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.

Yasmine H Tartor,Samah S Abdellatif,Hend M El Damaty,Amira S A Attia,Enas A Soliman,Rasha M A Gharieb,Hazem Ramadan,Norhan K Abd El-Aziz,Mosa M Bahnass,Mohammed Elbediwi,Yousry A El-Shazly,Shymaa Enany

doi:10.3389/fmicb.2021.770813

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 (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.

Highlights

Mastitis is one of the most prevalent diseases in dairy cows, producing well-recognized detrimental effects on animal health and profitability of dairy farms (Ruegg, 2017; Abd El-Aziz et al, 2021a)
The indiscriminate use of antimicrobials has led to the development of multidrug-resistant (MDR) Gram-negative bacteria in raw milk, in particular E. coli O157:H7, Klebsiella pneumoniae (K. pneumoniae), Aeromonas hydrophilia (A. hydrophilia), and Proteus mirabilis (P. mirabilis) (Zelalem and Bernard, 2006; Oliver et al, 2009; Garedew et al, 2012)
Polymerase chain reaction (PCR) of the amplified products of bacterial species could identify the isolates 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%)

Summary

Introduction

Mastitis is one of the most prevalent diseases in dairy cows, producing well-recognized detrimental effects on animal health and profitability of dairy farms (Ruegg, 2017; Abd El-Aziz et al, 2021a). Treatment of mastitis is often applied without prior knowledge of cause-related agents and mostly with broad-spectrum antimicrobials, which are known to enhance resistance evolution to a greater extent. This increases the selective pressure on potentially present pathogens and is considered a possible human health hazard (Neyra et al, 2014; Abd El-Aziz et al, 2021b). The indiscriminate use of antimicrobials has led to the development of multidrug-resistant (MDR) Gram-negative bacteria in raw milk, in particular E. coli O157:H7, Klebsiella pneumoniae (K. pneumoniae), Aeromonas hydrophilia (A. hydrophilia), and Proteus mirabilis (P. mirabilis) (Zelalem and Bernard, 2006; Oliver et al, 2009; Garedew et al, 2012)

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