotitre plate during the biofilm disruption assayswhere++correspondstoaTBO‐impregnatedpolyurethanepolymerexposedtothelaserlight‐+correspondstoaTBO‐impregnatedpolyurethanepolymernotexposedto the laser light +‐ corresponds to a polyurethane polymer exposed to the laserlightand ‐‐ corresponds toapolyurethanepolymernotexposed to the laser lightShadedcirclesrepresentwellsfilledwithfoil

7222 Scanningelectronmicroscopy

Afterthreehoursincubationat37degCthesampleswerepreparedforSEManalysisby

DrNickyMordanThesamplesunderwentaseriesof10minutesdehydrationstages

242

in increasing concentrations of alcohol (20 50 70 90 and 3x 100) before

immersioninhexamethyldisilazane(HMDS)(TAABLaboratoriesLtdReadingUK)for5

min followedbydryingon filterpaper for2 ‐3 hours toensure that theHMDShad

completely evaporated The samples were then fixed onto alumininum SEM stubs

(Agar Scientific) using carbon conducting cement (Neubauer Chemikalien Munster

Germany) as an adhesive before sputter‐coating with goldpalladium in a Polaron

E5000 Sputter Coater (Quorum Technologies Ltd Newhaven UK) A Cambridge

Stereoscan90B (LEO ElectronMicroscopyLtdCambridgeUK)wasused toview the

specimensoperatingat15kVandatleasttenfieldsofviewwereexaminedThei‐scan

2000software(ISSGroupManchesterUK)wasusedtocapturerepresentativedigital

imagesforeachsample

7223 Photo‐bleachingeffects

TheTBO‐impregnatedpolymerswereirradiatedwiththeHeNelasersourcedescribed

inSection243foreither90180or240secondsbeforeincubation inasterilepetri

dishfor24hoursat22degCThepolymerswerethenprocessedasdescribed inSection

2123polymerswhichhad been initially irradiated for 90 secondswereexposed to

another90 second laserdosepolymers irradiated for 180 secondswere re‐exposed

for180secondsandpolymersirradiatedfor240secondsweretreatedwithafurther

240 second light doseNaiumlve TBO‐impregnated polymerswere used as controls ie

TBO‐impregnated polymers that had been stored in the dark during the initial

irradiationstepThreeTBO‐impregnatedpolymersweretestedforeachexposuretime

andtheexperimentwasrepeatedthreetimestodemonstratereproducibility

243

73 Results


7311 Assessmentofbacterialattachment

The attachment of EMRSA‐16 to the surface of the Ag‐TiO2 thin filmswas assessed

using the flowcellmodelBacteriawere observed in thecirculatingbrothafter zero

hours in low numbers in Figure 73(a) and Figure 73(b) the cocciwere in constant

motionmoving in the direction of the flow suggesting that attachment had not yet

occurredAsimilarnumberofbacteriawerefoundontheAg‐TiO2thin filmsandthe

uncoated control slides After 6 hours the number of bacteria observed on both

coating typeshad increased substantiallyanda near complete coverageof the slide

was observed (Figure 74a and Figure 74b) Again there was no difference in the

attachment of bacteria to the irradiatedAg‐TiO2 thin film and the uncoated control

exposedtothesame lightconditionsAfter18hoursexposuretothewhite lightno

reductioninthenumberofbacteriawasobservedontheAg‐TiO2thinfilmsexposedto

thewhitelightandtherewasnovisualdifferenceinthenumberofbacteriaobserved

ontheAg‐TiO2thinfilmcomparedwiththeuncoatedcontrol (Figure75aandFigure

75b)

TheshrinkcrackswhichcanbeclearlyseenontheAg‐TiO2thinfilmsareafeatureof

the coating and are a result of the annealing process There was no greater than

bacterial attachment observed in these areas than on the non‐cracked areas of the

thinfilm

244

Figure73AttachmentofEMRSA‐16toeitheran(a)uncoatedslideor(b)Ag‐TiO2thinfilmafter0hexposuretothewhitelightsource



245


Astherewasnodifference intheattachmentofEMRSA‐16totheAg‐TiO2thinfilms

theviabilityofEMRSA‐16wasexaminedafterirradiationwithwhitelightItispossible

thatthephoto‐activatedthinfilmswerenotpreventingbacterialattachmentbutwere

inactivatingthebacteriathatdidadhereAnimmaturebiofilmofEMRSA‐16inPBSwas

grownonthesurfaceoftheAg‐TiO2thinfilmsandexposedtowhitelightfor24hours

a reduction in the viability of the attached bacterial cellswas observed Therewere

substantiallymore non‐viable cells on the Ag‐TiO2 thin films exposed towhite light

(Figure76)comparedthatobservedonthesurfaceoftheAg‐TiO2thinfilmsincubated

inthedark(Figure77)Thisdemonstratesthatwhite light irradiationoftheAg‐TiO2

thin films caused an increase in the permeability of the cell membrane to the

propidiumiodidestainandaccompanyingdamagetotheintegrityofthebacterialcell

membrane No antibacterial activity was observed in the absence of light which

suggests that the damage to the bacterial cell membranes was not caused by the

leakageofsilverionsfromthesurfaceofthethinfilm

246

Figure76ConfocalmicrographofEMRSA‐16inPBSontheAg‐TiO2thinfilmafter24hoursgrowthat37degC in thedarkand24hoursexposure towhite lightat22degC (xyprojection 300 x 300 μm) Viable bacterial cells are stained green and non‐viablecellsarestainedredThedepthofthebacterialgrowthisdisplayedunderneaththemainimage(xzprojection)

247

Figure77ConfocalmicrographofEMRSA‐16inPBSontheAg‐TiO2thinfilmafter24hours growth at 37degC in the dark and 24 hours incubation at 22degC in the dark (xyprojection 300 x 300 μm) Viable bacterial cells are stained green and non‐viablecellsarestainedredThedepthofthebacterialgrowthisdisplayedunderneaththemainimage(xzprojection)

248

Figure78andFigure79showtheattachmentofEMRSAonthesurfaceoftheAg‐TiO2

thin films after re‐suspension in the nutrient‐rich medium BHI with and without

exposure to the white light source respectively The photocatalytic antibacterial

activityof theAg‐TiO2 thin filmswasnotevidentonlya smallnumberofnon‐viable

cellswereobservedafter24hoursexposuretowhitelightandthesewerelocated in

smalldefinedareaswhereaswhenEMRSA‐16wasre‐suspendedinPBSandgrownon

thethin films thenon‐viablecellsweredispersedmoreevenlyacrossthesurfaceof

the sampleThecellsattached to these surfaceshad begun to coalesce thedistinct

single cells that were in abundance in the nutrient‐poor conditions were seen less

frequentlyandtheinitialstagesofabiofilmwerebeginningtodevelop

The continued viability of EMRSA‐16 observed in the presence of white light also

suggests that the damage to the cellmembrane seen in Figure 76was not a direct

effectofthewhitelightbutproducedduetothephotocatalyticactivityoftheAg‐TiO2

thinfilm

The thickness of the immature biofilms on the surface of theAg‐TiO2 thin films are

displayedat thebottomofeachconfocalmicrographThe immature biofilm formed

fromEMRSA‐16re‐suspendedinPBSandexposedtothewhitelight(Figure76)isless

thick than the biofilms formed when EMRSA‐16 was re‐suspended in PBS and

incubated for24hoursat22degC in thedarkorwhenEMRSA‐16was re‐suspended in

BHIandincubatedfor24hoursat22degCinthepresenceorabsenceoflight(Figure77

Figure78andFigure79)

249

Figure78ConfocalmicrographofEMRSA‐16inBHIontheAg‐TiO2thinfilmafter24hoursgrowthat37degC in thedarkand24hoursexposure towhite lightat22degC (xyprojection 300 x 300 μm) Viable bacterial cells are stained green and non‐viablecellsarestainedredThedepthofthebacterialgrowthisdisplayedunderneaththemainimage(xzprojection)

250

Figure79ConfocalmicrographofEMRSA‐16inBHIontheAg‐TiO2thinfilmafter24hours growth at 37degC in the dark and 24 hours incubation at 22degC in the dark (xyprojection 300 x 300 μm) Viable bacterial cells are stained green and non‐viablecellsarestainedredThedepthofthebacterialgrowthisdisplayedunderneaththemainimage(xzprojection)

251



The TBO‐impregnated polyurethane polymers were assessed for their ability to

preventthe initialattachmentofPaeruginosaPAO1after irradiationwiththeHeNe

laserTheTBO‐impregnatedpolymerswereinitiallyirradiatedwiththeHeNelaserfor

90secondsandthenincubatedinasuspensionofPaeruginosafor3hourstherewas

nosignificantdifference inbacterialattachmentcomparedwiththecontrolpolymers

incubatedinthedarkTheirradiationperiodwasdoubledto180secondsandtheanti‐

attachmentpropertiesofthepolymerwerenotimprovedThereforethefrequencyof

theirradiationdosingwasincreasedandthetimeofdosingaltered(Table71)

