)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/archive/2014/121367.pdf · 2019. 7. 31. ·...

Research ArticleMixture of Sodium Hypochlorite and HydrogenPeroxide on Adhered Aeromonas hydrophila to Solid Substratein Water Impact of Concentration and Assessment ofthe Synergistic Effect

Chreacutetien Lontsi Djimeli1 Antoine Tamsa Arfao1 Olive V Noah Ewoti1

Mireille Ebiane Nougang1 Marlyse L Moungang1 Geneviegraveve Bricheux2

Mose Nola1 and Teacutelesphore Sime-Ngando2

1 University of Yaounde I Laboratory of General Biology Hydrobiology and Environment Research UnitPO Box 812 Yaounde Cameroon

2 Laboratoire ldquoMicroorganismes Genome amp Environnementrdquo UMR CNRS 6023 Universite Blaise PascalComplexe Scientifique des Cezeaux 24 avenue des Landais BP 80026 63171 Aubiere Cedex France

Correspondence should be addressed to Moıse Nola moisenolayahoocom

Received 28 August 2013 Revised 22 December 2013 Accepted 13 January 2014 Published 3 March 2014

Academic Editor Rodrigo E Mendes

Copyright copy 2014 Chretien Lontsi Djimeli et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The synergistic effects of the combined treatments of NaOCl and H2O2on the elimination of A hydrophila adhered to polythene

under static and dynamic conditions were evaluatedThe concentrations 01 02 and 03permilNaOCl and 05 1 and 15permilH2O2were

used The contact periods were 180 360 540 and 720 minutes The abundance of cells adhered reached 247 and 227 units (log(CFUcm2)) respectively under static and dynamic conditions after action of the mixture of disinfectants whereas it reached 241and 339 units (log (CFUcm2)) after action of NaOCl and H

2O2alone respectively Increase in the incubation period resulted in a

significant decrease in the abundance of cells adhered when the mixture of 03permilNaOCl and 15permilH2O2was used (119875 lt 001) For

each cell growth phase there was a significant difference amongst the mean densities of cells adhered after action of the mixtureof disinfectants (119875 lt 005) Although the Freundlich isotherm parameters relatively varied from one experimental condition toanother the 119870

119891value registered in the exponential growth phase was relatively higher in static state than in dynamic regime cells

adhered under dynamic condition seem more sensitive to the synergistic action than those adhered under static condition

1 Introduction

The drinking water distribution network is a source of dis-quiet regarding the contamination of water during deliveryand regrowth of microorganisms that survive after treat-ment [1] It is often the scene of many physicochemicaland biological reactions resulting from interactions betweendisinfectants pipe walls and the free and fixed biomass[2] The presence of natural organic matter provides a foodsource for bacteria that can colonize the inner walls ofdistribution pipes forming biofilms that protect and supportthe growth of microorganisms some of which are associatedto hostile effect on human health [1] and others through their

interactions with disinfectants and pipe walls are sometimesthe cause of the deterioration of the organoleptic propertiesof the water supply [2 3]

In recent years World Health Organization recognizesA hydrophila as an opportunistic pathogen implicated as apathogenic agent in gastroenteritis septicemia cellulitis col-itis meningitis and respiratory infections [4ndash6] To preventbacterial regrowth a residual of a disinfectant is maintainedin the water distribution network Previous work has shownthat the bacteriumA hydrophila is widespread in the environ-ment especially in water intended for human consumption[7 8] Its concentration can sometimes reach 102 CFUmLat the outlet of treatment plants for drinking water This

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2014 Article ID 121367 13 pageshttpdxdoiorg1011552014121367

2 International Journal of Bacteriology

concentration may be higher in networks of drinking waterdistribution due to the growth of A hydrophila on biofilms[7 9]

The ingestion of water or contaminated food is thecommon way of progress in the case of Aeromonas infection[10] Numerous studies have been conducted in view of high-lighting the inactivation of various waterborne pathogensby various disinfectants including sodium hypochloritehydrogen peroxide ozone and chlorine dioxide [11]

The mixture of NaOCl and H2O2in water resulted in a

redox reaction which gave the following equations [12]H2O2H2O 177 v and ClO

2

minusClOminus 066 v

ClOminus + 2HOminus 997888rarr ClO2

minus+H2O + 2eminus (1)

H2O2+ 2H+ + 2eminus 997888rarr 2H

2O (2)

(1) and (2) ClOminus +H2O2+ 2HOminus + 2H+

997888rarr ClO2

minus+ 3H2O

(3)

ClOminus +H2O2+ 2H2O 997888rarr ClO

2

minus+ 3H2O (4)

ClOminus +H2O2997888rarr ClO

2

minus+H2O (5)

Na+ + ClOminus +H2O2997888rarr Na+ + ClO

2

minus+H2O (6)

NaClO +H2O2997888rarr NaClO

2+H2O (7)

NaClO2is a very unstable compound that gives NaCl +

1O2(singlet oxygen) It resulted in

NaClO +H2O2997888rarr NaCl + 1O

2+H2O (8)

The reaction between these disinfectants produces singletoxygen (1O

2) which is a powerful oxidant that rapidly kills

bacterial cells Singlet oxygen short lifespan (100 nanosec-onds in lipid media and 50 nanoseconds in the cytoplasm)can diffuse a short distance and reactwith certain amino acidsleading to structural and functional alteration of the mem-brane causing lipoperoxidation [13] Less data are availableon the bacterial behavior or bacterial metabolism when bothdisinfectants are dissolved in water at the same time Lessinformation are also available on the cell survival with respectto the both disinfectants concentrations

Most studies carried out so far provided some informa-tion on the doses of disinfectants and adequate contact dura-tion period to effectively control pathogens of public healthimportance that are commonly used to develop regulationsand strategies treatment Chemical disinfectants cause lethalor nonlethal changes in proteins [14] lipids [15] membrane[16] and DNA [17] of microorganisms In addition themechanisms of disinfection are also highly dependent on thetype of microorganism cell growth stage and disinfectant[18]

Other studies have considered the impact of disinfectantson A hydrophila adhered to the fragments of polytheneimmersed in water It appears that NaOCl ismore effective onA hydrophila adhered to polythene than H

2O2 In addition

A hydrophila adhered to polythene under dynamic conditionis more sensitive to each of the two disinfectants than that

adhered under static condition [18] However little data onthe combined effect of these disinfectants are available Thisstudy aims to evaluate in microcosm the synergistic effectof NaOCl and H

2O2on A hydrophila cells from different

cell growth phases and adhered to fragments of polytheneimmersed in water

2 Materials and Methods

21 Collection and Identification of A hydrophila The bac-terium A hydrophila was isolated from well water inYaounde (Cameroon) using membrane filtration techniqueon ampicillin-dextrin agar medium [19 20] Cell subculturewas performed on standard agar medium (Bio-Rad Labora-tories France) The cells were then identified using standardbiochemical methods [21] These cells are facultative anaero-bic nonsporulated Gram-negative bacilli and ferment man-nitol produce indole and are mobile They do not possessurease lysine decarboxylase (LDC) ornithine decarboxylase(ODC) and arginine dihydrolase (ADH) For the preparationof stocks of bacteria colonies are inoculated into 100mL ofnutrient broth (Oxford) for 24 hours at 37∘C Afterwardscells were harvested by centrifugation at 8000 rpm for 10minat 10∘C and washed twice with NaCl (85 gL) solution Thepellet was resuspended in NaCl (85 gL) solution and thentransferred to 300120583L tubes The stocks were then frozenstored

