Bazı ilaç moleküllerinin klinik acinetobacter baumannii biyofilm yapısı üzerine etkilerinin gerçek zamanlı hücre analizi yöntemi ile incelenmesi
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Dosyalar
Tarih
2019
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Acinetobacter baumannii, ventilatör ilişkili pnömoni ve kateter ilişkili bakteriyemi gibi ciddi nozokomiyal enfeksiyonlara neden olabilen, önemli bir fırsatçı patojendir. Bu bakterinin biyofilm oluşturma özelliği gibi virülans faktörleri ve son yıllarda hızla artan antibiyotik direnci, patogeneze katkı sağlayarak, enfeksiyonların tedavisini zorlaştırmaktadır. Farklı ülkelerde yürütülen çok sayıda araştırma, çoklu ilaç dirençli A.baumannii izolatlarının artışına ve bu izolatlarda biyofilm üretim kapasitesinin de yüksek olduğuna işaret etmektedir. Bu açıdan, antibakteriyel ve antibiyofilm etkinliğe sahip ilaç veya ilaç kombinasyonlarına yönelik araştırmalar değer kazanmaktadır. Ayrıca, tedavi başarısızlıklarında önemli bir rolü olan biyofilm oluşumunun hızlı ve doğru bir şekilde belirlenmesi, enfeksiyonlarla mücadele açısından kritik bir öneme sahiptir.
Çalışmamızda, tigesiklin (TGC), n-asetilsistein (NAS) ve asetilsalisilik asit (ASA) gibi çeşitli ilaç moleküllerinin, klinik A. baumannii izolatlarının biyofilm yapısı üzerine etkilerinin, konvansiyonel ve gerçek zamanlı hücre analizi (Real Time Cell Analyses-RTCA) yöntemleri ile incelenmesi amaçlanmıştır. Ayrıca, incelenen etken maddelerin A. baumannii suşlarında biyofilm ile ilişkili bazı genlerin ekspresyon seviyelerine etkisi de araştırılmıştır.
Ege Üniversitesi Hastanesi’nden izole edilen ve klonal olarak ilişkisiz 22 A.baumanni izolatının biyofilm oluşturma kapasiteleri öncelikle spektrofotometrik mikroplak-kristal viyole yöntemi ile belirlendi. Güçlü biyofilm üreticisi olduğu belirlenen altı temsilci kökenin biyofilm oluşturma kapasiteleri RTCA yöntemi ile de araştırıldı ve elde edilen sonuçların konvansiyonel yöntem ile uyumlu olduğu saptandı. RTCA yöntemi ile 8-10 saat içinde güçlü biyofilm üreticisi kökenler ile zayıf üretici ve negatif kontroller arasında istatistiksel olarak anlamlı fark bulundu. Tigesiklin, NAS ve ASA moleküllerinin tek başlarına ve kombine halde, kökenlerin biyofilm oluşumu ve olgun biyofilmleri üzerine etkileri her iki yöntemle de değerlendirildi ve sub-MİK konsantrasyonlarda TGC+NAS ve TGC+ASA kombinasyonlarının sinerjik etkili olduğu tespit edildi. İncelenen etken maddelerin A. baumannii kökenlerinin biyofilm oluşumu üzerine etkilerini saptamak açısından konvansiyonel ve RTCA yöntemleri paralel sonuçlar verirken, olgun biyofilm üzerine etkilerin değerlendirilmesinde RTCA yöntemi ile herhangi bir etki belirlenemedi. Temsilci A.baumannii kökenlerinde biyofilm oluşumu ile ilişkilendirilen bazı virülans genlerinin (bap, adeG, csuE, ompA) varlığı polimeraz zincir reaksiyonu ile araştırıldı. Altı kökenin tamamında csuE ve ompA genleri tespit edilirken, ikişer kökende bap ve adeG gen varlığı gözlendi. Fenotipik yöntemlerle sinerjik etkili olduğu saptanan ilaç kombinasyonlarının, bazı suşlarda csuE ve bap gen ekpresyon seviyelerinde düşüşe neden olduğu kantitatif gerçek zamanlı ters transkriptaz kantitatif polimeraz reaksiyonu (RT-qPCR) ile saptandı.
Bildiğimiz kadarıyla, A. baumannii kökenlerinde çeşitli ilaç moleküllerinin biyofilm yapısı üzerine etkilerinin RTCA yöntemi ile ilk olarak değerlendirildiği çalışmamızda, genel olarak konvansiyonel yöntem ile uyumlu sonuçlar alınmış, sub-MİK konsantrasyonlarda sinerjik etkili kombinasyonlar saptanmıştır. Ayrıca, incelenen etken maddelerin A. baumannii biyofilmi üzerinde görülen fenotipik etkilerinin, biyofilm ilişkili bazı genlerinin ekspresyon seviyelerindeki azalmaya bağlı olabileceği de ortaya konmuştur. RTCA yöntemi biyofilm üreticisi kökenleri saptamada hızı ve tekrar edilebilirliği ile öne çıkarken, etken maddelerin olgun biyofilm üzerine etkilerinin değerlendirilmesi için uygun gözükmemektedir.
