Molecular analysis of antibiotic resistance in carbapenem-resistant Pseudomonas aeruginosa isolates isolated from clinical samples

Pseudomonas aeruginosa is an opportunistic pathogen that causes morbidity and mortality in risky patient groups. The spread of carbapenemases among these strains causes difficulties to treat infectious diseases. In this thesis study, it was aimed to investigate the molecular epidemiology of 47 carbapenem-resistant P. aeruginosa (CRPA) isolates isolated from various clinical samples at Kocaeli University Research and Application Hospital Central Laboratory Bacteriology Unit between October 2021 and March 2023. Identification of the isolates performed with the VITEK® MS automated system. Sensitivity tests of the isolates against some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were performed with the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of isolates was investigated by the imipenem/meropenem-EDTA combined disk method. Conjugation experiments were carried out using the 'broth mating' method. Plasmid DNA isolation was performed by alkaline lysis method. Co-transferred antibiotic resistances in transconjugants were determined by the disk diffusion method. Carbapenemase genes (blaIMP, blaVIM, blaNDM, blaKPC and blaOXA-48), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were investigated by polymerase chain reaction (PCR). Clonal relationships of the isolates were investigated by evaluating DNA fingerprint analyzes obtained from ERIC (Enterobacterial Intergenic Repetitive Consensus)-PCR. The most resistance rate to the isolates was determined to be levofloxacin. The lowest resistance rate was observed against tobramycin, gentamicin and amikacin. MBL production was detected in twenty-five (53.2%) isolates. In conjugation experiments, conjugative resistance plasmids were detected in 12 (25.5%) isolates. Biofilm genes lasR and rhlR were detected by PCR in 90% of the isolates. The blaVIM gene was detected in six (12.8%) isolates, the blaNDM gene was detected in five (10.6%) isolates, and the blaOXA-48 gene was detected in three (6.4%) isolates. blaKPC and blaIMP genes were not detected. In integron-specific PCR, 39 (82.9%) isolates were positive for the intI1 gene, while 24 (51%) were determined to carry a class 1 integron gene cassette. Six of the isolates containing conjugative plasmids were found to carry the class 1 integron gene cassette. Class 2 integrons were not found in CRPA isolates. Dendrogram analysis of ERIC-PCR patterns determined that there was no clonal similarity between CRPA isolates and that the isolates did not spread by cross-contamination. As a result, it was observed that most of the CRPA isolates showed high resistance to other antibiotic groups other than carbapenems and could co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains, which have the potential to cause epidemics in the clinical arena.