A newly identified sequence type: genomic and pathogenic profiling of the highly virulent Acinetobacter baumannii ST3475 from bovine mastitis

Background Bovine mastitis causes substantial economic losses worldwide. Acinetobacter baumannii has emerged as a pathogen in bovine mastitis, raising considerable concerns due to its multidrug resistance and potential zoonotic transmission. However, characteristics of A. baumannii involved in bovine mastitis in China remain poorly understood, particularly with emergence of novel sequence types. Methods From 135 Acinetobacter spp. isolates isolated in China from bovine mastitis (2021-2024), we identified a novel sequence type, ST3475. Using the known bovine strain ST3256 for comparison, we conducted hybrid whole-genome sequencing, antimicrobial susceptibility testing, and multiple phenotypic pathogenicity assays (including biofilm formation, cell adhesion/invasion, and in vivo infection models). Results The A. baumannii ST3475 chromosome is 3,875,774 bp with 1 plasmid (158,806 bp); it has an expanded repertoire of 70 virulence genes, including csu and bap , and 18 antimicrobial resistance genes. Furthermore, its genome contains a complement of phage elements, carbohydrate-active enzymes, and a unique carbapenemase gene bla OXA-344 , indicating distinct evolution. Phenotypically, ST3475 demonstrated enhanced biofilm formation, adhesion, invasion, and cytotoxicity against bovine mammary epithelial cells. It also promoted secretion of IL-1β, IL-6, and TNF-α. In Galleria mellonella , the median lethal dose of ST3475 was 2 orders of magnitude lower than ST3256 (LD 50 = 1,378,077 CFU/larva). In a murine mammary infection model, ST3475 caused substantial alveolar destruction and inflammatory infiltration. Conclusion Phylogenetically, ST3475 belongs to clonal complex CC2 but represents a distinct, putative bovine-adapted lineage separate from major human clone CC1. Although currently susceptible to last-resort antimicrobials like carbapenems, carriage of high-risk resistance genes highlights livestock as a potential reservoir for clinically relevant resistance determinants. This study reveals ST3475 as a novel, virulent, and putative bovine-adapted lineage, underscoring the importance of One Health surveillance.