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Mixtures of Essential Oils in an Air ConditioningPrototype to Reduce the Prevalence of AirbornePathogenic BacteriaSupayang Piyawan Voravuthikunchai a b , Sukanda Minbutra b , Lavanya Goodla a , JenniferJefferies c & Somboon Voravuthikunchai da Natural Products Research Center, Faculty of Science , Prince of Songkla University , HatYai , Songkhla 90112 , Thailandb Department of Microbiology , Faculty of Science, Prince of Songkla University , Hat Yai ,Songkhla 90112 , Thailandc Increasing your Health, Wealth and Sanity , PO Box 867 , North Lakes QLD 4509 , Australiahttp://www.jenniferjefferies.comd Department of Mechanical Engineering , Faculty of Engineering, Prince of SongklaUniversity , Hat Yai , Songkhla 90112 , ThailandPublished online: 12 Mar 2013.

To cite this article: Supayang Piyawan Voravuthikunchai , Sukanda Minbutra , Lavanya Goodla , Jennifer Jefferies & SomboonVoravuthikunchai (2012) Mixtures of Essential Oils in an Air Conditioning Prototype to Reduce the Prevalence of AirbornePathogenic Bacteria, Journal of Essential Oil Bearing Plants, 15:5, 739-749, DOI: 10.1080/0972060X.2012.10644114

To link to this article: http://dx.doi.org/10.1080/0972060X.2012.10644114

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Mixtures of Essential Oils in an Air Conditioning Prototype toReduce the Prevalence of Airborne Pathogenic Bacteria

Supayang Piyawan Voravuthikunchai 1, 2,*, Sukanda Minbutra 2,Lavanya Goodla 1, Jennifer Jefferies 3, Somboon Voravuthikunchai 4

1Natural Products Research Center, Faculty of Science,Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.

2Department of Microbiology, Faculty of Science,Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.

3 www.jenniferjefferies.com, Increasing your Health,Wealth and Sanity, PO Box 867, North Lakes QLD 4509, Australia4 Department of Mechanical Engineering, Faculty of Engineering,Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

Abstract: The rationale for this work was to test the possibility of creating a protective atmosphere byusing natural compounds to minimize hazards from chemicals and to control the risk of common infections.The antimicrobial activities of essential oils of Agronis fragrans, Cinnamomum zeylanicum, Lavandulaangustifolia, Melaleuca alternifolia (tea tree), Melaleuca nesophila (honey myrtle), Pelargonium x asperum(geranium), Pogostemon cablin (patchouli), Thymus serpyllum, and Thymus vulgaris were evaluated againstan array of environmental-borne pathogenic bacteria. Gram-positive bacteria included Bacillus subtilis andStaphylococcus aureus; four Gram-negative bacteria covered Acinetobacter baumanii, Escherichia coli,Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, eleven isolates of methicillin-resistant S.aureus (MRSA) and a multidrug-resistant A. baumanii were incorporated. In agar disc diffusion tests, cinnamonhad shown the strongest activity that could inhibit all pathogens, followed by lemon thyme, honey myrtle andlavender, while pachouli exhibited the weakest inhibition. Though fragonia possessed a broad range of activityagainst all pathogens, it did not inhibit some of the MRSA isolates. Cinnamon and lemon thyme demonstratedstrong activity measured by minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations.In disk volatilization assay, thyme oil demonstrated the greatest inhibition, followed by lemon thyme, while noinhibition from patchouli was observed. Subsequently, the composition of the atmosphere generated by threedifferent oil blends in our invented portable air-conditioning prototype was assessed. The results showed thatmixture of oil blend No. 2 can produce good synergistic effect in reducing the prevalence of airborne pathogens,and thus preventing the spreading of infections.