Table72Resultsofthebacterialattachmentassayswhererow1denotesthatthesampleswere irradiatedwith theHeNe laseronce for 90 secondsat timepoint 0minuteswhichresultedina013logcfumlreductioninviablebacteria

Irradiationperiodsec

Irradiationfrequency

Irradiationdosingtimesmin

Logreductioncfuml‐1

90 1 0 013

180 1 0 000

180 2 090 058

180 3 060120 053

180 3 090180 156

A significant decrease in bacterial attachment was demonstrated when the TBO‐

impregnatedpolymerswere irradiatedthreetimesfor180secondsattimepoints0

60 and 180 minutes (Figure 710) A 973 reduction in bacterial attachment was

252

observed which corresponded to a 156 log reduction (p lt 0001) This therefore

demonstrates that increasing the dosing frequency improved the anti‐adhesive

propertiesoftheTBO‐impregnatedpolymerandfrequentdosesofthelaserlightwere

required to prevent the attachment of P aeruginosa to the TBO‐impregnated

polymers The laser light alone did not have a significant effect on P aeruginosa

attachment but a significant decrease in attachment was observed on the TBO‐

impregnatedpolymer in theabsenceof the laser light (p lt001) suggesting thatthe

presenceofthephotosensitiseralonedidhaveaneffectonbacterialattachment

Figure 710 Ability of the TBO‐impregnated polymers to prevent the initialattachment of P aeruginosa PAO1 TBO‐impregnated (S+) or non‐impregnatedcontrol(S‐)polymerswereeitherirradiatedwithlaserlight(L+)orincubatedinthedark(L‐)


The attachment of P aeruginosa to the TBO‐impregnated polymers was further

investigated by visualisation of bacterial attachment by SEM after the biofilm

disruption assay The most effective irradiation schedule was used (180 seconds

irradiation after 0 90 and 180 minutes) and the decrease in bacterial recovery

253

observed in Section 7312 was confirmed There were substantially less bacteria

adheredtothesurfaceoftheirradiatedTBO‐impregnatedpolymers(Figure711)than

theTBO‐impregnatedpolymersthatwerenotexposedtothelaserlight(Figure712)

Figure711SEMimageofPaeruginosaPAO1onthesurfaceofaTBO‐impregnatedpolymerafter irradiationwith themosteffective irradiation schedule (180 secondsirradiationafter090and180minutes)Thetotalincubationtimewas3hours

254

Figure712SEMimageofPaeruginosaPAO1onthesurfaceofaTBO‐impregnatedpolymerafter3hoursincubationintheabsenceoflaserlight


Duringthebacterialattachmentassay theTBO‐impregnatedpolymerswereexposed

tomultipledosesof laser lightwhichcausedthe intensityofthebluecolourationto

decreaseThedecreaseincolourationwasaccompaniedbyaconcomitantreductionin

antibacterial activity (Figure 713) It was shown in Chapter 6 that the antibacterial

activityoftheTBO‐impregnatedpolymerswasproportionaltotheirradiationtimeand

this was replicated in this experiment as the greatest reduction in P aeruginosa

255

recoveryfromtheTBO‐impregnatedpolymerswasobservedafter240secondsa185

log10 cfu polymer decrease was observed compared with the TBO‐impregnated

polymers incubated in the dark However this reductionwas significantly less than

thatobservedonthenaiumlveTBO‐impregnatedpolymersthatwerenotpre‐irradiated(p

lt0001)Thisreductioninantibacterialactivitywasobservedforalltimepointstested

andthedifferencesinrecoverywereallstatisticallysignificant(plt0001)

The reduction in P aeruginosa recovery observed on the naiumlve TBO‐impregnated

polymersafter180secondsirradiationinFigure713wasmuchgreaterthanthatseen

whenthisexperimentwasfirstconductedinChapter6a294log10cfupolymerwas

originally observed and a 356 log10 cfu polymerwas observed in this experiment

Moreover the reduction in P aeruginosa recovered from the TBO‐impregnated

polymers was greater after 180 seconds irradiation than 240 seconds This

demonstrates the intrinsic variation in activity of the TBO‐impregnated polymers

whichisalsoillustratedgraphicallybythelargeerrorbarsonthebarchart

256

Figure713Effectofphoto‐bleachingontheanti‐PaeruginosaactivityoftheTBO‐impregnatedpolymers

74 Discussion


It was previously shown that the Ag‐TiO2 thin films and TBO‐impregnated polymers

caused a significant decrease in the recovery of various bacterial species after

exposuretolightofanappropriatewavelengthTheviablecolonycountmethodwas

usedtoobservethephotocatalyticactivityofthematerialswhichwasestablishedby

showingchanges inbacterial recoveryafterexposure to the relevant light source In

this chapter visualisation techniques were employed to observe the antibacterial

effectofthelight‐activatedmaterialsInitiallythephoto‐inducedabilityoftheAg‐TiO2

thin films toprevent the initial attachmenteventwas investigated Ithadpreviously

been shown bacterial cells aremore susceptible to the photo‐induced effectswhen

theinoculumislower(Saitoetal1992Soetal2010)Thereforethehypothesiswas

257

if the thin filmswereable to reduceadhesion ofbacteria to the surface then there

maybefewerbacteriapresentonthesurfacetobetargetedbythe reactiveoxygen

speciesgeneratedTheflowcellwasusedtomonitorattachmentofEMRSA‐16tothe

Ag‐TiO2 thin films and no difference in bacterial attachmentwas observed between

the Ag‐TiO2 thin films and the uncoated controls after exposure to the white light

sourceThisresultwassurprisingasa34log10cfucm2decreaseinbacterialrecovery

wasdetectedbyaerobiccolonycountafter18hoursirradiationandthewatercontact

angle significantly decreased afterwhite light irradiation so a reduction in bacterial

attachmentwasexpected

Page et al (2009 2011) demonstrated increased attachment of S aureus on

irradiated titania‐containing thin films that had demonstrated photo‐induced

antibacterial activity however the bacterial cellsweremore dispersedwhich could

prove beneficial for photoinactivation of bacteria Liquid inoculated onto

superhydrophilicmaterialsliketheAg‐TiO2thinfilmsspreadoutasathinlayerwhich

means thatmoreof thebacterial suspension isexposed to the thin film resulting in

faster bacterial photo‐inactiavtion The group also examined the roughness of the

titania‐containingthinfilmsandalterationsinthesurfaceroughnessatthenanoscale

did not affect adhesion Increased surface roughness is commonly attributed to

increasedmicrobialadhesionbutthisisonamicroscalenotnanoscale(Verranetal

1991MorganandWilson2001Grayetal2003)LiandLogan(2005)demonstrated

decreasedattachmentofBsubtilisPaeruginosaEcoliandBurkholderiacepaciaon

titaniathinfilmsafter irradiationwithUV lightcomparedwithuncoatedglasswhich

wasascribedtophotoinducedsuperhydrophilicityonthe irradiatedtitania filmsThe

258

incident lightsourceused inthischaptersimilarlyused lightwithabandgapenergy

large enough to generate photocatalysis but a decrease in adhesion was not seen

Morerecentworkbythesamegroupusedspectralforceanalysistofurtherinvestigate

the adhesive properties of non‐irradiated TiO2 thin films and hypothesised that

increasedadhesionwasnotduetooverallsurfacepropertiessuchashydrophilicityor

surface charge but a small number of lsquosticky sitesrsquo present on the highly

heterogeneous surface (Maetal 2008)Applicationof thismethodology to theAg‐

TiO2 thin filmswould determinewhether the lsquosticky sitesrsquowere also presentwhich

couldcontributetowardsthepersistentadhesionofEMRSA‐16


Itwaspostulatedthatthebacterialcellshadremainedattachedtothesurfaceofthe

Ag‐TiO2 thin films but had been photo‐inactivated by the properties of thematerial

andwere non‐viable TheLive DeadBacLighttradeBacterial Viability kitwas therefore

used to stainbacterial cells inan immature 24 hourbiofilmofEMRSA‐16andCLSM

wasused tovisualise thecells EMRSA‐16was initially inoculated inPBSa nutrient‐

poor buffered solution and incubated at 37degC for 24 hours to allow attachment to

occurbefore24hoursirradiationwithwhitelightThereweresubstantiallymorered

cellspresentontheirradiatedthinfilmsthanthenon‐irradiatedfilmswhichindicated

an increase in the permeability of EMRSA‐16 cells to the propidium iodide stain

significant damage to bacterial cell membranes and a decrease in viability This

reductionintheviabilityofEMRSA‐16tothepropidiumiodidestainwasnotobserved

for EMRSA‐16 inoculated onto the surface of Ag‐TiO2 thin films incubated in the

absenceoflighttheirradiateduncoatedsamplesortheuncoatedsamplesincubated

259

inthedarkThissuggeststhatthedamageobservedwasdependentuponexposureto

boththeAg‐TiO2thinfilmsandwhitelight

Thepresenceofnon‐viablebacteriaon the surfaceof the thin film increases further

attachmentofbacterialcellsastheforcesattractingbacteriatoasurfacearegreater

when bacteria are already present on the surface compared with a bare surface

(Emerson and Camesano 2004) This would be a distinct disadvantage in a clinical

setting However after continued white light irradiation photoinduced oxidative

decomposition of the remaining bacterial cells should render the surface sterile

(Jacobyetal 1998)Lossofcellmembranepermeability isawell‐describedstage in

thephoto‐degradationof bacteria on the surfaceof titaniumdioxide basedcoatings

after exposure to appropriate wavelengths of light and this phenomenon is also

observed after bacterial exposure to silver ions or nanoparticles (Saito et al 1992

