This study investigated the presence, distribution, and antimicrobial resistance profiles of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in a dairy herd located in Northern Italy. The feces of clinically healthy calves, their mothers, and the cows treated for mastitis, as well as water, environmental samples, and waste milk were collected and subjected to bacteriological culture on CHROMagar^TM ESBL plates. A questionnaire was administered to identify risk factors. The isolates were identified as E. coli by MALDI-TOF MS and subjected to the double-disk synergy test (DDST) and minimal inhibitory concentration (MIC) assay. As a result, ESBL E. coli was isolated from the feces of 28 of 37 (75.67%) calves, the feces of 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and waste milk. All ESBL isolates showed multiple resistances and were categorized as multidrug-resistant (MDR). Several risk factors for ESBL E. coli selection and diffusion were identified, including lack of routine cleaning of calf feeding and housing equipment, administration of waste milk to male calves, and blanket dry cow therapy. In conclusion, this study highlighted the presence of MDR, ESBL E. coli in the feces of most dairy calves, and their association with different sample sources. Accordingly, adding to the prudent use of antibiotics, the adoption of adequate farm hygiene and biosecurity measures might also help prevent the spread and transmission of ESBL E. coli within the herd.

This study investigated the presence, distribution, and antimicrobial resistance profiles of extend-ed-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in a dairy herd located in Northern Italy. A questionnaire was administered, and the feces of clinically healthy pre-weaned dairy calves, their mothers, and the cows treated for mastitis, waste milk, water, and environmental samples, were collected and inoculated on Chromagar ESBL™ medium. The questionnaire identi-fied several risk factors including administration of waste milk to male calves, lack of routine cleaning of calf feeding and housing equipment, and blanket dry cow therapy. Escherichia coli was identified by MALDI-TOF MS in the feces of 28 of 37 (75.67%) calves, 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and the waste milk. Six of 14 (42.85%) environmental samples were also positive for ESBL Acinetobacter baumannii, while all fecal samples were negative. ESBL production was confirmed for all E. coli isolates based on the double disk susceptibility test (DDST), while none were carbapenemase producers. Upon minimal inhibitory concentration (MIC) analysis, all of them showed multiple resistances and were categorized as multi-drug resistant (MDR). This study confirms the widespread presence of MDR, ESBL-E. coli in dairy calves and highlights their diffusion in the farm environment.

Escherichia coli is a leading environmental cause of bovine mastitis, and continued genomic research in specialized dairy systems remains essential for understanding population dynamics and adaptive strategies. In this study, we analyzed 622 milk samples from clinical and subclinical mastitis quarters collected in the Arborea district (Sardinia, Italy) by bacteriological culture and MALDI-TOF MS. E. coli was the most prevalent pathogen (21.54% of positive samples), followed by Streptococcus uberis (12.31%) and Staphylococcus chromogenes (8.72%). Antimicrobial resistance (AMR) was limited to tetracycline (21.43%) and ampicillin (14.29%). Thirty-nine mastitis-associated E. coli (MAEC) obtained from six positive farms were subjected to Nanopore long-read whole-genome sequencing to characterize their population structure, virulome, resistome, and plasmidome. Genomic analysis revealed a polyclonal population with extensive sequence type (ST) diversity and no farm-specific clustering. Seven isolates carried acquired antimicrobial resistance genes (ARGs) (17.95%). Virulence analysis highlighted a conserved core repertoire for extraintestinal survival (adherence, motility, and iron acquisition), with a marked variation in accessory determinants. Comparison with E. coli genomes from mastitis cases and healthy cows isolated in Lombardy showed lineage interspersion rather than disease-specific clustering. Our data are in line with a model in which coliform mastitis in modern dairy farms is driven by heterogeneous environmental lineages sharing common colonization and iron sequestration traits.

This study investigated the growth of Bacillus cereus and the production of cereulide toxin in powdered infant formula (PIF) reconstituted according to two different protocols. B. cereus NCTC 11143 spores were inoculated in infant formula samples (approximately 1.5-2 log CFU/ml of final product), that were alternatively reconstituted following the WHO (using water pre-heated to 70°C) or the FDA method (using room-temperature water). Following reconstitution, samples were stored under ideal refrigeration (4°C), thermal abuse (10°C) and strong thermal abuse (20°C) until 72 hours to simulate different storage conditions of leftover formula. B. cereus total count, spore count and cereulide quantification were performed at settled times. Results demonstrated that B. cereus did not grow at 4 and 10°C. At 20°C, spores rapidly germinated and B. cereus grew to very high counts (over 8 Log CFU/ml) in both sample series. Formula prepared according to WHO protocol exhibited earlier growth, exceeding 7 Log CFU/g within 24 hours, whereas such level was reached in 36 h with the FDA protocol. In parallel, cereulide toxin was only detectable after 48 hours of storage at 20°C, when bacterial count exceeded 8 log CFU/g, with lower concentration in the FDA series. These findings suggest the particular nature of the risk posed by B. cereus in PIF if compared to other pathogens, and underscore the critical importance of adhering to proper hygiene, immediate feeding practices, and avoiding the storage of reconstituted formula at abuse temperatures to protect vulnerable infant populations from severe foodborne risks.