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Simple methods to screen efflux pump activity and efflux pump inhibitors in multidrug-resistant Escherichia coli

Abstract number: P1998

Martins M., Cardoso A., Viveiros M., Charepe N., Rodrigues L., Couto I., Amaral L.

Objectives. Over-expression of efflux pumps (EPs) in Gram-negative bacteria is associated with Multi-Drug Resistance (MDR). EPs inhibitors (EPIs) can be used to detect the activity of EPs and to reverse efflux-mediated MDR. In this study, 3 simple methods were developed to quantify efflux in MDR E. coli and screen for effective EPIs.

Methods. Thirty veterinary E. coli strains with MDR phenotype (resistance to 2 or more classes of antibiotics) were evaluated for ethidium bromide (EB) efflux activity by an agar-based method. Strains that showed efflux activity were selected for the screening of EPIs by the microplate method. Briefly, antibiotic discs were placed in 24-well plates containing media with/without the following EPIs (at half their MIC): Phe-Arg-napthylamide (PAN), thioridazine (TZ), chlorpromazine (CPZ) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). After 16 h, wells were examined for absence (reversal of resistance), partial (reduction of resistance) or full growth (no effect). Strains that showed reduction or reversal of resistance were then evaluated for efflux with/without EPIs by a semi-automated real-time fluorometric method. Expression of EPs genes was assayed by RT-PCR.

Results. The EB agar method correctly identified 4 strains with increased EPs activity, which was further quantified by the fluorometric assay. This efflux activity was reduced or even reversed to clinical levels by the EPIs PAN and TZ. The RT-PCR assay of these strains showed an increased level of expression of their major EPs.

Conclusion. The methodologies developed and to be described in detail can be used for detection of MDR strains whose phenotype and gene expression are consistent with increased efflux activity as well as for the screening of agents for EPI activity against collections of MDR clinical isolates. These methods are expected to be eventually used in the clinical bacteriology laboratory.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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