22 Assessment of Cell GrowthPhase On the basis of previousstudies regarding the different growth phases and biofilmformation the cell growth phases were assessed at 37∘CThe growth of A hydrophila in nonrenewed peptone liquidmedium gives 4 growth phases a lag growth phase from 0 to2 hours an exponential growth phase from 2 to 13 hours astationary growth phase from 13 to 22 hours and a declinegrowth phase which begins as from the 22th hour [18]

23 Disinfectants and Adsorbent Substrates Used The mix-ture of two disinfectants was used NaOCl which belongs tothe group of halogen derivatives and H

2O2which belongs

to the group of oxidants NaOCl and H2O2used are

respectively Colgate-Palmolive (USA) and Gilbert (France)brand The ease use of these two disinfectants in drinkingwater treatment justified their choice for this study Thecombination concentrations of each disinfectant used rangedfrom 01permil to 03permil and from 05permil to 15permil for NaOCland H

2O2 respectively These concentrations were evaluated

by simple method of dilution of crude solution obtaineddirectly from the supplier The choice of these combinationconcentrations is justified by their synergistic action Tocount the surviving bacteria after disinfection treatmentsterile NaCl solution (85 gL) was used as a diluent

The substrate used is high dense polythene It differs fromradical low dense polythene and linear low dense polytheneby the molecular structure of its sparsely branched chainsand its relatively high resistance to shocks high temperaturesand ultraviolet rays [22 23] It is a plastic piping material

International Journal of Bacteriology 3

obtained directly from the supplier and used in drinkingwater distribution

The high dense polythene is obtained by polymerizationof the macromolecules of polyolefin family This polymer-ization is obtained from gaseous ethylene according to thefollowing equation [24 25]

C CH H

H H

n

n

H2C C 2H ( )(9)

The polythene used in this study is commercialized byGoodfellow SARL (France)

24 Determination of Activity of Disinfectants Alone or inCombinations The protocol described by Maris [26] withsome modifications was applied The principle of this pro-tocol consists in preparation of the mixtures of NaOCl (A(B assoc)) and H

2O2(B (A assoc)) For it nine couples

of disinfectant concentrations (A (B assoc) B (A assoc))were studied simultaneously for the preparation of mixturesof disinfectants The disinfectant concentrations used aloneranged from 05permil to 15permil and from 5permil to 15permil for NaOCl(A alone) andH

2O2(B alone) respectivelyThe contaminated

substrates are getting in contact with these disinfectantconcentrations for 25 to 30min The disinfecting effect wasstopped by introducing substrates in 10mL of sterile salineAntimicrobial activity was assessed after culture of survivinggerms and appreciation of the reduction of the bacterial load

The effect of the association was estimated by calculatingthe fractional bactericidal concentration (FBC) according toMaris [26]

FBC = A (B assoc)A (alone)

+B (A assoc)B (alone)

(10)

wherein A (B assoc) and B (A assoc) are the respectiveconcentrations of NaOCl and H

2O2studied in the mixture

A (alone) and B (alone) are the respective concentrations ofthe two disinfectants studied alone

The synergy was then declared for a value of FBC lessthan or equal to 050 The study of this synergy was achievedat each stage of cell growth phase in stationary and dynamicregimes

25 Adhesion Protocol of Cells to Polythene On the basis ofprevious studies parallelepiped shaped fragments of poly-thenewith 1328 cm2 of total surface area suspendedwithwireof 01mmdiameterwere immersed in triplicate in the two setsA and B each in four flasks 250mL Duran A1 A11015840 and A110158401015840and B1 B11015840 and B110158401015840 A2 A21015840 and A210158401015840 and B2 B21015840 and B210158401015840A3 A31015840 and A310158401015840 and B3 B31015840 and B310158401015840 and A4 A41015840 and A410158401015840and B4 B41015840 and B410158401015840 each containing 99mL of NaCl solution(85 gL) Meanwhile the controls were made and coded A

01

A02 A03 and A

04 and B

01 B02 B03 and B

04 [27]The whole

was then autoclavedPrior to the experiments stocks frozen vial containing

A hydrophila cells were thawed at room temperature Then

100 120583L of the culture was transferred into test tubes contain-ing 10mL of nutrient broth (Oxford) and incubated at 37∘Cfor 24 hours Cells from a specific cell growth phase werethen harvested by centrifugation at 8000 rpm for 10min at10∘C and washed twice with sterile NaCl solution (85 gL)The pellets were then resuspended in 50mL of sterilizedNaCl solution (85 gL) After serial dilutions the initialconcentration of bacteria (data at 119905 = 0) in each solution wasadjusted to 6 times 108 CFUmL by reading the optical densityat 600 nm using a spectrophotometer (DR 2800) followed byculture on agar [27]

1mL of the suspension was added to 99mL of sterilizedNaCl solution (85 gL) contained in an Erlenmeyer flaskTriplicate flasks were incubated under dynamic condition for180 360 540 and 720min at a stirring speed of 60 revminusing a stirrer (Rotatest brand) In the same way anothertriplicate flasks were incubated under static condition for 180360 540 and 720min All these incubations were done atlaboratory temperature (25 plusmn 1∘C)

26 Disinfection Experiments After each incubation dura-tion fragments of polythene were drained for 10 seconds in asterile environment created by the Bunsen burner flame andthen introduced into test tubes containing 10mL of dilutedmixture of disinfectant of various concentrations Fragmentsremoved from flasks A1 A2 A3 A4 B1 B2 B3 and B4 wereintroduced in mixture disinfectant solutions of 01permil NaOCland 05permil H

2O2 Fragments removed from flasks A11015840 A21015840

A31015840 A41015840 B11015840 B21015840 B31015840 and B41015840 were introduced into mixturedisinfectant solutions of 02permilNaOCl and 1permilH

2O2 Simi-

larly those removed from flasks A110158401015840 A210158401015840 A310158401015840 A410158401015840 B110158401015840B210158401015840 B310158401015840 and B410158401015840 were introduced into mixture solutions of03permilNaOCl and 15permilH

2O2 Fragments of polythene flasks

from A01 A02 A03 and A

04 and B

01 B02 B03 and B

04 were

introduced into 10mL of sterile NaCl solution (85 gL) Theconcentration of the disinfectant has not been evaluated afterincubation

After 30min of incubation at room temperature andunder static condition each fragment was then drained outunder sterile condition Each fragment was then introducedinto 10mL of sterilized NaCl solution (85 gL) The unhook-ing of adherent cells was performed by vortex agitation atincreasing speeds for 30 seconds in three consecutive seriesof 10mL sterilized NaCl solution (85 gL) This techniqueallows for the unhooking of maximum adhered cells [28 29]The total volume of the suspension containing the unhookedbacterial cells was 30mL The isolation and enumeration ofunhooked cells were made by culture on ampicillin dextrinagar by using spread plat method followed by incubation onPetri dishes at 37∘C for 24 hours

27 DataAnalysis Thevariation of the abundance of adheredA hydrophila in each experimental condition was illustratedby semilogarithmic diagrams Standard deviations were notfitted because the curves were too close Spearman ldquo119903rdquocorrelation Test was used to assess the degree of correlationbetween the abundance of adhered cells and other parametersconsidered Kruskal-Wallis and Mann-Whitney tests were


Table 1 Value of fractional bactericidal concentration (FBC)obtained for each couple of disinfectants concentrations

Concentrations of disinfectant usedFBCDisinfectants in mixture Disinfectants alone

