| Summary: | The use of antibiotics to treat dairy calves may result in multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing <i>Escherichia coli</i>. This study investigated fluoroquinolone and macrolide resistance genes among ESBL-producing <i>E. coli</i> isolated from dairy calves. Fresh fecal samples from 147 dairy calves across three age groups were enriched to select for ESBL-producing <i>E. coli.</i> Plasmid-mediated fluoroquinolone (<i>qnrB</i>), macrolide (<i>mph</i>(A)), and beta-lactam (<i>bla</i><sub>CTX-M</sub> groups 1 and 9) resistance genes were identified by PCR and gel electrophoresis in ESBL-producing <i>E. coli</i>. Beta-lactamase variants and antibiotic resistance genes were characterized for eight isolates by whole-genome sequencing. Seventy-one (48.3%) samples were positive for ESBL-producing <i>E. coli</i>, with 159 (70.4%) isolates identified as <i>bla</i><sub>CTX-M</sub> variant group 1 and 67 (29.6%) isolates as <i>bla</i><sub>CTX-M</sub> variant group 9. Resistance gene <i>mph</i>(A) was more commonly associated with <i>bla</i><sub>CTX-M</sub> variant group 1, while resistance gene <i>qnrB</i> was more commonly associated with variant group 9. <i>E. coli</i> growth was quantified on antibiotic media for 30 samples: 10 from each age group. Significantly higher quantities of ceftriaxone-resistant <i>E. coli</i> were present in the youngest calves. Results indicate the dominant <i>bla</i><sub>CTX-M</sub> groups present in ESBL-producing <i>E. coli</i> may be associated with additional <i>qnrB</i> or <i>mph</i>(A) resistance genes and ESBL-producing <i>E. coli</i> is found in higher abundance in younger calves.
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