| BackgroundEnterobacteriaceae is an important opportunistic pathogen for community-acquired infections and nosocomial infections.It can cause respiratory infectionsurinary tract infections,and severe infections or even fatalities in patients with hypoimmunity.And the carbapenem antibiotics,including imipenem,meropenem,ertapenem,and so on,are currently the most powerful antibiotics for the treatment of Enterobacteriaceae infections.They were highly stable to most of the β-lactamase that were mediated by the plasmid or chromosome,and they were affinity with penicillin-binding protein(PBPs),can effectively penetrate the bacterial outer membrane,and performed continuous antibiotic effect.Carbapenems are widely used in the treatment of infection caused by bacteria producing extended-spectrum β-lactamase(ESBLs)and cephalosporinase(Amp C),because of its broad antimicrobial spectrum,strong antimicrobial activity,quick antimicrobial effect.It was reported that the extensive use of carbapenems has led to the increasing emergence of carbapenem-unsusceptible or carbapenem-resisitant to Enterobacteriaceae at home and abroad,which greatly limits the clinical treatment of carbapenems.The main reason for this phenomenon is that the strain has obtained the carbapenemase resistance gene.The carbapenemase genes carried by the plasmid are easy to transfer between different bacteria due to the presence of gene elements that mediate metastasis in its genetic environment,which resulting in extensive propagation of carbapenems resistance.It has become one of the hotspots in the field of clinical microbiology in recent years.In this study,the carbapenem-resisitant strains were identified and tested by the French Bio Mérieux Vitek2 automatic microbiological identification and susceptibility testing systems.The resistance genes were detected by multiplex PCR.Furthermore,plasmid conjugation experiment was performed on carbapenemase gene positive strain to explore the horizontal transmission patterns of the resisitance genes.The resistance genetic environment and mechanism of a bla IMP-26 producing Klebsiella pneumonia(KP)strain was then demonstrated by high-throughput sequencing and bioinformatics analyzed.ObjectiveTo understand the resistance features and carbapenemase status of the isolates of Enterobacteriaceae were isolated from our hospital,and to explore the mode of transmission and possible resistance mechanism of carbapenemase gene.And to provide the experimental basis for the clinical control of the spread of resistance genes.Methods1.Bacterial strains,antimicrobial susceptibility test The 18 isolates which were resistance to carbapenems were collected from different departments in different types in the Department of Microbiology of the First Affiliated Hospital of Guangzhou Medical University.All the strains were identified and tested by the French Bio Mérieux Vitek2 automatic microbiological identification and susceptibility testing systems.The drug susceptibility test was performed according to the Clinical and Laboratory Standards Institute(CLSI)M100-S22 2012 edition.2.Preparation of DNA template and carbapenemase gene detection The DNA template of the strain was prepared by boiling method;11 types of carbapenemase resistance genes bla IMP,bla SPM,bla AIM,bla VIM,bla OXA,bla GIM,bla BIC,bla SIM,bla NDM,bla DIM and bla KPC were detected by multiplex PCR.The positive PCR products were sent to sequence,and the results was aligned by BLAST to determine the subtype of the gene.3.Study on the transmission mode of drug resistance gene Plasmid conjugation experiment was performed on the carbapenemase genes positive strains.Then the conjugators were identified and tested by the French Bio Mérieux Vitek2 automatic microbiological identification and susceptibility testing systems.The drug susceptibility test was performed according to the Clinical and Laboratory Standards Institute(CLSI)M100-S22 2012 edition.MLST analysis of Klebsiella pneumoniae strains carrying carbapenemase gene was compared with those reported at home and abroad.4.The plasmid conjugation experiment was performed between KP DQ49 and EC600.The total DNA of the conjugator was extracted by bacteria genome DNA extraction kits and sequenced by Illumina Miseq platform.Sequencing data were assembled by Edena software and gene annotation was carried out by RAST server.Resistance genes were identified by Res Finder,and resistance gene environment was determined by NCBI BLAST.The plasmid incompatible group(Inc)was determined by Plasmid Finder.Sequence types(ST)were analyzed by MLST server.Results1.Drug susceptibility results 18 carbapenems-resistant strains include 12 strain of Klebsiella pneumonia,2 strains of Escherichia coli,2 strains of Enterobacter cloacae,1 strain of Laurus bacteria,1 strain of Citrobacter freundii.There were13 strains isolated from ICU,2 strains isolated from urology,1 strains isolated from the deparment of cardiovascular,1 strain isolated from hepatobiliary surgery,and 1 strain isolated from gastrointestinal surgery.According to the type of speci-men,they can be divided into 7 sputum specimens,2 midstream urine specimen-s,2 bile specimens,2 abdominal drainage fluid specimens,1 catheter tube head specimen,1 catheters specimen,1 blood specimen,1 secretion specimen,and the rest of specimen was unknown.After testing,18 strains were showed strong multiple resistance to carbapenems,penicillins,β-lactams,monocyc-lic lactams,but resistance was different in amikacin,gentamicin,nitrofurantoin,trimethoprim,tigecyclin,ciprofloxacin,levofloxacin.2.Detection of carbapenemase resistance gene by multiplex PCR 18 strains were detected by multiplex PCR and found to carry carbapenemase genes.All of them were sequenced.8 of them were bla NDM-1,8 were bla KPC-2,1 strain was bla VIM-1 type,1 strain was bla IMP-26 type.3.The mode of transmission of drug resistance genes and MLST results of Klebsiella pneumoniae In the conjugation experiment,10 strains successfully transferred the plasmid to the recipient EC600,including 7 strains of Klebsiella pneumonia,2 strains of Escherichia coli,1 strain of Enterobacter cloacae;8 strains were successfully transferred by electroporation,including 5 strains of Klebsiella pneumonia,1 strain of Enterobacter cloacae,1 strain of plant Laurus bacteria,1 strain of Citrobacter freundii.12 strains of Klebsiella pneumoniae were analysed by MLST,of which 10 strains was ST11,accounting for the majority;1 was ST20;1 strain was a new ST2460.4.The plasmid p IMP26_DQ49 carrying resistance gene bla IMP-26 was obtained by conjugation experiment,and then was sequenced.The sequencing results showed that p IMP26_DQ49 belonged to the Inc N group in the plasmid incompatible group(Inc),which was a 55179 bp circular plasmid with three resistance genes with a GC content of 50.4% and predicted 52 functional genes.BLAST found that p IMP26_DQ49 was similar to the reported p IMP_HZ1 as much as 99%,and the portable vector elements were highly similar.And the drug resistant gene environment contained IS903D、IS2、Tn2、Tn3、tnp、tnp A,which could lead to transposon events,and class 1 integrin gene int I1 which can capture and integrate exogenous gene.ConclusionsEnterobacteriaceae strains resistance to carbapenems all carry carbapenemase gene.All of these genes could be transferred to the same kind of bacteria by conjuga-tion or electroporation experiment.The p IMP26_DQ49 carried extended-spectrum β-lactamase gene blaTEM-1,quinolone resistance gene qnr S1 and metal carbapenemase gene bla IMP-26,the presence of these genes may be related to the spread of multiple drug resistance genes in Enterobacteriaceae. |
