Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk

Abstract

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance (<i>mcr</i>) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to <i>Enterobacteriaceae</i> (<i>n</i> = 90), <i>Pseudomonas aeruginosa</i> (<i>n</i> = 10), and <i>Aeromonas hydrophila</i> (<i>n</i> = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for <i>mcr</i>-<i>1-9</i> genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne <i>mcr</i> genes. The <i>mcr-1</i> gene was identified in 31.91%, <i>mcr-2</i> in 29.79%, <i>mcr-3</i> in 34.04%, and each of <i>mcr-4</i> and <i>mcr-7</i> in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were <i>E. coli</i>, 21.28% (10/47) <i>A. hydrophila</i>, 19.12% (9/47) <i>K. pneumoniae</i>, and 17.02% (8/47) <i>P. aeruginosa</i>. This is the first report of <i>mcr</i>-3 and <i>mcr</i>-7 in <i>P. aeruginosa</i>. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to <i>E. coli</i> J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance <i>mcr</i> gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of <i>mcr</i> genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria.