Cattle feces are a reservoir of diverse Acinetobacter species with potential to spread antibiotic resistance genes

Antibiotic resistance poses a major threat to human health, with antibiotic use in livestock contributing to the selection and spread of resistance genes. The genus Acinetobacter includes human- and animal-associated species capable of acquiring resistance, yet their diversity and resistance potential in livestock remain far less explored than in humans. In this study, we investigated Acinetobacter in cattle feces from 28 Czech farms with contrasting antibiotic use, aiming to assess species composition, resistance profiles, and the potential for resistance dissemination. We applied an integrative approach combining strain isolation and characterization, enrichment cultures, metabarcoding, and shotgun metagenomics. Cattle feces harbored diverse Acinetobacter species with A. indicus and A. pseudolwoffii being the core species based on both isolated strains and metabarcoding, while A. baumannii was less common. Acinetobacter species occurrence determined by metabarcoding was driven by multiple factors, including production type, herd size, and per-head antibiotic use, while their abundance was mostly influenced by sample type (higher in feces from the farm floor than in rectal samples) and production type (higher in dairy than in beef cattle). Remarkably, 37% of the 284 isolated strains could not be assigned to validly named species and represent at least 19 putative novel species. Decreased susceptibility due to acquired resistance was observed in 57 strains; notably, A. indicus and A. pseudolwoffii from antibiotic-using farms were less susceptible to streptomycin than those from antibiotic-free farms. Shotgun metagenomics revealed a greater richness of acquired resistance genes in antibiotic-using farms, including the clinically relevant carbapenemase gene blaOXA-58. This gene was located on putative plasmid contigs alongside streptomycin resistance determinants strA-strB, suggesting horizontal dissemination under streptomycin selection pressure. Strain analysis confirmed the co-localization of blaOXA-58 and strA-strB on a large plasmid in A. pseudolwoffii. Despite relatively strict regulations, Czech cattle farms constitute a reservoir of antibiotic-resistant Acinetobacter carrying mobile resistance genes of clinical concern. Commonly applied antibiotics likely co-select for such genes, posing an ongoing public health risk. Our findings reveal an unexpectedly high diversity of Acinetobacter spp. in cattle, highlighting the research bias toward human-associated species and underscoring the need for integrated One Health monitoring approaches.