Doğal kaynaklı bazı bileşiklerin Proteus mirabilis kökenleri üzerine antibakteriyel ve antivirülans etkinliklerinin incelenmesi
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Tarih
2018
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, terpenler ve fenolikler grubundan bazı fitokimyasalların, tipik bir üropatojen olan Proteus mirabilis'e antibakteriyel ve antivirülans (anti-patojenik) etkilerinin incelenmesi amaçlanmıştır. Bu kapsamda, Ege Üniversitesi Tıp Fakültesi Tıbbi Mikrobiyoloji Anabilim Dalı Bakteriyoloji Laboratuvarında izole edilmiş klinik P. mirabilis izolatları ve Mobley Araştırma Laboratuvarından (Ann Arbor, Amerika Birleşik Devletleri) temin edilmiş olan P. mirabilis HI4320 referans kökeninde, alfa-terpineol, bisabolol, dekanol, farnesol, hederasaponin, karvakrol, kuersetin, öjenol, ökaliptol, sinnamaldehit, terpinen-4-ol ve timolün minimum inhibitör konsantrasyonları (MİK) belirlenmiştir. Daha sonra bu bileşikler ve idrar yolu enfeksiyonlarında (İYE) kullanılan antibiyotiklerin (ko-amoksilav, ko-trimoksazol, meropenem, nitrofurantoin, seftazidim, siprofloksasin) kombine etkileri dama tahtası yöntemiyle incelenmiştir. Ayrıca, ilgili bileşiklerin referans kökende swarming (yayılma) hareketi, üreaz ekspresyonu ve biyofilm oluşumuna etkileri araştırılmıştır. Son olarak, inhibitör etkili bulunan bileşiklerin, virülans faktörlerinin regülasyonunda rol oynayan bazı genlerin (flaA, flhD, glnA, hisG, mrpJ, ompF, rcsB, rcsD, speB, umoC, ureD) ekspresyon düzeylerine etkileri real-time ters transkriptaz kantitatif polimeraz zincir reaksiyonu (gerçek zamanlı RT-kPZR) ile ortaya konmuştur. Mikrodilüsyon yöntemiyle belirlenen MİK değerlerine göre en güçlü antimikrobiyal etkinlik gösteren bileşikler karvakrol (128-256 µg/ml), sinnamaldehit (256-512 µg/ml) ve timoldür (128-512 µg/ml). Buna karşın, kombinasyon çalışmalarında antibiyotikler ve bileşikler arasında herhangi bir konsantrasyonda sinerjik etki tespit edilememiştir. Karvakrol, kuersetin, sinnamaldehit ve timolün yayılma hareketini dozla doğru orantılı olarak inhibe ettiği görülmüştür. Ayrıca, sinnamaldehit üreaz ekspresyonunu da dozla doğru orantılı olarak inhibe etmiştir. Yayılma hareketini baskılayan karvakrol ve timol biyofilm üretiminde inhibisyona, kuersetin ve sinnamaldehit ise aktivasyona neden olmuştur. Bu bileşiklerin seçilen genlerin ekspresyon düzeylerine etkileri değerlendirildiğinde, besiyeri formuna bağlı olarak etkilerinin de farklılaştığı görülmüştür. Bu tez çalışmasında, özgün yayılma hareketi, yoğun üreaz sentezi, kristal içerikli biyofilm oluşturması, idrar yolu ve böbrek taşlarına neden olması gibi ayırt edici özellikleriyle öne çıkan P. mirabilis model bakteri olarak kullanılmış ve yaygın olarak bilinen timol, karvakrol, sinnamaldehit, kuersetin gibi bileşikler bu tür özelliklere inhibitör etkili bulunmuştur. Söz konusu bileşikler, genellikle polimikrobiyal enfeksiyonlara yol açan bu bakteriyle birlikte, diğer üropatojenlere de etkili olabilir. Timol ve karvakrolün genel olarak toksik olduğu bilinmektedir. Buna karşın, kuersetin ve sinnamaldehit oldukça düşük toksisiteye sahip bileşiklerdir. Gıda olarak ya da fitoterapide kullanılan bu bileşikler çeşitli bitkilerde yaygın olarak bulunur. Bu tür avantajları göz önünde bulundurulduğunda, İYE proflaksisinde ilgili bileşiklerden yararlanılabileceği öngörülmektedir. Böylelikle, kullanılan antibiyotiklerin dozu azaltılarak, ekonomik anlamda kazanç edinilebilir ve direnç gelişimi belli ölçüde azaltılabilir. Ayrıca, bu tez kapsamında yapılan in vitro çalışmalara ek olarak gerçekleştirilecek in vivo çalışmalarla, ilgili bileşiklerin kaplama ajanı olarak kullanıldığında kateter ömrünü ne kadar uzattığı araştırılmalıdır.
