Publication: Assessment of carvacrol as a bacterial efflux pump inhibitor
Abstract
KARVAKROLÜN DIŞA ATIM POMPASI İNHİBİTÖRÜOLARAK DEĞERLENDİRİLMESİSon yıllarda antibiyotiklerin yanlış kullanımı sonucu çoklu ilaca dirençli bakterilerinsayısında bir artış olmuştur. Çoklu ilaç dirençli bakteriler antibiyotiklere karşı çeşitlidirenç mekanizmaları geliştirmişlerdir. Dışa atım pompaları bu dirençmekanizmalarından birisidir, bakteride verilen antibiyotiği hücre dışına atarak hücreiçindeki konsantrasyonu düşürür ve hücrenin hayatta kalmasını sağlar. Bu pompalarınıninhibisyonu ile söz konusu direnç mekanizması engellenebilir. Son zamanlarda çoklu ilaçdirençli bakteri enfeksiyonlarının tedavisinde mevcut antibiyotiklerle dışa atım pompasıinhibitörlerinin birlikte verilmesi söz konusudur. Kekik gibi aromatik bitkilerde bulunanve uçucu yağların bir bileşeni olan karvakrol son zamanlarda potansiyel bir dışa atımpompası inhibitörü olarak değerlendirilmektedir. Bu çalışmada, karvakrolün kendisininve antimikrobiyel etki gösteren substratlarla kombinasyonunun Bacillus subtilishücrelerindeki etkisi değerlendirilmiş ve hedef aldığı dışa atım pompası/ pompalarıaraştırılmıştır. Öncelikle bitki tabanlı alkaloit olan berberinle sinerjisi araştırılmış ve heriki bileşenin de birbirlerinin aktivitesine katkısı olduğu gözlemlenmiştir. Bileşenler içinbakteriyel büyüme eğrileri elde edilmiş, 75 μg/ mL berberin ve 50 μg/ mL karvakrolkombinasyonunun hücre ölümüne; 75 μg/ mL berberin ve 25 μg/ mL karvakrolkombinasyonunun ise büyümede gecikmeye sebep olduğu belirlenmiştir. In-vitro etkideğerlendirmesi ve gen ekspresyonu seviyesindeki değişimlerin belirlenmesi için 75μg/ mL berberin ve 25 μg/ mL karvakrol kombinasyonunun kullanılmasına kararverilmiştir. Her bir bileşenin tek başına ya da birlikte kullanımının hücre zarındabozulmalara yol açtığı belirlenmiştir. Yedi adet dışa atım pompası belirlenip qPCRyöntemi ile değerlendirilmiştir. Analiz sonucunda, seçilen tüm genlerde ekspresyon artışıbelirlenmiştir. Tüm bu sonuçlar değerlendirildiğinde, karvakrolün etkili bir antibakteriyelaktivite gösterdiği belirlenmiş, dışa atım pompası inhibitörü olabileceği düşünülmüştür.
ASSESSMENT OF CARVACROL AS AN EFFLUX PUMPINHIBITORLately, there has been an increase in the number of antimicrobial resistant bacteria, whichis commonly associated with the overuse and misuse of drugs, especially antibiotics.Emergence of this resistance constitutes an important issue in health care, resulting in thefailure of treatment of bacterial infections. One resistance mechanism acquired bymicroorganisms is the active efflux of drugs by efflux pumps. These proteins prevent theintracellular accumulation of the relevant drug, keeping it from reaching its target andthus the drug loses its effectiveness. Recently, the treatment of multidrug-resistantbacterial infections involves the administration of current antibiotics with efflux pumpinhibitors. Carvacrol, a component of essential oils found in aromatic plants such asthyme and oregano, has recently been considered as a potential efflux pump inhibitor. Inthis study, the effectiveness of carvacrol alone and its combination with berberine inBacillus subtilis cells was evaluated and their efflux pump targets were investigated.Firstly, the synergism between carvacrol and berberine was investigated by themicrodilution checkerboard technique and the synergism between the compounds wasfound to be additive. Bacterial growth profiles were obtained for both carvacrol andberberine. The combination of 75 μg/ mL berberine and 50 μg/ mL carvacrol resulted incell death and the combination of 75 μg/ mL berberine and 25 μg/ mL carvacrol was foundto cause retardation in cell growth. Subsequently, the combination of 75 μg/ mL berberineand 25 μg / mL carvacrol was selected to be used for further evaluation of the in-vitroeffect and determination of changes in gene expression levels. Each compound, alone orin combination, has been found to cause deterioration of the cell membrane. Seven effluxpumps were selected and their expressional changes have been evaluated by qPCR. As aresult, overexpression was identified in all selected genes and it was determined thatcarvacrol manifested an efflux pump inhibitory potential.
ASSESSMENT OF CARVACROL AS AN EFFLUX PUMPINHIBITORLately, there has been an increase in the number of antimicrobial resistant bacteria, whichis commonly associated with the overuse and misuse of drugs, especially antibiotics.Emergence of this resistance constitutes an important issue in health care, resulting in thefailure of treatment of bacterial infections. One resistance mechanism acquired bymicroorganisms is the active efflux of drugs by efflux pumps. These proteins prevent theintracellular accumulation of the relevant drug, keeping it from reaching its target andthus the drug loses its effectiveness. Recently, the treatment of multidrug-resistantbacterial infections involves the administration of current antibiotics with efflux pumpinhibitors. Carvacrol, a component of essential oils found in aromatic plants such asthyme and oregano, has recently been considered as a potential efflux pump inhibitor. Inthis study, the effectiveness of carvacrol alone and its combination with berberine inBacillus subtilis cells was evaluated and their efflux pump targets were investigated.Firstly, the synergism between carvacrol and berberine was investigated by themicrodilution checkerboard technique and the synergism between the compounds wasfound to be additive. Bacterial growth profiles were obtained for both carvacrol andberberine. The combination of 75 μg/ mL berberine and 50 μg/ mL carvacrol resulted incell death and the combination of 75 μg/ mL berberine and 25 μg/ mL carvacrol was foundto cause retardation in cell growth. Subsequently, the combination of 75 μg/ mL berberineand 25 μg / mL carvacrol was selected to be used for further evaluation of the in-vitroeffect and determination of changes in gene expression levels. Each compound, alone orin combination, has been found to cause deterioration of the cell membrane. Seven effluxpumps were selected and their expressional changes have been evaluated by qPCR. As aresult, overexpression was identified in all selected genes and it was determined thatcarvacrol manifested an efflux pump inhibitory potential.