Fecal Carriage and Whole-Genome Sequencing-Assisted Characterization of CMY-2 Beta-Lactamase-Producing Escherichia coli in Calves at Czech Dairy Cow Farm.

The study aimed to monitor the fecal shedding of cefotaxime-resistant Escherichia coli (CREC) in a cohort of healthy calves on a dairy farm with documented antimicrobial usage and to characterize selected AmpC beta-lactamase-producing E. coli isolates. Fecal samples from 13 suckling calves (1-63 d of age; 113 samples in total) were repeatedly collected and cultivated on MacConkey agar with cefotaxime (2 mg/L). Resistant colonies were counted, and one colony obtained from the highest dilution of each fecal sample was identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Susceptibility to antimicrobials and production of AmpC and extended-spectrum beta-lactamase (ESBL) were tested. No ESBL-producing E. coli was found, but representative AmpC-positive E. coli isolates were subjected to further typing and whole-genome sequencing (WGS) for the analysis of clonal relationships, resistance genes, virulence factors, and plasmid replicons. High amounts of CREC were detected in the feces of all 13 calves during the study. The number of CREC colonies varied from 1.0 log10 to 8.0 log10 colony-forming unit per gram. Drops in CREC density or its discontinued shedding were recorded at the end of the study period. A total of 82 (94%, n = 87) CREC isolates were confirmed as AmpC producers and all but one showed resistance to multiple antimicrobials. Twenty-nine selected AmpC-positive E. coli isolates belonged to 12 and 13 unique rep-PCR fingerprints and pulsed-field gel electrophoresis types, respectively, highlighting the variation in E. coli genotypes in individual calves. WGS of 10 selected isolates showed diverse antimicrobial resistance and virulence gene content and the presence of a blaCMY-2 gene carried by an IncK2 plasmid. Clinically important multiresistant E. coli isolates belonging to emerging extraintestinal pathogenic E. coli ST69 and ST648 lineages were found. Our findings reinforce the urgency of efforts to prevent the spread of ESBL-/AmpC-producing bacteria in dairy cow farms.