Background: Antimicrobial resistance is a pressing global concern affecting both human and animal health, with environment playing a key role in the dissemination of resistance determinants. This study aimed to investigate the presence of antimicrobial resistance genes (ARGs) associated with tetracyclines, β-lactams, macrolides, and sulfonamides in environmental matrices collected from 65 sheep and goat farms in central Portugal. Methods: Environmental samples, including water, soil, pasture, and bedding, were analyzed through qPCR for the detection of clinically relevant ARGs. Results: ARGs were detected in 83% of the samples, with over half exhibiting genes from three or more antibiotic classes, suggesting potential multidrug resistance. β-lactamase genes were the most prevalent, followed by those conferring resistance to tetracycline and sulfonamide resistance, while macrolide resistance genes were least frequent. The distribution of ARGs varied by farm type, host species, and municipality. Conclusions: These findings suggest that small ruminant farms serve as important reservoirs for ARGs. The results underscore the need for systematic surveillance and further research into the ecological and genetic factors driving ARG persistence and dissemination in extensive livestock systems, including proper waste management strategies to limit the spread and persistence of antibiotic resistance and mitigate broader public health risks.

Background: Antimicrobial resistance (AMR) is a growing global health concern, driven in part by antibiotic use in animal production systems. Despite its relevance, the microbiome and resistome of small ruminant farm environments remain largely underexplored. Methods: In this study, shotgun metagenomics was applied to environmental samples from 46 sheep, goat and mixed-species farms across 14 municipalities in central Portugal. Results: Microbial profiling revealed a well-preserved microbiome with Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota (syn. Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes respectively) as the most dominant phylum across different farm types. Regarding AMR, a total of 706 unique antimicrobial resistance genes (ARGs), covering 15 antibiotic classes, were detected. Tetracycline, aminoglycoside and macrolide resistance genes dominated across all samples, forming a conserved core resistome. While overall resistome profiles were broadly similar among farm types, significant differences were observed in specific ARG classes, such as pleuromutilin and fosfomycin. Conclusions: These findings highlight small ruminant farm environments as potential reservoirs of clinically relevant ARGs, including WHO highest priority critically important antimicrobial (HPCIA) resistance genes for macrolides (mph(c), erm(f), erm(b)) and fluoroquinolones (qnrD1), as well as critically important antimicrobial (CIA) resistance genes for glycopeptides (vanR-SC, vanR-O) and aminoglycosides (str, aadA), supporting the need to incorporate these environments into surveillance strategies.

Zoonotic bacteria, namely Campylobacter spp., Escherichia coli, Salmonella spp. and Staphylococcus aureus, are commonly implicated in human infections and can be transmitted from animals to humans through direct contact, the environment or the food chain. The emergence of antimicrobial resistance in these zoonotic bacteria, namely extended spectrum β-lactamases (ESBLs)-producing strains of E. coli and methicillin-resistant S. aureus (MRSA), has become a public health concern worldwide. This study aimed to determine the prevalence of fecal carriage of Campylobacter spp., Salmonella spp. and ESBL-producing strains of E. coli, as well as nasal carriage of MRSA, and to identify risk factors associated with the presence of these zoonotic bacteria in small ruminants from Portugal’s Central Region. A total of 732 animals, of which 432 sheep and 300 goats from 122 farms were sampled. Zoonotic enteric bacteria were isolated from individual fecal samples, while MRSA were isolated from pooled nasal swabs collected from each farm. Bacteria were isolated according to standard microbiological methods. The overall prevalence of Campylobacter spp. and Salmonella spp. at the animal level was 15.6% and 8.3%, respectively, with significantly higher prevalence in sheep (19.0% and 12.7%) compared to goat (10.7% and 2.0%). Presumptive ESBL-producing strains of E. coli was isolated from 5.2% of the animals. Presumptive MRSA was isolated from 5.7% of the farms. A cluster analysis was performed to identify farm clusters with similar characteristics based on the isolation of Campylobacter spp., Salmonella spp., presumptive ESBL-producing E. coli, and presumptive MRSA. Farms were grouped into three clusters: “Resistant”, “Campylobacter” and “Salmonella”. The likelihood of farms belonging to “Campylobacter” and “Salmonella” clusters in comparison to “Resistant” cluster was associated with animal species, farm location, and farmer’ practices regarding antibiotic use. This study reinforces the role of small ruminants as asymptomatic reservoirs of Campylobacter spp., Salmonella spp., E. coli and S. aureus and confirms, for the first time, their role as carriers of presumptive antibiotic-resistant zoonotic bacteria in Portugal’s Central Region.