Dibrovetal2002Luetal2003Kimetal2007Jungetal2008)

InterestinglythiseffectwasnotreplicatedwhentheimmatureEMRSA‐16wasgrown

in BHI a nutrient‐rich growthmedium lownumbers of single non‐viable cellswere

present after light exposure but the majority of attached cells fluoresced green

indicatingviabilityThecellspresentonthethinfilmhadalsobeguntoaggregateand

form microcolonies which is one of the initial stages of biofilm formation (Tolker‐

Nielsen et al 2000) Overall a greater number of cells were present after the

incubation period and faster bacterial growth was encouraged because of the

increasedlevelofnutrientsinthebacterialsuspensioncomparedwiththePBS‐based

experiment The additional proteins present in the growth medium could have

scavenged the reactive oxygen species generated shielding EMRSA‐16 from the

260

photocatalyticeffectsofthethinfilms(Blakeetal1999KomerikandWilson2002)

Furno et al (2004) observed a similar effect on the viability of S epidermidis

inoculated onto silver‐impregnated polymers after the addition of host‐derived

proteinsConverselyFuertesetal(2011)showeddecreasedantibacterialactivityofa

suspensionofsilica‐coatedsilvernanoparticlesagainstEcoli inPBScomparedwitha

standardgrowthmediaLuria‐Bertani(LB)brothTheauthorscitedthatthedecreased

activitywasduetoalargerzetapotentialofEcoliandthesilvernanoparticlesinthe

PBS solution compared with the LB broth This meant that the silver nanoparticles

immersed inPBSwere less likelyto interactwithEcolicomparedwiththeLBbroth

andtheantibacterialactivitywasdependentupontheproximitytothenanoparticles

Itisunlikelythatthezetapotentialhadalargeeffectonthephotocatalyticactivityof

the Ag‐TiO2 thin films described in this chapter as the silver nanoparticles were

immobilisedonthethinfilmratherthanfreeinsolutionasananoparticulatepowder

asdescribedintheFuertespaper


The ability of the TBO‐impregnated polymers to prevent initial attachment of P

aeruginosa PAO1 after irradiation with laser light was subsequently investigated

Repeatedexposuretothelaserlightwasneededtogenerateasignificantreductionin

bacterialattachmentandthemosteffectiveregimentestedwas3dosesoflaserlight

for 180 seconds in 90minute intervals A significant reduction in the viability ofP

aeruginosa PAO1 was also observed on the irradiated TBO‐impregnated polymers

comparedwiththeTBO‐impregnatedpolymers incubated intheabsenceof lightand

the non‐impregnated polymers regardless of the light exposure conditions The

261

endpointof the biofilmdisruptionassaywasenumeration ofbacterial coloniesafter

inoculation onto agar plates which only detects viable cells Therefore in order to

visualise all bacteria remaining on the surface of the polymers after irradiation the

sampleswereexaminedbySEMAreductionintheattachmentofPaeruginosaPAO1

to the surface of the irradiated TBO‐impregnated polymers was observed when

comparedwiththeTBO‐impregnatedpolymersincubatedintheabsenceoflaserlight

Theseresultscombinedsuggestthatthephoto‐activityofthepolymersinactivatedP

aeruginosa PAO1 which resulted in a decrease in the number of viable organisms

cultured and less bacteria remained adhered to the surface of the irradiated TBO‐

impregnatedpolymersasdemonstratedbySEM

TBO‐mediatedPDIhadbeendemonstratedtodisruptthearchitectureofSaureusand

Sepidermidis16‐hourbiofilmsreducingthecellnumbersandcausingdamagetothe

bacterial cell membranes (Sharma et al 2008) This was observed after treatment

withanaqueoussolutionofTBOwhichshouldinactivatebacteriaatafasterratethan

TBOimpregnated intoapolymerduetothe increasedsurfaceareatovolumeratio

OtherresearchgroupshavedescribedPDIofbacterialbiofilmsaftertreatmentwitha

solutionofTBOand irradiation(DobsonandWilson1992Sealetal 2002Zaninet

al 2006 Donnelly et al 2007 Nastri et al 2010) however to the authors

knowledge biofilm disruption has not been demonstrated on an irradiated TBO‐

impregnated polymer which makes this finding unique to this thesis However a

photo‐bleachingeffectwasnotedontheTBO‐impregnatedpolymersafterexposureto

thelaserlightwhichresultedinareductioninthephoto‐activityofthematerialThis

262

indicatesthatthelifespanofthephoto‐activityofthepolymercouldbelimitedwhich

wouldrestricttheclinicalapplicationofthematerial


The anti‐adhesive photo‐activity of each of the novel light‐activated materials was

assessedagainstonly justbacterial strainand theadhesivepropertiesof one isolate

cannot always be used to predict the adhesive properties of another isolate of the

samegenusorevenspeciesForexample thebapLgenewasfoundtoplayarole in

theattachmentofListeriamonocytogenes 10403s to inanimate surfaceshowever it

was absent from a number of Lmonocytogenes isolates from food sources so the

attachmentmechanismsfoundinLmonocytogenes10403scouldnotbeextrapolated

tootherstrains(Jordanetal2008)

The size and shape of bacterial cells can also affect the strength of the binding to

surfacessoattachmentoftheGram‐positivecoccusEMRSA‐16islikelytodifferfrom

thatoftheGram‐negativebacillusPaeruginosaMicroscopiccrackswereobservedon

the surfaceof theAg‐TiO2 thin filmsby lightmicroscopyandbacterial cells thatare

abletofitwithinthesecrackscouldescapephysicalremovalbycleaning(Verranetal

2010b)Howeverthisproblemwouldpotentiallybeovercomebythephotoactivityof

the thin films as silver nanoparticles were observed in these ridges and a photo‐

activated antibacterial effect would be exerted on these cells after irradiation with

whitelight

TheflowcellmodelwasusedtoinvestigatetheadhesionofEMRSA‐16totheAg‐TiO2

thin filmshowever itwouldbeunlikely that the thin filmswouldbeexposed to the

263

shear forces experienced in the flow cell during the proposed use in a hospital

environmentTheflowcellwasusedasitenabledaconstantbacterialinoculumtobe

passedoverthethinfilmandprovidedtheopportunityforattachment

Thebacterialgrowthatmospherecanalsoaffectsusceptibilitytothephoto‐activityof

theTBO‐impregnatedpolymersBacteriacolonisingtheoropharynxwillbeexposedto

higher concentrations of carbon dioxide than that found in atmospheric conditions

Wilcoxetal(1991)foundincreasedadherencetopolyurethaneandsiliconecatheters

by some strains of coagulase‐negative staphylococci after growth in 5 carbon

dioxidesuggestingthatcarbondioxidecouldbeusedbythecellsasatriggertoup‐

regulate genes involved in adhesion The isolates used in these experiments were

grown in atmospheric conditions so these candidate adhesion genes would not be

expressed

75 Conclusions

The anti‐adhesive properties of the novel antibacterial Ag‐TiO2 thin films and TBO‐

impregnated polymers were investigated A reduction in the viability of EMRSA‐16

adheredontothesurfaceoftheirradiatedAg‐TiO2thinfilmswasdemonstratedusinga

differential viability stain and fluorescencemicroscopy The reductionwas observed

when EMRSA‐16 was prepared in a buffered saline suspension but it was not

replicated when the bacterial inoculum was prepared in a nutrient‐rich medium

AdditionallytherewasnodifferenceinbacterialattachmentontheirradiatedAg‐TiO2

thin films compared to those incubated in the dark implying that the photo‐

inactivated cells remained adhered to the surface A significant reduction in the

264

adhesionofPaeruginosaontheTBO‐impregnatedpolymerswasobservedaftera3‐

stepirradiationscheduleThiseffectwasdeterminedusingabiofilmdisruptionassay

and confirmed by SEM The irradiation source caused photo‐bleaching of the TBO‐

impregnated polymers with a concomitant decrease in antibacterial activity which

wouldlimitthelifespanofthematerial

265

8 Concludingremarksandfuturework

Healthcare associated infections (HCAIs) remain a significant problem in healthcare

institutions and the near‐patient environment is known to harbour bacteria These

microorganismscanbe transferred from theenvironment toapatientand themost

common vehicle of transmission is via unwashed hands If themicrobial load of the

near‐patient could be decreased then the risk of bacterial transmission will be

reducedwhichmayinturnreducetheacquisitionandonwardstransmissionofHCAIs

Self‐cleaning coatings could be applied to hand‐touch surfaces in the vicinity of the

patientalongsideotherinfectioncontrolmeasurestoachievethisaim

A range of sampling methods was initially trialled to develop an optimal sampling

regimen for assessing the antibacterial activity of novel light‐activated coatings

