Genomic characterization, antimicrobial resistance, and biofilm formation of Elizabethkingia anophelis isolated from bovine mastitis cases in Japan

Elizabethkingia anophelis is an emerging opportunistic pathogen in human medicine, known for healthcare-associated outbreaks, intrinsic multidrug resistance, and persistence in aqueous environments. However, its occurrence and potential relevance in food-producing animals remain poorly understood. This study aimed to characterize E. anophelis isolates recovered from bovine mastitis cases and to assess their potential significance from a One Health perspective. Five isolates of E. anophelis were recovered from milk samples collected from clinical mastitis cases on a single dairy farm in Japan. Whole-genome sequencing was performed for species identification and comparative genomic analysis, including average nucleotide identity (ANI) and phylogenetic reconstruction. Antimicrobial susceptibility was determined using broth microdilution, and biofilm formation was evaluated using a crystal violet assay. All isolates showed extremely high genomic similarity (ANI > 99.99%), indicating that they likely originated from a single clonal strain. Phylogenetic analysis placed the isolates within E. anophelis subsp. endophytica, closely related to strains previously reported from animal-associated and environmental sources, while remaining genetically proximate to some human clinical isolates. The isolates showed high minimum inhibitory concentrations to multiple β-lactam antibiotics, including carbapenems, and harbored chromosomal β-lactamase genes (bla B , bla GOB , and bla CME ). In addition, all isolates demonstrated biofilm-forming capacity in vitro. These findings indicate that food production environments can harbor clonal E. anophelis strains carrying chromosomal β-lactamase genes and exhibiting high MIC values against multiple β-lactam antibiotics. Although a direct causative role in bovine mastitis remains unclear, the presence of such strains in milk suggests a potential interface between livestock and human health. This study highlights the importance of including agricultural settings in surveillance of emerging pathogens and supports a One Health approach to understanding their ecology and public health relevance.