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ANTIMICROBIAL ACTIVITY OF O. AMERICANUM AGAINST ORAL MICROORGANISMS

Vol 40 No. 5 September 2009 1025

Correspondence: Sroisiri Thaweboon, Depart-ment of Microbiology, Faculty of Dentistry,Mahidol University, 6 Yothi Road, Bangkok10400, Thailand.E-mail: [email protected]

IN VITRO ANTIMICROBIAL ACTIVITY OFOCIMUM AMERICANUM L. ESSENTIAL OIL AGAINST

ORAL MICROORGANISMS

Sroisiri Thaweboon and Boonyanit Thaweboon

Department of Microbiology, Faculty of Dentistry, Mahidol University Bangkok,Thailand

Abstract. The aim of the present study was to evaluate the efficacy of the essential oilof Ocimum americanum L. on in vitro activity against Streptococcus mutans, Lactobacilluscasei and Candida albicans. An agar disk diffusion method was employed for screeningantimicrobial activity. Minimum inhibitory concentration (MIC) and minimum cidalconcentration (MCC) values of the oil against planktonic cells were determined usingthe Millipore membrane method. The antimicrobial potential of the essential oil wasalso investigated with a biofilm model. The results indicate that essential oil has anti-microbial activity against all tested microorganisms. The MIC values of the oil againstthe three organisms was 0.04% v/v whereas the MCC values for S. mutans, L. casei andC. albicans were 0.08%, 0.3% and 0.08% v/v, respectively. S. mutans and C. albicans weremore sensitive to the essential oil than L. casei. With the biofilm assay, a 5-minute ex-posure to 3% v/v essential oil eliminated 3 log10 of the tested microorganisms. At alower concentration (0.3% v/v), a 2 log10 reduction in S. mutans and C. albicans wasobserved while the lactobacilli were more resistant. This finding indicates the possi-bility of using the essential oil of O. americanum L. in oral health care products forreducing these pathogenic microorganisms in the oral cavity.

more than those of other oral bacteria. Theyhave been shown to have cariogenic poten-tial in both humans and animals (Harper andLoesche, 1984; Loesche, 1986). S. mutans ap-pears to be important in the initiation of den-tal caries since its activities lead to coloniza-tion of the tooth surface, dental plaque ororal biofilm formation and demineralizationof tooth enamel. Lactobacilli, which aremainly found in carious dentin and cemen-tum, have been suspected to be secondaryinvaders that contribute to the progressionof lesions. Oral candidiasis is a commonopportunistic infection of the oral cavitycaused by Candida spp. The incidence variesdepending on age and certain predisposingfactors. Four types of oral condidiasis are

INTRODUCTION

Dental caries and candidiasis are com-mon oral health problems of people in manycountries. These conditions are caused bybacteria and yeast residing in the oral cav-ity. Dental caries is the destruction of thehard tissue of teeth. It is initiated by directdemineralization of the enamel of teeth dueto acids produced by bacteria. Streptococcusmutans and Lactobacillus spp are known bytheir acidogenic and acidophilic properties,

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common, namely pseudomembranous,erythematous, hyperplastic and angularcheilitis. In the debilitated, compromisedhost, oral candidal infection may spread tothe gastrointestinal tract, trachea, lungs, liverand central nervous system, which may re-sult in septicemia, meningitis, hepatosplenicdisease and endocarditis (Samaranayake,2000). The most frequent Candida spp isolateof the oral cavity is C. albicans (Samaranayake,2000). There is a large body of evidence indi-cating Candida can adhere and colonize oralsurfaces including the mucosa and acrylicdenture which represents the first step in theinfectious process. In addition, yeast cellshave the ability to co-aggregate with oral bac-teria (Cannon and Chaffin, 1999).