Key words: Acinetobacter baumanii, Air conditioning system, Essential oil, Staphylococcusaureus, Multidrug-resistant pathogenic bacteria

IntroductionDroplet infections have long been one of the

most deadly branches of infectious diseases.Airborne contamination continues as by far the

Journal of Essential Oil Bearing PlantsISSN Print: 0972-060X Online: 0976-5026www.jeobp.com

*Corresponding author (Supayang Piyawan Voravuthikunchai)E-mail: < supayang.v@psu.ac.th > © 2012, Har Krishan Bhalla & Sons

Jeobp 15 (5) 2012 pp 739 - 749 739

Received 18 January 2012; accepted in revised form 12 May 2012

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most important route by which infectious diseasesspread. In the 21st century, the infections are stilla major public health issue for both developingand developed countries. For developingcountries, poverty, lack of health infrastructureand sanitation, immigration, trade, globalizationcontributes to the spread of the diseases. Fordeveloped countries, infectious diseases are athreat on the horizon because of the problem ofnew and drug-resistant microorganisms migratingto the industrialized countries. Numbers of highlyantibiotic resistant bacterial strains are increasing.Methicillin-resistant Staphylococcus aureus(MRSA) has become increasingly widespread asa cause of both nosocomial and communityinfections. In 2002, vancomycin-resistant S.aureus (VRSA) strains emerged in the UnitedStates 3, followed by reports of these isolates fromother parts of the World 1,6. More recently,multidrug-resistant (MDR) Acinetobacter spp.have emerged as major causes of nosocomialinfections associated with significant morbidityand mortality rates 4,8,12,14.

Apparently concerns over the use of chemicalsthat could endanger people have been raised. Agrowing demand from consumers for safeproducts, desiring fewer synthetic substancestogether with their increased quality and safety,has resulted in extensive investigations fromresearchers to assess the feasibility of techniquesand to improve the quality and safety of products,while maintaining efficacy. There is a worldwidetrend to explore new alternatives to controlinfectious diseases, giving priority to methodsthat reduce disease incidence and avoid adverseeffects on human health. During the past decades,much attention has been paid to the use ofessential oils which are volatile oily liquidsobtained from different plant parts. Essential oilsfrom an estimated 3,000 plant species are known,of which about 300 are commercially produced,mainly for the flavours and fragrances market 2.

In spite of having been long recognized for theirantibacterial, antifungal, antiviral, insecticidaland antioxidant properties 11,17, the recent interestin alternative natural substances has led to a newscientific awareness of the value of essential oils.In other context, these substances have been

extensively studied for their antibacterialactivities against a wide range of microorganisms2,10,18. Some authors have suggested the use ofessential oils for prevention of the transmissionof resistant and harmful pathogens includingMRSA 16.

Selected essential oils commonly employed inaromatherapy were used in this study. Theseincluded Agronis fragrans J.R. Wheeler & N.G.Marchant (fragonia), Cinnamomum zeylanicumBlume Cheel (cinnamon), Lavandula angustifoliaMill. (Lavender), Melaleuca alternifolia (teatree), Melaleuca nesophila (honey myrtle),Pelargonium x asperum (geranium), Pogostemoncablin (Blanco) Benth. (Patchouli), Thymusserpyllum L. (lemon thyme), and Thymus vulgarisL. (thyme). The work presented in this communi-cation has three main aims: (i), to check theeffectiveness of selected essential oils againstdifferent pathogenic bacteria in solid phase bymeans of the disk diffusion method; (ii), toevaluate their effectiveness in vapour phase; and(iii), to apply the atmosphere generated by themost effective essential oils in a portable airconditioning prototype.

Materials and methodsEssential oilsEssential oils were extracted by hydro

distillation from the air dried plant material fromcinnamon (Cinnamomum zeylanicum), fragonia(Agonis fragrans), lemon thyme (Thymusserpyllum), patchouli (Pogostemon cablin(Blanco) Benth.), geranium (Pelargoniumxasperum), honey myrtle (Melaleuca nesophila),tea tree (Melaleuca alternifolia), lavender(Lavandula angustifolia), and thyme (Thymusvulgaris) using a Clevenger-type apparatus for 4hours. The oils were extracted with CHCl3 andthen were dried over anhydrous Na2SO4 andstored under N2 atmosphere at 20°C in a sealedvial until use.

