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)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae 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-type Enterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for total Enterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence of bla(CTX-M), bla(CMY), bla(SHV), bla(OXA), and bla(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 were Escherichia 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 genes bla(CTX-M-1), bla(CTX-M-14), bla(CTX-M-15), bla(SHV-12), and bla(CMY-2) were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 × 10(8) CFU/g feces. This study showed an abundance of ESBL-producing Enterobacteriaceae 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.