Genomic characterization of $Klebsiella$ spp. from bovine mastitis: dissemination of a conserved, highly transmissible $lac_{\mathrm{acq}}^{+}\ fec^{+}$ plasmid drives burden of disease

Bovine mastitis poses a significant health concern for dairy cattle and a major economic burden on the dairy industry. Klebsiella spp. are important mastitis pathogens, with previous studies identifying lactose-utilization ( lac $_{acq}$ ) and iron-acquisition ( fec ) systems as key determinants associated with pathogenicity. To investigate how these are acquired and shared, we genomically characterized 60 mastitis-associated Klebsiella isolates by fully resolving their chromosomes and plasmids. The isolates were identified as Klebsiella pneumoniae ( n = 46), Klebsiella michiganensis ( n = 8), Klebsiella quasipneumoniae ( n = 3), Klebsiella grimontii ( n = 2), and Klebsiella variicola ( n = 1). Phylogenetic analysis revealed diverse lineages, with sporadic transmission events within and across farms. Among unique Klebsiella spp. isolates, 48/55 (87.3%) and 53/55 (96.4%) harbored lac $_{acq}$ and fec , respectively. In K. pneumoniae , the lac $_{acq}$ operon was consistently found on plasmids alongside fec . Both horizontal and vertical transfer of lac $_{acq}$ $^{+}$ fec $^{+}$ plasmids were observed. Many phylogenetically diverse mastitis isolates from distant farms carried an identical conjugative 132 kb plasmid, suggesting recent acquisitions of a single circulating plasmid as a major driver of mastitis. K. pneumoniae ST107, a globally prevalent mastitis-causing lineage, carried large non-mobilizable plasmids with fec and two lac $_{acq}$ operons. In contrast to K. pneumoniae , mastitis-associated K. michiganensis often carried lac $_{acq}$ on integrative conjugative elements and inherently harbored a chromosomal fec -like gene cluster. Few isolates possessed antimicrobial resistance genes or virulence factors linked to pathogenicity in humans. Our results provide new insights into the genomic diversity of mastitis-associated Klebsiella and the role of mobile genetic elements. IMPORTANCE Understanding the genetic basis of Klebsiella -induced mastitis is essential for improving prevention and control strategies. Our study reveals that the key mastitis-associated traits—lactose utilization ( lac $_{acq}$ ) and iron acquisition ( fec )—are commonly encoded on plasmids. The discovery of an identical conjugative plasmid in diverse Klebsiella pneumoniae lineages highlights the potential for rapid and widespread dissemination of virulence traits, independent of clonal background. However, we also show that clonal spread—combined with the vertical inheritance of a lac $_{acq}$ ⁺ fec ⁺ plasmid—contributes to the success of K. pneumoniae ST107, a globally prevalent mastitis lineage. Together, our findings highlight the central role of mobile genetic elements in the ecology of mastitis-associated Klebsiella .