Longitudinal Study of Extended-Spectrum-β-Lactamase- and AmpC-Producing Enterobacteriaceae in Household Dogs

A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producingEnterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producingEnterobacteriaceae in feces. Fecal samples from 38 dogs were collected monthly for 6 months. Additional samples were collected from 7 included dogs on a weekly basis for 6 weeks. Numbers of CFU per gram of feces for non-wild-typeEnterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for totalEnterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence ofbla CTX-M ,bla CMY ,bla SHV ,bla OXA , andbla TEM gene families. Bacterial species were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis. PCR-negative isolates were tested by a double-disk synergy test for enhanced AmpC expression. A total of 259 samples were screened, and 126 samples were culture positive on MCC, resulting in 352 isolates, 327 of which wereEscherichia coli . Nine dogs were continuously positive during this study, and 6 dogs were continuously negative. Monthly or weekly shifts in fecal shedding were observed for 23 dogs. Genotyping showed a large variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. The ESBL genesbla CTX-M-1 ,bla CTX-M-14 ,bla CTX-M-15 ,bla SHV-12 , andbla CMY-2 were most frequently found. The mean number of CFU of non-wild-typeEnterobacteriaceae was 6.11 × 108 CFU/g feces. This study showed an abundance of ESBL-producingEnterobacteriaceae in dogs in the Netherlands, mostly in high concentrations. Fecal shedding was shown to be highly dynamic over time, which is important to consider when studying ESBL epidemiology.