Preparation of essential oil mixturesOil blend No. 1Cinnamon 10%Geranium 30%Tea tree 10%

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Thyme 50%Oil blend No. 2Cinnamon 20%Lemon thyme 30%Patchouli 30%Tea tree 20%Oil blend No. 3Cinnamon 23%Lavender 23%Lemon thyme 39%Thyme 15%

Tested bacterial strainsReference guidelines for culturing and

antibiotic sensitivity testing were used 5.Pathogenic Gram-positive and Gram-negativebacteria commonly found in the environmentincluding Acinetobacter baumanii, Bacillussubtilis, Escherichia coli, Klebsiella pneumoniae,Pseudomonas aeruginosa, and Staphylococcusaureus were tested for their sensitivity to theessential oils. In addition, eleven clinical isolatesof MRSA and a clinical isolate MDR A. baumaniiJVC1053 were included in our study. Clincallybacterial isolates were obtained from the RegionalMedical Sciences Center, Songkla andSonklagarind Hospital, Thailand. Each bacterialstrain was suspended in Mueller Hinton broth(MHB, Difco, Detroit, USA) and incubated at37°C for 18 h. Mueller Hinton agar (MHA, Difco)was used for antibacterial assay.

Paper disc agar diffusion methodSterile filter paper discs (6 mm) were soaked

with 10 ml of each undiluted essential oil. Thedisks were applied onto the surface of MHAplates seeded with 5-h broth culture of the testedbacteria. The plates were then incubated for 18 hat 35°C. The antibacterial activity was evaluatedby measuring the diameter of inhibition zone. Theexperiments were performed in triplicate and themean of the diameters of the inhibition zoneswere recorded. Antibiotic susceptibility discsincluding amikacin, ampicillin, gentamicin,kanamycin, and tetracyline (10-30 μg) wereincorporated as control.

Vapour diffusion assayMueller Hinton agar was inoculated with 5 h

broth culture of the tested bacteria. Each essentialoil (100 μl) at its minimal concentration wasadded onto 10 mm sterile blank filter discs andsubsequently placed on the medium-free coverof each Petri dish. The Petri dishes were placedwith the lid upside down, sealed with sterileadhesive tape and incubated at 35°C for 18 h 13.Mean growth measurements were calculated fromtriplicates of each bacterial species. The effecti-veness of the essential oil was calculated bymeasuring the diameter (in mm) of the zone ofmicroorganism growth inhibition above the disc.The size of the zone with visible growth reductionaround the inhibition zone was also measured.

Determination of minimal inhibitory con-centration (MIC) and Minimal bactericidalconcentration (MBC)A modified agar microdilution method 5 was

used to determine the MIC of essential oils thatproduced inhibition zones. One microliter of anovernight culture of each bacterial strain,containing approximately 104 CFU, was appliedonto MHA supplemented with the essential oilsat concentrations ranging from undiluted oil to1:128. The plates were incubated at 35°C for 18h. Observations were performed in triplicate andthe results were expressed as the lowestconcentration of essential oils that produced acomplete suppression of colony growth, MIC.Minimal bactericidal concentration using agardilution method in petri dishes with Milliporefilter 13 was performed with the essential oils thatshowed significant efficacy against each bacterialstrain. Amikacin, ampicillin, gentamicin,kanamycin, and tetracyline (10-32 μg/ml) wereincluded as reference standards.

Applications of oil blends in a portable airconditioning prototypeOil blend was applied to the system as indicated

in the diagram (Fig. 1). Each pathogen was grownin trypticase soy broth (TSB, Difco). An aliquotof 0.01 μl of diluted inoculum (approximately104 CFU/ml) was then swabbed on trypticase soyagar (TSA, Difco). In a closed system, theportable air conditioning prototype which wasapplied with different oil blends was used tocreate an environment of each oil blend. The

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petri-plates with each bacterial inoculum wereexposed to the environment created due to thevapours of each oil blend for 8h with a timeinterval of 1 h each. The bacterial numbers wereenumerated at time intervals after exposure to theoil blend of different formulas. The experimentswere performed in duplicate and the mean of thecolony counts were recorded after 24 h incubationperiod.

Results and discussionIn agar disc diffusion test (Table 1), cinnamon

was the most potent essential oil that inhibitedall pathogens, followed by lemon thyme andhoney myrtle that could affect most organisms,except Pseudomonas aeruginosa. In Contrast,lavender, geranium and tea tree oils showedmoderate inhibitory effect on most of the bacteriaand patchouli exhibited the weakest inhibition.Though fragonia seemed to possess a broad rangeof activity against most pathogens, it did notinhibit some of MRSA isolates. It is to be notedthat thyme oil produced large inhibition zonesagainst most bacteria including all drug-resistantorganisms except Klebsiella pneumoniae andPseudomonas aeruginosa. Cinnamon and lemonthyme demonstrated strong activity measured byminimal inhibitory concentrations (MICs) and

minimal bactericidal concentrations (Table 2). Anumber of interesting compounds including (E)-cinnamaldehyde, alcohol and acetate, R-phellandrene, and citronellal were detected incinnamon 13. Cinnamaldehyde is well-known tohave antimicrobial activity 7. Antimicrobialproperties of lemon thyme in vapour phase havealso been reported by other workers 15. Therefore,these two oils were selected as major componentsin our oil blends.