In this study, it was aimed to investigate the antibacterial and anti-pathogenic activities of some phytochemicals from terpenes and phenolics against Proteus mirabilis, a typical uropathogen. In this context, minimum inhibitor concentrations (MIC) of alpha-terpineol, bisabolol, decanol, farnesol, hederasaponin, carvacrol, quercetin, eugenol, eucalyptol, cinnamaldehyde, terpinen-4-ol and thymol were determined in clinical isolates of P. mirabilis isolated in Ege University Medical Faculty Medical Microbiology Department Bacteriology Laboratory and P. mirabilis HI4320 reference strain from Mobley Research Laboratories (Ann Arbor, USA). The combined effects of these compounds and antibiotics (co-amoxilav, co-trimoxazole, meropenem, nitrofurantoin, ceftazidime, ciprofloxacin) used in urinary tract infections (UTIs) were then examined using the checkerboard method. In addition, the effects of related compounds on the formation of swarming motility, urease expression and biofilm formation of the reference strain were investigated. Finally, the effects of compounds with inhibitory activity on the expression levels of certain genes (flaA, flhD, gnNA, HisG, mRpJ, ompF, rcB, rcSD, speB, umoC, ureD) that play a role in the regulation of virulence factors were examined by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). Compounds showing the strongest antimicrobial activity are carvacrol (128-256 μg/ml), cinnamaldehyde (256-512 μg/ml) and thymol (128-512 μg/ml) according to the MIC values determined by microdilution method. However, no synergistic effect was observed at any concentration between antibiotics and compounds in combination studies. It has been shown that carvacrol, quercetin, cinnamaldehyde and thymol inhibit the swarming motility in a döşe dependent manner. Furthermore, cinnamaldehyde has been found to be a dose dependent inhibitör against urease expression. Carvacrol and thymol, which suppress the swarming motility, caused inhibition in the production of biofilms, although quercetin and cinnamaldehyde cause activation. When the effects of these compounds on the expression levels of selected genes were assessed, their effects were different depending on the form of the medium. In this thesis study, P. mirabilis, a model bacterium which is distinguished by prominent characteristics such as swarming motility, intensive urease synthesis, formation of crystallized biofilm, causing urinary tract and kidney stones were used, and compounds that commonly known such as thymol, carvacrol, cinnamaldehyde, quercetin were found effective against such properties. These compounds may also be effective against other uropathogens, together with this bacterium, which generally leads to polymicrobial infections. Timol and carvacrol are generally known to be toxic. However, quercetin and cinnamaldehyde have a very low toxicity. These compounds, which are used as food or in phytotherapy, are widely available in various plants. Given these advantages, it is foreseen that the relevant compounds may be utilized in the UTIs prophylaxis. Thus, by reducing the amount of antibiotics used, economic gains can be gained and the development of resistance can be reduced to a certain extent. Furthermore, the extent to which the catheter's life is prolenged when the compounds are used as coating agents should be investigated by in vivo studies, in addition to the in vitro studies performed within the scope of this thesis.
In this study, it was aimed to investigate the antibacterial and anti-pathogenic activities of some phytochemicals from terpenes and phenolics against Proteus mirabilis, a typical uropathogen. In this context, minimum inhibitor concentrations (MIC) of alpha-terpineol, bisabolol, decanol, farnesol, hederasaponin, carvacrol, quercetin, eugenol, eucalyptol, cinnamaldehyde, terpinen-4-ol and thymol were determined in clinical isolates of P. mirabilis isolated in Ege University Medical Faculty Medical Microbiology Department Bacteriology Laboratory and P. mirabilis HI4320 reference strain from Mobley Research Laboratories (Ann Arbor, USA). The combined effects of these compounds and antibiotics (co-amoxilav, co-trimoxazole, meropenem, nitrofurantoin, ceftazidime, ciprofloxacin) used in urinary tract infections (UTIs) were then examined using the checkerboard method. In addition, the effects of related compounds on the formation of swarming motility, urease expression and biofilm formation of the reference strain were investigated. Finally, the effects of compounds with inhibitory activity on the expression levels of certain genes (flaA, flhD, gnNA, HisG, mRpJ, ompF, rcB, rcSD, speB, umoC, ureD) that play a role in the regulation of virulence factors were examined by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). Compounds showing the strongest antimicrobial activity are carvacrol (128-256 μg/ml), cinnamaldehyde (256-512 μg/ml) and thymol (128-512 μg/ml) according to the MIC values determined by microdilution method. However, no synergistic effect was observed at any concentration between antibiotics and compounds in combination studies. It has been shown that carvacrol, quercetin, cinnamaldehyde and thymol inhibit the swarming motility in a döşe dependent manner. Furthermore, cinnamaldehyde has been found to be a dose dependent inhibitör against urease expression. Carvacrol and thymol, which suppress the swarming motility, caused inhibition in the production of biofilms, although quercetin and cinnamaldehyde cause activation. When the effects of these compounds on the expression levels of selected genes were assessed, their effects were different depending on the form of the medium. In this thesis study, P. mirabilis, a model bacterium which is distinguished by prominent characteristics such as swarming motility, intensive urease synthesis, formation of crystallized biofilm, causing urinary tract and kidney stones were used, and compounds that commonly known such as thymol, carvacrol, cinnamaldehyde, quercetin were found effective against such properties. These compounds may also be effective against other uropathogens, together with this bacterium, which generally leads to polymicrobial infections. Timol and carvacrol are generally known to be toxic. However, quercetin and cinnamaldehyde have a very low toxicity. These compounds, which are used as food or in phytotherapy, are widely available in various plants. Given these advantages, it is foreseen that the relevant compounds may be utilized in the UTIs prophylaxis. Thus, by reducing the amount of antibiotics used, economic gains can be gained and the development of resistance can be reduced to a certain extent. Furthermore, the extent to which the catheter's life is prolenged when the compounds are used as coating agents should be investigated by in vivo studies, in addition to the in vitro studies performed within the scope of this thesis.
Açıklama
Anahtar Kelimeler
Proteus Mirabilis, Fitoterapötik, Kuersetin, Sinnamaldehit, Anti-Patojenik, Gerçek Zamanlı Polimeraz Zincir Reaksiyonu, Proteus Mirabilis, Phytotherapeutic, Quercetin, Cinnamaldehyde, Anti-Pathogenic, Real-Time Polymerase Chain Reaction
