| The live poultry market is an essential part of the poultry industry’s sales network,since it is where huge numbers of animals are collected for trade,producing a critical point of contact between people and animals.However,this also provides a conducive environment for the spread of pathogens and antibiotic resistance genes.According to research,the live poultry markets harbor a considerable number of pathogenic and drug-resistant bacteria that can be transmitted to humans through touch,consumption of food or water,and inhalation of air,which poses a severe threat to public safety and health.Carbapenem antibiotics are generally considered one of the last drugs to treat multidrug-resistant infections.Unfortunately,carbapenem resistance is becoming more prevalent due to misuse and overuse in clinical settings.Recently,there has been no systematic research on the molecular epidemiology and genomic characteristics of carbapenem resistance gene blaNDM in the live poultry market.Moreover,research on the distribution of microbiomes and resistomes across various niches in live poultry markets is scarce.This study focused on investigating the prevalence characteristics,and transmission mechanisms of the carbapenem resistance gene blaNDM in live poultry markets.In addition,the study exhaustively described the diversity and characteristics of microbiota and resistomes in these markets by quantifying specific antibiotic resistance genes and performing metagenomic sequencing.Initially,we identified 336 blaNDM-positive strains from 596 samples in two live poultry markets A and B(humans,environments,and animals)and discovered that the isolation rate of blaNDM-positive samples was higher in live poultry market B(45.06%).The majority of blaNDM-positive bacteria were Escherichia coli(266/336),whereas a small number were Klebsiella pneumoniae(62/336),Acinetobacter baumannii(3/336),Acinetobacter bereziniae(1/336),Enterobacter cloacae(1/336),Providencia rettgeri(1/336),Pseudomonas monteilii(1/336),and Pseudomonas otitis(1/336).The isolation rate of blaNDM-positive bacteria was greatest in wastewater samples(35/66,53.03%),followed by animal feces samples(139/395,35.19%).Based on antimicrobial susceptibility testing,all blaNDM-positive bacteria were resistant to many medicines,including meropenem.The findings of conjugation assay revealed that more than half of blaNDM had the capacity to transfer,including interspecies transmission,which posed a severe threat to public health.After that,we concentrated on the genomic features of 51 blaNDM-positive strains,and the findings revealed that there were five blaNDM variants,with blaNDM-5 being the most frequent dominant antibiotic resistance gene,accounting for as much as 82.35%of the total.Among these,blaNDM-27 was the novel variation that had never been reported.The MIC of blaNDM-27-positive conjugative donor strains and the blaNDM-27-positive transformative strains for meropenem are 2-4 times higher than those of the other variants.Simultaneously,phylogenetic analysis revealed clonal transmission of blaNDM-positive Escherichia coli and Klebsiella pneumoniae between different animals,as well as between animals and the environment in live poultry markets.By comparing sequences of blaNDM-positive isolates with NCBI databases,it was found that the SNP of the blaNDM-positive E.coli recovered in this study and the two blaNDM-positive E.coli isolated from the French medical institution was 181.It is speculated that the clonal transmission of blaNDM-positive E.coli has occurred between humans and animals.By analyzing the characteristics of blaNDM-positive plasmids,it was found that the insertion sequence played a very important role in mediating the transfer of blaNDM.Further analysis of the genetic environment found that the blaNDM genetic environment is conservative,and the horizontal transfer mechanism mediated by the core genetic environment G5(ΔTn2-IS3000-ISAba125-IS5-blaNDM-5-bleMBL-trpF-tat-IS26)is an important factor for the prevalence of blaNDM in live poultry markets.Moreover,this study carried out absolute quantification by targeting specific antibiotic resistance genes,mobile genetic elements,and plasmid replicons to analyze the abundance differences of different samples in live poultry markets.And we found that the abundance of antibiotic resistance genes and mobile genetic elements in animal samples was significantly higher than that of human and environmental samples,suggesting that animals may be a huge reservoir of antibiotic resistance genes.Moreover,there is a possibility risk of spreading antibiotic resistance genes to workers through air,water,and food chains.In addition,it was discovered that the mobile genetic elements IS613,Tp614,ISApl1 and intl1 in live poultry markets are strongly correlated with various antibiotic resistance genes,and the plasmid replicon also has a certain correlation with antibiotic resistance genes,indicating that antibiotic resistance genes can be disseminated in live poultry markets via mobile genetic elements and plasmid-mediated horizontal transfer mechanisms.Finally,metagenomic sequencing was used to analyze the diversity and different characteristics of microbiota and resistomes in humans,environments,and animals in live poultry markets,the different characteristics of gut organisms and resistomes in different live poultry markets,and similarities and differences in the distribution of resistomes in chicken samples from different regions.We found that the diversity of resistomes in animal samples was significantly higher than that in human samples and environmental samples in live poultry markets,and the results were consistent with the results of absolute quantification.It is worth noting that the microbiota and resistomes in live poultry market A are more diverse than those in live poultry market B.It is speculated that it may be caused by factors such as the relatively dense breeding environment,poor sanitation conditions,and the large size of the live poultry market A.At the same time,the distribution of the carbapenem resistance gene blaNDM was analyzed,and it was found that animal-derived Escherichia coli was the dominant strain of blaNDM.The results are consistent with those of the blaNDM isolation assay.This study comprehensively describes the epidemic distribution and transmission mechanism of carbapenem resistance gene blaNDM,and further analyzes epidemic characteristics and different distribution of microbiome and resistomes in the live poultry market from the perspectives of single bacteria isolation,whole genome analysis,absolute quantification of targeted genes,and metagenomic analysis,which provides a theoretical basis for controlling and slowing the spread of antibiotic resistance genes in the live trading markets. |
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