Abstract
BackgroundThe emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria. Here we aimed to determine the prevalence of colistin resistance, among enterobacteria isolated from poultry and the possible underlying colistin resistance mechanisms.MethodsA collection of 944 cloacal samples were obtained from poultry and screened for colistin resistance. To uncover the molecular mechanism behind colistin resistance, the presence of plasmid encoded colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4 was examined by PCR. The nucleotide sequences of the mgrB, pmrA, pmrB, phoP, phoQ, crrA and crrB genes were determined. The genetic relatedness of the colistin resistant (ColR) isolates was evaluated by Multilocus sequence typing. Three ColR mutants were generated in vitro by repetitive drug exposure.ResultsOverall from 931 enteric bacteria isolated from poultry samples obtained from 131 farms, nine ColR bacteria (0.96%) with high level colistin resistance (MICs ≥ 64 mg/L) were detected all being identified as K. pneumoniae. The 9 ColR bacteria originated from different farms and belonged to 7 distinct Sequence types including ST11 (22.2%) and ST726 (22.2%) being the most prevalent STs followed by ST37, ST74, ST485, ST525 and novel sequence type 3380 (11.1% each). mcr-type genes were not detected in any isolate. In 88.8% of the isolates (n = 8), MgrB was inactivated by Insertion of IS elements (IS1-like, IS3-like, IS5-like families, positions + 75, + 113, + 117, + 135) and nonsense mutations at codons 8, 16, 30. All ColR isolates harboured wild type PmrA, PhoP, PhoQ or polymorphic variants of PmrB. Sequence analysis of the CrrB revealed a familiar S195N and 4 novel I27V, T150R, F303S and K325R substitutions. PmrB T93N substitution and mgrB locus deletion were identified in two laboratory induced ColR mutants and one mutant lacked alteration in the studied loci. In one ColR isolate with wild type MgrB an A83V substitution was detected in CrrA.ConclusionIt is concluded from our results that colistin resistance in the studied avian K. pneumoniae isolates was mostly linked to alterations identified within the mgrB gene.
Highlights
The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria
Antimicrobial susceptibility testing and bacterial genotyping Overall 944 samples were collected from 131 farms located in 29 different cities of the East Azerbaijan province which included 503 samples from healthy broilers at slaughterhouse and 441 samples from dead poultry referred to clinic during 2017–2018 (Table 1)
The nine colistin resistant (ColR) K. pneumoniae (CRKP) isolates originated from different farms and belonged to 7 distinct Sequence types including ST11 (22.2%) and ST726 (22.2%) being the most prevalent STs followed by ST37, ST74, ST485, ST525 and novel sequence type 3380 (11.1% each) (Fig. 1)
Summary
The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria. The agricultural use of human antibiotics as growth promoters or as prophylactic agents in farm animals has been criticized for contributing to the magnitude of the global challenge of ABR [1]. This is considered as a potential threat to human health as commensal resistant organisms propagated in food-producing animals can be transmitted to humans indirectly along the food chain or following direct contact or may serve as reservoirs of transferable antibiotic resistance determinants that could be transferred to human pathogens [1, 2]. Acquisition of plasmid encoded mcr-type genes, which encode enzymes able to modify LPS by the addition of phosphoethanolamine has been found to contribute in colistin resistance in most of the E. coli, Salmonella and some K. pneumoniae isolates [7, 12, 13]
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