MIC reduction assay using meropenem.

<p>MIC reduction assay using meropenem.</p

Transcriptional Analysis of MexAB-OprM Efflux Pumps System of <i>Pseudomonas aeruginosa</i> and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India

<div><p>Carbapenem resistance presents severe threat to the treatment of multidrug resistant <i>Pseudomonas aeruginosa</i> infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of <i>mexA</i> gene in clinical isolates of <i>P</i>. <i>aeruginosa</i> from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (<i>P</i>. <i>aeruginosa</i>) and in a heterologous host (<i>E</i>. <i>coli</i>). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of <i>mexA</i> gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (<i>P</i>. <i>aeruginosa</i> PAO1) and heterologous host (<i>E</i>. <i>coli</i> JM107) but transcription level of <i>mexA</i> gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in <i>E</i>. <i>coli</i> JM107 and <i>P</i>. <i>aeruginosa</i> PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of <i>P</i>. <i>aeruginosa</i> where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance <i>mexA</i> transcriptions significantly, there might be a possibility that the increase in expression of efflux pump genes does not mediated by single antibiotic but rather by a combination of antipseudomonal drugs which are used during treatments. Early detection of efflux genes will help in selection of proper therapeutic options.</p></div

MIC₅₀ and MIC₉₀ of efflux mediated carbapenem resistant <i>P</i>. <i>aeruginosa</i> isolates.

<p>MIC₅₀ and MIC₉₀ of efflux mediated carbapenem resistant <i>P</i>. <i>aeruginosa</i> isolates.</p

Expression of <i>mexA</i> gene before and after single dose exposure in transformants.

<p>Expression of <i>mexA</i> gene before and after single dose exposure in transformants.</p

Figure describing different regions of mexAB-OprM operon which are amplified using different primers as shown.

<p>Figure describing different regions of mexAB-OprM operon which are amplified using different primers as shown.</p

Expression of <i>mexA</i> gene before and after single dose exposure with meropenem in bacteria.

<p>Expression of <i>mexA</i> gene before and after single dose exposure with meropenem in bacteria.</p

MIC reduction assay of isolates that demonstrated synergy between CCCP and one of the tested antibiotics (meropenem and ciprofloxacin).

<p>MIC reduction assay of isolates that demonstrated synergy between CCCP and one of the tested antibiotics (meropenem and ciprofloxacin).</p

REP PCR showing same REP types of eight <i>P</i>. <i>aeruginosa</i> isolates with mutated <i>mexR</i> gene.

<p>Lane 1: Hyper ladder (1 kb); lane 2 to 9: test samples.</p

Antibiogram profile of MDR <i>P</i>. <i>aeruginosa</i>.

<p>Antibiogram profile of MDR <i>P</i>. <i>aeruginosa</i>.</p

mRNA expression of <i>mexA</i> and <i>OprD</i> gene.

<p>mRNA expression of <i>mexA</i> and <i>OprD</i> gene.</p