| In recent years,the rapid and worldwide dissemination of Carbapenem-resistant Enterobacteriaceae(CRE)is of great concern.So far,CRE has been detected in animals,environment and vegetable samples that are closely related to humans.First reported in 2008,New Delhi metallo-?-lactamases(NDMs)are one of the main types of carbapenemases and confer resistance to almost all?-lactam antibiotics,including carbapenems.24variants of NDM enzymes(NDM-1 to NDM-24)have been widely reported.The purpose of this study is to investigate the distribution and abundance of bla NDMresistance genes in pig farm,pig slaughter production process and the environment in Jiangsu,analyze the drug resistance phenotype of bla NDM-positive Escherichia coli(E.coli)and reveal the molecular epidemiological characteristics of bla NDM-positive E.coli.Experiment 1.Epidemic characteristics of bla NDM-positive E.coli in JiangsuA total of 831 samples were collected,of which 46 were positive for bla NDM(5.54%),sample were from large-scale designated pig slaughterhouses and farm(including Nantong Changhou Taizhou Huaian and Yancheng)in Jiangsu province.The bla NDMwas detected in anal swab samples(4.12%)in pig farms,cecum contents in slaughterhouses(26.67%)and surface swabs after bleeding and before evisceration(8.84%).NDM resistance genes were not detected in the other surface wipe samples and environmental samples.160 E.coli,7 Proteus,4 Pseudomonas,9 Millerella Wisconsin,and 3 Morganella were recovered.Detection of bla NDM-positive E.coli in 13 types of extended-spectrum?-lactamase genes(ESBL),there were 5 dominant ESBL genes,including bla TEM,bla SHV,bla CTX-M-9,bla OXA-2,bla OXA-10.45 NDM-positive E.coli containing different sample sources,different ST types and multiple ESBL genes were used to determine the minimum inhibitory concentration(MIC)by antimicrobial susceptibility testing.The bla NDM-positive E.coli isolates were 100%resistant to cephalosporins(ceftiofur,ceftazidime),tetracycline and ampicillin.To meropenem the drug resistance rate was 55.56%(25/45),and to imipenem was 17.33%higher than that of meropenem.E.coli(bla NDM-5)has a MIC?8?g/m L meropenem,its higher than E.coli(bla NDM-1),which had a MIC?4?g/m L meropenem.And its estimated that the activity of NDM-5 was higher than that of NDM-1,which could induce stronger carbapenem resistance.In addition,45 NDM-positive E.coli showed different levels of drug resistance to amino acid glycoside and quinolones except tigecycline.Experiment 2.Molecular characteristics of bla NDM-positive E.coli in JiangsuA total of 15 sequencing types(STs)were identified in 160 bla NDM-positive E.coli.The most prevalent STs among NDM-positive isolates were ST410,ST48,ST10,ST206,and ST846.ST3076,ST4214,and ST1626 were first discoveried in the sudy.These strains usually carry ESBL genes,and they are widely reported in clinical and veterinary medicine.Pulsed field gel electrophoresis(PFGE)was performed on 15bla NDM-positive E.coli containing different NDM variants,ESBL genes,and STs,namely JSEwj-31-JSEwj-45.15 E.coli could be divided into 11 types of PFGE,showed overall genetic diversity and the partial were clonally related.Different ST types had the same PFGE pattern,the same ST type had different PFGE patterns.Although MLST and PFGE both belong to bacterial typing technology,PFGE uses restriction enzymes to cut specific sites,and then separates fragments of different sizes by changing electric fields.It is generally used for short-term outbreak analysis.MLST is determining different ST types by amplifying 7 housekeeping genes,and then compare with the Pub MLST database.The 7 housekeeping genes are relatively conservative and have a low frequency of mutation.Therefore,they are generally used for a long span and a wide range of epidemics medical investigation.According to this study,the ST846 and ST206 had B and H types PFGE patterns,while ST206 and ST846types had different PFGE patterns.This was prevalent molecule characteristic of bla NDM-positive E.coli from pigs in Jiangsu.Experiment 3.Mapping and analysis of genetic environment of NDM resistance genesFifteen bla NDM-positive E.coli were subjected to conjugation and transfer experiments(JSEwj-31-JSEwj-45).The bla NDMcould be conjugated to Escherichia coli recipient J53,and the frequency of conjugation and transfer was 5.38×10-07-2.2×10-02.S1-PFGE of the binder obtained in the membrane-bound transfer assay failed to locate the NDM resistance gene.Donor isolates with different plasmid PFGE types for binder were selected for electrotransformation,Six electrotransformants were obtained.S1-PFGE of electrotransformants binder showed that 4 E.coli NDM resistance genes were located on plasmids.There were two size of plasmid types,one is between?54kb and?76kb,and the other is?40kb.The surrounding genetic structure of NDM gene was obtained using WGS,It was found that bla NDM-positive E.coli carried 37 resistance genes,and these resistance genes conferred isolates on aminoglycoside,sulphonamide,phenicol,fosfomycin,rifampicin,oxazolidinone,trimethoprim,tetracycline,quinolone,macrolide,?-lactam,colisin resistantance to 12 drugs,and 9?-lactamase resistance genes had appeared.On the other hand,NDM resistance genes were found in three different genomic backbone context,in which 91.67%(11/12)of bla NDM-1was mainly located in the type I genomic backbone profiles,bla NDM-5(1/1)was mainly located in the second type II genetic environment.Type I possessed an identical 9401bp fragment,the 4318bp gene region of bla NDMwas100%identity to the bla NDM-carrying plasmid region from Klebsiella pneumoniae plasmid p Kp ANDM-1(Gen Bank:FN396876.1),such genetic environments was the most prevalent.The type II genomic backbone had a2180bp gene fragment carrying bla NDMthat was similar to the corresponding type III gene structure in?99.9%.In summary,the bla NDMresistance gene in this study mainly existed in the upstream pig farms and transportation environments,and was transferred to the slaughterhouse with the shift of pigs.Most bla NDMresistance genes were located on the plasmid and could be combined and transferred.The bla NDM-positive E.coli in Jiangsu carried 5 ESBL genes,and the phenomenon of multi-drug resistance was serious.The isolates showed overall genetic diversity.This study provides a scientific basis for the comprehensive monitoring and risk assessment of carbapenem-resistant bacteria from pig. |
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