Whole-Genome Sequencing of Gram-Negative Bacteria Isolated From Bovine Mastitis and Raw Milk: The First Emergence of Colistin mcr-10 and Fosfomycin fosA5 Resistance Genes in Klebsiella pneumoniae in Middle East

Abstract

Antimicrobial resistance is a major concern in the dairy industry. This study investigated the prevalence, antimicrobial resistance phenotypes, and genome sequencing of Gram-negative bacteria isolated from clinical (<i>n</i> = 350) and subclinical (<i>n</i> = 95) bovine mastitis, and raw unpasteurized milk (<i>n</i> = 125). <i>Klebsiella pneumoniae</i>, <i>Aeromonas hydrophila</i>, <i>Enterobacter cloacae</i> (100% each), <i>Escherichia coli</i> (87.78%), and <i>Proteus mirabilis</i> (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug-resistance (XDR) phenotype was found in <i>P. mirabilis</i> (30.30%) and <i>E. coli</i> (3.33%) isolates. Ten isolates (four <i>E. coli</i>, three <i>Klebsiella</i> species and three <i>P. mirabilis</i>) that displayed the highest multiple antibiotic resistance (MAR) indices (0.54-0.83), were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced <i>E. coli</i> isolates. Three <i>E. coli</i> 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 <i>E. coli</i> ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin <i>mcr</i>-<i>10</i> and fosfomycin <i>fosA</i>5 resistance genes in a <i>K. pneumoniae</i> (K1) isolate from bovine milk. Sequence analysis of the reconstructed <i>mcr-10</i> plasmid from WGS of K1 isolate, showed that <i>mcr-10</i> gene was bracketed by <i>xer</i>C and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including <i>mcr-10</i>-harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)]. This study reports the first emergence of <i>K. pneumoniae</i> co-harboring <i>mcr</i>-<i>10</i> and <i>fosA5</i> 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.