Investigation of Plasmid Mediated mcr Colistin Resistance Gene in Clinical Enterobacterales Isolates

Abstract

Carbapenems are used in the treatment of infections caused by multidrug-resistant bacteria and colistin (polymyxin E) is used as the last choice of antimicrobial agent in those resistant to carbapenems. The worldwide and increased use of colistin, which causes cell death by disrupting the permeability of the cytoplasmic membrane of gram-negative bacteria, raised the problem of resistance. The transferable colistin resistance enzyme mcr, is a phosphoethanolamine transferase that adds phosphoethanolamine to lipid A and modifies lipopolysaccharides, leading to polymyxin resistance. The aim of this study was to investigate some of the most prevalent plasmid mediated colistin and carbapenemase resistance genes in colistin resistant Enterobacterales isolates. Enterobacterales isolates which were isolated in the samples of patients treated in the clinical units between October 2016 and September 2018 in the Karadeniz Technical University Faculty of Medicine Farabi Hospital Medical Microbiology Laboratory were included in the study. In addition to conventional methods, isolates were identified to the species level by MALDI-TOF MS (Bruker Daltonics, Germany). The antibiotic susceptibilities of Enterobacterales isolates were studied by an automated microbiology system (Phoenix, Becton Dickinson, USA) and evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. In isolates that are resistant to colistin, and the isolates that are found to be sensitive but should be included in the patient report of the colistin susceptibility test, colistin susceptibility tests were repeated with liquid microdilution method in accordance with EUCAST standards. The presence of extended spectrum beta-lactamase (ESBL), AmpC beta-lactamase and carbapenemase were determined by phenotypic methods according to EUCAST recommendations in colistin resistant Enterobacterales isolates. Furthermore, resistance genes of mcr-1-5, blaOXA-48, blaKPC, blaNDM, blaVIM, blaIMP were detected by polymerase chain reaction (PCR) method, followed by nucleotide sequence analysis of the amplified products. In our study, 14657 Enterobacterales isolates belonging to 7535 patients treated in different clinical units were examined retrospectively. Escherichia coli 61.2% (n= 8968), Klebsiella pneumoniae 22.7% (n= 3334) and Enterobacter cloacae 6.9% (n= 1005) were the most prevalent isolates. Carbapenem resistance was detected in 894 isolates, and 5.8% (n= 412) of 7135 isolates isolated between October 2016 and September 2017; 6.4% (n= 482) of 7522 isolates between October 2017 and September 2018 were found to be resistant. Considering all isolates, colistin resistant isolates were 65 (0.9%) between October 2016 and September 2017 and 97 (1.3%) between October 2017 and September 2018. By including only the first isolates in the study for the same agent growths in different samples of the same patient, 46 colistin resistant isolates were selected. Six isolates which could not be cultivated from stock cultures were excluded from the study material. Thirteen (32.5%) of the 40 colistin resistant Enterobacterales isolates were isolated in 2017 and 27 (67.5%) were isolated in 2018. ESBL was detected in 22, AmpC beta-lactamase was detected in 6, carbapenem resistance was detected in 15 of them by phenotypic methods. As a result of PCR analysis, mcr-1 gene detected in 2 isolates, blaOXA-48 in 2 isolates, blaVIM in 1 isolate, blaKPC and blaOXA-48 in 1 isolate, blaNDM and blaOXA-48 in 5 isolates. These results were confirmed by sequencing of the PCR products. The mcr-1 genes were found in E.coli isolates grown in urine culture samples of 2 women over 65 years of age treated in our hospital. Among the antibiotics tested, only ampicillin resistance was observed in 1 of the patients, whereas ampicillin, amoxicillin-clavulanate and ciprofloxacin resistance were detected in the other. In conclusion, as far as we can reach in the literature our publication is the first study showing the presence of mcr-1 gene in clinical samples in our country and confirmed by DNA sequence analysis. The detection of mcr gene in isolates without multidrug resistance showed once again the importance of colistin susceptibility testing in the laboratories. In addition, the presence of isolates containing more than one resistance genes in our study, suggests that the spread of carbapenem and colistin resistance may be faster than expected.