Genetic diversity, virulence genes, antimicrobial resistance, and biofilm formation of <i>Klebsiella pneumoniae</i> isolated from bovine mastitis milk in South Korea

<i>Klebsiella pneumoniae</i>, a zoonotic agent, is a causative pathogen of bovine mastitis. Despite its clinical relevance in dairy farms, studies on <i>K. pneumoniae</i> in bovine mastitis remain limited. Additionally, studies on <i>K. pneumoniae</i>'s genetic diversity and virulence characteristics in South Korea remain limited. Therefore, in this study, we aimed to elucidate the genetic diversity, antimicrobial resistance, virulence genes, and biofilm-forming capacity of 29 <i>K</i>. <i>pneumoniae</i> strains isolated from bovine mastitis milk samples in South Korea between 2017 and 2023. Multilocus sequence typing revealed 23 sequence types, four of which were novel, indicating substantial genetic heterogeneity and the absence of a dominant clonal lineage. Excluding intrinsic resistance, the highest resistance rates were observed for tetracycline (34.5%) and sulfisoxazole (31.0%), whereas resistance to the other antibiotics tested ranged from 0% to 20.7%. In addition, multidrug resistance (MDR) was noted in 20.7% of isolates. Virulence gene analysis revealed that most isolates carried the <i>ureA</i>, <i>uge</i>, <i>wabG</i>, and <i>fimH</i> genes, whereas <i>allS</i>, <i>rmpA</i>, <i>iucB</i>, and <i>iroNB</i> were not detected. Two isolates exhibited a hypermucoviscous phenotype, and one belonged to the capsular serotype K2. All isolates demonstrated biofilm-forming ability, with moderate-to-strong production observed in over 89.0% of cases, indicating potential for persistence and treatment challenges. In conclusion, <i>K. pneumoniae</i> isolates from mastitis milk carried multiple virulence genes and showed MDR as well as robust biofilm formation. Therefore, continued surveillance and further characterization of <i>K. pneumoniae</i> are needed to support mastitis control and protect public health.IMPORTANCE<i>Klebsiella pneumoniae</i> is an emerging environmental pathogen associated with clinical mastitis in dairy cows, raising concerns regarding antimicrobial resistance and public health. To the best of our knowledge, this study provides the first comprehensive characterization of <i>K. pneumoniae</i> isolates from mastitis milk in South Korea, including analyses of genetic diversity, antimicrobial resistance, virulence factors, and biofilm formation. The findings advance our current understanding of <i>K. pneumoniae</i> associated with bovine mastitis and highlight the need for continued surveillance that will contribute to mastitis control efforts and safeguard public health.