Isolation and characterization of multidrug-resistant Klebsiella pneumoniae from raw cow milk in Jiangsu and Shandong provinces, China.

Antimicrobials are the most important therapy to bovine mastitis. Bacterial infection and antibiotic treatment of mastitis cycles frequently in dairy farms worldwide, giving rise to concerns about the emergence of multidrug-resistant (MDR) bacteria. In this study, we examined the microbial diversity and antibiotic resistance profiles of bacteria isolated from raw milk from dairy farms in Jiangsu and Shandong provinces, China. Raw milk samples were collected from 857 dairy cattle including 800 apparently healthy individuals and 57 cows with clinical mastitis (CM) and subjected to microbiological culture, antimicrobial susceptibility assay and detection of antibiotic-resistant genes by polymerase chain reaction (PCR) and sequencing. A total of 1,063 isolates belonging to 41 different bacterial genera and 86 species were isolated and identified, of which Pseudomonas spp. (256/1,063, 24.08%), Staphylococcus. spp. (136/1,063, 12.79%), Escherichia coli (116/1,063, 10.91%), Klebsiella spp. (104/1,063, 9.78%) and Bacillus spp. (84/1,063, 7.90%) were most frequently isolated. K. pneumoniae, one of the most prevalent bacteria, was more frequently isolated from the farms in Jiangsu (65/830, 7.83%) than Shandong (1/233, 0.43%) province, and showed a positive association with CM (P<0.001). The antimicrobial susceptibility assay revealed that four of the K. pneumoniae isolates (4/66, 6.06%) were MDR bacteria (acquired resistance to ≥ three classes of antimicrobials). Furthermore, among 66 isolates of K. pneumoniae, 21.21% (14/66), 13.64% (9/66) and 12.12% (8/66) were resistant to tetracycline, chloramphenicol and aminoglycosides, respectively. However, all K. pneumoniae isolates were sensitive to monobactams and carbapenems. The detection of antibiotic-resistant genes confirmed that the β-lactamase genes (blaSHV and blaCTX-M ), aminoglycoside modifying enzyme genes [aac(6')-Ib, aph(3')-I and ant(3'')-I], tetracycline efflux pump (tetA), and transposon genetic marker (intI1) were positive in MDR isolates. This study indicated that MDR K. pneumoniae isolates emerged in dairy farms in Jiangsu province and could be a potential threat to food safety and public health.