Abstract
BackgroundThe issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation. Therefore, here we attempted to find the role of two secondary resistance genes (SRG) ccdB and repA2 in carbapenem resistance in E. coli (CRE). In this context influential genes belonging to secondary resistome that act as helper to the primary resistance genes like blaNDM and blaCTX-M in aggravating β-lactam resistance were selected from an earlier reported in silico study. Transcriptional expression of the selected genes in clinical isolates of E.coli that were discretely harboring blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7 and blaCTX-M-15 with and without carbapenem and cephalosporin stress (2 μg/ml) was determined by real time PCR. Cured mutants sets that were lacking (i) primary resistance genes, (ii) secondary resistance genes and (iii) both primary and secondary resistance genes were prepared by SDS treatment. These sets were then subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method.ResultsOut of the 21 genes reported in the in silico study, 2 genes viz.repA2 and ccdB were selected for transcriptional expression analysis. repA2, coding replication regulatory protein, was downregulated in response to carbapenems and cephalosporins. ccdB, coding for plasmid maintenance protein, was also downregulated in response to carbapenems except imipenem and cephalosporins. Following plasmid elimination assay increase in diameter of zone of inhibition under stress of both antibiotics was observed as compared to uncured control hinting at the reversion of antibiotic susceptibility by the-then resistant bacteria.ConclusionSRGs repA2 and ccdB help sustenance of blaNDM and blaCTX-M under carbapenem and cephalosporin stress.
Highlights
The issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation
Transcriptional expression of ccdB and repA2 genes with and without antibiotic stress ccdB and repA2 genes showed distinct pattern of response in these selected isolates when exposed to carbapenems and cephalosporins (Fig. 1a, b, c, d)
Downregulation of repA2 was seen in isolates that were harboring blaCTX-M-15 both when no cephalosporin stress was given as well as when 2 μg/ml of cephalosporins viz. ceftazidime, cefotaxime and ceftriaxone stress was applied
Summary
The issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation. Here we attempted to find the role of two secondary resistance genes (SRG) ccdB and repA2 in carbapenem resistance in E. coli (CRE) In this context influential genes belonging to secondary resistome that act as helper to the primary resistance genes like blaNDM and blaCTX-M in aggravating β-lactam resistance were selected from an earlier reported in silico study. Morbidity and mortality rates due to Gram negative bacterial infections is increasing due to the global threat of antibiotic resistance [1]. This issue is a bane to health care industry as well as to economy [1, 2] and dearth in production of newer and efficient antibiotics is contributing to its escalation [3,4,5].
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