Colistin resistance has increased due to the increasing and inappropriate use of this antibiotic. The mechanism involves modification of lipid A with phosphoethanolamine (PEtN) and/or 4-amino-4deoxy-l-arabinose (L-Ara4N). EptA and eptB catalyze the transfer of phosphoethanolamine to lipid A. In this study, gene network was constructed to find the associated genes related to colistin resistance, and further in vitro validation by transcriptional analysis was performed. In silico studies showed that eptB gene is a highly interconnected node in colistin resistance gene network. To ascertain these findings twelve colistin-resistant clinical isolates of Escherichia coli were selected in which five were harboring the plasmid-mediated mcr-1. Screening for colistin resistance was performed by broth microdilution (BMD) method and Rapid polymyxin NP test. PCR confirmed the presence of the eptA and eptB genes in all isolates and five isolates were harboring mcr-1. Transcriptional expression in five isolates harboring mcr-1, showed an enhanced expression of eptB when exposed under sub-inhibitory colistin stress. The present study for the first time highlighted genetic interplay between mcr-1 and eptA and eptB under colistin exposure.
Objective: The polymyxin group of antibiotics is considered to be one of the most effective antimicrobial agents against many serious pathogenic bacteria, but the excessive use of these antibiotics has led to the development of drug resistance among bacteria. This study was designed to characterize polymyxin-resistant P. aeruginosa and to explore the role of PmrB and arnA in resistant phenotype. Methods: mRNA and cDNA of five selected polymyxin-resistant strains representing different MIC range; isolated under normal condition of strain growth, after treating sample/media with FeCl3 and MgCl2 alone, or after treating with FeCl3 and polymyxin antibiotic. The transcriptional expression was observed for PmrB and arnA by quantitative real time RT-PCR in reference to P. aeruginosa PAO1. The presence of plasmid mediated colistin resistance determinants mcr-1 was screened by PCR. Susceptibility of the strains was determined by disc-diffusion method and DNA fingerprinting was carried out by performing REP-PCR. Results: A down regulated expression of PmrB and arnA was observed even after the unique induction with FeCl3 and MgCl2. All the isolates were found to be resistant against cefepime and different clonal patterns of resistance were found among the isolates. Conclusion: This study has drawn a new insight into polymyxin resistance which will help in the detection and control of infections caused by multidrug resistant P. aeruginosa. The low susceptibility rate to aminoglycoside, piperacllin-tazobactam and ciprofloxacin was found and in addition, detection of PmrB and arnA as molecular markers in the follow up of infections caused by multidrug resistant P. aeruginosa.
The increased and inappropriate use of colistin led to the emergence of its resistance among Gram-negative bacterial isolates and the most common mechanism of colistin resistance in Gram-negative bacteria is the modification of the lipopolysaccharide mediated by two-component regulatory systems, PhoPQ and PmrAB. The aim of the present study was to investigate the transcriptional expression of the PhoPQ system against colistin stress in clinical isolates of Escherichia coli with colistin-resistant phenotype. Six colistin-resistant E. coli isolates were obtained from Silchar Medical College and Hospital, Silchar that were of clinical origin and received for routine culture and sensitivity testing. Screening for colistin resistance was done by broth microdilution method and further screened for the presence of the different types of plasmid-mediated colistin resistance mcr genes namely, mcr-1 to mcr-10 by polymerase chain reaction (PCR). The screened positive isolates were subjected to PCR assay targeting phoP and phoQ genes and their expression was measured by quantitative real-time PCR. The results of this study revealed that two E. coli isolates (TS2 and TS4) were found to carry the mcr-1 gene. PhoP and PhoQ gene amplification was observed in all the isolates. Transcriptional analysis showed that the isolates harboring the mcr-1 gene showed an enhanced level of expression in the PhoP, PhoQ genes in the presence of a subinhibitory concentration of colistin whereas no significant expression was observed for the isolates which were devoid of the mcr gene. This study demonstrates the involvement of mcr-1 in the PhoPQ system in clinical isolates of colistin-resistant E. coli which will help in designing a molecular marker for detecting colistin-resistant E. coli and contribute to the assessment of resistance burden and infection control strategy.
This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.
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