| Objectives:In order to provide experimental data for the prevention and control of the outbreak and spread of resistant strains in community,investigating the fecal carriage and molecular epidemiology of extended-spectrumβ-lactamase-producing Escherichia coli（ESBL-EC）and carbapenemase-producing Escherichia coli（CP-EC）from healthy children in Changsha.Methods:Three school were selected from Kaifu District,Yuelu District,and Changsha County in Changsha,China,which was based on the geographical location of the schools.Stool samples were collected from students who were also asked to fill out questionnaires.ESBL-EC and CP-EC were screened using CHROMagar TMchromogenic plates.ESBL and carbapenemase production was confirmed using the double-disc synergy test and a modified carbapenem inactivation method,respectively.Antimicrobial susceptibility was tested using TDR susceptibility plates.Resistance determinants,virulence factors,and phylogenetic groups were determined by PCR and sequencing.Multi-locus sequence typing（MLST）was performed on the phylogenic group B2 E.coli to detect high-risk clonal strains such as ST131 E.coli.Then,ST131 were characterized based on ST131 clades,O-type,and fim H alleles.The biofilm-forming ability and serum resistance were also examined.Then the differences in virulence gene,resistance gene,film-forming ability and serum resistance ability between ST131 and non-ST131 were compared.Finally,conjugation experiments were performed on CRE and E.coli with mcr-1.The transconjugants was measured including its growth,resistance,plasmid replication and plasmid stability.Results:1.A total of 420 feces from students were collected.Then based on the results of the questionnaire,a total of 330 healthy children were included in this study,and 118 ESBL-EC and 3 CP-EC were isolated.2.ESBL-EC isolates were highly resistant to ampicillin,cefuroxime,cefazolin and ceftriaxone while were sensitive to meropenem,tigecycline,nitrofurantoin,piperacillin/tazobactam and cefoperazone/sulbactam.CP-EC isolates were sensitive to amikacin,tigecycline,colistin,and minocycline.3.bla CTX-Mwas the most common genotype（27.1%）,identified in all ESBL-EC,except one,which carried bla SHV-12.The most common bla CTX-Msubtype was bla CTX-M-14（27.1%）,followed by bla CTX-M-15（24.6%）,bla CTX-M-27（24.6%）.Three types of the plasmid-mediated quinolone resistance（PMQR）genes were found:qnr S（22.0%）,aac（6’）-Ib（3.4%）,and qnr B（1.7%）.One isolate with mcr-1 was found among ESBL-EC,whereas all three CP-EC carried bla NDM-1.4.For ESBL-EC,the most frequent virulence genes in their genome were fim H（88.1%）,fyu A（72.9%）,tra T（65.3%）,kps MTII（61.0%）,and iut A（54.2%）.And approximately 52.5%of the isolates were extra-intestinal pathogenic bacteria（Ex PEC）.For CP-EC,the most frequent virulence genes observed were fim H（66.7%）,kps MTII（66.7%）,and tra T（66.7%）,and two of them were Ex PEC.5.The results of the conjugation experiments showed that the bla NDM-1gene could be transferred horizontally.The growth of the transconjugants was consistent with that of the recipient bacteria,and the bla NDM-1gene could exist stably in the transconjugants.6.The majority of isolates belonged to group B2.And the predominant sequence type（ST）clones in group B2 were ST131（30.0%）and ST1193（27.5%）.12 strains of ST131 E.coli belonged to the ST131lineage including clade A（n=8）and clade C1-M27（n=4）.Serotype results showed that 7 E.coli strains were O16-ST131 and 5 E.coli strains were O25b-ST131.The fim H alleles of all O16-ST131 isolates were fim H41,and the fim H alleles of O25b-ST131 isolates were fim H30（n=4）and fim H89（n=1）.The ST131 isolates had a significantly higher prevalence rate of bla CTX-M-27,kps MTII,tra T,iut A,fyu A,and PAI compared with that of non-ST131 isolates（P<0.05）.However,there was no significant difference in terms of film-forming ability and serum resistance between them.Conclusions:In this study,high carriage rate of ESBL-EC was found among healthy children,and there are many subtypes of CTX-M,among which CTX-M-14 is the most dominant.There are highly resistant strains carrying bla NDM-1and mcr-1 genes in the intestine of healthy children,and the bla NDM-1gene can be transferred horizontally between strains.The percentage of ST131 was high in group B2 E.coli.And ST131 E.coli were divided into two clade:clade A and clade C1-M27.To the best of our knowledge,this is the first systematic study focusing on the fecal carriage of ESBL-EC and CP-EC in healthy children from a community in China,and this is the first report of clade C1-M27 in China,which provide experimental data for the prevention and control of the outbreak and spread of resistant strains in community.14 figures,20 tables,97 references... |
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