NaOCl (permil) H2O2 (permil) NaOCl (permil) H2O2 (permil)01 05 05 5 0302 1 1 10 0303 15 15 15 0301 2 05 5 0602 3 1 10 0503 4 15 15 046025 5 05 5 1505 6 1 10 11075 8 15 15 103

used to compare the mean abundance of cells adhered fromone experimental condition to another

The data from absorption experiments were analyzedusing the Freundlich isotherm model This isotherm waschosen because of the number and the relevance of theinformation it provides on the real adsorption mechanismson one hand and its remarkable ability to match doses ofadsorption on the other hand The Freundlich isotherm isdescribed by the following equation [30 31]

119862119904= 119870119891sdot 119862119897119899 (11)

where 119862119904is the quantity of cells adsorbed in the presence of

the mixture of disinfectant solutions 119862 is the concentrationof cells adsorbed in the absence of mixture of disinfectantsolutions 119870

119891is the Freundlich coefficient adsorption which

is connected to the adsorption capacity 119897119899 is coefficientlinearity and 119899 is the intensity of adsorption Here 119862119904is expressed as the number of adherent cellsmixture ofdisinfectant concentration and 119862 is the number of adherentcellscm2 of polythene Constructing linear regression log 119862119904versus log 119862 results in a line of slope 119897119899 which intercepts the119910-axis log119870

119891

3 Results

31 Fractional Bactericidal Concentration (FBC) The FBCvalues were calculated using the formula indicated aboveThe different FBC obtained is given in Table 1 To ensure thesynergistic action of the two disinfectants only disinfectantconcentrations giving FBC equal to 03 were used for thepreparation of mixture of disinfectants

32 Abundance of Cells Adhered to Polythene after Actionof the Association of Disinfectants in Stationary Regime Thedensities of cells adhered ranged from 030 to 229 units (log(CFUcm2)) after the action of the mixture of NaOCl andH2O2under static condition The maximum abundance of

cells adhered was recorded in the presence of the mixture of01permilNaOCl and 05permilH

2O2and this is after 720 minutes

with cells harvested from the lag growth phase Adhered cellswere always partially decimated by themixture of NaOCl andH2O2(Figure 1)

With cells coming from the lag phase the abundance ofcells adhered under static condition to the control substratevaried throughout from 202 to 319 units (log (CFUcm2))and was always superior to those of fragments tested fordisinfection In addition they increase with the incubationduration Maximum cell density was recorded after an adhe-sion test of 720 minutes After the action of the mixture ofNaOCl and H

2O2 the densities of cells adhered ranged from

030 to 229 units (log (CFUcm2)) The effectiveness of themixture of NaOCl and H

2O2decreased with the length of

the adhesion duration testThemaximumcell abundancewasrecorded in the presence of the mixture of 01permilNaOCl and05permilH

2O2after an adhesion test of 720 minutes The lowest

density of adhered cells was observed in the presence of themixture of 03permilNaOCl and 15permilH

2O2with cells coming

from the adhesion tests of 180 minutes (Figure 1)The abundance of cells under static condition adhered to

the control substrate during the exponential growth phasewas lower than that tested for disinfection in the lag growthphase under the same condition They generally fluctuatedbetween 230 and 291 units (log (CFUcm2)) After disinfec-tion test it was noted that the effectiveness of the mixture ofNaOCl and H

2O2decreased when the duration of adhesion

test increased Abundance of cells adhered ranged between070 to 181 units (log (CFUcm2)) (Figure 1) The highestcell abundance was recorded in presence of the mixture of01permilNaOCl and 05permilH

2O2after an adhesion test of 720

minutes The lowest density of adhered cells was observed inthe presence of the mixture of 03permilNaOCl and 15permilH

2O2

with cells coming from the adhesion tests of 180 minutes(Figure 1)

The stationary growth phase shows the abundance of cellsin static regime adhered to the control substrate which variesfrom 192 to 249 units (log (CFUcm2)) They remainedhigher than those of the fragments tested for disinfectionAfter disinfection test abundance of cells adhered rangedbetween 090 and 189 units (log (CFUcm2)) As the durationof adhesion test increased it was noted that the effectivenessof the mixture of NaOCl and H

2O2decreased The highest

density of cells adhered to the polythene was recorded inthe presence of the mixture of 03permilNaOCl and 15permilH

2O2

after 720 minutes incubation duration The lowest densityof adhered cells was observed in the presence of mixture of03permilNaOCl and 15permilH

2O2after 180 minutes incubation

duration (Figure 1)The abundance of cells adhered in static regime to the

control substrate during the decline growth phase variedfrom 195 to 248 units (log (CFUcm2)) Adhered cellsafter the action of NaOCl relatively increased (Figure 1) Themaximum density of cells adhered to the polythene wasrecorded in the presence of the mixture of 03permilNaOCland 15permilH

2O2after 720 minutes incubation duration The

minimum density of adhered cells was observed in thepresence of mixture of 03permilNaOCl and 15permilH

2O2after 180

minutes incubation (Figure 1)


Control (NaCl)05permil (NaOCl)1permil (NaOCl)15permil (NaOCl)

(01permil NaOCl + 05permil H2O2)(02permil NaOCl + 1permil H2O2)(03permil NaOCl + 15permil H2O2)

Control (NaCl)5permil(H2O2)10permil (H2O2)15permil (H2O2)

(01permilNaOCl + 05permilH2O2)(02permilNaOCl + 1permilH 2O 2)(03permil NaOCl + 15permil H2O2)

Cells coming from lag phase(disinfectant H2O2)

Cells coming from lag phase(disinfectant NaOCl)

Cells coming from exponential(disinfectant H2O2)

Cells coming from exponential(disinfectant NaOCl)

Cells coming from stationary(disinfectant H2O2)

Cells coming from stationary(disinfectant NaOCl)

Cells coming from decline(disinfectant H2O2)

Cells coming from decline(disinfectant NaOCl)

0

1

2

3

4

5

180 360 720

Incubation period (min)

0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

0

1

2

3

4

5

180 360 540 540720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


growth phase growth phase

growth phasegrowth phase


540540

540 540

540540

Figure 1 Temporal evolution of cells adhered under static condition after the action of NaOCl and H2O2alone and in the mixture of the two

disinfectants at different concentrations


33 Abundance of Cells Adhered to Polythene after Action ofAssociation of Disinfectants in Dynamic Regime The abun-dances of cells adhered ranged from 085 to 227 units (log(CFUcm2)) after the action of the mixture of NaOCl andH2O2under dynamic condition The maximum abundance

of cells adhered was recorded in the presence of mixture of01permilNaOCl and 05permilH


with cells harvested from the lag growth phaseThe density of cells adhered under dynamic condition

to the control substrate varied throughout from 235 to325 units (log (CFUcm2)) from the lag phase and wasalways superior to those fragments tested for disinfectionIn addition they increase with the incubation duration Themaximum cell abundance was recorded in the presence of themixture of 01permilNaOCl and 05permilH

2O2after an adhesion

test of 720 minutes The lowest density of adhered cells wasobserved in the presence of the mixture of 03permilNaOCl and15permilH

2O2with cells coming from the adhesion tests of 180

minutes (Figure 2) After action of the mixture of NaOCl andH2O2 the densities of cells adhered ranged from 085 to 227

units (log (CFUcm2)) The effectiveness of the mixture ofNaOCl and H

2O2decreased with the length of the adhesion

test durationAbundance of cells adhered under dynamic condition to

control substrate during the exponential growth phase waslower than that tested for disinfection in the lag growth phaseunder the same conditionThey generally fluctuated between247 and 319 units (log (CFUcm2)) After disinfection testit was noted that the effectiveness of the mixture of NaOCland H

