Evaluation of virulence factors of hospital-acquired Pseudomonas aeruginosa strains for quorum sensing and their effects on epithel cells

Pseudomonas aeruginosa (P. aeruginosa) is an important cause of morbidity and mortality in individuals with suppressed immünity system. The quorum sensing (QS) mechanism and virulence factors which it possesses produce epithelial tissue damage, causing systemic infections. In this study, virulence factors of clinic isolates resistant to ceftazidime were evaluated in terms of QS and their effects on epithelial cells were investigated. Antibiotic susceptibility of 100 isolates of Pseusomonas aeruginosa resistant to ceftazidime among hospital samples were analyzed with the disk diffusion and the microdilution method, and their virulence factors were analyzed with phenotypic tests in terms of formation of biofilm, slime factor, homoserin lactone existence, and production of pyocyanin, elastase and protease. The existence of QS genes (intact and internal lasI, lasR, rhlI and rhlR) that involved in the regulation of virulence factors was shown with PCR method. Effects of N-3-oxododecanoly-L-homoserin lakton (3-oxo-C12-HSL) which is one of the virulence factor of P. aeruginosa, on the cytotoxicity and Cox-2 and IL-8 expression were investigated by the MTT, real time PCR and Western Blotting method in hTERT-HPNE cells. Intensive care units were found to be at the first rank with 38%, when the distribution of the isolates was evaluated according to hospital services. A significant difference was found between hospital services and antibiotic resistance (p<0.05). It was found that 76% of isolates formed biofilm, 57% slime factor and 51% homoserin lactone, 65% of isolates produced pyocyanin, 66% elastas, and 88% protease and they were found to be mobile. In QS analysis, lasI gene was found to be 96% intact and 95% internal and lasR gene was found to be 91% intact 95% internal for las system; rhlI gene was found to be 91% intact 90% internal and rhlR gene was found to be 80% intact and 90 % internal for rhl system. 3-oxo-C12-HSL was determined to be cytotoxic on hTERT-HPNE cells (IC50 75 μM). 3-oxo-C12-HSL increased the mRNA level by 2-5 fold and IL-8 mRNA level by 1.5-2 fold. Cox-2 and IL-8 proteins were also increased. Consequently, this study shows that the antibiotic resistance profile and virulence factor levels of clinical isolates were high and also they increase the levels of proinflammatory cytokines in hTERT-HPNE cells. This study shows that P. aeruginosa in the pancreas causes colonization and induces inflammation, and emphasizes the importance of developing targeted drugs to prevent this inflammation. Keywords: Pseudomonas aeruginosa, Virulence factors, hTERT-HPNE, Real Time PCR, Western Blotting.