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

Infections nosocomiales chez le brûlé (étude épidémiologique sur cinq ans)

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

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

Susceptibilities of the <i>E. aerogenes</i> isolates (1995) 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

Percentages of <i>Enterobacter aerogenes</i> resistant strains.

<p>Antimicrobial agent abbreviations: IMI, Imipenem; CEF, Cefepime; TET, Tetracycline; CHL, Chloramphenicol; NAL, Nalidixic Acid; NFL, Norfloxacin; OFL, Ofloxacin; SPA, Sparfloxacin.</p

Effect of EPI on chloramphenicol susceptibility of <i>E. aerogenes</i> strains.

<p>I, R ^a: Intermediate, Resistant.</p><p>EP ^b: Efflux Pump susceptible when PAßN induces a decrease of at least 3 dilutions.</p

PFGE of <i>Xba</i>I-digested genomic DNA preparations from various clinical <i>Enterobacter aerogenes</i> isolates.

<p>1995 isolates: 6582, 106701, 313, 139, 120, 31, 70, 17, 49, 130, 109, 183, 52. 2003 isolates: 101979, 121653, 109688, 181243, 138074, 121435, 109709, 121652, 128500, 116621. ATCC13048 and EA27, used as reference strains. Molecular weight markers are indicated: a, 48.5 kb; b, 97 kb; c, 145.5 kb; d, 194 kb; e, 242.5 kb; f, 291 kb; g, 340 kb; h, 388 kb; i, 437 kb; j, 485 kb; h, 534 kb.</p