# Whole genome sequencing reveals environmental pathogen misidentification and potential for cross-phylum antimicrobial resistance gene transfer in bovine mastitis: a pilot genomic study

## TL;DR

This pilot study shows that whole genome sequencing can detect misidentified environmental bacteria in bovine mastitis and reveals their potential for spreading antibiotic resistance across different bacterial groups.

## Contribution

The study demonstrates the potential for cross-phylum antimicrobial resistance gene transfer in bovine mastitis through genomic analysis of misidentified environmental pathogens.

## Key findings

- Whole genome sequencing identified Stutzerimonas stutzeri, an environmental pathogen misclassified by conventional methods.
- Both isolates showed diverse antimicrobial resistance genes from multiple bacterial phyla, indicating horizontal gene transfer potential.
- Mobile genetic elements like plasmids and integrons were found, supporting the spread of resistance genes.

## Abstract

The diagnosis of bovine mastitis relies predominantly on conventional microbiological methods optimized for common pathogens, potentially ignoring environmental bacteria with complex antimicrobial resistance profiles.

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. The limited sample size reflects the pilot nature of this proof-of-concept study. Analysis of antimicrobial resistance genes was performed using BLAST searches against the Comprehensive Antibiotic Resistance Database.

Of 330 samples, 202 (61.2%) tested positive for mastitis. WGS revealed misidentification of critical species of an environmental pathogen (Stutzerimonas stutzeri) and comparative analysis with E. coli (included as a control for a known mastitis pathogen). An isolate originally characterized as Gram-positive with Staphylococcus-like morphology was definitively identified as Stutzerimonas stutzeri by genomic analysis. Both isolates harbored diverse antimicrobial resistance genes with phylogenetic origins spanning multiple bacterial orders and phyla (Enterobacterales, Bacillales, Pseudomonadales, Enterococcales), suggesting a potential for horizontal gene transfer. Mobile genetic elements such as plasmids, integrons and insertion sequences were identified in both genomes, consistent with the ability for gene mobility. Phylogenetic analysis revealed that resistance genes originated from Proteobacteria (61%) and Firmicutes (39%), indicating cross-phylum gene exchange.

This pilot study provides preliminary evidence that whole genome sequencing can identify bacterial species that may be missed by traditional diagnostic methods. Analysis of two isolates revealed evidence of horizontal gene transfer potential in mastitis-associated bacteria. The environmental pathogen S. stutzeri may represent a poorly recognized opportunistic mastitis pathogen with significant resistance potential. Based on these exploratory findings from two cases, our results suggest the potential utility of genomic surveillance approaches in veterinary diagnostic microbiology, necessitating larger validation studies.

The online version contains supplementary material available at 10.1186/s12917-025-05280-z.

i. WGS identified misclassified environmental pathogen in bovine mastitis.

ii. S. stutzeri showed extensive AMR genes despite conventional ID failure.

iii. Phylogenetic diversity and mobile elements suggest horizontal gene transfer potential.

iv. Genomic analysis revealed 287-294 resistance genes per isolate.

v. Pilot study demonstrates need for molecular diagnostic approaches.

The online version contains supplementary material available at 10.1186/s12917-025-05280-z.