Natural products have been used as folkmedicine for many years. Among them,hoary basil has attracted increased interestdue to its antimicrobial activity against awide range of pathogenic microorganisms.Ocimum americanum L., hoary basil or mos-quito plant is an annual herbaceous plantnative to Asia and Africa. It is a robust, aro-matic plant reaching 0.7 m high with an erectstem and very green, ovate leaves, grayish-green beneath, and white, greenish or pur-plish pink-white flowers. The whole planthas a strong aromatic scent and three distinctchemotypes: flora-lemony, camphoreceousor spicy. Essential oil is obtained by steamdistillation of the leaves and floweringtops. The oil is colorless to pale yellow andeasily evaporates. From a pharmacologicalperspective, essential oil obtained fromO. americanum L. has shown antibacterial ac-tivity against Staphylococcus aureus, Strepto-myces pyogenes, Escherichia coli and Salmonellatyphosa. Antitubercular activity against My-cobacterium spp was found at a concentrationof 100 ppm. The oil has antifungal activityagainst a great number of fungi includingsome human pathogens (Ntezurubanza et al,1986).

Even though the antimicrobial activityof essential oil against microorganisms hasbeen investigated by many researchers formedical purposes, little information isavailable regarding its efficacy against oralpathogens. The aim of the present study wasto evaluate the efficacy of the essential oil ofO. americanum L. on the in vitro activityagainst the oral microorganisms S. mutans,L. casei and C. albicans as planktonic cells andin a biofilm model.

MATERIALS AND METHODS

Microorganisms

The tested microorganisms were ob-tained from the culture collections of theDepartment of Microbiology, Faculty of Den-tistry, Mahidol University, Thailand. Theorganisms were: S. mutans KPSK2, L. caseiATCC 6363 and C. albicans ATCC 13803. Theywere maintained on Brain Heart Infusion(BHI) agar (BBL, USA). Overnight cultureswere prepared by inoculating approximately2 ml Mueller Hinton broth (Difco, USA) with2-3 colonies of each organism taken fromBHI agar. Broth was incubated overnight at37C. Inocula were prepared by dilutingovernight cultures in saline to approximately108 cfu/ml for bacteria and 107 cfu/ml foryeast. These suspensions were further usedfor antimicrobial determination.

Essential oil

The extraction of the essential oil wascarried out at the laboratory of the ThailandInstitute of Scientific and Technological Re-search by steam distillation of O. americanumleaves.

Disk diffusion method

The microbial growth inhibitory poten-tial of the oil was determined using the diskdiffusion method as described by Washing-ton (1981). Freshly prepared bacterial and



yeast suspensions were spread on MuellerHinton agar. Essential oil (15 l) wasdropped onto sterile paper disks (6 mm di-ameter) and the same volume of 0.2%chlorhexidine gluconate and saline solutionserved as positive and negative controls, re-spectively. The disk was dispensed onto theMueller Hinton agar and incubated at 37Cfor 24-48 hours. All tests were performed intriplicate and zones of inhibition were mea-sured after incubation.

Millipore membrane method

The minimum inhibitory concentration(MIC) and minimum cidal concentration(MCC) values of essential oil were deter-mined using the Millipore method as de-scribed by Tantaoui-Elaraki et al (1992). Inbrief, serial dilutions of essential oil, rang-ing from 2% v/v to 0.04% v/v, were preparedin Mueller Hinton agar incorporated with0.5% v/v Tween-20 to enhance oil solubility.Cellulose acetate membrane filters (0.45 mporosity) (Sartorius, Germany) were placedon the surface of the agar plate and 5 l ofeach microbial suspension was droppedonto each filter. Mueller-Hinton agar with0.5% v/v Tween-20 without essential oil(15 l) was used as a positive growth con-trol. The plates were incubated at 37C for24-48 hours. The MICs were determined asthe lowest concentration of essential oil in-hibiting the visible growth of each organismon the membranes.

For the determination of MCCs, cellu-lose acetate filters without any microbialgrowth were transferred into BHI broth andincubated at 37C for 24-48 hours. The low-est concentration of essential oil that showedno visible growth of the organisms in thetube was interpreted as the MCC.

