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 (mcr) 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 Enterobacteriaceae (n = 90), Pseudomonas aeruginosa (n = 10), and Aeromonas hydrophila (n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr-1–9 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 mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli, 21.28% (10/47) A. hydrophila, 19.12% (9/47) K. pneumoniae, and 17.02% (8/47) P. aeruginosa. This is the first report of mcr-3 and mcr-7 in P. aeruginosa. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli 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 mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr 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.

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

“…Since the first colistin-resistant mutant that was reported in 1981 ( Vaara et al, 1981 ), colistin resistance has been evolving in several microorganisms like A. baumannii ( Potron et al, 2019 ), K. pneumoniae ( Shankar et al, 2019 ; Tartor et al, 2021b ), and E. cloacae ( Huang et al, 2019 ). A recent study has reported mcr -harboring isolates from animals and humans ( Luo et al, 2020 ) and another group has isolated mcr -positive E. coli and K. pneumoniae from cancerous patients in Egypt ( Zafer et al, 2019 ).…”

An Update of Mobile Colistin Resistance in Non-Fermentative Gram-Negative Bacilli

“…Because of the low incidence of the mcr gene in Pseudomonas compared to other microorganisms in ESKAPE pathogens, this raises the question of the fitness barrier or transferability properties of the mcr gene among Pseudomonas. The transmissibility of mcr genes in P. aeruginosa was demonstrated by Tartor et al via conjugation with E. coli J53 (Tartor et al, 2021). Four mcr genes, mcr-1, mcr-2, mcr-3, and mcr-7, were able to transfer into the recipient bacteria and increased the MIC of the recipient cells up to 64 mg/ml (Tartor et al, 2021).…”

“…Ahmed et al collected fecal samples from migratory birds in Egypt during the winter season and detected MCR-1 in P. aeruginosa (Ahmed et al, 2019). Some studies have detected mcr genes in cow's milk, animal meat, and soil (Fujihara et al, 2015;Javed et al, 2020;Tartor et al, 2021). It is noteworthy that the oldest specimen was retrieved from the environment in 1983 but was never recognized until the WGS era (Fujihara et al, 2015).…”

Longitudinal Monitoring Reveals Persistence of Colistin-Resistant Escherichia coli on a Pig Farm Following Cessation of Colistin Use

“…The mcr genes encode phosphoethanolamine transferases enzymes which change the lipid A portion of the lipopolysaccharides (LPS), suppressing colistin binding ( 9 ). The mcr genes have been reported not only from various livestock origins (pigs, poultry, bovine) ( 10 , 11 ) and food products ( 12 ) but also from the environment as well as from humans ( 13 ). Since the extensive usage of colistin in livestock farms played a major role in the occurrence of colistin resistant mcr genes, controlling the dissemination of these resistant genes from farms to the environment has become a critical concern ( 14 ).…”