In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

International audienceInfections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen

Identification and Evolution of Drug Efflux Pump in Clinical Enterobacter aerogenes Strains Isolated in 1995 and 2003

BACKGROUND: The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed 'multidrug resistance' (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (beta-lactams, quinolones, ...). METHODOLOGY/PRINCIPAL FINDINGS: Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAbetaN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAbetaN-susceptible efflux system was also identified in resistant E. aerogenes strains. CONCLUSIONS/SIGNIFICANCE: For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum beta-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in the evolution of mechanism in E. aerogenes

Antimicrobial Drug Efflux Pumps in Enterobacter and Klebsiella

International audienc

Rejuvenating the Activity of Usual Antibiotics on Resistant Gram-Negative Bacteria: Recent Issues and Perspectives

Antibiotic resistance continues to evolve and spread beyond all boundaries, resulting in an increase in morbidity and mortality for non-curable infectious diseases. Due to the failure of conventional antimicrobial therapy and the lack of introduction of a novel class of antibiotics, novel strategies have recently emerged to combat these multidrug-resistant infectious microorganisms. In this review, we highlight the development of effective antibiotic combinations and of antibiotics with non-antibiotic activity-enhancing compounds to address the widespread emergence of antibiotic-resistant strains

Susceptibilities of the <i>E. aerogenes</i> isolates (2003) to different antibiotics (MIC µg/mL)^a.

a<p>The values are the means of three independent assays.</p><p>Antimicrobial agent abbreviations: IMI, Imipenem; CEF, Cefepime; TET, Tetracycline; CHL, Chloramphenicol; NAL, Nalidixic acid; NFL, Norfloxacin; OFL, Ofloxacin; SPA, Sparfloxacin.</p

Antimicrobial susceptibilities and chloramphenicol accumulation in <i>E. aerogenes</i> strains.

a<p>Ratio of chloramphenicol accumulated at 600 s with and without CCCP.</p>b<p><i>E. aerogenes</i> control strains used: ATCC 15038 reference strain that expresses a normal level of the AcrAB efflux system; EAEP294 (<i>acrA⁻</i> deleted mutant)and EAEP289 that exhibits overexpression of the AcrAB efflux system.</p>c<p>N.D: not determined.</p><p>Antimicrobial agent abbreviations: IMI, Imipenem; CEF, Cefepime; TET, Tetracycline; CHL, Chloramphenicol; NAL, Nalidixic Acid; NFL, Norfloxacin; OFL, Ofloxacin; SPA, Sparfloxacin; CIP, Ciprofloxacin. Values in parentheses were determined in the presence of efflux pump inhibitor PAßN at 26.3 µg/ml.</p

Genome of <i>E</i>. <i>aerogenes</i> G7.

<p>This figure was generated with GCviewer (<a href="http://stothard.afns.ualberta.ca/cgview_server/" target="_blank">http://stothard.afns.ualberta.ca/cgview_server/</a>) and shows the map of the chromosome and plasmid pGPN1 (174 Kbp) of <i>E</i>. <i>aerogenes</i> isolate G7 obtained by <i>de novo</i> sequencing and assembly.</p