Surveillance Of Resistance To Carbapenem Resistant Enterobacteriaceae And Its Genotype

Background:Enterobacteriaceae is widely distributed in the human intestinal tract and hospital environment,one of the most common opportunistic pathogens in clinical practice,can lead to a variety of different types of infection.Carbapenem antimicrobials have the characteristics of broad antibacterial spectrum,rapid bactericidal activity and strong antibacterial activity.With the increasing pressure of choice of antibiotics,there have been carbapenem antibiotics resistant to Enterobacteriaceae bacteria,will have an unprecedented stress on human life and health.CRE resistance mechanism is mainly for the production of KPC,NDM,VIM,IMP and OXA-48 carbapenemase resistance gene,which produces NDM type carbapenemase CRE can be a potent hydrolysis of various carbapenem antibacterial drug.At present,the research focus of CRE strain is carbapenem-resistant Enterobacter cloacae.Miyoshi-Akiyama T and others established MLST molecular typing scheme to make the traceability of multiple resistant Enterobacter cloacae tend to be standardized,combined with PFGE high-resolution Enterobacter cloacae genomic map,can fully understand the production of NDM-type cloning transmission mechanism.At the same time,the accessibility of the resistant plasmid makes it possible to spread the level of the drug resistance gene located in the plasmid.Therefore,it is significant to analyze the drug resistance of CRE strain,to clarify the mechanism of CRE resistance to carbapenems,and to explore the mechanism of clonal and horizontal transmission.Objective:1.This study was to investigate the clinical distribution and drug resistance of carbapenem-resistant Enterobacteriaceae from 2012 to 2016.2.In this study,the resistant phenotypes and drug resistance genes of carbapenem-resistant Enterobacteriaceae in Wuhan were analyzed.3.Cloning and horizontal transmission mechanism of carbapenem-resistant Enterobacter cloacae were analyzed.Methods:1.This research collected the susceptibility information of the CRE strain isolated from Wuhan University People's Hospital from January 2012 to December 2016.The strains were identified by BD PhoenixTM 100 automatic bacterial identification and drug sensitivity analyzer.The strain was identified by biochemical reaction.The antimicrobial susceptibility test was carried out on 12 kinds of antibiotics by micro-broth dilution method,and the susceptibility test was supplemented by KB(Kirby-Bauer)paper diffusion method.2.Carba NP experiments were used to confirm the in vitro presence of carbapenemase phenotypes.Five kinds of carbapenemase resistance genes,KPC,NDM,IMP,VIM and OXA-48,were detected by PCR.The PCR products were subjected to 2.0%agarose gel electrophoresis.Purification product was sequenced and Blast was used to verify the amplification product gene subtype.3.The clonal transmission mechanism of carbapenem antibiotic resistance strains of Enterobacter cloacae was tested by pulsed-field gel electrophoresis(PFGE)and multilevel sequence typing(MLST),And the horizontal transmission mechanism was verified by plasmid binding experiments.4.Data analysis using Whonet 5.6,SPSS 21.0,Excel 2013 software,PFGE data using BioNumerics software.The MLST serial number is assigned by the site database.Results:1.The specimens of the hospital were mainly composed of sputum(114,42.9%)and urine(94,35.3%).Klebsiella pneumoniae(155,58.3%)is the main constituent of CRE.The Department of Neurology(56,21.1%),ICU(43,16.2%)and neurosurgery(39,14.7%)were mainly investigated.The resistance rate of CRE to imipenem and meropenem were 90.6%and 87.6%,respectively.The resistant rates of the remaining P-lactam antibiotics were higher than 92.8%.The resistance rate to paediatric compound sulfamethoxazole and amikacin were 46.4%and 41.1%,respectively.2.The positive rate of Carba NP was 86.4%(76/88)in 88 strains of carbapenem antibiotics resistant to Enterobacteriaceae.There were 29 strains with NDM-1 resistance gene,the positive rate was 33%;There were 24 strains with KPC-2 resistance gene,the positive rate was 27.3%,two groups had no statistically significant difference(?2=0.68,P>0.008).There were 9 strains with VIM-19 resistance gene,the positive rate was 10.2%,the IMP-4 resistance gene was 14,the positive rate was 15.9%,the difference between the two groups was not statistically significant(?2 = 1.25,P>0.008).No OXA-48 gene was found;3 strains of Klebsiella pneumoniae were used to produce KPC-2 and NDM-1 genes.3.The positive rate of Carba NP was 55.6%(5/9)in 9 strains of carbapenem antibiotic resistant Enterobacter cloacae.PCR and sequencing analysis showed that one strain of NDM-1 combined with VIM-19 Enzyme,3 strains of NDM-1 carbapenemase,and 1 strain of VIM-19 carbapenemase.4 strains of NDM-type carbapenemase Enterobacter cloacae and drug-resistant plasmids were successfully transferred to Escherichia coli C600,which were grown in imipenem(4 ?g/ml)and rifampicin(100 ?g/ml)on a double-resistant plate.The sequence type of these strains was ST145,ST508 and ST509 by MLST experiments.The similarity coefficient of strain No.6 and No.7 was 92.3%,which was the same clonal strain.The similarity coefficient of strain 6 was 66.7%,and the similarity coefficient of strain No.1 was 59.9%,namely,different clonal strains.Conclusion:1.The distribution of CRE specimens shows that they are common in respiratory and urinary tract infections.Clinicians should focus on the drug resistance of carbapenem Klebsiella pneumoniae,timely adjust the empirical medication and target the treatment as soon as possible.The CRE distribution shows that it is common in patients with neurology,ICU and neurosurgical wards.CRE is resistant to most antimicrobial agents.SXT and amikacin for the hospital is currently relatively sensitive to the antibiotics CRE.2.Carbapenemases is the main reason of CRE in carbapenem resistance.Resistance gene by NDM-1 and KPC-2,followed by IMP-4 and VIM-19,CRE strain has the characteristics of combined part of carbapenemases.3.NDM-1 carbapenem enzyme is an important cause of Enterobacter cloacae resistance to carbapenem antibiotics.NDM-1 and/or VIM-19 carbapenemase genes can be propagated horizontally through drug-resistant plasmids.ST508,ST509 and ST 145 were three different clones,which were scattered in the clonal distribution.ST509 carrying NDM-1 resistance gene was the potential clonal strain of the hospital.