Scrapie (classical and atypical) susceptibility in sheep is strongly influenced by PRNP gene polymorphisms. In Portugal, limited data exist for native breeds such as Serra da Estrela, despite their relevance to animal conservation and food production. The full coding region of PRNP gene of 92 Serra da Estrela sheep was sequenced and SNP frequencies were analysed. The predicted functional impact of nonsynonymous SNPs was assessed using PolyPhen-2 and AMYCO. A total of 27 SNPs were identified, including 20 nonsynonymous variants. Thirteen major haplotypes were observed. The ARR allele, which provides resistance to classical scrapie, was present in 58.7% of the population, with 18.5% of animals being homozygous. Several previously unreported SNPs were identified, and their impact on prion protein aggregation propensity and structure was explored. The high frequency of the ARR allele without full ARR fixation suggests that no selective breeding for scrapie resistance has been applied. These results support the adoption of gradual selection strategies that preserve genetic variability and promote farmer compliance, while increasing classical and atypical scrapie resistance.

There is growing concern that antimicrobial use (AMU) in livestock may contribute to antimicrobial resistance (AMR) in humans and lead to the consumption of animal-derived foods contaminated with antimicrobial residues. As stakeholders in the livestock industry, farmers must participate in the joint effort to reduce AMU. This cross-sectional study, based on a survey questionnaire, was conducted to evaluate the biosafety measures implemented on small ruminant farms and to assess the knowledge, attitudes and practices (KAP) of small ruminant farmers regarding AMU, AMR and residues. The mean biosafety score obtained was 8.4 points on a 0–17 scale. Some biosafety measures appeared difficult to implement, namely vehicle disinfection, requiring visitors to change clothing and footwear at the farm entrance, cleaning and disinfecting farm facilities, using high-pressure washing equipment, and requiring employees to change clothing and footwear upon entering the farm. Although farmers self-reported moderate levels of knowledge (4.9 points on a 0–7 scale) and positive attitudes (5.8 points on a 0–7 scale), significant gaps in knowledge about antibiotics and antimicrobial stewardship persisted. Practices received lower scores (4.7 on a 0–7 scale), especially regarding medication recording, leftover antibiotic management, and waste disposal. Cluster analysis identified distinct farmer profiles with different patterns of knowledge and practices. These findings underscore the importance of considering farmer heterogeneity when designing interventions aimed at improving AMU.

Mikrobiološka kvaliteta mlijeka odražava zdravlje vimena i naknadnu kontaminaciju koja može nastati putem izravnih i neizravnih rezervoara. Cilj ovog istraživanja bio je identificirati bakterijsku raznolikost u uzorcima mlijeka ovaca s mastitisom i u mlijeku iz spremnika te istražiti njihove profile osjetljivosti i otpornosti na antibiotike. Analizirano je ukupno 232 uzorka mlijeka. Iz pojedinačnih uzoraka mlijeka izolirane su 22 bakterijske vrste, među kojima se ističu Staphylococcus aureus (20,3%) i Staphylococcus epidermidis (18,6%). Raznolikost bakterijskih vrsta izoliranih iz spremnika bila je veća i obuhvaćala je 44 vrste, s prevladavanjem E. coli (20,6%), S. aureus (7,8%) i Hafnia alvei (7,2%). Testiranje osjetljivosti na antimikrobne lijekove provedeno je na 217 bakterijskih izolata. E. coli izdvojena iz pojedinačnih uzoraka mlijeka pokazala je visoke stope otpornosti na peniciline i tetracikline, dok je S. aureus pokazao visoke stope otpornosti na aminoglikozide, polimiksin, sulfonamide i tetraciklin, ali i visoku razinu osjetljivosti na cefalosporine treće generacije, linkozamide, neke makrolide, peniciline, fenikole i pleuromutiline. Profil otpornosti bakterija izoliranih iz mlijeka iz spremnika zabrinjavajući je u odnosu na uzorke mlijeka ovaca s mastitisom. Ovi rezultati ukazuju na potrebu za strukturnim poboljšanjima na farmama i podizanjem svijesti o jačanju biosigurnosti, medicinskoj profilaksi i kontroli pristupa veterinarskim lijekovima kako bi se poboljšala kvaliteta mlijeka i smanjila upotreba antibiotika.