Genomic characterization of cefotaxime-resistant Proteobacteria isolated from a bat-harboring cave in Zambia

Bats are widely recognized as reservoirs of emerging and re-emerging pathogens, and their ecological interactions with humans and livestock present important opportunities for the transmission of infectious agents and antimicrobial resistance (AMR). However, little is known about the occurrence of resistant bacteria in bat-associated environments in Zambia or their potential role in the maintenance of AMR outside clinical and agricultural settings. This study investigated the genomic characteristics of cefotaxime-resistant Proteobacteria isolated from bat fecal droppings collected at Leopards Hill Cave, an established hotspot for zoonotic pathogens. Four hundred bat fecal samples were cultured on cefotaxime-supplemented MacConkey agar, and those exhibiting bacterial growth were subjected to antimicrobial susceptibility testing and whole-genome analysis. Of the 400 samples processed, four (1 %) yielded growth, resulting in three bacterial species: <i>Pseudomonas aeruginosa</i> (n = 1), <i>Enterobacter mori</i> (n = 1), and <i>Brucella intermedia</i> (formerly <i>Ochrobactrum intermedium</i>) (n = 2). Genomic screening revealed that <i>P. aeruginosa</i> strain CB_234 harbored <i>bla</i> _OXA-50, <i>aph(3')-IIb</i>, and <i>catB7</i>, which confer resistance to β-lactams, aminoglycosides, and chloramphenicol, respectively. It also possessed multiple virulence determinants involved in adherence, motility, and secretion systems that enhance host colonization and environmental persistence. Core genome phylogenetic analysis placed CB_234 within a clade exclusively composed of clinical isolates from Nigeria, Thailand, Russia, Kenya, and Ghana, indicating a shared evolutionary lineage among globally dispersed hospital-associated strains. Conversely, environmental isolates from plant and aquatic sources, along with a dog-associated isolate, were phylogenetically distant, highlighting the distinct evolutionary origins. The <i>E. mori</i> isolate carried <i>bla</i> _ACT and <i>qnrE</i> resistance genes and plasmid replicons, suggesting potential mobility of resistance traits through horizontal gene transfer. In contrast, the two <i>B. intermedia</i> isolates did not harbor any known AMR genes or plasmid replicons. However, this species is increasingly recognized as an opportunistic pathogen. The detection of AMR-associated bacterial species in a natural bat habitat supports the evidence of resistance determinants circulating in wildlife environments in Zambia. Given that bats are unlikely to encounter clinical antibiotics directly, the persistence of such genes in their environment suggests that natural ecosystems may play an underappreciated role in maintaining AMR reservoirs independent of direct antimicrobial pressure. These findings underscore the importance of incorporating wildlife and environmental niches into national and global AMR surveillance frameworks under a One Health approach to better understand the ecological dimensions of AMR emergence and dissemination.