Extended-Spectrum Beta-Lactamase and AmpC-producing Enterobacteriaceae in household dogs: a longitudinal study

A longitudinal study was performed (i) to investigate continuity of shedding of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in dogs without clinical signs, (ii) to identify dominant plasmid-mediated ESBL genes and (iii) to quantify ESBL-producing Enterobacteriaceae in feces. Fecal samples of 38 dogs were collected monthly for 6 months. From 7 included dogs, additional samples were collected on a weekly basis for 6 weeks. CFU/g feces were determined for non-wild-type Enterobacteriaceae on MacConkey agar supplemented with 1 mg/L cefotaxime (MCC) and total number of Enterobacteriaceae on MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for presence of blaCTX-M, blaCMY, blaSHV, blaOXA and blaTEM gene families. Bacterial species were identified by MALDI-TOF MS analysis. PCR-negative isolates were tested by double disk synergy test for enhanced AmpC expression. 259 samples were screened, 126 samples were culture-positive on MCC, resulting in 352 isolates of which 327 isolates 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 in 23 dogs. Genotyping showed high variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. ESBL genes blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaSHV-12 and blaCMY-2 were most frequently found. Mean 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 showed to be highly dynamic over time which is important to consider when studying ESBL epidemiology.