Comparison of antimicrobial activities invapour phase is quite difficult since there is nostandard method. Although there are manymethods used by different authors, not all of themare suitably adapted for fast screening of largequantities of samples. One of the most promisingmethods for this purpose seems to be diskvolatilization test. Hence, we decided to assessantimicrobial properties of each essential oil atits minimal concentration by this method. Theresults demonstrated that thyme oil possessed thebest activity, followed by lemon thyme (Table 3).Patchouli did not produce inhibition zones in anyof the pathogenic bacteria. Differences among thevolatiles in the essential oils and their hydro-phobicity may be responsible for the differencesin their antimicrobial patterns in two differenttypes of contact test. Thyme oil at absolute

Fig. 1. Diagram of a Portable Air Conditioning Prototype (A) Front view and (B) Back view

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Tabl

e 1.

Ant

ibac

teri

al a

ctiv

ity te

stin

g of

ess

entia

l oils

aga

inst

pat

hoge

nic

bact

eria

by

agar

diff

usio

n m

etho

d

Bac

teri

al s

trai

nM

ean

valu

es o

f inh

ibiti

on z

ones

± st

anda

rd e

rror

s (m

m) p

rodu

ced

by e

ssen

tial o

ils (1

0 m

l/dis

c)A

FC

ZM

AM

NPA

PCT

ST

VL

A

Gra

m-p

ositi

ve b

acte

riaBa

cillu

s su

btili

s PS

SCM

I007

535

±131

±114

±113

±116

022

±828

±248

±115

±2St

aphy

loco

ccus

aur

eus A

TCC

259

2310

±134

±127

±229

±217

±229

531

±264

±018

±1M

ethi

cilli

n-re

sist

ant S

. aur

eus

(11

isol

ates

)8±

3*33

±024

±232

±222

±310

±435

258

±112

±1

Gra

m-N

egat

ive

bact

eria

Acin

etob

acte

r ba

uman

iiATC

C 1

9606

14±2

30±3

15±2

16±2

9±2

-23

±134

±014

±2M

ultid

rug-

resi

stan

t A. b

aum

anii

JVC

105

318

±229

±317

±014

±211

±4-

23±2

18±0

-Es

cher

ichi

a co

li AT

CC

259

2220

±223

±115

±112

±111

1-

19±2

32±1

22±1

Kle

bsie

lla p

neum

onia

PSSC

MI 0

031

12±1

24±2

-10

±1-

-14

±1-

12±1

Pseu

dom

onas

aer

ugin

osaP

SSC

MI 0

048

10±1

24±5

--

--

--

13±2

* =

only

9 is

olat

es d

emon

stra

ted

inhi

bitio

n zo

nes,

- = N

o in

hibi

tion

zone

;Va

lues

are

mea

n ±

S.E

AF:

Ago

nis

frag

rans

(Fra

goni

a);

CZ:

Cin

nam

omum

zey

lani

cum

(Cin

nam

on;

MA

: Mel

aleu

ca a

ltern

ifolia

(Tea

tree

);M

N: M

elal

euca

nes

ophi

la(H

oney

myr

tle);

PA: P

elar

goni

um x

asp

erum

(Ger

aniu

m);

PC: P

ogos

tem

on c

ablin

(Pat

chou

li);

TS: T

hym

us s

erpy

llum

(Lem

on th

yme)

;TV

: Thy

mus

vul

gari

s (T

hym

e);

LA: L

avan

dula

ang

ustif

olia

(Lav

ende

r);

Supayang Piyawan Voravuthikunchai et al. / Jeobp 15 (5) 2012 739 - 749 743

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Tabl

e 2.

