| Carbapenems are one of the most important antibiotics in the treatment of severe bacterial infections in human medicine.However,carbapenem-resistant bacteria have spread widely around the world,leading to fewer effective treatment options.Carbapenemase is the main mechanism of carbapenem resistance in Enterobacteriaceae.Although carbapenems are not approved in animals in China,reports on carbapenemase-producing Enterobacteriaceae（CPE）in food animals are emerging.CPE of food animal source might further spread to consumers via the food chain,posing a serious threat to human health and safety.Therefore,it is necessary to have an enhanced monitoring system toward the prevalence of CPE in food chain.The current study revealed the molecular epidemiology of CPE recovered from retailed meat in Guangzhou city.It analyzed the mechanism mediating the spread of bla NDMamong Enterobacteriaceae from retailed meat,which proved the necessity of prevention on resistance genes.Between 2016 and 2018,110 strains of imipenem-insensitive bacteria were isolated from 794 commercial meat samples collected from 7 districts of Guangzhou.The imipenem-insensitive bacteria included Escherichia coli（n=94）,Klebsiella pneumoniae（n=10）,Enterobacter cloacae（n=2）and Proteus mirabilis（n=4）.The genes encoding carbapenemases were detected by PCR methods.The results showed that all 110 strains carried bla NDMgene.In total,three NDM variants were detected,i.e.,bla NDM-5（n=98）,bla NDM-1（n=11）and bla NDM-7（n=1）whereas other genes of carbapenmeases were unavailable.We observed marked increases in carbapenemase-producing Enterobacteriaceae detection in retail meat,from 9.4%（2016）,and 11.1%（2017）to 22.2%（2018）.In addition,bla CTX-M,mcr-1,fos A3,rmt B and flo R genes were detected in bla NDMpositive strains,with flo R representing the highest rate（85.4%）.The positive rates of bla CTX-M-1Gand bla CTX-M-9Gwere 3.6%（n=4）and 15.4%（n=17）,respectively,whereas the positive rates of fos A3 and rmt B were 12.7%（n=14）and 2.7%（n=3）,respectively.14.5%were positive for mcr-1,with a significant decrease among NDM producers from 2016（24.3%）to 2018（8.6%）.Their sensitivity to 15 antibiotics was determined by agar dilution method.The results showed that the resistance rate of Escherichia coli to ampicillin,doxycycline,florfenicol and sulfamethoxazole were all higher than 80%.However,they remained to be sensitive to tigecycline and amikacin,with corresponding resistance rates less than 10%.The phylogenetic relationship of NDM-positive strains was studied by multi-site sequence typing（MLST）.A total of 51 MLST types were obtained from 94 NDM-positive E.coli strains,among which ST48（n=13）and ST10（n=12）were dominant clones.To further confirmed the cloning relationship of ST48 and ST10 E.coli,pulsed-field gel electrophoresis（PFGE）was carried out.It showed a low similarity and indicated that the spread of bla NDMin E.coli was not based on clonal transmission.10 strains of NDM-positive Klebsiella pneumoniae were classified by MLST,which divided them into 8ST-types and confirmed the dominance of ST1（n=3）.For those that were clonally related,the similarities were above 99%.Through conjugation,replicon typing,S1-PFGE and Southern hybridization experiments,79.1%of the 110 bla NDMpositive bacteria were found located on IncX3 type plasmids with size of～50 kb,including bla NDM-5（n=80）,bla NDM-1（n=6）and bla NDM-7（n=1）,indicating the major role of IncX3 in the widespread of bla NDM.In the other isolates,bla NDMwere located on the 60～280 kb plasmid（n=21）or on the chromosome（n=2）.Several NDM-positive strains were randomly selected for whole-genome sequencing.The results showed that the IncX3 plasmid backbone region was conserved,and the umu D gene was inserted within variable region.In general,the backbone was 99%similar to p NDM5＿IncX3 recovered from K.pneumoniae.Through comparative analysis,a total of 7different genetic environments of variable region were obtained,81.6%of which was the structure(bla NDM-△ISAba125-IS5-△ISAba125-IS3000-ΔTn2).There are five strains encoding the structure(bla NDM-△ISAba125-IS5-△IS3000-△Tn2),the remaining five structures have not been reported.In summary,there is CPE contamination existing in the retailed meat in Guangzhou,with bla NDM-5gene as the prevailing type.Coexistence of mcr-1,flo R,fos A3,rmt B and multiple drug resistance genes was also observed in the isolates.Carbapenem-resistant strains in retail meat can be directly transmitted to humans,posing a great threat to human health and safety.Therefore,China should strengthen the monitoring of CPE in animal food. |
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