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

Abstract

Klebsiella pneumoniae, a zoonotic agent, is a causative pathogen of bovine mastitis. Despite its clinical relevance in dairy farms, studies on K. pneumoniae in bovine mastitis remain limited. Additionally, studies on K. pneumoniae'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 K. pneumoniae 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 ureA, uge, wabG, and fimH genes, whereas allS, rmpA, iucB, and iroNB 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, K. pneumoniae isolates from mastitis milk carried multiple virulence genes and showed MDR as well as robust biofilm formation. Therefore, continued surveillance and further characterization of K. pneumoniae are needed to support mastitis control and protect public health.IMPORTANCEKlebsiella pneumoniae 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 K. pneumoniae 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 K. pneumoniae associated with bovine mastitis and highlight the need for continued surveillance that will contribute to mastitis control efforts and safeguard public health.

Keywords: Antimicrobial resistance; Klebsiella pneumoniae; biofilm; bovine mastitis; multilocus sequence typing; virulence factors.

Fig 1

MLST-based phylogenetic tree of Klebsiella pneumoniae isolated from bovine mastitis milk. A phylogenetic tree of the 29 Klebsiella pneumoniae isolates was constructed using MLST sequences and the neighbor-joining method with the Jukes-Cantor model. Bootstrap analysis with 1,000 replicates was performed. The isolate names, collection years, and geographic origins were also included. NT, non-typeable. *These isolates represent the novel STs identified in this study.

Fig 2

Minimum spanning tree analysis of Klebsiella pneumoniae isolates from human infection-derived and bovine mastitis milk based on MLST allelic profiles. A minimum spanning tree of K. pneumoniae isolates was generated using the geoBURST algorithm in PhyloViz 2.0, based on multilocus sequence typing allelic profiles and the single-locus variant criterion. Each node represents a unique sequence type (ST), and the node size is proportional to the number of isolates sharing the same ST. Colors indicate the origin of isolates: blue represents human infection-derived isolates, red represents bovine mastitis isolates, and green represents bovine mastitis isolates. Edges connect STs that differ in only one of the seven housekeeping loci.

Fig 3

Antimicrobial susceptibility of Klebsiella pneumoniae isolated from bovine mastitis milk. Superscript letters (a–c) following the antimicrobial names on the x-axis indicate statistically significant differences in antibiotic resistance rates (P < 0.05). Ampicillin resistance was excluded from statistical analysis because of the intrinsic resistance of K. pneumoniae to this antimicrobial. AMC, amoxicillin/clavulanic acid; AMP, ampicillin; TAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; MER, meropenem; CHL, chloramphenicol; CIP, ciprofloxacin; NAL, nalidixic acid; COL, colistin; GEN, gentamicin; TET, tetracycline; FIS, sulfisoxazole; SXT, trimethoprim/sulfamethoxazole.