Investigation of the effects of some drug molecules on clinical Acinetobacter baumannii biofilm structure by real-time cell analysis

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.