Barbosa, Joana

Teixeira, Paula

English

Repositório Institucional da Universidade Católica Portuguesa

In vitro antimicrobial activity of variousessential oils against Clostridioides difficileJoana Barbosa, Paula TeixeiraUniversidade Católica Portuguesa, CBQF—Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Porto, PortugalAcknowledgementsIntroductionClostridioides difficile is a Gram-positive, endospore-forming, and toxin-producinganaerobic organism responsible for mild-to-severe forms of gastrointestinalinfections, ranging from asymptomatic intestinal colonization, self-limiting milddiarrhea to severe or life-threatening pseudomembranous colitis, toxicmegacolon, sepsis, and death.[1,2] Since C. difficile infection is associated with theantibiotics use and hypervirulent strains have shown antibiotic resistances,alternative therapeutics are urgently required. Plant-derived essential oils arenatural antimicrobials found in many plants and could be capable of inhibitingthe growth and survival of some microorganisms.[3] Essential oils in aromaticplants are among the most significant active compounds of herbs and spices.Various biological characteristics, such as digestive, anti-inflammatory, sedative,antioxidant, antimicrobial, antiviral. Cytotoxic activities have also been attributedto the essential oils.[4]ObjectivesThe objective of this study was to determine in vitro antimicrobial activity ofvarious essential oils against C. difficile isolates, aiming for a future anti-C. difficilestrategy.Table 1. Zones of growth inhibition (mm; mean±standard deviation) showinganticlostridial activity of testedConclusions- The greatest anticlostridial activity was obtained for essential oils of oregano (oregano V. and oregano C.E) and thyme, being this last one the best, with translucent halozones with diameters higher than 85 mm. Also good inhibitions were achieved for several essential oils, such as of basil, bay, cumin, nutmeg or parsley. In contrast, noinhibition was obtained for essential oils of garlic and anise against all C. difficile isolates.- The highest inhibitions observed in the disk diffusion assay were in accordance with the lowest MIC and MBC values obtained. Essential oils of oregano and thymepresented MIC and MBC values ranging from 0.39 to 3.13% (v/v). Also essential oils of basil, bay, coriander, parsley and rosemary allowed inhibiting C. difficile isolates withlow MIC and MBC values.- The evaluation of antimicrobial activity of essential oils observed against C difficile isolates revealed that several essential oils are potentially useful as alternative toantibiotics on the treatment and/or prevention of C. difficile infection.ResultsEssential oil CdH63866 CdI805452 CdI805937 CdV315639Anise NI NI NI NIBasil 30.5 ± 2.1 28.5 ± 2.1 26.5 ± 2.1 29.5 ± 0.7Bay 26.5 ± 3.5 41.5 ± 12.0 40.0 ± 0.0 41.0 ± 7.1Carrot 11.5 ± 0.7 18.0 ± 2.8 19.0 ± 5.7 16.0 ± 5.7Cloves 35.0 ± 2.8 25.0 ± 5.7 22.0 ± 0.0 27.0 ± 7.1Coriander 31.0 ± 8.5 25.5 ± 7.8 30.0 ± 2.8 19.0 ± 1.4Cumin 36.5 ± 4.9 35.0 ± 1.4 36.0 ± 5.7 43.5 ± 9.2Fennel 10.0 ± 2.8 10.0 ± 1.4 13.5 ± 4.9 16.0 ± 5.7Garlic NI NI NI NIGinger 19.0 ± 1.4 19.0 ± 1.4 18.0 ± 2.8 11.0 ± 1.4Lemon 21.0 ± 1.4 21.5 ± 0.7 11.0 ± 1.4 11.5 ± 0.7Nutmeg 27.0 ± 7.1 19.5 ± 2.1 36.0 ± 5.7 30.5 ± 0.7Oregano V. 63.0 ± 7.1 77.5 ± 10.6 75.0 ± 7.1 79.0 ± 8.5Oregano C.E. 60.5 ± 7.8 55.0 ± 4.2 62.5 ± 3.5 66.0 ± 2.8Parsley 26.5 ± 7.8 36.5 ± 4.9 39.0 ± 4.2 33.0 ± 7.1Pepper mint 13.5 ± 3.5 17.5 ± 3.5 11.5 ± 2.1 11.0 ± 1.4Rosemary 29.0 ± 4.2 11.0 ± 1.4 13.0 ± 4.2 35.5 ± 7.8Sage 34.5 ± 7.8 27.0 ± 4.2 