2O2decreased when the duration of adhesion test

increased Abundance of cells adhered ranged between 095and 209 units (log (CFUcm2)) (Figure 2) The maximumcell abundance was recorded in presence of mixture of01permilNaOCl and 05permilH


minutesTheminimumdensity of adhered cells was observedin the presence of mixture of 03permilNaOCl and 15permilH

2O2


The abundance of cells adhered in dynamic regime tothe control substrate varied from 235 to 274 units (log(CFUcm2)) during the stationary growth phase It remainedhigher than those of fragments tested for disinfection Afterdisinfection test abundance of cells adhered ranged between130 and 213 units (log (CFUcm2)) As the duration ofadhesion test increased it was noted that the effectiveness ofthe mixture of NaOCl and H

2O2decreased The maximum


2O2

after 720minutes incubation durationwhereas theminimumdensity was observed in the presence of the mixture of03permilNaOCl and 15permilH


duration (Figure 2)Density of cells adhered in dynamic condition to the

control substrate during the decline growth phase variedfrom 210 to 271 units (log (CFUcm2)) Cells adhered afterthe action of NaOCl were relatively high (Figure 2) Themaximum density of cells adhered to the polythene wasrecorded in the presence of the mixture of 03permilNaOCl and

15permilH2O2after 720 minutes incubation duration and the

minimum in the presence of the mixture of 03permilNaOCl and15permilH

2O2after 180 minutes incubation (Figure 2)

34 Freundlich Isotherms of Cells Adsorption Freundlichisotherms were constructed by considering only the combi-nation concentrations the number of cells adhered to thesubstrate subjected to the test of disinfection and obtainedwithout exposure to the mixture of disinfectants for eachstage of cell growth and each experimental condition TheFreundlich isotherms are shown in Figure 3 It can be notedthat no matter which growth stage cells are the appearanceof the isotherms differs from one incubation condition toanother The linearity coefficient 119897119899 which is related to theadsorption intensity ranged from 001 to 021 and from 002to 015 respectively under static and dynamic incubationconditions The adsorption coefficient 119870

119891which is related to

the adsorption capacity ranged between 2 and 53 and between2 and 54 cells adhered respectively under static and dynamicincubation conditions The adsorption coefficient for thelag growth phase ranged between 4 and 53 and between 2and 54 cells adhered respectively under static and dynamicconditions (Table 2) The lowest adsorption coefficient afterthe mixture of disinfectant treatment was obtained with cellharvested from the lag growth phase (Table 2)

When considering each experimental condition theadsorption coefficient of cells harvested from the lag phasewas relatively higher after the mixture of disinfectant treat-ment than that of cell harvested from the other cells growthphases (Table 2) It was also noted that for the whole cellgrowth phases and the whole incubation conditions theadsorption coefficient values were relatively higher with themixture of 01permilNaOCl and 05permilH

2O2concentration than

those of the two other mixture of disinfectant concentrations(Table 2)

35 Correlation Coefficients between the Abundance of CellsAdhered and Incubation Durations and Concentrations ofDisinfectants Spearman ldquo119903rdquo correlation coefficients betweenthe abundances of cells adhered and incubation durations foreach concentration ofmixture of disinfectant and each exper-imental condition were assessed and are presented in Table 3It is noted that the increase in the incubation durations causeda significant decrease in the efficiency of 03permilNaOCl and03permilH

2O2mixture of disinfectant concentration (119875 lt 001)

This could result in higher abundance of cells adhered as theduration of the cell adhesion process increased

Spearman ldquo119903rdquo correlation coefficients between abundanceof cells adhered and concentrations of the mixture dis-infectants for each incubation duration and under eachexperimental condition were also assessed (Table 4) Understatic as well as dynamic condition it was noted that theeffectiveness of the mixture of disinfectant concentrations oncells adhered to polythene increased leading to a significantdecrease (119875 lt 001) in the abundance of bacteria adheredafter disinfection treatment

The degrees of relationship between the mixture ofdisinfectant concentrations and abundance of cells adhered



(01permilNaOCl + 05permilH2O2)(02permilNaOCl + 1permilH2O2)(03permilNaOCl + 15permilH2O2)

Control (NaCl)5permil (H2O2)10permil (H2O2)15permil (H2O2)

(01permil NaOCl + 05permilH2O2)(02permil NaOCl + 1permilH2O2)(03permil NaOCl + 15permilH2O2)









180 360 720


180 360 720

Incubation period (min)180 360 720


180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))




Figure 2 Temporal evolution of cells adhered under dynamic condition after the action of NaOCl and H2O2alone and in the mixture of the

two disinfectants at different concentrations


01 NaOCl + 05permil H2O2 (stat)02 NaOCl + 1permil H2O2 (stat)03 NaOCl + 15permil H2O2 (stat)

0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375

01 NaOCl + 05permil H2O2 (dyna)02 NaOCl + 1permil H2O2 (dyna)03 NaOCl + 15permil H2O2 (dyna)

0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2

Abundance of cells adheredcm2 in theNaCl solution






Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Figure 3 Freundlich isotherms for cells absorption under static (A1 B1 C1 and D1) and dynamic (A2 B2 C2 and D2) conditions in thepresence of the mixture of NaOCl and H

2O2(lag growth phase (A1 A2) exponential growth phase (B1 B2) stationary growth phase (C1

C2) and decline growth phase (D1 D2))


Table 2 Values of adsorption coefficient (119870119891) (adhered A hydrophilamL of mixture of disinfectant) and linearity coefficient (119897119899) of

isotherms under static and dynamic conditions when using different disinfectants concentrations

Disinfectant concentrations and static or dynamic condition Freundlich isotherm coefficients according to the cell growth phaseAdsorption coefficient (cells adheredcm2) Linearity coefficient

Disinfectant concentrations Condition Lag Expo Stat Decl Lag Expo Stat Decl

01permil NaOCl + 05permil H2O2Static 53 15 18 27 009 006 021 014

Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004

Table 3 Spearman ldquo119903rdquo correlation coefficients between the abundances of adhered A hydrophila and incubation durations for eachconcentration of mixture of disinfectant and each experimental condition

Experimental condition Mixtures of disinfectant concentrations01permil NaOCl + 05permil H2O2 02permil NaOCl + 1permil H2O2 03permil NaOCl + 03permil H2O2

Static 0800 minus0200 minus0400lowastlowast

Dynamic 0400 0632 minus0949lowastlowast

lowastlowast119875 lt 001 ddl = 15

Table 4 Spearman ldquo119903rdquo correlation coefficients between the abun-dance of adheredA hydrophila and concentration of mixture of dis-infectant for each incubation duration and under each experimentalcondition

Experimental condition Incubation durations180min 360min 540min 720min

Static 1000lowastlowast 1000lowastlowast 1000lowastlowast 1000lowastlowast

Dynamic 1000lowastlowast 1000lowastlowast 1000lowastlowast 1000lowastlowastlowastlowast119875 lt 001 ddl = 15

harvested from each growth stage were also assessed (Table5) It resulted that an increase in the mixture of disinfectantconcentration significantly increased (119875 lt 001) the abun-dance of cells adhered to the substrate with cell harvestedfrom each cell growth phase