Min

imal

Inh

ibiti

on C

once

ntra

tion

(MIC

) and

Min

imal

Bac

teri

cida

lC

once

ntra

tion

(MB

C) o

f Ess

entia

l Oils

on

Path

ogen

ic B

acte

ria

Bac

teri

al s

trai

nD

eter

min

atio

n of

MIC

/MB

C (d

ilutio

n)A

FC

ZM

AM

NPA

PCT

ST

VL

A

Gra

m-p

ositi

ve b

acte

riaBa

cillu

s su

btili

s1:

8/1:

161:

64/1

:64

1:8/

1:8

1:32

/1:3

21:

3/1:

321:

16/1

:81:

32/1

:32

1:16

/1:1

61:

18/1

:16

PSSC

MI 0

075

Stap

hylo

cocc

us a

ureu

s1:

8/1:

21:

64/1

:64

1:8/

1:2

1:64

/1:3

21:

16/1

:21:

4/>U

1:32

/1:3

21:

16/1

:41:

16/1

:8 A

TCC

259

23M

ethi

cilli

n-re

sist

ant S

. aur

eus

1:8/

1:4

1:64

/1:6

41:

8/1:

21:

16/1

:16

1:32

/1:1

61:

4/>U

1:32

/1:3

21:

16/1

:41:

8/1:

8N

PRC

001

Gra

m-N

egat

ive

bact

eria

Acin

etob

acte

r ba

uman

ii1:

1/1:

81:

64/1

:64

1:8/

1:8

1:16

/1:1

61:

8/1:

4N

A1:

32/1

:32

1:8/

1:8

1:16

/ 1:1

6AT

CC

196

06M

ultid

rug-

resi

stan

t1:

8/1:

41:

64/1

:64

1:16

/1:1

61:

16/1

:16

1:8/

1:8

NA

1:32

/1:3

21:

8/1:

81:

8 / 1

:4A.

bau

man

ii JV

C 1

053

Esch

eric

hia

coli

1:8/

1:8

1:64

/1:6

41:

8/1:

81:

16/1

:81:

8/1:

8N

A1:

32/1

:32

1:8/

1:8

1:16

/ 1:

16AT

CC

259

22K

lebs

iella

pne

umon

iae

1:4/

1:2

1:64

/1:3

21:

8/1:

81:

8/1:

8N

AN

A1:

16/1

:16

1:8/

1:8

1:8/

1:8

PSSC

MI 0

031

Pseu

dom

onas

aer

ugin

osa

U/>

U1:

64/1

:64

1:2/

1:2

U/>

UN

AN

A1:

16/1

:81:

2/1:

21:

4 / 1

:4PS

SCM

I004

8

NA

= N

ot a

pplic

able

,U

= u

ndilu

ted

oil.

AF:

Ago

nis

frag

rans

(Fra

goni

a);

CZ:

Cin

nam

omum

zey

lani

cum

(Cin

nam

on;

MA

: Mel

aleu

ca a

ltern

ifolia

(Tea

tree

);M

N: M

elal

euca

nes

ophi

la(H

oney

myr

tle);

PA: P

elar

goni

um x

asp

erum

(Ger

aniu

m);

PC: P

ogos

tem

on c

ablin

(Pat

chou

li);

TS: T

hym

us s

erpy

llum

(Lem

on th

yme)

;TV

: Thy

mus

vul

gari

s (T

hym

e);

LA: L

avan

dula

ang

ustif

olia

(Lav

ende

r);

Supayang Piyawan Voravuthikunchai et al. / Jeobp 15 (5) 2012 739 - 749 744

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Tabl

e 3.

