The Molecular Epidemiology Of The NDM And MCR-1 Positive E.Coli From Poultry Production Chain

Carbapenems are considered as one of the most critically important antimicrobials for clinical treatments against multi-drug resistant gram-negative bacterium,in view of the important role of carbapenem antibiotics in the prevention and treatment of infectious diseases,it was not approved for the drug used in animal,but the carbapenem resistant gram-negative bacteria have been spreaded worldly with increasingly useage of the drug in clinic.The polymyxin has been use as an animal feed additive in livestock of China since 1980s,and is regarded to be the "last line of defense" for the treatments of infection caused by carbapenem resistant bacteria.The colistin resistance gene mcr-l,which was discovered in 2016 from the livestock of china and mediated by the plasmid with the ability of horizontal transfer,hense the resistance of colistin has caught the world's attention.Carbapenem and colistin resistant E.coli have been detected in chicken from China.However,no data have been reported on the systematic resistome analysis for the whole poultry production chain.The aim of this study was to investigate the carbapenem and colistin resistome from poultry production chain,and illuminate the mode of transmission of resistome in human and animal foods.A total of 739 non-duplicate samples were collected from a poultry industry chain,including hatchery farms,commercial farms,slaughterhouse and supermarkets,between November 2014 and August 2015,and the samples were from chicken,dogs,flies,faecal swabs/nests of swallow or sparrow,sewage and farmer.A total of 245 CRE were recovered on carbapenem resistant selective medium,of which blaNDM was identified,using PCR screening,in 161,55 and 29 E.coli,Klebsiella pneumoniae and Enterobacter cloacae,respectively,of these,37 NDM positive E.coli were also carrying MCR-1.However,Direct sample testing(DST)for blaNDM and mcr-1 on the poultry industry chain revealed considerably higher levels of positive samples than the blaNDM and mcr-1-positive E.coli,indicating that other bacteria,which cannot be routinely cultured,were carrying MCR-1 and NDM.However,samples were blaNDM-negative,and CREC were not recovered the from the hatchery farm,but isolation of CREC was compared to the DST for blaNDM,from commercial farms,slaughterhouse and supermarkets.and found at the rates ranging from 10%-29.2%and 33.3%-62%,respectively.In contrast,DST for mcr-1 were positive,and MCRPE were recovered from the hatchery farm.DST for mcr-1 was compared to the isolation of MCRPE from the whole poultry production chain,and found at the following rates ranging from 85.8%-97.0%and 25%-49.7%respectively(Fig.2A).A total of 41 known sequencing types(STs)were identified in 161 CREC and 55 CSEC,23 E.coli were carrying new alleles,which were submitted to the E.coli MLST database for assignment of 11 new STs.The most prevalent STs among blaNDM-positive isolates were ST156(n=31),STI01(n=19)and ST746(n=13),the ST746 E.coli were only carrying NDM-5,without other types of NDM variants.Overall,MLST analysis showed the most common between strains from all samples,typified by genotypes ST10 and ST156,and ST156 were also the predominant type(48.6%)among the E.coli strains carrying both blaNDM and mcr-1 genes.Core-genome SNP-based phylogenetic analysis were performed on the 174 E.coli(161 CREC and 13 randomly selected CSEC),which have been whole genome sequenced,and 12 CREC from six countries(the genome data were downloaded from GenBank),the phylogenetic relationship were closely related found in some isolates from all sectors of poultry production chain,and some isolates from chicken,swallow,farmers,dogs,flies and sewage,and between some of the 12 CREC and some isolates in this study,suggesting that the CREC could spread among the whole poultry production chain,different hosts of commercial farms,and even different countries and regions.In total,69.6%(112/161)blaNDM and 83.8%(31/37)mcr-l genes were located on plasmids ranging in size between 30-280 kb,while the co-existence of blaNDM and mcr-1 on the same plasmid was not observed.Conjugation assays were performed on the 37 E.coli isolates carrying both NDM and MCR-1,and showed that blaNDM and mcr-1 could be successfully transferred from 20 and 24 donor strains at frequencies of 9.0×10-10-5.0×10-3 and 3.7×10-5-3.2×10-1 cells per donor cell,respectively.Co-transfer of blaNDM and mcr-1 was also observed in five strains from flies(n=2),cloacae(nO2)from farm B,and cecum(n=1)from the slaughterhouse.Three types of blaNDM genomic backbone profiles were obtained by WGS from 161 CREC,and containing four NDM variants,NDM-5(n=84),NDM-9(n=55),NDM-1(n=21)and NDM-7(n=1),and blaNDM-1,blaNDM-5 and blaNDM-9 genes were mainly located on the type ?(85.7%),type ?(98.8%)and type I(92.7%)genomic backbone profiles,respectively.The 44 mcr-1 positive contigs,originating from 37 CREC and 7 CSEC WGS,were classified into four types.161 genomes were interrogated for blaNDM genetic contexts and were subsequently correlated to the phylogenetic lineages,and indicated that the blaNDM are disparately disseminated among the 161 tested E.coli strains.In conclusion,the mcr-1-carrying E.coli could spread from hatchery to supermarket along the poultry production chain,suggesting that the emergency of the mcr-1 gene was due to the mass use of colistin in the poultry industry.Isolation rates of CREC from chicken,dogs,birds,flies and farmers from commercial farm was extremely high,and the CREC could spread to downstream of the food production chain,but the samples were blaNDM-negative,and CREC were not recovered the from the hatchery farm.The phylogenetic relationship was closely related among CREC from the sectors of poultry industry chain and the CREC from clinical isolates from six countries(Australian,Singapore,Norway,United States,Columbia and China),suggesting the blaNDM gene presented in the chicken production chain were originated from environment,and could spread via migratory birds,flies and human contact.The blaNDM are disparately disseminated among the 161 tested E.coli strains.This study has elucidated the origin of the drug resistance genes and its transmission rules in the poultry production chain,these results will provide scientific basis for the originating,transmission,control,and risk assessment of antimicrobial resistance in bacteria of animal origin.