Whole Genome Sequencing Reveals Environmental Pathogen Misidentification and Cross- Phylum Antimicrobial Resistance Gene Transfer in Bovine Mastitis: A Pilot Genomic Study

Background: Bovine mastitis diagnosis relies predominantly on conventional microbiological methods optimized for common pathogens, potentially overlooking environmental bacteria with complex antimicrobial resistance profiles. Methods: This pilot study combined conventional identification with whole-genome sequencing (WGS) analysis of bovine mastitis isolates. A total of 330 milk samples were analyzed using standard microbiological methods, followed by comprehensive genomic characterization of two representative multidrug-resistant isolates using Illumina NovaSeq 6000 sequencing. Antimicrobial resistance gene analysis was performed using BLAST searches against the Comprehensive Antibiotic Resistance Database. Results: Of 330 samples, 202 (61.2%) tested positive for mastitis. WGS revealed critical species misidentification: one isolate initially characterized as gram-positive with Staphylococcus-like morphology was definitively identified as Stutzerimonas stutzeri through genomic analysis. Both sequenced isolates harbored extensive antimicrobial resistance gene repertoires distributed across 8-10 resistance classes, with evidence of horizontal gene transfer across bacterial orders. Phylogenetic analysis revealed resistance genes originated from Proteobacteria (61%) and Firmicutes (39%), indicating cross-phylum gene exchange. Conclusions: This pilot study demonstrates that WGS can identify bacterial species missed by conventional diagnostic methods and reveals complex horizontal gene transfer networks in mastitis-associated bacteria. The environmental pathogen S. stutzeri represents a potentially underrecognized opportunistic mastitis agent with extensive resistance potential. These findings validate the need for genomic surveillance approaches in veterinary diagnostic microbiology.