Ant

ibac

teri

al A

ctiv

ity T

estin

g of

Ess

entia

l Oils

aga

inst

Pat

hoge

nic

Bac

teri

a in

Vap

our

Con

tact

Bact

eria

l str

ain

Mea

n va

lues

of i

nhib

ition

zone

s ± st

anda

rd er

rors

(mm

) pro

duce

d by

esse

ntia

l oils

at m

inim

al in

hibi

tion

conc

entr

atio

nA

FC

ZM

AM

NPA

PCT

ST

VL

A

Gra

m-p

ositi

ve b

acte

riaBa

cillu

s su

btili

s PS

SCM

I 007

5N

D43

±118

±1N

D40

±0-

29±3

38±0

30±2

Stap

hylo

cocc

us a

ureu

s ATC

C 2

5923

ND

--

ND

48±1

-35

±160

±318

±1M

ethi

cilli

n-re

sist

ant S

. aur

eus

(11

isol

ates

)N

D-

-N

D-

-29

±352

±312

±1

Gra

m-N

egat

ive

bact

eria

Acin

etob

acte

r ba

uman

ii AT

CC

196

06N

D30

±089

±1N

D-

-29

±140

±0-

Mul

tidru

g-re

sist

ant A

. bau

man

ii JV

C 1

053

ND

19±1

-N

D-

-18

±150

±119

±1Es

cher

ichi

a co

li AT

CC

259

22N

D22

±165

±2N

D-

-29

±185

±2-

Kle

bsie

lla p

neum

onia

e PS

SCM

I 003

1N

D-

13±0

ND

--

-33

±320

±6Ps

eudo

mon

as a

erug

inos

a PS

SCM

I 004

8N

D-

90±0

ND

--

55±2

85±2

15±1

ND

= N

ot d

eter

min

ed;

- =

No

inhi

bitio

n zo

ne;

Val

ues a

re m

ean

± S.

E,A

F: A

goni

s fr

agra

ns (F

rago

nia)

;C

Z: C

inna

mom

um z

eyla

nicu

m (C

inna

mon

;M

A: M

elal

euca

alte

rnifo

lia (T

ea tr

ee);

MN

: Mel

aleu

ca n

esop

hila

(Hon

ey m

yrtle

);PA

: Pel

argo

nium

x a

sper

um (G

eran

ium

);PC

: Pog

oste

mon

cab

lin(P

atch

ouli)

;TS

: Thy

mus

ser

pyllu

m (L

emon

thym

e);

TV: T

hym

us v

ulga

ris

(Thy

me)

;LA

: Lav

andu

la a

ngus

tifol

ia (L

aven

der)

;

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concentration could not inhibit Klebsiella pneu-moniae and Pseudomonas aeruginosa in agardiffusion test, but the activity is enhanced invaporization assay, resulting in inhibition of allpathogenic bacteria tested, even at the MIC.

A synergistic effect on antimicrobial activitymay be produced from combinations of subs-tances. It has been recently demonstrated thatcombination of essential oils in vapour phasecould wield a synergistic effect on the inhibitionof Listeria monogenes, Bacillus cereus, andYersinia enterocolitica 9. Taken together theresults from previous experiments, we decidedto formulate oil blends. According to the resultsobtained in Table 4 and Table 5, oil blends No. 2and 3 were further selected to incorporate in ourportable air conditioning prototype. Antibacterialefficacy in the atmosphere generated by selectedoil blends in our invented air conditioning systemwas assessed. Exposure of bacterial colonies tothe system resulted in a marked decrease incounts, especially with the Gram-positive bacteria(Fig. 2). Both Gram-positive and Gram-negative

bacteria demonstrated noticeably smaller size intheir colony appearance towards the end of thefirst hour. This was due to cells being injured fromvaporization. Oils in the vapour phase wereshown to have significant antimicrobial activity.Thus, the vapour-phase approach appears to bepromising as a control protocol and could beapplied in the ventilation, creating a protectiveatmosphere with no risk effects from chemicals.

In conclusion, our results demonstrated that thisair conditioning prototype, incorporated with oilblend No. 2 is of advantage in preventingspreading of infections.

Its application is useful, especially for use inrisk environment such as hospital wards, theatres,conference rooms and other public spaces.

AcknowledgementsAuthors are grateful to The Australian Trade

Commission, Thailand. This work was supportedby the Thailand Research Fund, Fiscal year 2012-2014.

Time (hours)

Num

ber o

f col

onie

s (C

FU)

Num

ber o

f col

onie

s (C

FU)

Time (hours)

Fig. 2. Antibacterial efficacy of oil blends in air conditioning prototype. During the first hour,all bacteria demonstrated noticeably smaller size in their colony appearance. Reduction in numbersof colony counts: Bacillus subtilis ( ), Staphylococcus aureus ( ), methicillin-resistant S. aureus( ), Acinetobacter baumanii ( ), multidrug-resistant A. baumanii ( ), Escherichia coli ( ),Klebsiella pneumoniae (Δ), and Pseudomonas aeruginosa (Ο) after incorporation of oil blend No. 2(cinnamon 20%, lemon thyme 30%, patchouli 30%, tea tree 20%) (A) and oil blend No. 3 (cinnamon23%, lavender 23%, lemon thyme 39%, thyme 15%) (B).