36 Comparison of the Mean Abundance of Cells Adheredamongst the Different Stages of Cell Growth The 119867 test ofKruskal-Wallis was performed in order to compare the meanabundance of cells adhered harvested from different cellgrowth stages and considering each mixture of disinfectantsconcentrations It showed that there is an overall significantdifference (119875 lt 005) between the mean abundance of cellsadhered to polythene for each mixture of disinfectant con-centration at different cell growth stagesThe pair two-by-twocomparisons of the mean abundances were then performedusing the 119880 test of Mann-Whitney It was noted that at eachcell growth stage there was a significant difference (119875 lt 005)amongst themean abundance of cells adhered after the actionof various mixture of disinfectant concentrations with cellscoming from each cell growth phase With the mixture of01permilNaOCl and 05permilH

2O2and that of 03permilNaOCl and

15permilH2O2 a nonsignificant difference was observed only

with cells harvested from the stationary cell growth phase(119875 ge 005) (Table 6)

4 Discussion

The aim of this study was to determine the synergistic effectof NaOCl and H

2O2on A hydrophila adhered to polythene

immersed in water under static and dynamic conditions Bycontrast most previous studies have indicated only the effectof NaOCl on one hand and that of H

2O2on the other hand

on the adhesion of A hydrophila to polythene [18 32 33]From the 9 pairs of concentration of disinfectants used forthe preparation of mixture of disinfectants three couples(01permilNaOCl + 05permilH

2O2 02permilNaOCl + 1permilH

2O2 and

03permilNaOCl + 15permilH2O2) were used to evaluate the syn-

ergy as they presented an FBC equal to 03 A synergy isdeclared when a value of FBC is less than or equal to 050[26]

The present study showed that the overall abundance ofcells adhered to polythene after the action of the mixtureof two disinfectants was lower than that obtained afterthe action of H

2O2alone Abundance of cells adhered to

polythene ranged from 030 to 229 and 085 to 227 units(log (CFUcm2)) after the action of the mixture of NaOCland H

2O2under static and dynamic conditions respectively

Previous studies showed that they sometimes reached 241and 339 units (log (CFUcm2)) after the action of NaOCland H

2O2 respectively [18] These results suggest that the

combination of NaOCl and H2O2leads to a significant

synergy in eliminating cells adhered to polythene This hasbeen also suggested in previous studies [34]

Abundance of cells adhered to polythene after the actionof the mixture of NaOCl and H

2O2was relatively higher

than those obtained after the action of NaOCl alone


Table 5 Spearman ldquo119903rdquo correlation coefficients between the abundance of adhered A hydrophila and incubation durations for eachconcentration of the mixture of disinfectant and each cell growth phase

Cell growth phase Mixtures of disinfectant concentrations01permil NaOCl + 05permil H2O2 02permil NaOCl + 1permil H2O2 03permil NaOCl + 03permil H2O2

Lag 0947lowastlowast 0950lowastlowast 0981lowastlowast

Exponential 0970lowastlowast 0964lowastlowast 0905lowastlowast

Stationary 0955lowast 0920lowastlowast 0694lowastlowast

Decline 0980lowastlowast 0930lowastlowast 0945lowastlowastlowastlowast119875 lt 001 lowast119875 lt 005 ddl = 31

Table 6 Comparison amongst abundance of A hydrophila harvested from different cell growth stages in the presence of each mixture ofdisinfectant concentrations


Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast

Exponential 119875 = 0050lowast

119875 = 0001lowast

119875 = 0038lowast

Stationary 119875 = 0161 119875 = 0003lowast

119875 = 0065

Decline 119875 = 0007lowast

119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92

The maximum abundance of cells adhered to polythene wasrecorded under static condition in the presence of themixtureof 01permilNaOCl and 05permilH


with cells obtained in the lag growth phase (Figures 1 and 2)That obtained after the action of NaOCl was recorded duringthe lag phase under dynamic condition in the presence of05permilconcentrations of NaOCl and this is after an adhesiontest of 720minutes By cons the abundance of cells adhered topolythene after the action of the mixture of NaOCl andH

2O2

was considerably lower than those obtained after the actionof H2O2

The maximum abundance of cells adhered after theaction of H

2O2was recorded during the stationary growth

phase under static condition in the presence of 5permilH2O2

concentration after the same period of adhesion test Due toits highly oxidizing capacity-based production of free radicalsthat affect the biofilmsmatrix H

2O2was chosen to fight effec-

tively against biofilms formation [35 36] In addition H2O2

was chosen as it is highly effective disinfectant in inhibitingbiofilms formation at a concentration of 005 It can alsodestroymature biofilms at concentrations between 008 and02 [37] The reaction between NaOCl and H

2O2produces

singlet oxygen (1O2) which is a powerful oxidant that rapidly

kills bacterial cells In addition oxygen singlet short lifespan(100 nanoseconds in lipid media and 50 nanoseconds in thecytoplasm) can diffuse a short distance and react with certainamino acids leading to structural and functional alterationof the membrane causing lipoperoxidation [13] NaOCl andH2O2inhibit the Brownianmotion and control the growth of

the microbial population [34]The adhesion of microorganisms to surfaces is the first

step in biofilms formation which is a form of microbiallife in aquatic environments [38] The latter is the sourceof problems bioburden in various fields such as health

environment food industry and water purification [31 3940] Adhesion is governed by physicochemical interactionsof the Van Der Waals and Lewis acid-base types Fluctuatingvelocities of adhesion of cells observed during differentstages of growth in stationary and dynamic regimes couldbe explained by changes in the physiology of bacteriumat each stage of growth [41 42] There are three strategiesagainst biofilms formation (i) the disinfection time beforethe biofilms develop (ii) the disinfection of biofilms usingaggressive disinfectants and (iii) inhibition fixing microbeschoosing surface materials that do not promote adherence[43]

By considering separately each condition it was notedthat the increase in incubation durations resulted in a signifi-cant decrease (119875 lt 001) in the effectiveness of the mixtureof 03permilNaOCl and 15permilH

2O2(Table 3) This resulted in

higher abundance of cells Indeed a biofilm can be developedwithin in a few hours allowing bacteria therein to becomeresistant to external agents causing any contamination [4445] In static as well as dynamic condition increasing theeffectiveness of the mixture concentration of NaOCl andH2O2on cells adhered to polythene resulted in a significant

decrease in abundance of cells adhered after disinfection test(119875 lt 001) (Figures 1 and 2) The treatment of biofilms bycombining antimicrobial agents has a synergistic effect on theremoval of adherent bacterial cells [34] Furthermore thisvariation of the reaction of cells against the combination ofdisinfectants may be related to changes in the surface due toa change in their growth phase [46]

It was also noted that for each incubation period and eachcell growth phase a rise in the concentration of disinfectantmixture increases significantly (119875 lt 001) the abundance ofcells adhered to the substrate (Table 4) Face with antimicro-bial agent bacteria develops biofilm formation as a coping


strategy [47 48] For each cell growth phase a significantdifference was observed between the mean densities of cellsadhered after the action of the different concentrations ofthe mixture of disinfectants (119875 lt 005) The effectiveness ofany method of disinfection depends on biotic factors such asthe physiological state and the intrinsic microbial resistanceto lethal agents [49] The age of the culture also plays animportant role since the adhesion of the bacterium is betterduring exponential growth phase than stationary growthphase [50]

It is important to remember that bacteria in a biofilmhave very different characteristics from their planktoniccounterparts including the production of exopolymers [51]a significant increase in antimicrobial resistance and envi-ronmental stress [52 53] The matrix of exopolymers whichpresents itself as a mechanical barrier reducing the penetra-tion of environmental compounds through the biofilms thusprotects bacterial cells embedded in biofilmThis explains thefact that the increase in the concentration of the mixture ofdisinfectants for each stage of growth leads to a significantincrease (119875 lt 001) in abundance of cells adhered to thesubstrates The adsorption coefficient (119870

