The Molecular Resistance Mechanismof Carbapenem-Resistant Citrobacter .spp Recovered From Clinical Isolates In China

Objective To study the resistant phenotype,genotype,homology and plasmid-mediated resistance mechanism of carbapenem resistant Citrobacters.spp so as to provide etiology and epidemiological data for the treatment of infection caused by them.Methods 25 carbapenem antibiotic-resistant Citrobacters isolates were collected from several hospitals in Shandong Province.The strains were identified by ANI（Average Nucleotide Identity）.Carbapenemase were detected by the modified Hodge test and the imipenem-EDTA E-test.The carbapenemase and others genes genes were confirmed byusing PCR and sequencing.The clonal relatedness of these strains were detected by pulsed-field gel electrophoresis.The transfer characteristics of plasmids was determined by conjugation tests and electrotransformation assay.Bacterial genomic DNA was extracted using the Wizard Genomic DNA Purification Kit,followed by the MiSeq and MinION sequencing.Mobile elements,resistance genes,and other features were annotated by INTEGRALL,ISfinder,ResFinder,PlasmidFinder,and the Tn Number Registry online databases.Comparisons of the multiple and paired sequences were conducted using MUSCLE 3.8.31 and BLASTN,respectively.Gene organization diagrams were drawn in Inkscape0.48.1.Results 25 Citrobacter.spp strains were identified by ANI,including 9 Citrobacter freundii strains,9 Citrobacter portucalensis strains,6 Citrobacter braakii strains,and 1Citrobacter werkmaniistrain.These strains are resistant to penicillins,cephalosporins and carbapenems,aminoglycosides and quinolones antibiotics.Out of 25 strains,24 were positive in the modified Hodge tests,while 18 were positive in the MBL E-test tests.A number of carbapenem-resistantgenes in these isolates,including bla_NDM-1 gene in 16strains,bla_KPC-2 in 6 strains and bla _IMP-4 in two strains,were detected by using PCR and sequencing.Five ST171 staines,four ST116,three ST85,two ST17,one ST367,one ST252,one ST134,one ST62,one ST257 and six novel ST（ST400,ST401,ST402,ST403,ST405 and ST406）,were found in 25 strains.While there were no clonal relatedness between these 25 strains.The S1 nuclease pulsed-field gel electrophoresis showed that each isolates habored a couple of plasmids.Bioinformatics analysis showed that a novel IncR/IncP6 hybrid plasmid pCRE3-KPC was founed in CRE3 isolate and that it contained the bla_KPC-2 gene cluster and a novel Tn6626transposon,which comprised the novel Tn3 family element ISCfr16,the truncated aacC2-tmrB region,and ISEc21.Conclusion Our results revealed that these 25 Citrobacter.spp strains were multi-drug resistant phenotype and carried a number of multi-drug resistant genes.To the best of our knowledge,this is the first report in worldwide that the C.portucalensis stainesharboring carbapenem-resistant gene were recovered from clinical samples and that the pCRE3-KPC in clinical Citrobacter.spp CRE3 isolate was a novel IncR/IncP6 hybrid plasmid.Therefore,there is a need on monitoring the prevalence status of carbapenem-resistant Enterobacteriaceaes and enhancing the reasonable use of antibiotics.