Reports of the use of ATP bioluminescence to assess the efficiency of cleaning

regimens within the healthcare environment are increasing so this technology was

applied to provide an accurate estimate of concentration of bacteria on the test

surfacesHowevertheviablecounttechniquewasshowntobesuperiorandthiswas

especially apparent at lowbacterial concentrationswhen theATP bioluminescence‐

basedtechniqueswereunabletoconsistentlyconfirmthepresenceofsmallnumbers

ofbacteria

Aseriesof light‐activatedantibacterialmaterialsweregenerated Initially twonovel

nitrogen‐dopedtitaniumdioxide(TiO2)basedthinfilmsweresynthesisedbychemical

vapour deposition (CVD) titanium oxynitride and nitrogen‐doped titania These thin

filmsexhibitedmarkedantibacterialactivityagainstEcoliafter irradiationwithboth

266

ultravioletlight(UV)andwhitelightActivationofthethinfilmswithincidentlightof

anincreasedwavelengthdemonstratedashiftinthebandonsetofthematerialfrom

the UV to the visible portion of the electromagnetic spectrum The photocatalytic

propertiesoftheN‐dopedthinfilmsweregreaterthanthatobservedonthetitanium

oxynitride thin films White‐light activated sulfur‐doped thin films were also

synthesisedandasignificantphotocatalyticactivitywasobservedagainstEcoliThe

greatestantibacterialactivitywasgeneratedontheN‐dopedthinfilmsafter24hours

irradiationwithwhite lightanda25 log10 cfu sampledecreasewasobservedThe

durabilityofthethinfilmswasassessedbyexposuretosuccessivecyclesofuseand

decontamination and the integrity of the coating remained intact A longer‐term

evaluation of the effect on wear and successive cleaning cycles in addition to an

assessment of the toxicity against eukaryotic cells would be warranted as these

coatingswouldneedtobeextremelyrobustandnon‐toxicifappliedontohand‐touch

surfacesinahealthcareenvironment

HoweveritwasdifficulttosynthesisereproduciblethinfilmsusingtheCVDmethodof

depositionandsoanalternativemethodwasusedtogenerateasecondseriesofthin

films Silver‐coated TiO2 thin films were synthesised by the sol gel method of

deposition and the addition of the silver nanoparticles induced a shift in the band

onset of the thin films to enable white light activation The thin films displayed

photochromicbehaviourandachangeintheoxidationstatewasinducedbydifferent

storage conditions After storage in the dark silver was oxidised to silver oxide

resultinginapurplecolouredfilmextendedexposuretoindoorlightingconditionsor

indeed UV light induced photoreduction of the silver oxide back to silver which

267

resultedinanorangecolouredfilmAUVfilterwasappliedtothewhitelightsourceto

filterouttheminimalUVcomponentemittedduringilluminationandtruevisiblelight

photocatalysiswasdemonstratedbyphoto‐oxidationofstearicacidareductioninthe

water contactangleand significantantibacterial activityagainst twomicroorganisms

implicated in HCAIs E coli and EMRSA E coli was shown to display increased

susceptibility to the antibacterial activity of the silver‐coated TiO2 thin films via a

light‐independentmechanism In contrast the photo‐induced destruction of EMRSA

wasduetoreactiveoxygenproducedbyTiO2drivenbywhitelightphotocatalysis in

turn driven by silver This is the first example of unambiguous visible light

photocatalysis and photo‐induced superhydrophilicity alongside a titanium dioxide

controlthatshowednoactivation

Assessment of the silver‐coated TiO2 thin films against non‐vegetative cells such as

bacterialsporesandviruseswoulddeterminewhethertheactivityobservedwasbroad

spectrumwhichwouldfurtherincreasethepotentialuseofthethinfilmsIntroducing

organic soil into the bacterial inoculum would establish whether the presence of

non‐bacterial contaminants affected the activity of the thin films and altering the

length and duration of the irradiation times would mimic the hospital lighting

schedules and assess the effect of day‐time activation and night‐time deactivation

These further translational tests replicate conditions similar to that foundwithin the

hospital environment which would provide more detailed information on the

potentialactivityofthethinfilmsinahealthcaresetting

The anti‐adhesive properties of the silver‐coated TiO2 were also explored and the

viability of an immature biofilm EMRSAon the surface of the thin filmwas reduced

268

afterwhite light irradiationThisreductionwasobservedwhenEMRSAwasprepared

inabufferedsalinesolutionbutwasnotrepeatedwhenthebiofilmwasgrown ina

nutrientrichmediumWhitelightexposuredidnotreducebacterialattachmenttothe

thin films which suggested that the photo‐inactivated bacterial cells remained

attachedtothesurfaceThisfeaturewouldbedetrimentaltothefunctionalityofthe

thinfilminaclinicalsettingasfurtherattachmentofviablebacteriatothenon‐viable

attached cells would be greater than attachment to a naive surface which would

impactonreducingthebacterialloadinthenearpatientenvironment

Further investigationintothemechanismcausingincreasedbacterialadhesionwould

be useful to increase understanding in this area Spectral force analysis has

demonstrated that the possession of numerous lsquosticky sitesrsquo can contribute towards

the adhesion of bacteria to titania thin films rather than surface charge or

hydrophilicitywhichhavepreviouslybeenthoughttobethemainfactorsinvolvedin

attachment Identificationofthe reactiveoxygenspeciesgeneratedbythethin films

would fully elucidate themechanismof the observed antibacterial activity Thiswas

attemptedunsuccessfullywithvariousspecificfluorescentprobesandfurtheranalysis

intothisareawouldbeofgreatinterest

Finally a light‐activated polyurethane polymer was synthesised by the swell

encapsulation method for potential use in endotracheal tubes (ETTs) The

photosensitiser toluidine blue (TBO) was impregnated into the polymer and the

antibacterialactivityofthematerialwasassessedusingapanelofpathogensknownto

cause ventilator‐associated pneumonia A type II photosensitisation reaction

generated the significant dose‐dependent antibacterial activity observed against all

269

tested bacterial strains A clinical isolate of P aeruginosa displayed decreased

susceptibilitytothephoto‐activityoftheTBO‐impregnatedpolymerscomparedwitha

laboratory strain which suggests that the laboratory‐adapted strain may have lost

virulence factorsnecessary forwithstandingattack fromsingletoxygenA significant

reduction in the recovery of a clinical isolate of C albicans was also observed

demonstratingthatthelight‐inducedeffectwasnotrestrictedtobacteriaAsignificant

reduction in the adhesion of P aeruginosa was demonstrated on the irradiated

TBO‐impregnated polymers however a photo‐bleaching effect was noted which

reduced the antibacterial activity of the polymers Thiswould impact on the clinical

application of the product and reduce the lifespan of the material so further

modification of the polymerwould be necessary to prevent this leaching effect and

retainthephotosensitiserwithinthepolyurethanematrix

270

9 Publicationsarisingfromthiswork

91 Peer‐reviewedPublications

bull AikenZAWilsonMampPrattenJ(2011)EvaluationofATPbioluminescence

assays for potential use in a hospital setting Infection Control and Hospital

Epidemiology32507‐509

bull DunnillCWPageKAikenZANoimarkSHyettGKafizasAPratten

JWilsonMamp Parkin I P (2011)Nanoparticulate silver coated‐titania thin

films‐Photo‐oxidativedestructionofstearicacidunderdifferent lightsources

and antimicrobial effects under hospital lighting conditions Journal of

PhotochemistryandPhotobiologyAChemistry220113‐123

bull AikenZAHyettGDunnillCWWilsonMPrattenJampParkinIP(2010)