119891) was relatively

higher in the static than in the dynamic regime no matter thecell growth phase or presence of a well-defined concentrationof the mixture of disinfectant Cells adhered to polytheneunder dynamic condition were more sensitive than thatobtained with the two combined disinfectants under staticconditionThis could be explained by the structure of adheredbacteria which depends on the hydrodynamic regime [54]Enzymes produced byA hydrophila are essentially proteasesesterases and lyases Although these enzymes often remainqualitatively unchanged with bacterial growth phase [55]they would quantitatively be modified from one cell growthstage to another

5 Conclusion

This study showed that the combination of NaOCl andH2O2has a synergistic effect on cells adhered to polythene

Abundance of cells adhered to polythene after the action ofthe mixture of NaOCl and H

2O2is relatively higher than

that obtained after the action of NaOCl alone By cons it issignificantly lower than that obtained after the action ofH

2O2

alone Under static as well as dynamic condition an increasein the effectiveness of the concentrations of the mixture ofNaOCl and H

2O2on cells adhered is noted For each cell

growth phase the densities of cells adhered differed from agiven concentration of a mixture of disinfectants to anotherAlthough the adsorption coefficient (119870

119891) obtained from the

Freundlich isotherm is relatively higher in static state than indynamic regime cells adhered to polythene in the presence ofthemixture of the two disinfectants under dynamic conditionseem more sensitive than under static condition

Conflict of Interests

The authors declare that they have no conflict of interests thatcould inappropriately influence this work

References

[1] Comite federal-provincial-territorial sur lrsquoeau potable (Canada)ldquoConseils sur les bacteries pathogenes drsquoorigine hydriquerdquo 2012httpwwwhc-scgccaewh-semtalt formatspdfconsult2012bacterial-bacteriesbacterial-bacteries-frapdf

[2] P Mouchet A Montiel and S Rigal ldquoDegradations physico-chimiques de lrsquoeau dans les reseaux de distributionrdquoTSM LrsquoEauvol 87 pp 299ndash306 1992

[3] D Schoenen ldquoRole of disinfection in suppressing the spreadof pathogens with drinking water possibilities and limitationsrdquoWater Research vol 36 no 15 pp 3874ndash3888 2002

[4] K Krovacek A Faris S B Baloda T Lindberg M Peterz andI Mnsson ldquoIsolation and virulence profiles of Aeromonas sppfrom different municipal drinking water supplies in SwedenrdquoFood Microbiology vol 9 no 3 pp 215ndash222 1992

[5] A A Gavriel J P B Landre and A J Lamb ldquoIncidence ofmesophilicAeromonaswithin a public drinking water supply inNorth-East Scotlandrdquo Journal of Applied Microbiology vol 84no 3 pp 383ndash392 1998

[6] J Michael Janda and S L Abbott ldquoEvolving concepts regardingthe genus Aeromonas an expanding panorama of speciesdisease presentations and unanswered questionsrdquo ClinicalInfectious Diseases vol 27 no 2 pp 332ndash344 1998

[7] C Chauret C Volk R Creason J Jarosh J Robinson andC Warnes ldquoDetection of Aeromonas hydrophila in a drinking-water distribution system a field and pilot studyrdquo CanadianJournal of Microbiology vol 47 no 8 pp 782ndash786 2001

[8] G E El-Taweel and A M Shaban ldquoMicrobiological quality ofdrinking water at eight water treatment plantsrdquo InternationalJournal of Environmental Health Research vol 11 no 4 pp 285ndash290 2001

[9] P Payment E Franco and J Siemiatycki ldquoAbsence of rela-tionship between health effects due to tap water consumptionand drinking water quality parametersrdquo Water Science andTechnology vol 27 no 3-4 pp 137ndash143 1993

[10] R H W Schubert ldquoAeromonads and their significance aspotential pathogens in waterrdquo Journal of Applied Bacteriologyvol 70 supplement pp 131Sndash135S 1991

[11] M Cho J Kim J Y Kim J Yoon and J-H Kim ldquoMechanismsof Escherichia coli inactivation by several disinfectantsrdquo WaterResearch vol 44 no 11 pp 3410ndash3418 2010

[12] S Rondinini and A Vertova ldquoElectroreduction of halogenatedorganic compoundsrdquo in Electrochemistry For the Environmentpp 279ndash306 2010

[13] T Karu L Pyatibrat and G Kalendo ldquoIrradiation with He-Nelaser increases ATP level in cells cultivated in vitrordquo Journal ofPhotochemistry and Photobiology B vol 27 no 3 pp 219ndash2231995

[14] O J Sproul R M Pfister and C K Kim ldquoThe mechanism ofozone inactivation of water borne virusesrdquo Water Science andTechnology vol 14 no 4-5 pp 303ndash314 1982

[15] P-C Maness S Smolinski D M Blake Z Huang E JWolfrum and W A Jacoby ldquoBactericidal activity of photo-catalytic TiO

2reaction toward an understanding of its killing

mechanismrdquo Applied and Environmental Microbiology vol 65no 9 pp 4094ndash4098 1999

[16] S B Young and P Setlow ldquoMechanisms of killing of Bacillussubtilis spores by hypochlorite and chlorine dioxiderdquo Journal ofApplied Microbiology vol 95 no 1 pp 54ndash67 2003

[17] K Oguma H Katayama H Mitani S Morita T Hirata andS Ohgaki ldquoDetermination of pyrimidine dimers in Escherichia


coli and Cryptosporidium parvum during UV light inactivationphotoreactivation and dark repairrdquo Applied and EnvironmentalMicrobiology vol 67 no 10 pp 4630ndash4637 2001

[18] C Lontsi Djimeli M Nola A Tamsa Arfao et al ldquoEffect ofdisinfectants on adhered Aeromonas hydrophila to polytheneimmersed in water under static and dynamic conditionsrdquoInternational Journal of Research in BioSciences vol 2 pp 33ndash48 2013

[19] N Marchal J L Bourdon and C Richard Culture Media ForIsolation and Biochemical Identification of Bacteria Doin ParisFrance 1991

[20] APHA Standard Methods for the Examination of Water andWastewater American Public Health Association WashingtonDC USA 21st edition 2005

[21] G Holt N R Krieg P H A Sneath J T Staley and ST Williams Bergeyrsquos Manual of Determinative BacteriologyLipponcott Williams and Wilkins Philadelphia Pa USA 9thedition 2000

[22] K L Coeyrehourcq Etude de methodes rapides drsquoanalyse de lastructure moleculaire du polyethylene [These de Doctorat] Ecoledes Mines de Paris Specialite Science et Genie des Materiaux2003

[23] N Boutaleb Etude de la formation de biofilms sur les materiauxcouramment utilises dans les canalisations drsquoeaux potables [Thesede Doctorat] Universite de Bretagne-sud 2007

[24] B D Ratner ldquoPlasma deposition of organic thin film-control offilm chemistryrdquo Polymer Preprints vol 34 pp 643ndash644 1993

[25] B D Ratner ldquoSurface modification of polymers chemicalbiological and surface analytical challengesrdquo Biosensors andBioelectronics vol 10 no 9-10 pp 797ndash804 1995

[26] P Maris ldquoModes of action of disinfectantsrdquo in DisinfectantsActions and Applications H A McDaniel Ed pp 47ndash55 1995

[27] O V Noah Ewoti M Nola L M Moungang M E NougangF Krier and N E Chihib ldquoAdhesion of Escherichia coli andPseudomonas aeruginosa on rock surface in aquaticmicrocosmassessment of the influence of dissolved magnesium sulfate andmonosodium phosphaterdquo Research Journal of Environmentaland Earth Sciences vol 3 no 4 pp 364ndash374 2011

