Molecular analysis of the effect of antimicrobial subinhibitory concentrations on Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is the leading opportunistic pathogen which causes nosocomial infections. Release of some virulence factors of P. aeruginosa is controlled via a mechanism called Quorum Sensing (QS) which is dependent on cell density. Biofilm formation, which is an important virulence factor of bacteria, cause difficulties in treatment of P. aeruginosa infections.The aim of this study is to determine the the change in the ability of P. aeruginosa strains isolated from various clinic samples at Gazi University Medical Faculty Hospital, Microbiology and Clinic Microbiology Laboratory, to produce biofilm in the presence of antimicrobial subinhibitory concentrations. In addition, we aim to investigate the presence of QS genes related with biofilm formation and to determine whether there is a relationship between biofilm formation intensity and QS genes.24 P. aeruginosa strains used in the study were isolated from: 2 sputum samples (8.3%), 2 tracheal aspirate samples (8.3%), 5 urine samples (20.9%), 6 wound samples (25%) and 9 (37.5%) various clinic samples (blood, biopsy, feces etc.). P. aeruginosa ATCC 27853 strain was used as control.According to biofilm absorbance values of P. aeruginosa ATCC 27853, 5 strains (20.8%) formed a biofilm at a rate of %50 and below (weak), 6 strains (25%) at a rate of 50%-100% (intermediate), 13 strains (54.2%) at a rate over 100% (strong).MIC values of 24 strains studied was determined via microdulition method to be in the range of 0.125-32 ?g/ml for ciprofloxacin and 2-256 ?g/ml for piperacillin/tazobactam. For both antibiotics, at sub-MIC concentrations (MIC/2, MIC/4, MIC/8) biofilm formation was reduced in a dose-dependent manner when compared to the case without antibiotics. At sub-MIC doses, after treating with antibiotics, the concentration which provided the highest reduction in the biofilm formation for both antibiotics was determined to be MIC/2. This was validated using scanning electron microscope.Polimerase chain reaction was used to show the presence of genes lasI, lasR, rhlI ve rhlR which play a role in Quorum sensing. All of the strains (100%) which form strong and intermediate biofilm compared to P. aeruginosa ATCC 27853, were found to have lasI/lasR gene, 4 in 5 strains (80%) which form weak biofilm had lasI/lasR genes. rhlI gene was determined in all (100%) strains studied. rhlR gene was determined in 6 strains (46.2%) which produce strong biofilm, in 4 strains which produce intermediate biofilm (66.7%) and in 3 strains (60%) which produce weak biofilm. Absence of RhlR gene in some of the strains is attributed to mutations in this gene. Further investigation should be performed for the identification of these mutations.As a result, considering subinhibitory concentrations of antimicrobials as new treatment strategies is thought to provide positive outcomes during the recovery period of P. aeruginosa infections. In addition, QS system in P. aeruginosa has important relationship with biofilm formation however biofilm formation is not only dependent on the presence of QS genes, and environmental and strain characteristics also effect biofilm formation mechanisms.