Antimicrobial activity in thin films of pseudobrookite‐structured titanium

oxynitride under UV irradiation observed for Escherichia coli Chemical Vapor

Deposition1619‐22

bull DunnillCWAikenZAPrattenJWilsonMampParkinIP(2010)Sulfur‐

and Nitrogen‐doped titania biomaterials via APCVD Chemical Vapor

Deposition1650‐4

bull DunnillCWAikenZAPrattenJWilsonMMorganDJampParkinIP

(2009) Enhanced photocatalytic activity under visible light in Nitrogen‐doped

TiO2 thin films produced by APCVD preparations using t‐butylamine as a

nitrogen source and their potential for antibacterial films Journal of

PhotochemistryandPhotobiologyAChemistry207(2‐3)244‐53

bull DunnillCWAikenZAKafizasAPrattenJWilsonMMorganDJamp

Parkin I P (2009)White light induced photocatalytic activity of sulfur‐doped

TiO2 thin films and their potential for antibacterial application Journal of

MaterialsChemistry198747‐54

271

bull Dunnill C W Aiken Z A Pratten J Wilson M amp Parkin I P (2009)

Nitrogendoped titania thin filmspreparedbyatmosphericpressure chemical

vapour deposition Enhanced visible light photocatalytic activity and anti‐

microbialeffectsECSTransactions2565‐72

92 Posterpresentations

bull Aiken Z A Parkin I P Dunnill C W Pratten J amp Wilson M (2009)

Evaluationofanovelantibacterialcoatingactivatedbywhite lightSocietyof

GeneralMicrobiologyConferenceHarrogateUK

bull AikenZAWilsonMampPrattenJ(2008)Evaluationoftechniquestodetect

surface‐associated pathogens Society of General Microbiology Conference

DublinIreland

93 Otherpublications

bull AikenZA ‐ChristinePhilphotprizeessay‐ lsquoTheroleoftheenvironment in

theacquisitionofhealthcare‐associatedinfectionsrsquo2010ACMNews

bull Aiken Z A ndash Press release on EurekAlert ndash lsquoLight‐activated antibacterial

coating is new weapon in fight against hospital‐acquired infectionsrsquo 2009

httpwwweurekalertorgpub_releases2009‐03sfgm‐lac032709php

[Accessedon280610]

272

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2

Declaration

I Zoie Alexandra Aiken confirm that the work presented in this thesis is my own

WhereinformationhasbeenderivedfromothersourcesIconfirmthatthishasbeen

indicatedinthethesis

3






















4























5











images







statisticalanalysis




6



continuedsupport










readthis

7


Abstract 3

Acknowledgements 5

Tableofcontents 7

Listoffigures 13

Listoftables 19

1 Introduction 20


















152 Sol‐gel 71



161 Swabbing 73

8

162 Dipslides 73

163 Airsampling 74














24 Lightsources 87















9










31 Introduction 110





33 Results 115




34 Discussion 130




35 Conclusions 138


41 Introduction 139



10




43 Results 142







44 Discussion 161




45 Conclusions 168


51 Introduction 171





53 Results 173




54 Discussion 195



11


55 Conclusion 203


61 Introduction 204




63 Results 207






64 Discussion 226




65 Conclusions 234


71 Introduction 235




73 Results 243


12


74 Discussion 256





75 Conclusions 263






10 References 272

13
























14


















15
















16

















17


















18








19

Listoftables
















20

1 Introduction























usualniches

21
























22


2003‐20042004)




















23











theaffectedpatient













24






transmissionofGRE

1113 Cdifficile
















25


















2010)






26




















27









28












29




















2011)




30












31



belinkedto






patientsandvisitors













32






















33





andRutala2011)



















34























35





(Dentonetal2004)










colonycount









36






2011)


















37























38






















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40










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al2011)

41















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42
























43
























44























45























46
























47





Etris1997)













wererequired





48























49




materials










13311 Conductors



Electronhole


50





category


13312 Insulators



aninsulator


Electronhole


51















Electronhole


Bandgap

CB

VB

52
















Electronhole


Lightin

CB

VB

53












Normaltransition


conductionband

CB

VB

Normaltransition


CB

VB

54







55










56




TiO2 h+vb+e‐cb







e‐cb+O2 O2‐

2O2‐+2H2O 2HO+2OH‐+O2







λlt385nm

57





iii)









source









58






















59










2009)










60
























61























62
























63























64












2005)











(Berraetal2004)

65
















2009Torresetal2009)








66























67










68










69






membrane


















70























71








152 Sol‐gel





72


catalyse the reaction (West 1999 Rampaul et al 2003 009) A viscous gel




















73






161 Swabbing













162 Dipslides




74









163 Airsampling















75





















luciferase

76








plates
















77
























78




















79
















1983)






80
















1997)





81












point











82






















83























84

























85








86


21 Targetorganisms






















87

22 Growthconditions













mL



(BPW)wasused

24 Lightsources




88















89















mW







90





















91























92











BioProbeluminometer










93























94













adherentbacteria

A

B

C

D

95


intothesamebijou


intoseparatebijoux















freefinish

96























97



















N3





98






















cooled

99





additionalcontrol
















overnightinthedark

100












101














thinfilms









102








(Pernietal2009b)















103























104
























105


materials














depositionmethod


sol‐gel




106











107




















108
























109





110


31 Introduction





















111
























112




project

















113























114















Section213

115

33 Results










116












117


3321 Saureus













118



cfucm2


Clean‐Trace

25x105 16 62 52 21

25x104 20 64 70 29

25x103 27 51 62 35

25x102 44 158 137 133




119

















3322 Ecoli






120










121


cfucm2Ecoli


25x105 14 85 52 32

25x104 23 67 32 36

25x103 15 254 58 54

25x102 13 0 98 104















122






















123




log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions


124

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions








125

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions







samplewereobtained

126














127













Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


128

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


129









Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


130



(log10cfusample)

SaureusNCTC6571 56

EcoliATCC25922 02

Efaecalis 01


EMRSA‐16 09

EMRSA‐15 15

MRSA43300 46


34 Discussion













131























132











counts












133























134
























135












environment











136
























137


(Hamblinetal2005)





















138

35 Conclusions














139


41 Introduction


















140
















etal2010)







141




Section213














Taiwan)

142

43 Results










log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


143









$ amp$$()$$+-$(-

0123)45$6-+-3


144











Figure43





throughout








145




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146




A‐L‐)






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147














thelightsource







148

log 10cfuthinfilm

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log 10cfuthinfilm

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149

$ amp$$()$$+-$(-

0123)45$6-+-3






thinfilmsN1N2andN3






150







log 10cfuthinfilm

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log 10cfuthinfilm

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151
















152

log 10cfuthinfilm

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log 10cfuthinfilm

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153



log 10cfuthinfilm

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log 10cfuthinfilm

Exposureconditions











154





werepresent

(a) (b)

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

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log10

cfuthinfilm

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log10

cfuthinfilm

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log10

cfuthinfilm

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log10

cfuthinfilm

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155










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157

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entirely

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159















160

log 1

0 cfu

t

hin

film

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log 1

0 cfu

t

hin

film


161





TiON1 06 plt001


N1 25 plt0001

N2 11 Nil(pgt005)

N3 05 Nil(pgt005)


S2 17 pgt0001


44 Discussion









162




photo‐activated












dopingexperiments







163














technique










164






observed


















165























166























bacterialspecies

167















2003)








168













45 Conclusions










169




170


51 Introduction




















171






















172























173





53 Results










174







formula






hv+TiO2

air

175


reaction










176

0

10

20

30

40

50

60

70

80

90

100

200 700 1200 1700 2200

Wavelength

T

Qaurtz

TiO2

Ag-TiO2













177

0

20

40

60

80

100

120

140

160

180

200

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2320 eV29 eV

0

50

100

150

200

250

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2

320 eV










178










UV 24(2)deg

TiO2 None 64(2)deg

UV 8(2)deg



UV 8(2)deg


179

0

10

20

30

40

50

60

70

80

90

100

200 300 400 500 600 700 800 900 1000 1100

Wavelength nm

T















180


















181

-002

000

002

004

006

008

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28002850290029503000

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tion

0

5

24

29

48

53

72

-002

000

002

004

006

008

010

28002850290029503000

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tion

0

5

24

29

-002

000

002

004

006

008

010

28002850290029503000

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tion

0

5

24

29


a

b

c

182

-002

000

002

004

006

008

010

012

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

016

018

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h


a

b

c

183


a

b

c

184


TiO2 Ag‐TiO2



Rate

UVndash254nm 332x1016 114x1015 330x1016 114x1015

Whitelight 149x1016 155x1014 405x1016 422x1014
















185



186
















187



188















189







190












191










192















193












194














516)

195

54 Discussion






(Ag)+e‐ e‐Ag
















196



















generatedthinfilms

197


thinfilms














2009)








