Isolation Rate, Antimicrobial Resistance, Virulence-Associated Genes, and Multilocus Sequence Types of Klebsiella pneumoniae Isolated from Cattle with Bovine Respiratory Disease in Henan Province, China

Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in cattle, and Klebsiella pneumoniae (K. pneumoniae) has emerged as an opportunistic pathogen that can colonize the bovine respiratory tract. However, epidemiological data on K. pneumoniae from cattle with BRD in Henan Province, central China, remain scarce. This study aimed to investigate the isolation rate, antimicrobial resistance profiles, resistance genes, virulence-associated genes, and genetic diversity of K. pneumoniae from BRD-affected calves in Henan. From 2021 to 2026, deep nasal swabs were collected once from 517 calves with clinical signs of BRD (Wisconsin score ≥5) across 38 farms in 13 cities over six years. A total of 87 K. pneumoniae isolates were obtained, giving an overall isolation rate of 16.8% (87/517), ranging from 13.8% to 22.6% across five geographic regions. Antimicrobial susceptibility was determined by disk diffusion using CLSI VET01S and CLSI M100-S26 criteria for 15 antimicrobials belonging to six classes. Resistance and virulence-associated genes were screened by PCR, and multilocus sequence typing (MLST) was performed. MLST revealed 34 sequence types (STs), with ST950 being the most prevalent (6.9%), followed by ST262, ST187, and ST306. Antimicrobial resistance was highest against ampicillin (94.3%), sulfisoxazole (79.3%), cefoxitin (73.6%), cefazolin (70.1%), and enrofloxacin (67.8%). Multidrug resistance (resistance to ≥3 classes) was observed in 96.6% (84/87; 95% CI 90.3–98.8%) of isolates. The most common resistance genes were blaTEM (72.4%), sul1 (39.1%), blaSHV (37.9%), and qnrB (14.9%). After Firth’s penalized logistic regression with farm-level cluster-robust standard errors and Benjamini–Hochberg FDR correction, no resistance gene was significantly associated with phenotypic resistance (all q > 0.05). Virulence-associated genes fimH, mrkD, uge, wabG, entB, iutA, and ureA were highly prevalent (>90%), whereas hypervirulence markers (rmpA and rmpA2) were absent. The most common virulence-associated gene combination (eight genes) was present in 35.6% of isolates. These findings indicate high genetic diversity, widespread multidrug resistance, and a broad virulence-associated gene repertoire among K. pneumoniae isolated from cattle with BRD in Henan. Because no healthy comparison group or etiological confirmation was included, the study does not establish K. pneumoniae as the causative agent of BRD. The high resistance rates to β-lactams, sulfonamides, and quinolones highlight the need for prudent antimicrobial use. Florfenicol showed the lowest in vitro resistance rate among the tested agents; however, clinical efficacy, treatment outcome, and safety were not evaluated. Continuous surveillance and molecular monitoring are essential to manage this pathogen in cattle populations.