Biofilm model

The antimicrobial activity of essential oilwas also evaluated on biofilm against S.mutans KPSK2, L. casei ATCC 6363 and C.

albicans ATCC 13803 on the bottom of amicrotiter plate. Biofilm assays were doneusing a protocol modified from Loo et al(2000) and Guggenheim et al (2004). Briefly,all microorganisms were cultivated in fluidthioglycolate medium (Difco, USA) supple-mented with 0.3% glucose for 7 hours. Eachmicrobial suspension (106 cfu/ml) was com-bined and placed at a cold temperature for 6hours. Approximately 500 ml of unstimulatedsaliva was collected from three volunteers 2hours after eating, drinking or having a hy-giene procedure. The pooled saliva was cen-trifuged at 20,000g for 30 minutes at 4C. Thesupernatant was filtered through a 0.45 m-pore membrane (Sartorius, Germany). Ab-sence of any viable microorganisms in thesaliva was checked by culturing on bloodagar. Salivary components were absorbedonto polystyrene, flat-bottom 96-wellmicrotiter plates for 4 hours at room tem-perature in order to form an acquired pel-licle. After removal of excess saliva, thecoated microtiter plates were inoculatedwith a mixture of saliva, thioglycollate me-dium supplemented with 3% glucose and apool of microorganisms. The final density ofinoculum in each well of the microtiter platewas 105 cfu/ml. The microtiter plates werethen incubated at 37C for 24 hours underanaerobic conditions.

Saline solution and 0.2% chlorhexidinegluconate solution were used as negativeand positive controls, respectively. Thebiofilms were exposed to controls and essen-tial oil (0.3% and 3% v/v) for 5 minutes andincubated for 24 hours. After removal of themedia and unattached cells, the biofilmswere washed twice, scrapped and sonicatedin PBS. Serial dilutions of sonicated cellswere cultivated in Mitis-Salivarius Bacitra-cin agar (Difco, USA), Rogosa SL agar (Difco,USA) and Sabouraud dextrose agar (Difco,USA) to determine the number of S. mutans,L. casei and C. albicans, respectively.



RESULTS

Using the disk diffusion method as ascreening test, the essential oil of O.americanum showed antimicrobial activityagainst all of the tested microorganisms witha zone of growth inhibition > 30-19 mm(Table 1). Greater inhibition zones were ob-served when S. mutans and C. albicans wereexposed to the oil compared with L. casei.

The MICs of essential oil obtained bythe method described by Tantaoui-Elarakiet al (1992) are also shown in Table 1. All thetested organisms were inhibited at 0.04%v/v of the oil, whereas the MCC values for

S. mutans, L. casei and C. albicans were 0.08%,0.3% and 0.08% v/v, respectively. S. mutansand C. albicans were more sensitive to essen-tial oil than L. casei.

We also employed the highest MCCvalue of the tested organisms (0.3% v/v) and3% v/v which was 10 x the MCC concentra-tion when investigating the antimicrobialeffect of essential oil against biofilm formedby the species in this study. Table 2 showsthe antimicrobial effect of essential oilagainst multi-species biofilm. After 5 min-utes of exposure, the essential oil at MCCvalue (0.3% v/v) showed a 2 log10 reductionin S. mutans and C. albicans compared to

Zones of inhibition (mm) MIC (% v/v) MCC (% v/v)

1. Essential oilS. mutans 28 0.04 0.08L. casei 19 0.04 0.30C. albicans >30 0.04 0.08

2. 0.2% chlorhexidineS. mutans 21 0.062 0.062L. casei 20 0.125 0.125C. albicans 16 0.062 0.062

Table 1Antimicrobial activity of the essential oil of O. americanum against oral microorganisms in

planktonic state.

MIC, minimum inhibitory concentrationMCC, minimum cidal concentration

Microorganisms 0.3% v/v 3% v/v 0.2% Chlorhexidine Normal salineEssential oil Essential oil gluconate solution

S. mutans 7.2 x 104 2.9 x 103 1.7 x 103 8.5 x 106

L. casei 5.1 x 105 6.3 x 103 2.5 x 103 6.0 x 106

C. albicans 4.5 x 104 3.4 x 103 2.1 x 103 2.8 x 106

Table 2Antimicrobial activity of the essential oil of O. americanum against oral microorganisms in

a biofilm model.

Data presented as cfu/ml of microorganisms



standard growth of biofilm with the nega-tive control. L. casei was found to be moreresistant. At a higher concentration, a 3 log10reduction in S. mutans, L. casei and C.albicans was observed which is similar tothe results obtained from chlorhexidine so-lution.

