| In recent years,with the emergence and widespread of multidrug-resistant gram-negative bacteria,tigecycline has been regarded as one of the few drugs of choice for the treatment of infections caused by carbapenem-resistant Klebsiella pneumoniae and other strains.However,with the extensive use of antibiotics in clinical settings,the problem of tigecycline resistance has become increasingly serious.The mechanism of tigecycline resistance is commonly recognized,including overexpression of the chromosome-mediated RND-type efflux pumps and mutations of tetracycline efflux pumps and mutations in the ribosomal binding site and Tet（X）enzymatic inactivation,but little is known about the involvement of plasmid-mediated RND efflux pumps in tigecycline.Here,we identified a plasmid-mediated RND efflux pump that confers resistance to tigecycline and other drugs from five pandrug-resistant K.pneumoniae isolates of chickens,and we further analyzed the plasmid characteristics and prevalence,so as to provide scientific data support for guiding clinical rational drug use and new drug research.Five pandrug-resistant K.pneumoniae strains（AH6I,AH8I,AH25I,AH28I and AH33I）were isolated from a chicken farm in Anhui province in 2017.The minimum inhibitory concentration of tigecycline of these isolates was 16μg/m L and were classified as sequence type 1（ST1）.The effect of NMP,an efflux pump inhibitor,was detected and the results showed that NMP could restore the sensitivity of the strain to tigecycline.A pandrug-resistant strain AH8I was randomly selected for whole genome and three-generation sequencing analysis.It was found that the strain carried 30 known resistance genes such as bla NDM-1,mcr-1,mcr-8,but no gene mutation（tet（A）,ram R,acr R and rps J）related to tigecycline resistance was detected.AH8I carried five plasmids,of which one 12,196 bp Inc FIA plasmid named p HNAH8I-1.The plasmid p HNAH8I-1 carried an RND efflux pump gene cluster,which had78%nucleotide sequence homology with mex CD-opr J gene cluster on the chromosome of Pseudomonas aeruginosa,designated tmex CD1-topr J1.The amino acid sequence analysis showed that TMex C1,TMex D1,TOpr J1 were closely related（64.5%～77.8%identity）to Mex C,Mex D,Opr J.The tmex CD1-topr J1 gene cluster was cloned into a low-copy vector and could be transferred to K.pneumoniae,Escherichia coli and Salmonella.It was determined that the efflux pump could confer resistance to glycyclines（tigecycline）,tetracyclines,quinolones,cephalosporins,aminoglycosides and other drugs（MIC value increased by 4 to 32 fold）,which indicated that tmex CD1-topr J1 could be considered as a novel plasmid-mediated quinolone resistance determinant.The plasmid can be successfully transferred to the recipient strain with a conjugation frequency of about 10-6～10-7 and conferred tigecycline resistance.The genetic environment analysis of tmex CD1-topr J1 gene showed that it lay adjacent to two genes encoding site-specific integrases,which might be responsible for its acquisition from chromosome to a plasmid of some pseudomonas.The results of the tigecycline treatment experiment in a mouse infection model showed that mice infected tmex CD1-topr J1-positive K.pneumoniae had a higher mortality rate.The bacterial growth curve experiment results showed that tmex CD1-topr J1 could significantly reduce the growth of E.coli and S.Typhimurium,but not K.pneumoniae.Stability experiments were carried out on the original bacteria AH8I and transformants,and it was found that the plasmid was stable in K.pneumoniae,but loss rate in E.coli J53 after 15 days of passaging without antibiotics was 29%.Samples of 2326 chicken cloaca,142 pig rectal swabs,643 chicken caecum and 699retail meat were collected from chicken farms,pig farms and farmer’s markets in 10provinces in China.PCR was used to determine the dissemination and occurrence of tmex CD1-topr J1.The results showed that the tmex CD1-topr J1-positive enterobacteriaceae isolates accounted for 14.3%in chicken fecal and 3.4%in retail meat samples.The results of tmex CD1-topr J1-positive strain identification showed that tmex CD1-topr J1 was widely distributed in K.pneumoniae,and also found in Klebsiella oxytoca and Raoultella planticola.In addition,The positive rate of tmex CD1-topr J1 detected from 2575 K.pneumoniae strains isolated from 18 top three hospitals in 15 provinces in China from 2016 to 2018 was 0.08%,which indicated that tmex CD1-topr J1 might be widely disseminated in chicken samples.We firstly identified a plasmid-encoded multidrug efflux pump tmex CD1-topr J1 which could confer tigecycline resistance and was located on Inc FIA type plasmid and lay adjacent to site-specific integrase.TMex CD1-TOpr J1 had been widely found in K.pneumoniae from chicken samples.Although the isolation rate of tmex CD1-topr J1 in human was relatively low,considering the transferability of the tmex CD1-topr J1 gene cluster and the broad substrate spectrum of tmex CD1-topr J1,it might promote the dissemination of this mobile tigecycline resistance and seriously threaten human health.Therefore,measures are urgently needed to monitor and control its further spread. |
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