| Carbapenem resistance is a significant global public health problem,and Gram-negative bacteria are one of the main reasons for the widespread of it.Studies have shown that although carbapenem antibiotics are prohibited in livestock farming,bacteria resistant to carbapenem still have emerged in food-producing animals in China,and related reports are gradually increasing.In addition to hindering the healthy growth of animal husbandry,drug-resistant bacteria from food animals can transfer to humans through the food chain and other pathways,posing a severe threat to human health.Therefore,we need to do more research on carbapenem-resistant bacteria of food animal origin to elucidate the potential hazards,which are of great importance for domestic and global public health.In this study,food animal slaughterhouses and farms in different regions of Jiangsu and Henan provinces were selected for random sampling.We isolated and identified carbapenem-resistant Gram-negative bacteria of different food animal regions(swine,dairy cow and chicken),comparing their differences in resistance and prevalence,and also exploring the transmission mechanism,in order to provide more data supports for the epidemiological characteristics of carbapenem-resistant bacteria in China.The following results were acquired:1)A total of 126 carbapenem-resistant strains were isolated from all 512 samples collected,with an overall isolation rate of 24.6%,among which the highest isolation rate(25.90%)was found in samples of swine origin.There were 43 carbapenem-resistant strains of swine origin,covering seven genera,including Escherichia fergusii,Escherichia coli,Shigella,Enterobacter cloacae,Pseudomonas,Bacillus immobilis,Bacillus subtilis,and Stenotrophomonas maltophilia,of which Escherichia coli accounted for the highest proportion.Altogether 13 carbapenem-resistant isolates from dairy cow samples,including 3 genera:Shewallena,Acinetobacter,and Stenotrophomonas maltophilia accounting for the majority of these isolates.A total of 70 carbapenem-resistant strains of chicken origin contained 5 genera:Escherich ia coli,Providence,Pseudomonas,Proteus and Klebsiella pneumoniae,of which Escherichia coli constituted the highest percentage.The drug sensitivity test results showed that all the above strains were multi-drug resistant bacteria.2)The isolates in this study have two resistance mechanisms to carbapenems:carbapenemase-producing and the lack of porin.Multiple carbapenem resistance genes were detected in 123 strains,including blaNDM,blaEBR,blaVIM,blaPST,and blaOXA,with the carriage rate of blaNDM(117/126)being much higher than the other resistance genes.blaNDM-5 was prevalent in carbapenem-resistant strains of swine origin,and blaNDM-1 was prevalent in carbapenem-resistant strains of dairy cow origin.However,there were more than one blaNDM variant prevalent in chicken carbapenem-resistant strains,including blaNDM-1 blaNDM-5,blaNDM-7 and blaNDM-13.In addition,among the 126 carbapenem-resistant isolates,there were 20 strains co-harboring blaNDM and mcr,8 strains co-harboring blaNDM and tet(X),2 strains co-harboring blaNDM and tmexCD-toprJ strains,2 strains co-harboring b1aPST and tmexCD-toprJ strains,1 strain co-harboring blaVIM and tmexCD-toprJ strain,and 1 blaNDM,mcr,and tmexCD-toprJ triple genes coexisting strain.No carbapenem resistance genes were detected in three Pseudomonas strains,and further study confirmed that their resistance to carbapenem antibiotics was caused by lacking gene encoding OprD2 porin.3)Conjugation assay,MLST and SNPs analysis showed that horizontal transmission of resistance genes mediated by mobile genetic elements such as plasmids and vertical transmission of clonal strains were present in carbapenem-resistant isolates of swine,dairy cow and chicken origin.There were 53.85%of isolates from cow and 52.38%of isolates from swine successfully transferred carbapenem-resistant strains.The MICs of meropenem for transconjugants were stable in the range of 16-32 mg/L.The success rate of conjugation in chicken strains.was 44.29%,and the MICs of meropenem for corresponding transconjugant ranged from 8 to 128 mg/L.Carbapenem-resistant ST3856 Shigella disseminated in slaughterhouses,and 18 chicken-derived Escherichia coli strains(through whole genome sequencing)contained a total of 9 known ST types,reflecting the diversity of carbapenem-resistant Escherichia coli of chicken origin.4)The genetic environments of carbapenem resistance genes in different food animal sources is relatively conservative,similar to that of different strains in the NCBI database.ISCR1,ISCR2,ISAba125,and IS3000 are important mobile genetic elements for the horizontal transfer of carbapenem resistance genes.The formation of circular intermediate[ΔISCR2-tet(X3)-blaNDM-1]suggests that two resistance genes could be transmitted together so that bacteria would acquire both tigecycline and carbapenem resistance phenotypes.The carbapenem resistance gene-positive plasmids isolated in this study were structurally diverse,and some of them had high homology with plasmids from other sources in the NCBI database,suggesting that carbapenem resistance genes can be mediated by plasmids and widely disseminated through horizontal transfer.Class Ⅰ integrons were often found in the MDR region containing carbapenem resistance genes,which may also mediate the insertion of carbapenem resistance genes.In summary,this study investigated the prevalence characteristics and transmission mechanisms of carbapenem-resistant Gram-negative bacteria in various food animal sources by describing the distribution of genera,the characteristics of carbapenem resistance genes,the backbone structure of positive plasmids and the relatedness among strains,enriching the genomic characteristics of carbapenem-resistant genes,and providing a data support for preventing the epidemic of drug-resistant bacteria.Although carbapenem antibiotics are prohibited in animal husbandry,carbapenemases have been widely spread across the"human-environment-animal" cycle and can hydrolyze almost all β-lactam antibiotics.The ability of food animal-derived bacteria to transmit drug resistance between animal and human cannot be ignored,stronger surveillance and regulatory measures are needed.We expect this study to call for enhanced public investigation and surveillance of carbapenem-resistant bacteria from different sources.In order to slow down and inhibit transmission of carbapenem-resistant bacteria for the health of animals and humans,more extensive and in-depth studies on their causes and emergence were need. |
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