[28] S Dukam P Pirion and Y Levi ldquoModelisation du developpe-ment des biomasses bacteriennes libres et fixees en reseau dedistribution drsquoeau potablerdquo in Adhesion des Microorganismesaux Surfaces M N Bellon-Fontaine and J Fourniat Eds pp149ndash160 1995

[29] O V Noah Ewoti Retention des bacteries dans le sol et sur desfragments de roches en milieu aquatique influence du type decellule et de quelques parametres chimiques de lrsquoenvironnement[These] Universite de Yaounde I 2012

[30] M J Miller M M Critchley J Hutson and H J FallowfieldldquoThe adsorption of cyanobacterial hepatotoxins from wateronto soil during batch experimentsrdquoWater Research vol 35 no6 pp 1461ndash1468 2001

[31] I-W Wang J M Anderson M R Jacobs and R E MarchantldquoAdhesion of Staphylococcus epidermidis to biomedical poly-mers contributions of surface thermodynamics and hemo-dynamic shear conditionsrdquo Journal of Biomedical MaterialsResearch vol 29 no 4 pp 485ndash493 1995

[32] V SingamaneniGMadiraju andH Sura ldquoIn vitro effectivenessof different endodontic irrigants on the reduction of Enterococ-cus faecalis in root canalsrdquo Clinical and Experimental Dentistryvol 2 no 4 pp 169ndash172 2010

[33] K Tote T Horemans D Vanden Berghe L Maes and PCos ldquoInhibitory effect of biocides on the viable masses andmatrices of Staphylococcus aureus and Pseudomonas aeruginosabiofilmsrdquo Applied and Environmental Microbiology vol 76 no10 pp 3135ndash3142 2010

[34] J-H Ha S-H Jeong and S-D Ha ldquoSynergistic effects ofcombined disinfection using sanitizers and uv to reduce thelevels of Staphylococcus aureus in oyster mushroomsrdquo Journalof Applied Biological Chemistry vol 54 no 3 pp 447ndash453 2011

[35] C C C R de Carvalho ldquoBiofilms recent developments on anold battlerdquo Recent patents on biotechnology vol 1 no 1 pp 49ndash57 2007

[36] C C C R De Carvalho andM M R Da Fonseca ldquoAssessmentof three-dimensional biofilm structure using an optical micro-scoperdquo BioTechniques vol 42 no 5 pp 616ndash620 2007

[37] M N N N Shikongo-Nambabi B Kachigunda and S NVenter ldquoEvaluation of oxidising disinfectants to control Vibriobiofilms in treated seawater used for fish processingrdquoWater SAvol 36 no 3 pp 215ndash220 2010

[38] R M Donlan ldquoBiofilms microbial life on surfacesrdquo EmergingInfectious Diseases vol 8 no 9 pp 881ndash890 2002

[39] N Y Jayasekara G M Heard J M Cox and G H FleetldquoAssociation of micro-organisms with the inner surfaces ofbottles of non-carbonated mineral watersrdquo Food Microbiologyvol 16 no 2 pp 115ndash128 1999

[40] B A Jucker H Harms and A J B Zehnder ldquoAdhesionof the positively charged bacterium Stenotrophomonas (Xan-thomonas) maltophilia 70401 to glass and teflonrdquo Journal ofBacteriology vol 178 no 18 pp 5472ndash5479 1996

[41] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[42] S Parot Electroactifs formation caracterisation et mecanismes[These] Institut National polytechnique de Toulouse 2007

[43] B Meyer ldquoApproaches to prevention removal and killing ofbiofilmsrdquo International Biodeterioration and Biodegradationvol 51 no 4 pp 249ndash253 2003

[44] I B Beech and C L M Coutinho ldquoBiofilms on corrodingmaterialsrdquo in Biofilms in Medicine P Lens A P Moran TMahony P Stoodley and V OrsquoFlaherty Eds 2003

[45] I B Beech and J Sunner ldquoBiocorrosion towards understandinginteractions between biofilms and metalsrdquo Current Opinion inBiotechnology vol 15 no 3 pp 181ndash186 2004

[46] R BriandetMaıtrise de lrsquohygiene des surfaces par la creation desbiofilms-Aspects physico-chimiques [These de Doctorat] EcoleNationale Superieure Agronomique de Rennes Rennes France1999

[47] S Stepanovic I Cirkovic V Mijac and M Svabic-VlahovicldquoInfluence of the incubation temperature atmosphere anddynamic conditions on biofilm formation by Salmonella spprdquoFood Microbiology vol 20 no 3 pp 339ndash343 2003

[48] S Stepanovic I Cirkovic L Ranin and M Svabic-VlahovicldquoBiofilm formation by Salmonella spp and Listeria monocyto-genes on plastic surfacerdquo Letters in AppliedMicrobiology vol 38no 5 pp 428ndash432 2004

[49] R Patel ldquoBiofilms and antimicrobial resistancerdquo ClinicalOrthopaedics and Related Research no 437 pp 41ndash47 2005

[50] P M Stanley ldquoFactors affecting the irreversible attachment ofPseudomonas aeruginosa to stainless steelrdquo Canadian Journal ofMicrobiology vol 29 no 11 pp 1493ndash1499 1983


[51] M R Parsek and E P Greenberg ldquoAcyl-homoserine lac-tone quorum sensing in Gram-negative bacteria a signalingmechanism involved in associations with higher organismsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 97 no 16 pp 8789ndash8793 2000

[52] T-F C Mah and G A OrsquoToole ldquoMechanisms of biofilmresistance to antimicrobial agentsrdquo Trends in Microbiology vol9 no 1 pp 34ndash39 2001

[53] C Campanac L Pineau A Payard G Baziard-Mouysset andC Roques ldquoInteractions between biocide cationic agents andbacterial biofilmsrdquoAntimicrobial Agents andChemotherapy vol46 no 5 pp 1469ndash1474 2002

[54] M Klausen M Gjermansen J-U Kreft and T Tolker-NielsenldquoDynamics of development and dispersal in sessile microbialcommunities examples from Pseudomonas aeruginosa andPseudomonas putida model biofilmsrdquo FEMS Microbiology Let-ters vol 261 no 1 pp 1ndash11 2006

[55] D Buttner and U Bonas ldquoGetting across Bacterial type IIIeffector proteins on their way to the plant cellrdquo The EMBOJournal vol 21 no 20 pp 5313ndash5322 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


concentration may be higher in networks of drinking waterdistribution due to the growth of A hydrophila on biofilms[7 9]

The ingestion of water or contaminated food is thecommon way of progress in the case of Aeromonas infection[10] Numerous studies have been conducted in view of high-lighting the inactivation of various waterborne pathogensby various disinfectants including sodium hypochloritehydrogen peroxide ozone and chlorine dioxide [11]

The mixture of NaOCl and H2O2in water resulted in a

redox reaction which gave the following equations [12]H2O2H2O 177 v and ClO

2

minusClOminus 066 v

ClOminus + 2HOminus 997888rarr ClO2

minus+H2O + 2eminus (1)

H2O2+ 2H+ + 2eminus 997888rarr 2H

2O (2)

(1) and (2) ClOminus +H2O2+ 2HOminus + 2H+

997888rarr ClO2

minus+ 3H2O

(3)

ClOminus +H2O2+ 2H2O 997888rarr ClO

2

minus+ 3H2O (4)

ClOminus +H2O2997888rarr ClO

2

minus+H2O (5)

