We aimed to investigate the prevalence, molecular characteristics and risk factors of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) shedding in horses. A prospective study included three cohorts: (i) farm horses (13 farms, n = 192); (ii) on hospital admission (n = 168) and; (iii) horses hospitalized for ≥72 h re-sampled from cohort (ii) (n = 86). Enriched rectal swabs were plated, ESBL-production was confirmed (Clinical and Laboratory Standards Institute (CLSI)) and genes were identified (polymerase chain reaction (PCR)). Identification and antibiotic susceptibility were determined (Vitek-2). Medical records and owners’ questionnaires were analyzed. Shedding rates increased from 19.6% (n = 33/168) on admission to 77.9% (n = 67/86) during hospitalization (p < 0.0001, odds ratio (OR) = 12.12). Shedding rate in farms was 20.8% (n = 40/192), significantly lower compared to hospitalized horses (p < 0.0001). The main ESBL-E species (n = 192 isolates) were E. coli (59.9%, 115/192), Enterobacter sp. (17.7%, 34/192) and Klebsiella pneumoniae (13.0%, 25/192). The main gene group was CTX-M-1 (56.8%). A significant increase in resistance rates to chloramphenicol, enrofloxacin, gentamicin, nitrofurantoin, and trimethoprim-sulpha was identified during hospitalization. Risk factors for shedding in farms included breed (Arabian, OR = 3.9), sex (stallion, OR = 3.4), and antibiotic treatment (OR = 9.8). Older age was identified as a protective factor (OR = 0.88). We demonstrated an ESBL-E reservoir in equine cohorts, with a significant ESBL-E acquisition, which increases the necessity to implement active surveillance and antibiotic stewardship programs.