DISCUSSION

Plant essential oils are valuable naturalcomponents used as raw materials in manyproducts, perfumes, cosmetics, spices, treat-ments, aromatherapy, phytotherapy, spicesand in the field of nutrition (Buchbauer,2000). The oils are complex mixtures com-prised of many compounds. Each constitu-ent contributes to the beneficial or adverseeffects of these oils.

O. americanum has long been used as amedicinal and aromatic plant in many coun-tries. Previous studies reveal the major com-pound found in the essential oil of this plantis eugenol, reaching nearly 30% of the com-pound. Methyl chavicol is the second mostcommon compound and accounted for 17%of the compound. Trepinol has been identi-fied as the third main constituent in the oilcomprising 15% of the compound (Shadiaet al, 2007).

In recent years, due to the widespreadand often indiscriminate use of antimicro-bial drugs, many microorganisms have ac-quired resistance to specific antibiotic treat-ment (Evans, 1999). This has created majorclinical problems in the treatment of infec-tious disease (Davis, 1994). Antibiotics aresometimes associated with adverse effectsincluding hypersensitivity reactions, reduc-tion of beneficial gut and mucosal microor-ganisms, immunosuppression and allergicreactions (Idose et al, 1968). Research of me-dicinal plants has increased and their anti-microbial activity has been evaluated inmany studies (Koo et al, 2000; Smullen et al,

2007; More et al, 2008).A variety of methods have been used to

determine antimicrobial activity of essentialoils depending on the type of essential oiland target organism. Frequently used meth-ods include agar disk diffusion (Rafii andShahverdi, 2007; Fontenelle et al, 2008; Moreet al, 2008) agar well diffusion (Singh et al,2006; Fontenelle et al, 2008) and incorpora-tion of the essential oil in agar media priorto inoculation (Rosato et al, 2008). In thepresent study, essential oil from O. ameri-canum was first screened for its antimicro-bial activity using the agar disk diffusionmethod and then the MIC and MCC wereexamined using the method reported byTantaoui-Elaraki et al (1992). In this method,the organisms were cultured on Milliporemembranes of 0.45 m porosity placed onagar media containing different concentra-tions of the oil.

Agar disk diffusion is the method mostfrequently used to screen plant extracts forantimicrobial activity. However, the useful-ness of this method is limited to the genera-tion of preliminary, qualitative data only, asthe hydrophobic nature of most essential oilsprevents the uniform diffusion of these sub-stances through agar medium (Janssen et al1987; Hili et al 1997). Moreover, essential oilsare poorly soluble in water. This causesmany problems in studying their biologicaland pharmacological properties. To over-come these problems, many researchers haverecommended the use of various solvents inthe dilution of essential oils such as acetone,alcohol, ethylene glycol, ethanol, methanoland DMSO, or using an emulsifier detergentor Tween 20 as was chosen to use in thepresent study (Lahlou, 2004). In addition,their activities show some challenges, forexample, essential oils present a complexchemical composition, some of their con-stituents are volatile and have to be used inlow doses.



According to Coller et al (1989), any an-timicrobial agent is considered effective if thesize of inhibition zone produced, measuredafter deduction of well or disk diameter, is 2mm or more. In the present study, the resultobtained from the disk diffusion methoddemonstrated the essential oil of O.americanum possessed strong antimicrobialactivity since the zones of inhibition pro-duced by the oil against S. mutans, L. caseiand C. albicans were 28, 19 and >30 mm, re-spectively (disk diameter was 6 mm). Eventhough the essential oil seems to be moreeffective than chlorhexidine solution, itshould be noted that the concentration ofchlorhexidine used was 0.2% whereas the oilhad not been diluted. The antifungal activ-ity of the oil against C. albicans was similarto that reported by Tajo and Thoppil (1999)and Hammer et al (1999). However, the MICvalue found in the current study (0.04% v/v)was much lower than the previous report(0.5% v/v). The difference may be from theexperimental conditions (method, tempera-ture, photoperiod, etc), materials, themethod of extraction of the essential oil,which influence the chemical composition,the dissolution and dispersion of the oil andthe tested organisms. Furthermore, the com-position of plant oils is known to vary ac-cording to local climatic and environmentalconditions (Janssen et al, 1987; Sivropoulouet al, 1995).