198
























199
























200















spectrum








201
























202
























203





55 Conclusion















204


61 Introduction





















205











206




inthischapter







polymers










207


63 Results












208

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


209

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


210

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


211

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions




212











cfuperpolymer

30 044 639

60 049 679

90 141 961

120 209 992

150 282 9985

180 294 9989

210 305 9991

240 333 9995





213










wavelength









214

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


215

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions












polymers

216




















seconds

217



Exposuretimeseconds





90 106 913 141 961

180 170 980 294 9989

240 155 972 333 9995








218

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


$amp())+-

01+2()amp3456532


219

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














220












221

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Exposureconditions

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer


222

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














223












log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


224

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


225
























226



Exposuretimeseconds

Paeruginosa

PAO1

Paeruginosa

clinicalisolate


Smaltophilia

clinicalisolate


90 141 106 172 096 054

180 294 170 190 282 148

240 333 155 416 312 179

64 Discussion














227












sameirradiationtime












228
























229
























230
























231














typestrain










232


















effectsgenerated





233























2010)

234













2010)

65 Conclusions









235


71 Introduction




















236













237














238
























239























240
























241










disruption





242











imagesforeachsample












243

73 Results
















75b)




thinfilm

244




245
















246


247


248














thinfilm








249


250


251
















90 1 0 013

180 1 0 000

180 2 090 058

180 3 060120 053

180 3 090180 156




252















253





254









255















256


74 Discussion












257
























258
























259
























260
























261























262




















whitelight



263












expressed

75 Conclusions










264






265


















ofbacteria





266
























267
























268
























269













270














Deposition1619‐22



Deposition1650‐4










271











DublinIreland







[Accessedon280610]

272

10 References












273












274












275













276













277














278












279












280











281












282













283












284












285














286











287












288












289













290













291













292













293












294













295











296












297













298











299












300














301













302












303














304













305








3






















4























5











images







statisticalanalysis




6



continuedsupport










readthis

7


Abstract 3

Acknowledgements 5

Tableofcontents 7

Listoffigures 13

Listoftables 19

1 Introduction 20


















152 Sol‐gel 71



161 Swabbing 73

8

162 Dipslides 73

163 Airsampling 74














24 Lightsources 87















9










31 Introduction 110





33 Results 115




34 Discussion 130




35 Conclusions 138


41 Introduction 139



10




43 Results 142







44 Discussion 161




45 Conclusions 168


51 Introduction 171





53 Results 173




54 Discussion 195



11


55 Conclusion 203


61 Introduction 204




63 Results 207






64 Discussion 226




65 Conclusions 234


71 Introduction 235




73 Results 243


12


74 Discussion 256





75 Conclusions 263






10 References 272

13
























14


















15
















16

















17


















18








19

Listoftables
















20

1 Introduction























usualniches

21
























22


2003‐20042004)




















23











theaffectedpatient













24






transmissionofGRE

1113 Cdifficile
















25


















2010)






26




















27









28












29




















2011)




30












31



belinkedto






patientsandvisitors













32






















33





andRutala2011)



















34























35





(Dentonetal2004)










colonycount









36






2011)


















37























38






















39





















40










Carlingetal2010)












al2011)

41















Lvetal2010)









42
























43
























44























45























46
























47





Etris1997)













wererequired





48























49




materials










13311 Conductors



Electronhole


50





category


13312 Insulators



aninsulator


Electronhole


51















Electronhole


Bandgap

CB

VB

52
















Electronhole


Lightin

CB

VB

53












Normaltransition


conductionband

CB

VB

Normaltransition


CB

VB

54







55










56




TiO2 h+vb+e‐cb







e‐cb+O2 O2‐

2O2‐+2H2O 2HO+2OH‐+O2







λlt385nm

57





iii)









source









58






















59










2009)










60
























61























62
























63























64












2005)











(Berraetal2004)

65
















2009Torresetal2009)








66























67










68










69






membrane


















70























71








152 Sol‐gel





72






















73






161 Swabbing













162 Dipslides




74









163 Airsampling















75





















luciferase

76








plates
















77
























78




















79
















1983)






80
















1997)





81












point











82






















83























84

























85








86


21 Targetorganisms






















87

22 Growthconditions













mL



(BPW)wasused

24 Lightsources




88















89















mW







90





















91























92











BioProbeluminometer










93























94













adherentbacteria

A

B

C

D

95


intothesamebijou


intoseparatebijoux















freefinish

96























97



















N3





98






















cooled

99





additionalcontrol
















overnightinthedark

100












101














thinfilms









102








(Pernietal2009b)















103























104
























105


materials














depositionmethod


sol‐gel




106











107




















108
























109





110


31 Introduction





















111
























112




project

















113























114















Section213

115

33 Results










116












117


3321 Saureus













118



cfucm2


Clean‐Trace

25x105 16 62 52 21

25x104 20 64 70 29

25x103 27 51 62 35

25x102 44 158 137 133




119

















3322 Ecoli






120










121


cfucm2Ecoli


25x105 14 85 52 32

25x104 23 67 32 36

25x103 15 254 58 54

25x102 13 0 98 104















122






















123




log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions


124

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions








125

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions







samplewereobtained

126














127













Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


128

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


129









Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


130



(log10cfusample)

SaureusNCTC6571 56

EcoliATCC25922 02

Efaecalis 01


EMRSA‐16 09

EMRSA‐15 15

MRSA43300 46


34 Discussion













131























132











counts












133























134
























135












environment











136
























137


(Hamblinetal2005)





















138

35 Conclusions














139


41 Introduction


















140
















etal2010)







141




Section213














Taiwan)

142

43 Results










log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


143









$ amp$$()$$+-$(-

0123)45$6-+-3


144











Figure43





throughout








145




$ amp$$()$$+-$(-

0123)45$6-+-3










146




A‐L‐)






$ amp$$()$$+-$(-

0123)45$6-+-3


147














thelightsource







148

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions












149

$ amp$$()$$+-$(-

0123)45$6-+-3






thinfilmsN1N2andN3






150







log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions






151
















152

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions













153



log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions











154





werepresent

(a) (b)

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions










155










$ amp$$()$$+-$(-

0123)45$6-+-3


156


















157

$

amp

(

)


+-012345406

78096

990454lt

=284gt934-8

01A6

)06

06



thinfilms










158








entirely

$ amp$$()$$+-$(-

0123)45$6-+-3





159















160

log 1

0 cfu

t

hin

film

Exposure conditions


Exposure conditions

log 1

0 cfu

t

hin

film


161





TiON1 06 plt001


N1 25 plt0001

N2 11 Nil(pgt005)

N3 05 Nil(pgt005)


S2 17 pgt0001


44 Discussion









162




photo‐activated












dopingexperiments







163














technique










164






observed


















165























166























bacterialspecies

167















2003)








168













45 Conclusions










169




170


51 Introduction




















171






















172























173





53 Results










174







formula






hv+TiO2

air

175


reaction










176

0

10

20

30

40

50

60

70

80

90

100

200 700 1200 1700 2200

Wavelength

T

Qaurtz

TiO2

Ag-TiO2













177

0

20

40

60

80

100

120

140

160

180

200

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2320 eV29 eV

0

50

100

150

200

250

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2

320 eV










178










UV 24(2)deg

TiO2 None 64(2)deg

UV 8(2)deg



UV 8(2)deg


179

0

10

20

30

40

50

60

70

80

90

100

200 300 400 500 600 700 800 900 1000 1100

Wavelength nm

T















180


















181

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

48

53

72

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29


a

b

c

182

-002

000

002

004

006

008

010

012

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

016

018

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h


a

b

c

183


a

b

c

184


TiO2 Ag‐TiO2



Rate

UVndash254nm 332x1016 114x1015 330x1016 114x1015

Whitelight 149x1016 155x1014 405x1016 422x1014
















185



186
















187



188















189







190












191










192















193












194














516)

195

54 Discussion






(Ag)+e‐ e‐Ag
















196



















generatedthinfilms

197


thinfilms














2009)








198
























199
























200















spectrum








201
























202
























203





55 Conclusion















204


61 Introduction





















205











206




inthischapter







polymers










207


63 Results












208

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


209

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


210

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


211

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions




212











cfuperpolymer

30 044 639

60 049 679

90 141 961

120 209 992

150 282 9985

180 294 9989

210 305 9991

240 333 9995





213










wavelength









214

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


215

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions












polymers

216




















seconds

217



Exposuretimeseconds





90 106 913 141 961

180 170 980 294 9989

240 155 972 333 9995








218

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


$amp())+-

01+2()amp3456532


219

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














220












221

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Exposureconditions

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer


222

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














223












log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


224

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


225
























226



Exposuretimeseconds

Paeruginosa

PAO1

Paeruginosa

clinicalisolate


Smaltophilia

clinicalisolate


90 141 106 172 096 054

180 294 170 190 282 148

240 333 155 416 312 179

64 Discussion














227












sameirradiationtime












228
























229
























230
























231














typestrain










232


















effectsgenerated





233























2010)

234













2010)