30.5 ± 7.8 20.5 ± 2.1Thyme 71.0 ± 0.0 >85 ± 0.0 >85 ± 0.0 >85 ± 0.0Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) of essential oils tested (results are expressed in % v/v)Essential oil CdH63866 CdI805452 CdI805937 CdV315639BasilMIC 25 12.5 25 25MBC 25 6.25 50 25BayMIC 3.13 6.25 6.25 6.25MBC 6.25 6.25 6.25 6.25CarrotMIC 50 50 50 50MBC >50 50 >50 >50ClovesMIC 50 50 50 50MBC >50 >50 >50 >50CorianderMIC 12.5 6.25 6.25 12.5MBC 12.5 12.5 12.5 12.5CuminMIC 25 25 25 25MBC 50 50 50 50FennelMIC 50 25 25 50MBC >50 50 50 >50GingerMIC >50 >50 >50 >50MBC >50 >50 >50 >50LemonMIC 50 50 50 >50MBC >50 >50 >50 >50NutmegMIC 50 50 25 25MBC >50 >50 50 50Oregano V.MIC 3.13 0.39 0.39 0.39MBC 0.39 0.39 0.39 0.39Oregano C.E.MIC 0.39 0.39 0.39 0.39MBC 0.39 0.39 0.39 0.39ParsleyMIC 3.13 6.25 6.25 6.25MBC 6.25 6.25 6.25 6.25Pepper mintMIC 50 50 25 50MBC 50 50 25 50RosemaryMIC 25 12.5 50 6.25MBC 50 25 50 12.5SageMIC 25 12.5 12.5 12.5MBC 25 25 25 25ThymeMIC 3.13 0.39 0.39 0.39MBC 0.39 0.39 0.39 0.39Methods1. Essential oils and target C. difficile2. Disk diffusion assay3. Determination of minimum inhibitory and bactericidal concentrationsAnise (Pimpinella anisum), basil (Ocimum basilicum), bay (Laurusnobilis L.) and fennel (Foeniculum vulgare), carrot (Daucus carrot L.),cloves (Syzygium aromaticum), coriander (Coriandrum sativum), cumin(Cuminum cyminum), nutmeg (Myristica fragrans), parsley(Petroselinum crispum), oregano (Origanum vulgare L.), rosemary(Rosmarinus officinalis), pepper mint (Mentha piperita L.) and sage(Salvia officinalis) lemon (Citrus limon), garlic (Allium sativum), ginger(Zingiber officinale), oregano (Origanum vulgare) and thyme (Thymusvulgaris)23 plantessentialoilsC. difficile H63866C. difficile I805452C. difficile I805937C. difficile V315638 (gently provided by Hospital de S. Marcos, Braga) 4 clinicalC. difficilestrainsSpread100 μlC. difficile suspension Brain Heart Infusion (0.7% w/v agar) + 0.1% sodium thaurocolate37 ºC for 24 to 48h, anaerobiosisFilter paperdisks5 μl of essential oil Diameter oftranslucenthalo (mm)Concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39% (v/v)For translucenthalos >10 mmReferences[1] Kachrimanidou M., Malisiovas N. (2011). Clostridium difficile infection: a comprehensivereview. Crit Rev Microbiol, 37: 178-187.[2] Rupnik M., Wilcox M.H., Gerding D.N. (2009). Clostridium difficile infection: newdevelopments in epidemiology and pathogenesis. Nat Rev Microbiol 7: 526-536.[3] Calo J.R., Crandall P.G., O'Bryan C.A., Ricke S.C. (2015). Essential oils as antimicrobials infood systems – A review. Food Control, 54: 111-119.[4] Raut J.S., Karuppayil S.M. (2014). A status review on the medicinal properties of essentialoils. Ind Crops Prod, 62: 250-264.This work was supported by funding from the National Funds from the Fundação para a Ciência e a Tecnologia (FCT) through project UIDB/50016/2020. Financialsupport for author J. Barbosa was provided by a post-doctoral fellowship SFRH/BPD/113303/2015 (FCT).100 μl100 μlDilutions in Müeller-HintonBroth (1:1)C. difficile suspension37 ºC for 24 to 48h, anaerobiosisEssential oils solutionMBC10 μl of each well (with no microbial growth) was spread into MHA and incubated under the same conditionsMIClowest concentration of essential oil which prevented growth

In vitro antimicrobial activity of various essential oils against clostridioides difficile

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In vitro antimicrobial activity of various essential oils against clostridioides difficile

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