Mechanisms of Fluoroquinolone Resistance in Escherichia coli Isolates from Food-Producing Animals

Abstract

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 g/ml and MICs of ciprofloxacin ranging from 4 to >32 g/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalaninearginine-␤-naphthylamide (PA␤N) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 3 Ala), ParC (Ser80 3 Trp), and ParE (Glu460 3 Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PA␤N the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

Molecular Characterization of Fluoroquinolone Resistance of Methicillin –Resistant Clinical Staphylococcus aureus Isolates from Rawalpindi, Pakistan

“…Efflux pump activity was monitored by the EtBr uptake and measurement of the fluorescence in the presence and absence of EPIs as described previously [15] with some modifications. The bacteria were grown overnight at 37 o C in LB broth containing 1% sodium chloride and the cells were centrifuged for 5 min at 18,000 X g in an Eppendorf centrifuge (Centrifuge 5417C, rotor F45-30-11) (Eppendorf North America, Hauppauge, NY, USA).…”

Mechanisms of Quinolone Resistance in Vibrio parahemolyticus strains Isolated from Imported Shrimp

“…Mutation at position 68 on the GyrA results in loss of charge, bulkiness and stability of GyrA and increases the MIC values to these drugs. Point mutations exclusively in the QRDR regions of topoisomerase genes may not be the sole contributing factor for bacterial resistance to quinolone antibiotics, suggesting that other possible mechanisms may also play a role in conferring resistance to these antibiotics (Baranwal et al, 2002;Ghosh et al, 1998;Hopkins et al, 2005Karczmarczyk et al, 2011Poole, 2000;Webber and Piddock, 2003). Since none of the isolates examined in this study contained any of the plasmid-mediated quinolone resistance genes (qnr), high levels of quinolone resistance could be a function of decreased accumulation of the antibiotics due to active efflux pumps.…”

“…In Gramnegative bacteria, resistance to quinolones is mainly due to chromosomal mutations in the quinolone resistance determining region (QRDR) of genes encoding the drug target enzymes (DNA gyrase and topoisomerase IV) and a majority of mutations in these bacteria have been found within the N termini of GyrA (between codons Ala67-Gln106), GyrB (between codons Asp426-Lys447) and ParC proteins (between codons 83 and 87). Recently several reports have indicated resistance through efflux pumps (Poole, 2000;Webber and Piddock, 2003) that are involved in the extrusion of toxic substrates from within cells into external environment and plasmid-mediated quinolone resistance (Hopkins et al 2005;Karczmarczyk et al 2011). However, limited information is available on the characterization of quinolone resistance mechanisms in V. parahaemolyticus.…”

“…s 2.6. Determination of efflux pump activity in the quinolone-sensitive and resistant strains of V. parahaemolyticus: Efflux pump activity was determined as described previously (Baranwal et al, 2002;Karczmarczyk et al, 2011). For determination of the presence of active efflux, ethidium bromide at a final concentration of 2 µg/ml was added immediately before the reading in a Synergy 2 Multi-Mode Microplate Reader (BIOTEK Instruments, Winooski, VT).…”

“…Thus, U.S. regulatory agencies, such as United States Food and Drug Administration (USFDA) and Centers for Disease Control and Prevention (CDC), want to limit the prevalence of quinoloneresistant bacteria in food-producing animals to protect the efficacy of the quinolone drugs for human use (USFDA, 1997). Several different kinds of mechanisms confer bacterial resistance to quinolone antibiotics (Baranwal et al, 2002;Ghosh et al, 1998;Hopkins et al, 2005;Karczmarczyk et al, 2011;Poole, 2000;Ruiz, 2003;Yoshida et al, 1991). In Gramnegative bacteria, resistance to quinolones is mainly due to chromosomal mutations in the quinolone resistance determining region (QRDR) of genes encoding the drug target enzymes (DNA gyrase and topoisomerase IV) and a majority of mutations in these bacteria have been found within the N termini of GyrA (between codons Ala67-Gln106), GyrB (between codons Asp426-Lys447) and ParC proteins (between codons 83 and 87).…”