65 Conclusions









235


71 Introduction




















236













237














238
























239























240
























241










disruption





242











imagesforeachsample












243

73 Results
















75b)




thinfilm

244




245
















246


247


248














thinfilm








249


250


251
















90 1 0 013

180 1 0 000

180 2 090 058

180 3 060120 053

180 3 090180 156




252















253





254









255















256


74 Discussion












257
























258
























259
























260
























261























262




















whitelight



263












expressed

75 Conclusions










264






265


















ofbacteria





266
























267
























268
























269













270














Deposition1619‐22



Deposition1650‐4










271











DublinIreland







[Accessedon280610]

272

10 References












273












274












275













276













277














278












279












280











281












282













283












284












285














286











287












288












289













290













291













292













293












294













295











296












297













298











299












300














301













302












303














304













305








4























5











images







statisticalanalysis




6



continuedsupport










readthis

7


Abstract 3

Acknowledgements 5

Tableofcontents 7

Listoffigures 13

Listoftables 19

1 Introduction 20


















152 Sol‐gel 71



161 Swabbing 73

8

162 Dipslides 73

163 Airsampling 74














24 Lightsources 87















9










31 Introduction 110





33 Results 115




34 Discussion 130




35 Conclusions 138


41 Introduction 139



10




43 Results 142







44 Discussion 161




45 Conclusions 168


51 Introduction 171





53 Results 173




54 Discussion 195



11


55 Conclusion 203


61 Introduction 204




63 Results 207






64 Discussion 226




65 Conclusions 234


71 Introduction 235




73 Results 243


12


74 Discussion 256





75 Conclusions 263






10 References 272

13
























14


















15
















16

















17


















18








19

Listoftables
















20

1 Introduction























usualniches

21
























22


2003‐20042004)




















23











theaffectedpatient













24






transmissionofGRE

1113 Cdifficile
















25


















2010)






26




















27









28












29




















2011)




30












31



belinkedto






patientsandvisitors













32






















33





andRutala2011)



















34























35





(Dentonetal2004)










colonycount









36






2011)


















37























38






















39





















40










Carlingetal2010)












al2011)

41















Lvetal2010)









42
























43
























44























45























46
























47





Etris1997)













wererequired





48























49




materials










13311 Conductors



Electronhole


50





category


13312 Insulators



aninsulator


Electronhole


51















Electronhole


Bandgap

CB

VB

52
















Electronhole


Lightin

CB

VB

53












Normaltransition


conductionband

CB

VB

Normaltransition


CB

VB

54







55










56




TiO2 h+vb+e‐cb







e‐cb+O2 O2‐

2O2‐+2H2O 2HO+2OH‐+O2







λlt385nm

57





iii)









source









58






















59










2009)










60
























61























62
























63























64












2005)











(Berraetal2004)

65
















2009Torresetal2009)








66























67










68










69






membrane


















70























71








152 Sol‐gel





72






















73






161 Swabbing













162 Dipslides




74









163 Airsampling















75





















luciferase

76








plates
















77
























78




















79
















1983)






80
















1997)





81












point











82






















83























84

























85








86


21 Targetorganisms






















87

22 Growthconditions













mL



(BPW)wasused

24 Lightsources




88















89















mW







90





















91























92











BioProbeluminometer










93























94













adherentbacteria

A

B

C

D

95


intothesamebijou


intoseparatebijoux















freefinish

96























97



















N3





98






















cooled

99





additionalcontrol
















overnightinthedark

100












101














thinfilms









102








(Pernietal2009b)















103























104
























105


materials














depositionmethod


sol‐gel




106











107




















108
























109





110


31 Introduction





















111
























112




project

















113























114















Section213

115

33 Results










116












117


3321 Saureus













118



cfucm2


Clean‐Trace

25x105 16 62 52 21

25x104 20 64 70 29

25x103 27 51 62 35

25x102 44 158 137 133




119

















3322 Ecoli






120










121


cfucm2Ecoli


25x105 14 85 52 32

25x104 23 67 32 36

25x103 15 254 58 54

25x102 13 0 98 104















122






















123




log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions


124

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions








125

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions







samplewereobtained

126














127













Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


128

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


129









Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


130



(log10cfusample)

SaureusNCTC6571 56

EcoliATCC25922 02

Efaecalis 01


EMRSA‐16 09

EMRSA‐15 15

MRSA43300 46


34 Discussion













131























132











counts












133























134
























135












environment











136
























137


(Hamblinetal2005)





















138

35 Conclusions














139


41 Introduction


















140
















etal2010)







141




Section213














Taiwan)

142

43 Results










log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


143









$ amp$$()$$+-$(-

0123)45$6-+-3


144











Figure43





throughout








145




$ amp$$()$$+-$(-

0123)45$6-+-3










146




A‐L‐)






$ amp$$()$$+-$(-

0123)45$6-+-3


147














thelightsource







148

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions












149

$ amp$$()$$+-$(-

0123)45$6-+-3






thinfilmsN1N2andN3






150







log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions






151
















152

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions













153



log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions











154





werepresent

(a) (b)

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions










155










$ amp$$()$$+-$(-

0123)45$6-+-3


156


















157

$

amp

(

)


+-012345406

78096

990454lt

=284gt934-8

01A6

)06

06



thinfilms










158








entirely

$ amp$$()$$+-$(-

0123)45$6-+-3





159















160

log 1

0 cfu

t

hin

film

Exposure conditions


Exposure conditions

log 1

0 cfu

t

hin

film


161





TiON1 06 plt001


N1 25 plt0001

N2 11 Nil(pgt005)

N3 05 Nil(pgt005)


S2 17 pgt0001


44 Discussion









162




photo‐activated












dopingexperiments







163














technique










164






observed


















165























166























bacterialspecies

167















2003)








168













45 Conclusions










169




170


51 Introduction




















171






















172























173





53 Results










174







formula






hv+TiO2

air

175


reaction










176

0

10

20

30

40

50

60

70

80

90

100

200 700 1200 1700 2200

Wavelength

T

Qaurtz

TiO2

Ag-TiO2













177

0

20

40

60

80

100

120

140

160

180

200

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2320 eV29 eV

0

50

100

150

200

250

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2

320 eV










178










UV 24(2)deg

TiO2 None 64(2)deg

UV 8(2)deg



UV 8(2)deg


179

0

10

20

30

40

50

60

70

80

90

100

200 300 400 500 600 700 800 900 1000 1100

Wavelength nm

T















180


















181

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

48

53

72

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29


a

b

c

182

-002

000

002

004

006

008

010

012

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

016

018

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h


a

b

c

183


a

b

c

184


TiO2 Ag‐TiO2



Rate

UVndash254nm 332x1016 114x1015 330x1016 114x1015

Whitelight 149x1016 155x1014 405x1016 422x1014
















185



186
















187



188















189







190












191










192















193












194














516)

195

54 Discussion






(Ag)+e‐ e‐Ag
















196



















generatedthinfilms

197


thinfilms














2009)








198
























199
























200















spectrum








201
























202
























203





55 Conclusion















204


61 Introduction





















205











206




inthischapter







polymers










207


63 Results












208

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


209

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


210

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


211

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions




212











cfuperpolymer

30 044 639

60 049 679

90 141 961

120 209 992

150 282 9985

180 294 9989

210 305 9991

240 333 9995





213










wavelength









214

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


215

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions












polymers

216




















seconds

217



Exposuretimeseconds





90 106 913 141 961

180 170 980 294 9989

240 155 972 333 9995








218

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


$amp())+-

01+2()amp3456532


219

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














220












221

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Exposureconditions

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer


222

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














223












log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


224

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


225
























226



Exposuretimeseconds

Paeruginosa

PAO1

Paeruginosa

clinicalisolate


Smaltophilia

clinicalisolate


90 141 106 172 096 054

180 294 170 190 282 148

240 333 155 416 312 179

64 Discussion














227












sameirradiationtime












228
























229
























230
























231














typestrain










232


















effectsgenerated





233























2010)

234













2010)

65 Conclusions









235


71 Introduction




















236













237














238
























239























240
























241










disruption





242











imagesforeachsample












243

73 Results
















75b)




thinfilm

244




245
















246


247


248














thinfilm








249


250


251
















90 1 0 013

180 1 0 000

180 2 090 058

180 3 060120 053

180 3 090180 156




252















253





254









255















256


74 Discussion












257
























258
























259
























260
























261























262




















whitelight



263












expressed

75 Conclusions










264






265


















ofbacteria





266
























267
























268
























269













270














Deposition1619‐22



Deposition1650‐4










271











DublinIreland







[Accessedon280610]

