Genomic Characterization of Multidrug-Resistant Klebsiella pneumoniae Strains from Bovine Mastitis Reveals Extensive Resistome and Virulome Arsenals
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Klebsiella pneumoniae , a major bovine clinical mastitis (CM) pathogen, carries multidrug resistance (MDR) and virulence factor genes (VFGs), posing serious animal and public health threats. This study screened 27 K. pneumoniae isolates (19 from CM milk and 8 from feces) through culture, biochemical tests, and 16S rRNA -gene sequencing. An overall prevalence of K. pneumoniae was 22.5% (27/120), with a higher rate in milk (27.14%) than feces (16.0%). Antibiogram profiling revealed that all isolates were multidrug-resistant, with high resistance to doxycycline, tetracycline, nalidixic acid, and ampicillin. Three highly resistant isolates (MBBL2, MNH_G2C5, MNH_G2C5F) underwent whole-genome sequencing for comprehensive genomic analysis. Sequence typing (ST), phylogenetic and pangenome analyses assigned MBBL2 and MNH_G2C5F to ST273 and MNH_G2C5 to ST101, clustering with global human- and animal-derived K. pneumoniae strains, and carrying notable strain-specific accessory genes (MNH_G2C5:123; MBBL2/MNH_G2C5F:826). Functional annotation identified abundant genes for carbohydrate metabolism (~ 10%), amino acid transport (~ 9%), and transcription (~ 9%). Resistome analysis identified 29–41 resistance genes, covering 12 antibiotic classes, metals, biocides, and acid stress. Virulence profiling identified 44–60 VFGs involved in adherence, biofilm formation, effector delivery, immune modulation, and metabolism. Genomic plasticity analysis revealed 27–34 variable regions, multiple prophages, 46–58 insertion sequences, and four plasmid replicons. Conserved exopolysaccharide/capsule clusters, secondary metabolites, and high pathogenicity scores (> 0.9) underscored both animal and human pathogenic potential. This study demonstrates that dairy cattle are a reservoir for high-risk MDR clones of K. pneumoniae carrying an extensive resistome and virulome arsenal, highlighting the urgent need for strengthened surveillance and control measures.
