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

Abstract

BackgroundThe 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 (ß-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 (PAßN) 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 PAßN-susceptible efflux system was also identified in resistant E. aerogenes strains.Conclusions/SignificanceFor 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 ß-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.

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…They also identified an increased efflux of fluoroquinolones and chloramphenicol in some multidrug-resistant strains (260), which is likely to be due to AcrB (261). Finally, frequencies of chloramphenicol-resistant isolates susceptible to the AcrB inhibitor PA␤N appeared to have increased between 1995 and 2003, suggesting a larger role for the broad-specificity efflux mechanism in recent years (262).…”

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases

“…We used the twofold broth dilution method with Mueller-Hinton broth according to the CLSI guidelines: the tests were carried out with or without the broad-spectrum efflux pump inhibitor (EPI) phenylalanine arginine ␤-naphthylamide (PA␤N). This diamine compound, used at a low concentration (26.3 mg/liter), can increase the activities of chloramphenicol, tetracycline, norfloxacin, and sparfloxacin against various enterobacterial isolates which overproduce efflux pumps such AcrAB-TolC (4,9,16). The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1).…”

“…The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1). Efflux pump activity was identified when the PA␤N addition induced a threefold decrease in the MIC of an antibiotic molecule (4,9). For the two fluoroquinolones, the PA␤N addition had better efficiency in decreasing the MICs of sparfloxacin than those of norfloxacin.…”

“…The PA␤N showed an effect similar to that of CCCP on chloramphenicol accumulation when added during accumulation in selected isolates (data not shown). It was reported previously that the overproduction of AcrA and TolC pump elements is involved in the resistance levels of clinical isolates (4,8,9). The membrane proteins of selected isolates were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with polyclonal antibodies directed against AcrA and TolC components (9,18).…”

Caracterização molecular dos mecanismos de resistência aos carbapenêmicos de isolados clínicos de Enterobacter aerogenes e Enterobacter cloacae

“…É considerado o terceiro patógeno mais comumente recuperado do trato respiratório em algumas casuísticas e frequentemente é isolado na urina e no trato gastrointestinal de pacientes hospitalizados. Também é responsável por 8% dos casos de bacteremias nosocomias, com mortalidade de 29%, apresentando alta resistência a antibióticos de largo espectro (6,27,28) . (7,29,30) .…”