Molecular Epidemiology and Antimicrobial Resistance of Outbreaks of Klebsiella pneumoniae Clinical Mastitis in Chinese Dairy Farms

Abstract

Klebsiella pneumoniae is an opportunistic pathogen that causes serious infections in humans and animals. However, the availability of epidemiological information on clinical mastitis due to K. pneumoniae is limited. To acquire new information regarding K. pneumoniae mastitis, data were mined about K. pneumoniae strains on dairy cattle farms (farms A to H) in 7 Chinese provinces in 2021. Hypermucoviscous strains of K. pneumoniae were obtained by the string test. MICs of antimicrobial agents were determined via the broth microdilution method. Ten antimicrobial resistance genes and virulence genes were identified by PCR. The prevalence of K. pneumoniae was 35.91% (65/181), and 100% of the bacteria were sensitive to enrofloxacin. Nine antimicrobial resistance genes and virulence genes were identified and compared among farms. The hypermucoviscous phenotype was present in 94.44% of isolates from farm B, which may be a function of the rmpA virulence gene. Based on these data, the multidrug-resistant strains SD-14 and HB-21 were chosen and sequenced. Genotypes were assayed for K. pneumoniae isolates from different countries and different hosts using multilocus sequence typing (MLST). Ninety-four sequence types (STs) were found, and 6 STs present a risk for spreading in specific regions. Interestingly, ST43 was observed in bovine isolates for the first time. Our study partially reveals the current distribution characteristics of bovine K. pneumoniae in China and may provide a theoretical basis for the prevention and treatment of bovine K. pneumoniae mastitis. IMPORTANCE K. pneumonia is ubiquitous in nature and infects a wide range of hosts, including animals, and humans. It is one of the leading inducements of clinical mastitis (CM) in dairy cows, a prevalent and costly disease that is predominantly associated with bacterial infection. In general, CM caused by Gram-negative bacteria is more difficult to cure than that associated with Gram-positive pathogens, with an average cost per case of 211.03 U.S. dollars (USD) for Gram-negative bacterial infections compared with 133.73 USD for Gram-positive bacterial CM cases. After Escherichia coli, K. pneumoniae is the second most common Gram-negative cause of bovine CM, but it is the most detrimental in terms of decreased milk yield, discarded milk, treatment costs, death, and culling. In view of the economic implications of K. pneumoniae infection in dairy farming, research into population structure and antibiotic resistance is particularly important.

Keywords: Klebsiella pneumoniae; antimicrobial resistance; bovine clinical mastitis; molecular epidemiology.

FIG 1

Percentage of K. pneumoniae in samples from cows with clinical mastitis in eight farms. The circle sizes indicate the detection rate of K. pneumoniae.

FIG 2

Bacterial resistance. (A) Resistance rate of K. pneumoniae to 12 antibiotics. (B) Percentages of MDR K. pneumoniae isolates resistant to different numbers of antibiotics. (C) Percentages of MDR K. pneumoniae isolates resistant to 8 or more antibiotics in the north and south regions of China. (D) Percentages of MDR K. pneumoniae isolates resistant to 8 or more antibiotics on different farms. (E) Rate of detection of resistance genes.

FIG 3

Results of the string test and bacterial virulence genes. (A) Colony morphology of hypermucoviscous K. pneumoniae. (B) Rate of hypermucoviscosity in K. pneumoniae isolates from different farms. (C) Rate of detection of virulence genes.

FIG 4

Proportions of 94 STs in 104 strains of K. pneumoniae. Different colors indicate percentages of the indicated STs in 104 K. pneumoniae isolates.

FIG 5

Burst map of 104 K. pneumoniae STs. Each circle represents one sequence type labeled with ST name; sizes indicate the total number of strains. Branch lengths indicate phylogenetic distances between STs. Different colors indicate percentages of certain STs in different countries.