(A) (B)

Supayang Piyawan Voravuthikunchai et al. / Jeobp 15 (5) 2012 739 - 749 746

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Tabl

e 4.

Ant

ibac

teri

al a

ctiv

ity te

stin

g of

ble

nded

ess

entia

l oils

aga

inst

pat

hoge

nic

bact

eria

by

agar

diff

usio

n m

etho

d

Bac

teri

al s

trai

nM

ean

valu

es o

f inh

ibiti

on z

ones

± s

tand

ard

erro

rs (m

m) p

rodu

ced

by b

lend

ed o

ils (1

0 m

l/dis

c)O

il bl

end

1O

il bl

end

2O

il bl

end

3(C

inna

mon

10%

,(C

inna

mon

20%

,(C

inna

mon

23%

,G

eran

ium

30%

,L

emon

thym

e 30

%,

Lav

ende

r 23

%,

Tea

Tree

10%

, P

atch

ouli

30%

,L

emon

thym

e 39

%,

Thy

me

50%

)Te

a tr

ee 2

0%)

Thy

me

15%

)

Gra

m-p

ositi

ve b

acte

riaBa

cillu

s su

btili

s PS

SCM

I 007

529

±124

±125

±1St

aphy

loco

ccus

aur

eus A

TCC

259

2331

±129

±128

±1M

ethi

cilli

n-re

sist

ant S

. aur

eus

(11

isol

ates

)24

±119

±125

±1

Gra

m-N

egat

ive

bact

eria

Acin

etob

acte

r ba

uman

ii AT

CC

196

0620

±123

±123

±1M

ultid

rug-

resi

stan

t A. b

aum

anii

JVC

105

318

±122

±122

±1Es

cher

ichi

a co

li AT

CC

259

2216

±116

±120

±1K

lebs

iella

pne

umon

iae

PSSC

MI 0

031

11±1

15±1

-Ps

eudo

mon

as a

erug

inos

a PS

SCM

I 004

810

±121

±110

±1

Valu

es a

re m

ean

±S.E

Supayang Piyawan Voravuthikunchai et al. / Jeobp 15 (5) 2012 739 - 749 747

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Tabl

e 5.

Min

imal

inhi

bitio

n co

ncen

trat

ion

(MIC

) and

min

imal

bac

teri

cida

lco

ncen

trat

ion

(MB

C) o

f ble

nded

oils

aga

inst

pat

hoge

nic

bact

eria

Bac

teri

al s

trai

nD

eter

min

atio

n of

MIC

/MB

C (d

ilutio

n)O

il bl

end

1O

il bl

end

2O

il bl

end

3(C

inna

mon

10%

,(C

inna

mon

20%

,(C

inna

mon

23%

,G

eran

ium

30%

,L

emon

thym

e 30

%,

Lav

ende

r 23

%,

Tea

Tree

10%

, P

atch

ouli

30%

,L

emon

thym

e 39

%,

Thy

me

50%

)Te

a tr

ee 2

0%)

Thy

me

15%

)

Gra

m-p

ositi

ve b

acte

riaBa

cillu

s su

btili

s PS

SCM

I 007

51:

16/1

:16

1:32

/1:3

21:

32/1

:16

Stap

hylo

cocc

us a

ureu

s ATC

C 2

5923

1:32

/1:8

1:64

/1:3

21:

16/1

:16

Met

hici

llin-

resi

stan

t S.a

ureu

s N

PRC

001

1:16

/1:8

1:16

/1:1

61:

16/1

:32

Gra

m-N

egat

ive

bact

eria

Acin

etob

acte

r ba

uman

ii AT

CC

196

061:

16/1

:81:

16/1

:16

1:32

/1:1

6M

ultid

rug-

resi

stan

t A. b

aum

anii

JVC

105

31:

16/1

:81:

16/1

:16

1:16

/1:1

6Es

cher

ichi

a co

li AT

CC

259

221:

8/1:

81:

16/1

:81:

16/1

:16

Kle

bsie

lla p

neum

onia

PSS

CM

I 003

11:

8/1:

81:

8/1:

81:

4/1:

2Ps

eudo

mon

as a

erug

inos

a PS

SCM

I 004

81:

4/>U

1:2/

1:2

1:8/

1:2

U =

und

ilute

d oi

l.

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