The emergence of Clostridioides difficile as the main agent of antibiotic-associated diarrhoea has raised concerns about its potential zoonotic role in different animal species. The use of antimicrobials is a major risk factor for C. difficile infection. Here, we provide data on C. difficile infection in dairy and beef calves in Umbria, a region in central Italy. This cross-sectional study focuses on prevalence, risk factors, ribotypes, toxinotypes and antimicrobial resistance profiles of circulating ribotypes. A prevalence of 19.8% (CI95%, 12–27.6%) positive farms was estimated, and the prescription of penicillins on the farms was associated with C. difficile detection (OR = 5.58). Eleven different ribotypes were found, including the ST11 sublineages RT-126 and -078, which are also commonly reported in humans. Thirteen isolates out of 17 showed resistance to at least one of clindamycin, moxifloxacin, linezolid and vancomycin. Among them, multiple-drug resistance was observed in two isolates, belonging to RT-126. Furthermore, RT-126 isolates were positive for tetracycline resistance determinants, confirming that tetracycline resistance is widespread among ST11 isolates from cattle. The administration of penicillins increased the risk of C. difficile in calves: this, together with the recovery of multi-resistant strains, strongly suggests the need for minimising antibiotic misuse on cattle farms.

Poultry is the most likely source of livestock-associated Extended Spectrum Beta-Lactamase (ESBL) and plasmid-mediated AmpC (pAmpC)-producing E. coli (EC) for humans. We tested the hypothesis that farming methods have an impact on the load of ESBL/pAmpC-EC in the gut of broilers at slaughter. Isolates (n = 156) of antibiotic-free (AF), organic (O), and conventional (C) animals were characterized for antibiotic susceptibility and antibiotic resistance genes. Thirteen isolates were whole-genome sequenced. The average loads of ESBL/pAmpC-EC in cecal contents were 4.17 Log CFU/g for AF; 2.85 Log CFU/g for O; and 3.88 Log CFU/g for C type (p < 0.001). ESBL/pAmpC-EC isolates showed resistance to antibiotic classes historically used in poultry, including penicillins, tetracyclines, quinolones, and sulfonamides. Isolates from O and AF farms harbored a lower proportion of resistance to antibiotics than isolates from C farms. Among the determinants for ESBL/pAmpC, CTX-M-1 prevailed (42.7%), followed by TEM-type (29%) and SHV (19.8%). Avian pathogenic E. coli (APEC), belonging to ST117 and ST349, were identified in the collection. These data confirm the possible role of a broiler as an ESBL/AmpC EC and APEC reservoir for humans. Overall, our study suggests that antibiotic-free and organic production may contribute to a reduced exposure to ESBL/AmpC EC for the consumer.

The impact of soil fertilization with animal manure on the spread and persistence of antibiotic resistance in the environment is far from being fully understood. To add knowledge about persistence and correlations between antibiotic residues and antibiotic resistance genes (ARGs) in fertilized soil, a longitudinal soil mesocosm study was conducted. Soil samples were collected from the mesocosms immediately before spreading and then afterward at fifteen time points during a 320-day observation period. Eight ARGs (ermB, sul1, tetA, tetG, tetM, cfr, fexA, and optrA) and the class 1 integron-integrase gene, intI1, were determined in both pig slurry and soil, as well as residues of 36 antibiotics. Soil chemical and biochemical parameters were also measured. Twelve antibiotics were detected in the slurry in the range of 3 µg kg⁻¹–3605 µg kg⁻¹, with doxycycline, lincomycin, and tiamulin being the most abundant, whereas ermB, sul1, and tetM were the predominant ARGs. Before spreading, neither antibiotic residues nor ARGs were detectable in the soil; afterwards, their concentrations mirrored those in the slurry, with a gradual decline over the duration of the experiment. After about three months, the effect of the amendment was almost over, and no further evolution was observed.

Antimicrobial resistance (AMR) among commensal Escherichia coli from poultry is a growing concern for food safety and public health. This study investigated AMR patterns in E. coli isolated from broiler neck skin at slaughter, comparing organic, antibiotic-free (ATB-free), and conventional production systems. A total of 375 samples were collected from two Italian slaughterhouses and tested by broth microdilution following EU protocols. E. coli was recovered from 358 samples, and 37.9% were presumptively positive for ESBL/AmpC-producing strains. Conventional broilers showed the highest resistance to ampicillin (73.8%), sulfonamides (72.5%), and fluoroquinolones (nalidixic acid, 62.5%; ciprofloxacin, 67.5%), while organic and ATB-free systems showed significantly lower levels. Intermediate resistance occurred for trimethoprim (21.4–47.9%) and tetracycline (36–54%), and low prevalence (<10%) was found for gentamicin, tigecycline, and third-generation cephalosporins. No relevant resistance was detected to colistin or carbapenems (≤1.2%). Total E. coli counts did not differ among systems, suggesting differences in resistant strain proportions rather than bacterial load. ATB-free flocks processed after conventional batches displayed higher resistance, indicating possible cross-contamination during slaughter. These results highlight the influence of farming practices and slaughterhouse hygiene on AMR dissemination, underscoring the need for integrated farm-to-slaughter control strategies.