Acinetobacter baumannii is an important opportunistic pathogen that can cause serious nosocomial infections such as ventilator-associated pneumonia and catheter-associated bacteremia. Virulence factors such as biofilm formation and the rapidly increasing antibiotic resistance in recent years contribute to the pathogenesis of this bacterium and making it difficult to treat infections. Many studies conducted in different countries point to an increased prevalence of multidrug-resistant A.baumannii isolates and a high biofilm production capacity in these isolates. In this respect, research on drugs or drug combinations with antibacterial and anti-biofilm activity gains importance. Furthermore, the rapid and accurate identification of biofilm formation, which plays an important role in treatment failures, is critical to combating infections. We aimed to investigate the effects of various drug molecules such as tigecycline (TGC), n-acetylcysteine (NAS) and acetylsalicylic acid (ASA) on the biofilms of clinical A. baumannii isolates by conventional and real time cell analysis (RTCA) methods. In addition, the effect of the examined active substances on the expression levels of some biofilm-related genes in A. baumannii strains was also analyzed. Biofilm formation capacities of 22 clonally unrelated A.baumanni strains isolated from Ege University Hospital, were first determined by spectrophotometric microplate-crystal violet method. The biofilm formation properties of six representative strains identified as strong biofilm producers were also investigated by the RTCA method and the results were found to be compatible with the conventional method. Statistically significant differences were found between strong biofilm producer strains and weak producer and negative controls within 8-10 hours by RTCA method. The effects of tigecycline, NAS and ASA molecules alone and in combination on the biofilm formation and mature biofilms of the strains were evaluated by both methods and TGC + NAS and TGC + ASA combinations were found to be synergistic at sub-MIC concentrations. Conventional and RTCA methods gave parallel results in determining the effects of the examined drug molecules on the biofilm formation of A. baumannii strains. However, no effect was detected by RTCA method in evaluating the activities of same molecules on mature biofilms. The presence of some virulence genes (bap, adeG, csuE, ompA) associated with biofilm formation was investigated by PCR in representative A.baumannii strains. CsuE and ompA genes were detected in all six strains, while bap and adeG genes were observed in two strains. Exposure to drug combinations that were found to be synergistic by phenotypic methods, was found to cause a decrease in csuE and bap gene expression levels in some strains by quantitative real-time reverse transcriptase polymerase reaction (RT-qPCR). To the best of our knowledge, the effects of various drug molecules on the biofilm structure of A. baumannii strains were evaluated by RTCA method for the first time in this study. In general, concordant results with conventional method and RTCA were obtained, and synergistic drug combinations were found at sub-MIC concentrations. Furthermore, it has been shown that the phenotypic effects of the drug combinations studied on A. baumannii biofilms may be due to the decrease in expression levels of some biofilm related genes. While the RTCA method stands out with its speed and reproducibility in detecting biofilm-producing strains, it does not seem to be suitable for evaluating the effects of active substances on mature biofilms.
Acinetobacter baumannii is an important opportunistic pathogen that can cause serious nosocomial infections such as ventilator-associated pneumonia and catheter-associated bacteremia. Virulence factors such as biofilm formation and the rapidly increasing antibiotic resistance in recent years contribute to the pathogenesis of this bacterium and making it difficult to treat infections. Many studies conducted in different countries point to an increased prevalence of multidrug-resistant A.baumannii isolates and a high biofilm production capacity in these isolates. In this respect, research on drugs or drug combinations with antibacterial and anti-biofilm activity gains importance. Furthermore, the rapid and accurate identification of biofilm formation, which plays an important role in treatment failures, is critical to combating infections. We aimed to investigate the effects of various drug molecules such as tigecycline (TGC), n-acetylcysteine (NAS) and acetylsalicylic acid (ASA) on the biofilms of clinical A. baumannii isolates by conventional and real time cell analysis (RTCA) methods. In addition, the effect of the examined active substances on the expression levels of some biofilm-related genes in A. baumannii strains was also analyzed. Biofilm formation capacities of 22 clonally unrelated A.baumanni strains isolated from Ege University Hospital, were first determined by spectrophotometric microplate-crystal violet method. The biofilm formation properties of six representative strains identified as strong biofilm producers were also investigated by the RTCA method and the results were found to be compatible with the conventional method. Statistically significant differences were found between strong biofilm producer strains and weak producer and negative controls within 8-10 hours by RTCA method. The effects of tigecycline, NAS and ASA molecules alone and in combination on the biofilm formation and mature biofilms of the strains were evaluated by both methods and TGC + NAS and TGC + ASA combinations were found to be synergistic at sub-MIC concentrations. Conventional and RTCA methods gave parallel results in determining the effects of the examined drug molecules on the biofilm formation of A. baumannii strains. However, no effect was detected by RTCA method in evaluating the activities of same molecules on mature biofilms. The presence of some virulence genes (bap, adeG, csuE, ompA) associated with biofilm formation was investigated by PCR in representative A.baumannii strains. CsuE and ompA genes were detected in all six strains, while bap and adeG genes were observed in two strains. Exposure to drug combinations that were found to be synergistic by phenotypic methods, was found to cause a decrease in csuE and bap gene expression levels in some strains by quantitative real-time reverse transcriptase polymerase reaction (RT-qPCR). To the best of our knowledge, the effects of various drug molecules on the biofilm structure of A. baumannii strains were evaluated by RTCA method for the first time in this study. In general, concordant results with conventional method and RTCA were obtained, and synergistic drug combinations were found at sub-MIC concentrations. Furthermore, it has been shown that the phenotypic effects of the drug combinations studied on A. baumannii biofilms may be due to the decrease in expression levels of some biofilm related genes. While the RTCA method stands out with its speed and reproducibility in detecting biofilm-producing strains, it does not seem to be suitable for evaluating the effects of active substances on mature biofilms.
Açıklama
Anahtar Kelimeler
Acinetobacter Baumannii, Biyofilm, İlaç Etkileşimleri, Elektrik Empedansı, Cihaz Yeni Kullanım Alanı, Biofilms, Drug Interactions, Electric Impedance, Equipment Reuse
