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.

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 (n = 350) and subclinical (n = 95) bovine mastitis, and raw unpasteurized milk (n = 125). 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. Ten isolates (four E. coli, three Klebsiella species and three P. mirabilis) 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 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)]. 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.

Antimicrobial Resistance of Salmonella enteritidis and Salmonella typhimurium Isolated from Laying Hens, Table Eggs, and Humans with Respect to Antimicrobial Activity of Biosynthesized Silver Nanoparticles.

Salmonella enterica is one of the most common causes of foodborne illness worldwide. Contaminated poultry products, especially meat and eggs are the main sources of human salmonellosis. Thus, the aim of the present study was to determine prevalence, antimicrobial resistance profiles, virulence, and resistance genes of Salmonella Enteritidis (S. enteritidis) and Salmonella Typhimurium (S. Typhimurium) isolated from laying hens, table eggs, and humans, in Sharkia Governorate, Egypt. The antimicrobial activity of Biosynthesized Silver Nanoparticles (AgNPs) was also evaluated. Salmonella spp. were found in 19.3% of tested samples with laying hens having the highest isolation rate (33.1%). S. Enteritidis) (5.8%), and S. Typhimurium (2.8%) were the dominant serotypes. All isolates were ampicillin resistant (100%); however, none of the isolates were meropenem resistant. Multidrug-resistant (MDR) was detected in 83.8% of the isolates with a multiple antibiotic resistance index of 0.21 to 0.57. Most isolates (81.1%) had at least three virulence genes (sopB, stn, and hilA) and none of the isolates harbored the pefA gene; four resistance genes (blaTEM, tetA, nfsA, and nfsB) were detected in 56.8% of the examined isolates. The AgNPs biosynthesized by Aspergillus niveus exhibit an absorption peak at 420 nm with an average size of 27 nm. AgNPs had a minimum inhibitory concentration of 5 µg/mL against S. enteritidis and S. typhimurium isolates and a minimum bactericidal concentration of 6 and 8 µg/mL against S. enteritidis and S. typhimurium isolates, respectively. The bacterial growth and gene expression of S. enteritidis and S. typhimurium isolates treated with AgNPs were gradually decreased as storage time was increased. In conclusion, this study indicates that S. enteritidis and S. typhimurium isolated from laying hens, table eggs, and humans exhibits resistance to multiple antimicrobial classes. The biosynthesized AgNPs showed potential antimicrobial activity against MDR S. enteritidis and S. typhimurium isolates. However, studies to assess the antimicrobial effectiveness of the biosynthesized AgNPs in laying hen farms are warranted.

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

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.