Antimicrobial resistance is a major concern in the dairy industry.
Aim: This study aimed to: (1) investigate the occurrence and resistance phenotypes of the Gram-negative bacteria in mastitic cow’s milk as well as raw unpasteurized milk that consumed by the public; (2) identify the multilocus sequence types (MLST), resistance genes, and plasmid replicon types of MDR and XDR Gram-negative isolates; (3) determine the genetic environment of mcr-10 gene and epidemiological relatedness between the study isolate and the publicly available genomes of K. pneumoniae isolates harboring mcr genes.
Material and methods: A total of 570 milk samples were collected from clinical (n = 350) and subclinical (n = 95) bovine mastitis, and raw unpasteurized milk (n = 125) for isolation of Gram-negative bacteria. They were identified depending on culture characters, gram stain reaction, biochemical tests, serological tests, and PCR. The antibiogram of all recovered Gram-negative bacteria was determined by the disc diffusion method. Whole-genome sequencing (WGS) was performed for 10 representative MDR and XDR isolates that exhibited unique antibiogram and the highest MAR indices.
Results: A total of 184 Gram-negative bacteria were isolated. They were identified as E. coli (48.91%), K. pneumoniae (13.59%), P. mirabilis (17.93%), A. hydrophila (9.24%), E. cloacae (8.15%), and C. freundii (2.17%). Klebsiella pneumoniae, Aeromonas hydrophila, Enterobacter cloacae (100% each), Escherichia coli (87.78%), and Proteus mirabilis (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug resistance (XDR) phenotype was found in P. mirabilis (30.30%) and E. coli (3.33%) isolates. There were ten isolates (four E. coli, three Klebsiella species and three P. mirabilis) displayed the highest multiple antibiotic resistance (MAR) indices (0.54–0.83). they were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced E. coli isolates. Three E. coli isolates (two from clinical mastitis and one from raw milk) belonging to ST2165 showed similar profile of plasmid replicon types: IncFIA, IncFIB, IncFII, and IncQ1 with an exception to an isolate that contained IncR, whereas E. coli ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin mcr-10 and fosfomycin fosA5 resistance genes in a K. pneumoniae (K1) isolate from bovine milk. Sequence analysis of the reconstructed mcr-10 plasmid from WGS of K1 isolate, showed that mcr-10 gene was bracketed by xerC and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including mcr-10-harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)].
Conclusion: This study reports the first emergence of K. pneumoniae co-harboring mcr-10 and fosA5 genes from bovine milk in the Middle East, which constitutes a public health threat and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required. |
