Investigation of plasmid-mediated quinolone resistance genes in clinical Escherichia coli isolates

The susceptibility to quinolone group antibiotics used in the treatment of infections caused by Escherichia coli (E. coli) is gradually decreasing. Accordingly, it is seen that quinolone resistant isolates are reported more frequently around the world. The development of quinolone resistance in bacteria may be of chromosomal or plasmid origin. The qnr genes, aac(6′)-Ib-cr gene and qepA/oqxAB genes are responsible for plasmid-mediated quinolone resistance. It has been determined that these genes, which reduce susceptibility to quinolones, also play a triggering role in the formation of chromosomal mutations in bacteria, and this situation causes high level of quinolone resistance and complicates the treatment. It has been shown that horizontal transmission of these genes, which are usually carried on conjugative plasmids, between bacteria by conjugation plays an important role in the rapid spread of quinolone resistance. Molecular detection and monitoring of the prevalence of these plasmid-mediated quinolone resistance which cannot be detected by phenotypic tests, are extremely important in terms of preventing the spread of quinolone resistance in hospitals. In this thesis, it was aimed to investigate the presence of plasmid-mediated qnrA, qnrB, qnrS and aac(6')-Ib-cr genes in ciprofloxacin-resistant clinical E. coli isolates and to determine whether these genes are carried by conjugative plasmids. For this purpose, 59 clinical E. coli isolates which were isolated from clinical samples of the patients hospitalized in the service and intensive care units of Kırşehir Ahi Evran University Training and Research Hospital (KAEÜEAH) between September 2018 and January 2019, were obtained from the Medical Microbiology Laboratory. E. coli isolates, stored in the hospital culture collection following their identification and the antibiotic resistance determination by the Vitek-2 automated system, were revived in the appropriate medium in our laboratory. Total DNAs of the isolates were obtained and the presence of qnrA, qnrB, qnrS and aac(6')-Ib-cr, which are the most common plasmid-mediated quinolone resistance genes, were investigated by PCR method. Conjugation experiments were performed to detect the presence of conjugative plasmids in the isolates and to determine the transferability of resistance genes. While none of the 59 E. coli isolates (100%) examined carried the qnrA, qnrB and qnrS genes, 39 (66.1%) isolates were positive for the aac(6′)-Ib-cr gene. This finding was in agreement with the results of national and international data showing that E. coli isolates carrying the aac(6′)-Ib-cr gene were significantly more common than isolates carrying the qnr genes. The results of the conjugation experiments showed that 10 (16.9%) of the 59 E. coli isolates, namely Ec15, Ec20, Ec22, Ec64, Ec82, Ec87, Ec131, Ec104, Ec117 and Ec146, carried a conjugative plasmid. Then, antibiotic susceptibility patterns of E. coli J53-2 transconjugant cells in which conjugative plasmids were transferred from these E. coli isolates were investigated by disk diffusion method. The results showed that each transconjugant cell phenotypically exhibited the same antibiotic resistance profile as its wild-type E. coli isolate. Finally, to investigate the presence of aac(6')-Ib-cr gene in conjugative plasmids transferred from E. coli isolates into E. coli J53-2 transconjugate cells, plasmids were isolated from transconjugate cells and aac(6')-Ib-cr gene was amplified by PCR. The results showed that 7 transconjugant cells were aac(6')-Ib-cr positive and the other 3 cells were negative. Antibiogram and PCR findings together showed that aac(6')-Ib-cr gene was located on conjugative plasmids with multiple antibiotic resistance in aac(6')-Ib-cr positive Ec15, Ec20, Ec22, Ec64, Ec82, Ec131 and Ec146 isolates. On the other hand, although quinolone resistance was detected phenotypically in E. coli J53-2 transconjugant cells into which conjugative plasmids were transferred from aac-(6')-Ib-cr negative Ec87, Ec104 and Ec117 isolates, aac-(6')-Ib-cr gene was not present on conjugative plasmids. This observation suggested that the conjugative plasmids of these 3 isolates had quinolone resistance gene or genes different from the aac-(6')-Ib-cr gene found in the other 7 isolates. This thesis is the first study to investigate plasmid-mediated quinolone resistance in clinical E. coli isolates causing infection in our hospital. This thesis is the first study to comprehensively investigate plasmid-mediated quinolone resistance in clinical E. coli isolates causing infection in our hospital. The results showed that the prevalence of isolates containing the plasmid-mediated aac(6′)-Ib-cr gene was quite high (66.1%). In addition, aac(6′)-Ib-cr gene was found to be carried together with multiple antibiotic-resistance genes on conjugative plasmids in some isolates. The presence of isolates containing conjugative plasmids carrying multiple antibiotic-resistance genes is an important problem that may limit the treatment options of E. coli infections in our hospital. Finally, our work emphasizes that it would be beneficial to use genotypic tests as well as phenotypic tests in determining the antibiotic resistance profiles of infectious microorganisms. Keywords: E. coli, PCR, Plasmid-mediated quinolone kinolon resistance (PMQR), Conjugative plasmid, qnrA, qnrB, qnrS, acc-6'-Ib-cr