Although the antimicrobial activity ofessential oil of O. americanum has alreadybeen reported for various pathogenic micro-organisms, no data has been obtained re-garding the activity against oral bacteria,especially S. mutans and L. casei. The resultsof this study demonstrate, in free form orplanktonic state, the oil has antibacterial ac-tivity against S. mutans and L. casei. S. mutansis more susceptible to the oil than L. casei,the MCC value for S. mutans was 0.08% v/vbut the L. casei was 0.3% v/v. The components

of the essential oil are known to act on thecell membrane of both gram-positive andgram-negative bacteria. Essential oil wasfound to be composed of mainly eugenol,methyl chavicol and trepinol (Shadia et al,2007). Previous studies revealed the mecha-nisms of action of eugenol and trepinol ondisrupting bacterial outer membranes andmitochondria as well as disturbing cell struc-tures (Oyedemi et al, 2009). In addition, theinhibition of energy generation by inhibitionof glucose uptake and glucose utilization hasbeen observed (Gill and Holley, 2004). Forchavicol, the proposed antimicrobial actionalso involved the alteration of the bacterialcell membrane structures which result in thedisruption of membrane permeability(Sharma et al, 2009).

In the oral cavity, most colonizing andinfecting microorganisms are found not assingle-living cells but rather as complexstructured microbial communities. They areoften encapsulated within a matrix ofexopolymeric material and attached to bi-otic or abiotic surfaces (Kolenbrander,2000). These communities are referred to asbiofilms. Of clinical relevance is the factthat biofilm is more resistant to immunedefense mechanisms and less susceptible toantimicrobial agents, which can display en-hanced pathogenicity (Quave et al, 2008).Many microbiological studies tested oralantimicrobial agents against growing cellsin a planktonic state. The use of an in vitrobiofilm model provides an alternative ap-proach for examining the antimicrobial ef-fects of agents against oral biofilm which isa key to the existence and survival of thesemicroorganisms with inter- and intra-spe-cies interactions in the oral cavity. We em-ployed an in vitro biofilm formed by threeoral pathogens (S. mutans, L. casei and C.albicans) more suitable for testing the agentaims to treat biofilm-related diseases in theoral cavity.



Our results show, despite the inhibitoryand effects on the tested microorganisms inplanktonic state, the essential oil from O.americanum leaves exhibited a less pro-nounced effect upon these microorganismsin biofilm form. Exposure to the essentialoil at 0.3% v/v, the highest MCC values inthe planktonic state, for 5 minutes elimi-nated only 2 log10 of S. mutans and C.albicans (Table 2). However, at a higher con-centration (3% v/v), the effect was similarto that of chlorhexidine gluconate (a 3 log10reduction in all tested microorganisms).This indicates that microorganisms in thebiofilm are strongly protected and less sus-ceptible to antimicrobial agents than inplanktonic cells. The cells in biofilm exhibitproperties distinct from those of planktoniccells, including resistance to antimicrobialagents, physiological properties and inter-action with host tissues (Wilson, 1996). Ex-periments comparing biofilms of oral bac-teria strains with broth cultures usingchlorhexidine gluconate have demon-strated that much higher concentrations ofchlorhexidine are required to significantlyinhibit existing biofilm cells (Pratten et al,1998; Shani et al, 2000).

Taken together, our results indicate theessential oil of O. americanum acts as a po-tent antimicrobial agent that can disrupt ex-isting biofilm. However, this study was con-ducted on planktonic and artificial biofilmin vitro. A clinical study may be required toconfirm the validity of the results obtained.Additionally, further investigations of thetoxic or irritant properties are imperativewhen considering their use in humans.

In summary, the results of the presentstudy suggest the essential oil of O.americanum at a concentration of 3% v/v maybe a beneficial component of oral health careproducts, such as mouthrinse and tooth-paste, that provide prevention against cari-

ogenic bacteria and candidal infection. Theuse of this oil would depend on cost consid-erations, odor and flavor.

ACKNOWLEDGEMENTS

The authors would like to thank DrSiripen Jarikasem and Dr Bhusita Vannis-sorn of the Thailand Institute of Scientificand Technological Research for providingthe essential oil for this study.

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