The Role of gyrA, Efflux Pump and Integrons in Mediating the Emergence of Multi-Drug Resistance among Canine and Feline Pathogenic Clinical Escherichia coli

Abstract

A series of experiments was performed to elucidate the emergence of fluoroquinolone (FQ)/multi-drug resistance (MDR) and the contribution of selected mechanisms of resistance to MDR (i.e. mutations in gyrA, efflux pump, and the integrons) in canine and feline pathogenic Escherichia coli. E. coli isolates (n= 377) were collected from dogs or cats with spontaneous infection between May and September 2005 for phenotypic (i.e. E-test®) and genotypic (i.e PFGE) characterization. Of isolates expressing resistance to any drug, two isolates expressed single drug resistance (SDR) only to enrofloxacin. The remaining 109 isolates expressing resistance to enrofloxacin also expressed MDR, with resistance to all 7 drugs (Z phenotype) representing the largest proportion (18.3%; 20/109). The drugs most commonly involved in MDR phenotypes (n=109) were amoxicillin (96.3 %; 105/109), amoxicillin-clavulanic acid (85%; 93/109) and enrofloxcin (61.5%; 67/109). Genotypically, isolates were extensively diverse, regardless of resistance phenotype, and phenotypes and genotypes were not related. For the first time, a FRET PCR was developed to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codon 83 and 87 of gyrA. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine with a very short time (1-2 hrs). For the 70 isolates tested, the sensitivity of the test was 87.5% (95% CI = 75% to 95.3%) (n=42/48) whereas specificity was 100% (95% CI = 87.3% to 100%) (n=22/22). MICs for E. coli isolates (n= 536) were determined for enrofloxacin and five other drug classes using broth micro-dilution. FQ resistance was significantly associated with the MDR phenotype compared to non FQ-R isolates. The results suggest that the double mutation in gyrA confers high level of resistance to the majority of FQ-R isolates. Furthermore, the impact of an efflux pump was studied. Blocking the action of the AcrAB efflux pump (i) decrease the intrinsic level of the MICs to FQ in susceptible isolates, (ii) decrease the MICs below the susceptible break point for FQ-R isolates even with single mutation in gyrA, (iii) decrease the magnitude of resistance in highly resistance isolates in the presence of double mutations in gyrA (IV) decrease the MICs below the susceptible break point for some of the highly FQ-R isolates without gyrA mutations, and (V) decrease the resistance to structurally unrelated drugs, thus reducing the incidence of MDR. Integrons of Class I and 2 were identified in 27% of the isolates; thus only 2.4% of the isolates carried Class 2 integrons. All integron positive isolates were MDR, compared to 56.6% of integron negative isolates. Resistance resolved in the 3 MDR integron positive isolates subjected to plasmid curing (using 10 % SDS). This is the first report of class 2 integrons in E. coli from companion animals in US.