PREVALENCE, LONGITUDINAL DYNAMICS, AND FACTORS ASSOCIATED WITH THE FAECAL SHEDDING OF EXTENDED-SPECTRUM Β-LACTAMASE-PRODUCING BACTERIA IN ITALIAN NEONATAL DAIRY CALVES

Antimicrobial resistance (AMR) is a major One Health threat affecting humans, animals, and the environment. Among the most resistant bacteria of concern, extended-spectrum β-lactamase (ESBL)-producing Enterobacterales are particularly relevant because they limit the efficacy of critically important β-lactam antimicrobials and can disseminate across the animal–human–environment interface. In dairy production systems, pre-weaned calves are considered important reservoirs of ESBL-producing Enterobacterales, yet information on their occurrence, associated risk factors, environmental distribution, and shedding dynamics remains limited in Italy. This thesis investigated the epidemiology of ESBL-producing Enterobacterales in dairy calves, with a particular focus on high-density dairy production systems in Northern Italy. The specific aims were to assess prevalence, identify farm-level and animal-level factors associated with an increased shedding, evaluate seasonal variation, describe environmental occurrence, characterise antimicrobial resistance profiles and ESBL-encoding genes, and investigate longitudinal faecal shedding dynamics during the first months of life. To address these aims, a series of observational studies were conducted. Faecal samples from neonatal or pre-weaned Holstein–Friesian calves, together with selected environmental samples, were cultured on selective media for ESBL-producing Enterobacterales. Isolates were identified by MALDI-TOF MS, phenotypic ESBL production was confirmed by double-disk synergy testing, antimicrobial susceptibility was assessed by disk diffusion, and blaCTX-M, blaTEM, and blaSHV genes were investigated by multiplex PCR. Farm management and biosecurity practices were evaluated through questionnaires. The results consistently showed a high occurrence of ESBL-producing Enterobacterales in dairy calves. In Northern Italy, prevalence reached 86.9% in neonatal calves from a high-density dairy production area. Repeated cross-sectional analyses confirmed that ESBL-producing Enterobacterales were endemic at the farm level and identified management-related factors associated with prevalence, including hygiene of milk feeding equipment and administration of cephalosporins for mastitis treatment. Seasonal sampling suggested a marked reduction in prevalence during winter. Longitudinal monitoring further showed that ESBL-producing E. coli shedding was frequent during the first months of life, decreased markedly around 4–5 months of age, and increased again at later time points, suggesting a fluctuating rather than linear age-related pattern. Finally, a pilot study on transition milk feeding did not show an association with reduced ESBL-producing Enterobacterales shedding prevalence, but suggested a possible relationship with lower antimicrobial resistance-class diversity among isolates. Overall, this thesis demonstrates that dairy calves represent important reservoirs of ESBL-producing Enterobacterales in Italian dairy systems. The findings highlight the multifactorial nature of colonisation and shedding, with management practices, hygiene measures, environmental exposure, and seasonal factors all contributing to the epidemiology of these resistant bacteria. From a One Health perspective, improving calf-rearing hygiene, feeding-equipment sanitation, biosecurity, and antimicrobial stewardship may help reduce the on-farm circulation of ESBL-producing Enterobacterales and their resistance determinants.