RESUMEN
OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , beta-Lactamasas/genética , Animales , Antibacterianos/farmacología , Técnicas de Tipificación Bacteriana , Cefoxitina/farmacología , Ceftazidima/farmacología , Cefalosporinas/farmacología , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Dermatitis Atópica/microbiología , Perros , Enrofloxacina , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/tratamiento farmacológico , Heces/microbiología , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Otitis/microbiología , Plásmidos/genética , Resistencia betalactámica/genéticaRESUMEN
OBJECTIVES: This study investigated the prevalence of vancomycin-resistant enterococci (VRE) in the broiler production environment after the avoparcin ban and their epidemiological relationship with human clinical VRE from the same geographical regions in Greece. METHODS: Caecal contents from broilers (nâ=â500) from eight livestock farms and faecal samples from poultry slaughterers (nâ=â50), all collected in two slaughterhouses during 2005-08, were analysed for species and vancomycin resistance gene identification using multiplex PCR. Sixty-three human clinical vancomycin-resistant Enterococcus faecium (VREF) isolates, obtained during 2006-09, were also examined. Discriminant analysis (DA) was used to establish the relationship of antimicrobial resistance profiles (ARPs) among broiler, poultry slaughterer and human clinical VREF. PFGE was conducted to study the genetic relatedness among VREF from the different sources. RESULTS: A total of 120 VRE were recovered from 113 (22.6%) broiler samples. VREF carrying the vanA gene were predominant, being recovered from 72 (14.4%) samples from five (62.5%) broiler farms. Concerning poultry slaughterers, VREF were recovered from 10 (20%) samples. Susceptibility testing revealed that broiler VREF were consistently resistant to tetracycline, whereas 93.7% of clinical VREF were resistant to ampicillin. Furthermore, 92.1% of clinical VREF compared with 54.4% of broiler VREF were multiresistant (resistant to at least five antimicrobial classes). DA classified broiler and human clinical VREF into their corresponding source with high classification rates (100% and 85.7%, respectively), while the classification rate of poultry slaughterer VREF was relatively low (50%), with 40% of them classified closely to broiler VREF. PFGE patterns were clearly related to the source of the VREF, with broiler isolates being clustered distinctly from all human isolates. CONCLUSIONS: A remarkable persistence of VREF was observed in the broiler production environment even >10 years after the avoparcin ban. Human and broiler VREF belonged to clearly unrelated populations, strongly indicating no clonal spread of VREF among the different sources, even between broilers and poultry slaughterers, despite them sharing common ARPs, as also supported by DA.