Na+ + ClOminus +H2O2997888rarr Na+ + ClO

2

minus+H2O (6)

NaClO +H2O2997888rarr NaClO

2+H2O (7)

NaClO2is a very unstable compound that gives NaCl +

1O2(singlet oxygen) It resulted in

NaClO +H2O2997888rarr NaCl + 1O

2+H2O (8)

The reaction between these disinfectants produces singletoxygen (1O

2) which is a powerful oxidant that rapidly kills

bacterial cells Singlet oxygen short lifespan (100 nanosec-onds in lipid media and 50 nanoseconds in the cytoplasm)can diffuse a short distance and reactwith certain amino acidsleading to structural and functional alteration of the mem-brane causing lipoperoxidation [13] Less data are availableon the bacterial behavior or bacterial metabolism when bothdisinfectants are dissolved in water at the same time Lessinformation are also available on the cell survival with respectto the both disinfectants concentrations

Most studies carried out so far provided some informa-tion on the doses of disinfectants and adequate contact dura-tion period to effectively control pathogens of public healthimportance that are commonly used to develop regulationsand strategies treatment Chemical disinfectants cause lethalor nonlethal changes in proteins [14] lipids [15] membrane[16] and DNA [17] of microorganisms In addition themechanisms of disinfection are also highly dependent on thetype of microorganism cell growth stage and disinfectant[18]

Other studies have considered the impact of disinfectantson A hydrophila adhered to the fragments of polytheneimmersed in water It appears that NaOCl ismore effective onA hydrophila adhered to polythene than H

2O2 In addition

A hydrophila adhered to polythene under dynamic conditionis more sensitive to each of the two disinfectants than that

adhered under static condition [18] However little data onthe combined effect of these disinfectants are available Thisstudy aims to evaluate in microcosm the synergistic effectof NaOCl and H

2O2on A hydrophila cells from different

cell growth phases and adhered to fragments of polytheneimmersed in water

2 Materials and Methods

21 Collection and Identification of A hydrophila The bac-terium A hydrophila was isolated from well water inYaounde (Cameroon) using membrane filtration techniqueon ampicillin-dextrin agar medium [19 20] Cell subculturewas performed on standard agar medium (Bio-Rad Labora-tories France) The cells were then identified using standardbiochemical methods [21] These cells are facultative anaero-bic nonsporulated Gram-negative bacilli and ferment man-nitol produce indole and are mobile They do not possessurease lysine decarboxylase (LDC) ornithine decarboxylase(ODC) and arginine dihydrolase (ADH) For the preparationof stocks of bacteria colonies are inoculated into 100mL ofnutrient broth (Oxford) for 24 hours at 37∘C Afterwardscells were harvested by centrifugation at 8000 rpm for 10minat 10∘C and washed twice with NaCl (85 gL) solution Thepellet was resuspended in NaCl (85 gL) solution and thentransferred to 300120583L tubes The stocks were then frozenstored

22 Assessment of Cell GrowthPhase On the basis of previousstudies regarding the different growth phases and biofilmformation the cell growth phases were assessed at 37∘CThe growth of A hydrophila in nonrenewed peptone liquidmedium gives 4 growth phases a lag growth phase from 0 to2 hours an exponential growth phase from 2 to 13 hours astationary growth phase from 13 to 22 hours and a declinegrowth phase which begins as from the 22th hour [18]

23 Disinfectants and Adsorbent Substrates Used The mix-ture of two disinfectants was used NaOCl which belongs tothe group of halogen derivatives and H

2O2which belongs

to the group of oxidants NaOCl and H2O2used are

respectively Colgate-Palmolive (USA) and Gilbert (France)brand The ease use of these two disinfectants in drinkingwater treatment justified their choice for this study Thecombination concentrations of each disinfectant used rangedfrom 01permil to 03permil and from 05permil to 15permil for NaOCland H

2O2 respectively These concentrations were evaluated

by simple method of dilution of crude solution obtaineddirectly from the supplier The choice of these combinationconcentrations is justified by their synergistic action Tocount the surviving bacteria after disinfection treatmentsterile NaCl solution (85 gL) was used as a diluent

The substrate used is high dense polythene It differs fromradical low dense polythene and linear low dense polytheneby the molecular structure of its sparsely branched chainsand its relatively high resistance to shocks high temperaturesand ultraviolet rays [22 23] It is a plastic piping material




C CH H

H H

n

n

H2C C 2H ( )(9)










(10)






01

A02 A03 and A

04 and B

01 B02 B03 and B

04 [27]The whole








2O2 Simi-




04 and B

01 B02 B03 and B

04 were










119862119904= 119870119891sdot 119862119897119899 (11)





119891

3 Results




















2O2





2O2







2O2after 180















0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

0

1

2

3

4

5

180 360 540 540720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720





540540

540 540

540540





















2O2





2O2
































180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




C CH H

H H

n

n

H2C C 2H ( )(9)










(10)






01

A02 A03 and A

04 and B

01 B02 B03 and B

04 [27]The whole








2O2 Simi-




04 and B

01 B02 B03 and B

04 were










119862119904= 119870119891sdot 119862119897119899 (11)





119891

3 Results




















2O2





2O2







2O2after 180















0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

0

1

2

3

4

5

180 360 540 540720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720





540540

540 540

540540





















2O2





2O2
































180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







119862119904= 119870119891sdot 119862119897119899 (11)





119891

3 Results




















2O2





2O2







2O2after 180















0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

0

1

2

3

4

5

180 360 540 540720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720





540540

540 540

540540





















2O2





2O2
































180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720


Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

0

1

2

3

4

5

180 360 540 540720


0

1

2

3

4

5

180 360 720


0

1

2

3

4

5

180 360 720





540540

540 540

540540





















2O2





2O2
































180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















2O2





2O2
































180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














180 360 720


180 360 720



180 360 720



180 360 720



0

1

2

3

4

5

6

180 360 720


0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3

4

5

6

540 540

540 540

540 540

540 540

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))

Cell

s adh

ered

(log

(CFU

cm

2))








0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

10

20

30

40

50

60

70

150 300 450 600 750 900

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325

0

10

20

30

40

50

60

70

80

90

75 125 175 225 275 325 375


0

20

40

60

80

100

120

140

200 500 800 1100 1400 1700

0

20

40

60

80

100

120

140

160

200 300 400 500 600

0

10

20

30

40

50

60

70

80

90

100

100 250 400 550

B1 B2

C1 C2

D1D2







Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

sAb

unda

nce o

f cel

ls ad

here

dcm

2in

the

mix

ture

of d

isinf

ecta

nts

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

0

50

100

150

200

250

100 600 1100 1600

0

20

40

60

80

100

120

140

160

180

200

100 600 1100 1600 2100

A1 A2



Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s

Abun

danc

e of c

ells

adhe

red

cm2

in th

em

ixtu

re o

f disi

nfec

tant

s










Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Dynamic 54 41 7 44 007 005 002 009


Dynamic 3 20 10 7 005 004 015 011


Dynamic 2 4 5 7 005 002 010 004















4 Discussion







2O2 and























Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Lag 119875 = 0015lowast

119875 = 0000lowast

119875 = 0005lowast


119875 = 0001lowast

119875 = 0038lowast


119875 = 0065


119875 = 0000lowast

119875 = 0021lowast

lowast119875 lt 005 ddl = 92




2O2









2O2produces
















5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusion





2O2








References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/archive/2014/121367.pdf · 2019. 7. 31. ·...

Documents

Transcript of )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/archive/2014/121367.pdf · 2019. 7. 31. ·...