272

10 References












273












274












275













276













277














278












279












280











281












282













283












284












285














286











287












288












289













290













291













292













293












294













295











296












297













298











299












300














301













302












303














304













305








5











images







statisticalanalysis




6



continuedsupport










readthis

7


Abstract 3

Acknowledgements 5

Tableofcontents 7

Listoffigures 13

Listoftables 19

1 Introduction 20


















152 Sol‐gel 71



161 Swabbing 73

8

162 Dipslides 73

163 Airsampling 74














24 Lightsources 87















9










31 Introduction 110





33 Results 115




34 Discussion 130




35 Conclusions 138


41 Introduction 139



10




43 Results 142







44 Discussion 161




45 Conclusions 168


51 Introduction 171





53 Results 173




54 Discussion 195



11


55 Conclusion 203


61 Introduction 204




63 Results 207






64 Discussion 226




65 Conclusions 234


71 Introduction 235




73 Results 243


12


74 Discussion 256





75 Conclusions 263






10 References 272

13
























14


















15
















16

















17


















18








19

Listoftables
















20

1 Introduction























usualniches

21
























22


2003‐20042004)




















23











theaffectedpatient













24






transmissionofGRE

1113 Cdifficile
















25


















2010)






26




















27









28












29




















2011)




30












31



belinkedto






patientsandvisitors













32






















33





andRutala2011)



















34























35





(Dentonetal2004)










colonycount









36






2011)


















37























38






















39





















40










Carlingetal2010)












al2011)

41















Lvetal2010)









42
























43
























44























45























46
























47





Etris1997)













wererequired





48























49




materials










13311 Conductors



Electronhole


50





category


13312 Insulators



aninsulator


Electronhole


51















Electronhole


Bandgap

CB

VB

52
















Electronhole


Lightin

CB

VB

53












Normaltransition


conductionband

CB

VB

Normaltransition


CB

VB

54







55










56




TiO2 h+vb+e‐cb







e‐cb+O2 O2‐

2O2‐+2H2O 2HO+2OH‐+O2







λlt385nm

57





iii)









source









58






















59










2009)










60
























61























62
























63























64












2005)











(Berraetal2004)

65
















2009Torresetal2009)








66























67










68










69






membrane


















70























71








152 Sol‐gel





72






















73






161 Swabbing













162 Dipslides




74









163 Airsampling















75





















luciferase

76








plates
















77
























78




















79
















1983)






80
















1997)





81












point











82






















83























84

























85








86


21 Targetorganisms






















87

22 Growthconditions













mL



(BPW)wasused

24 Lightsources




88















89















mW







90





















91























92











BioProbeluminometer










93























94













adherentbacteria

A

B

C

D

95


intothesamebijou


intoseparatebijoux















freefinish

96























97



















N3





98






















cooled

99





additionalcontrol
















overnightinthedark

100












101














thinfilms









102








(Pernietal2009b)















103























104
























105


materials














depositionmethod


sol‐gel




106











107




















108
























109





110


31 Introduction





















111
























112




project

















113























114















Section213

115

33 Results










116












117


3321 Saureus













118



cfucm2


Clean‐Trace

25x105 16 62 52 21

25x104 20 64 70 29

25x103 27 51 62 35

25x102 44 158 137 133




119

















3322 Ecoli






120










121


cfucm2Ecoli


25x105 14 85 52 32

25x104 23 67 32 36

25x103 15 254 58 54

25x102 13 0 98 104















122






















123




log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions


124

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions








125

log 10cfusample

Exposureconditions

log 10cfusample

Exposureconditions







samplewereobtained

126














127













Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


128

Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


129









Exposureconditions

log 10cfusample

Exposureconditions

log 10cfusample


130



(log10cfusample)

SaureusNCTC6571 56

EcoliATCC25922 02

Efaecalis 01


EMRSA‐16 09

EMRSA‐15 15

MRSA43300 46


34 Discussion













131























132











counts












133























134
























135












environment











136
























137


(Hamblinetal2005)





















138

35 Conclusions














139


41 Introduction


















140
















etal2010)







141




Section213














Taiwan)

142

43 Results










log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions


143









$ amp$$()$$+-$(-

0123)45$6-+-3


144











Figure43





throughout








145




$ amp$$()$$+-$(-

0123)45$6-+-3










146




A‐L‐)






$ amp$$()$$+-$(-

0123)45$6-+-3


147














thelightsource







148

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions












149

$ amp$$()$$+-$(-

0123)45$6-+-3






thinfilmsN1N2andN3






150







log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions






151
















152

log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions













153



log 10cfuthinfilm

Exposureconditions

log 10cfuthinfilm

Exposureconditions











154





werepresent

(a) (b)

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions

log10

cfuthinfilm

Exposureconditions










155










$ amp$$()$$+-$(-

0123)45$6-+-3


156


















157

$

amp

(

)


+-012345406

78096

990454lt

=284gt934-8

01A6

)06

06



thinfilms










158








entirely

$ amp$$()$$+-$(-

0123)45$6-+-3





159















160

log 1

0 cfu

t

hin

film

Exposure conditions


Exposure conditions

log 1

0 cfu

t

hin

film


161





TiON1 06 plt001


N1 25 plt0001

N2 11 Nil(pgt005)

N3 05 Nil(pgt005)


S2 17 pgt0001


44 Discussion









162




photo‐activated












dopingexperiments







163














technique










164






observed


















165























166























bacterialspecies

167















2003)








168













45 Conclusions










169




170


51 Introduction




















171






















172























173





53 Results










174







formula






hv+TiO2

air

175


reaction










176

0

10

20

30

40

50

60

70

80

90

100

200 700 1200 1700 2200

Wavelength

T

Qaurtz

TiO2

Ag-TiO2













177

0

20

40

60

80

100

120

140

160

180

200

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2320 eV29 eV

0

50

100

150

200

250

00 05 10 15 20 25 30 35 40Energy eV

(ah

v)1

2

320 eV










178










UV 24(2)deg

TiO2 None 64(2)deg

UV 8(2)deg



UV 8(2)deg


179

0

10

20

30

40

50

60

70

80

90

100

200 300 400 500 600 700 800 900 1000 1100

Wavelength nm

T















180


















181

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

48

53

72

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29

-002

000

002

004

006

008

010

28002850290029503000

Wavenumber cm-1

Absorb

tion

0

5

24

29


a

b

c

182

-002

000

002

004

006

008

010

012

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

016

018

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h

-002

000

002

004

006

008

010

012

014

28002850290029503000

Wavenumber cm-1

Absorb

tion

0 h

24 h

48 h

72 h

96 h


a

b

c

183


a

b

c

184


TiO2 Ag‐TiO2



Rate

UVndash254nm 332x1016 114x1015 330x1016 114x1015

Whitelight 149x1016 155x1014 405x1016 422x1014
















185



186
















187



188















189







190












191










192















193












194














516)

195

54 Discussion






(Ag)+e‐ e‐Ag
















196



















generatedthinfilms

197


thinfilms














2009)








198
























199
























200















spectrum








201
























202
























203





55 Conclusion















204


61 Introduction





















205











206




inthischapter







polymers










207


63 Results












208

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


209

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


210

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


211

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions




212











cfuperpolymer

30 044 639

60 049 679

90 141 961

120 209 992

150 282 9985

180 294 9989

210 305 9991

240 333 9995





213










wavelength









214

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


215

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions












polymers

216




















seconds

217



Exposuretimeseconds





90 106 913 141 961

180 170 980 294 9989

240 155 972 333 9995








218

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


$amp())+-

01+2()amp3456532


219

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














220












221

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


Exposureconditions

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer


222

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions














223












log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


224

log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


log 10cfupo

lymer

Exposureconditions

log 10cfupo

lymer

Exposureconditions


225
























226



Exposuretimeseconds

Paeruginosa

PAO1

Paeruginosa

clinicalisolate


Smaltophilia

clinicalisolate


90 141 106 172 096 054

180 294 170 190 282 148

240 333 155 416 312 179

64 Discussion














227












sameirradiationtime












228
























229
























230
























231














typestrain










232


















effectsgenerated





233























2010)

234













2010)

65 Conclusions









235


71 Introduction




















236













237














238
























239























240
























241










disruption





242











imagesforeachsample












243

73 Results
















75b)




thinfilm

244




245
















246


247


248














thinfilm








249


250


251
















90 1 0 013

180 1 0 000

180 2 090 058

180 3 060120 053

180 3 090180 156




252















253





254









255















256


74 Discussion












257
























258
























259
























260
























261























262




















whitelight



263












expressed

75 Conclusions










264






265


















ofbacteria





266
























267
























268
























269













270














Deposition1619‐22



Deposition1650‐4










271











DublinIreland







[Accessedon280610]

272

10 References












273












274












275













276













277














278












279












280











281












282













283












284












285














286











287












288












289













290













291













292













293












294













295











296












297













298











299












300














301













302












303














304













305








Measuring the susceptibility and adhesion of microorganisms to light-activated antimicrobial

Documents

Transcript of Measuring the susceptibility and adhesion of microorganisms to light-activated antimicrobial