Analysis Of Antibiotic Resistance And Clinical Infection Characteristics Of IMP Type Metalloenzyme Producing Enterobacteriaceae

BackgroundEnterobacteriaceae is an important pathogen for nosocomial infections and community-acquired infections.In recent years,with the increasing use of cephalosporins in the clinical,extended-spectrum P-lactamase(ESBLs)and AmpC enzyme enterobacteriaceae bacteria rate increased sharply.Carbapenem antibiotics have been used as the most effective drugs for the treatment of these severe infections.However,with the widespread use of imipenem and meropenem,carbapenem--resistant enterobacteriaceae(CRE)is also increasing year by year.Once the emergence of CRE strains of infection,clinical treatment of drug selection is very limited.And patients with infection often have concurrent disease,which have brought great difficulty for the clinical anti-infective treatment and nosocomial infection control.The study have confirmed that the main mechanism of resistance to CRE is carbapenemase.The enzyme types in the enterobacteriaceae that have been detected are mainly Ambler A(KPC),B type(VIM,IMP,NDM-1)and Type D(OXA-48).And the KPC is the most common type.KPC has appeared in many countries and regions in the world,such as the United States,France,Italy,India and China,but the other carbapenem types are relatively few reported.Class B carbapenems,also known as metallo-beta-lactamases(MBLs),referred to as metalloenzyme,is a class of active sites containing metal ions of P-lactamase.These enzymes efficiently hydrolyze almost all ?-lactam antibiotics except monocyclic antibacterials.IMP carbapenemase is a common type of enzyme in MBLs,the resistance gene is mediated by chromosomes or plasmids and has been widely spread in non-fermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii.In recent years,strains of IMP-producing MBLs have also been found in enterobacteriaceae,arousing the concern of all parties in the clinic.Molecular biology techniques have always been the gold standard for detecting of carbapenemase.However,the method is cumbersome,time-consuming,and requires complex instrumentation,which is not conducive to the rapid reporting of enzyme-producing strains.The relatively simple phenotypic detection technique is more helpful to early-prompt the production of carbapenemase and timely control of infected strains.The CLSI(Clinical&Laboratory Standards Institute)standard recommends the modified Hodge test(MHT)for the detection of the carbapenemase phenotype,but the standard mentions only the detection of the KPC enzyme,not for the other types of enzymes.In addition,the antibacterial treatment of CRE mainly focused on the infection of strains producing KPC,and a large sample study on the treatment of IMP-type CRE infection has not been reported.This study collected CRE strains detected in three general hospitals in the Liaocheng area during the past seven years,the drug resistance,phenotypes,genotypes,homology,and clinical infection characteristics was study in order to provide a theoretical basis for clinical anti-infective treatment and prevent the further dissemination of resistant strains.Part I Distribution Characteristics and Drug Resistance of Carbapenem-resistant EnterobacteriaceaeObjective To study the distribution characteristics and drug resistance trend of CRE in Liaocheng region,analyze the overall drug resistance and the differences in drug resistance among different CRE spp.Methods The non-repeat clinical isolates of CRE were collected form three comprehensive hospital in Liaocheng city,including Liaocheng People's Hospital,Liaocheng Chinese Medicine Hospital,Liaocheng Third People's Hospital between January 2010 and December 2016.The bacterial identification was performed using a Vitek MS automated mass spectrometry detection system,and antibiotic susceptibility was operated by Vitek 2 compact automated system.Then strains with reduced sensitivity(medium or resistance)to carbapenem antibiotics were screened.Statistical analysis was performed on the strain type,total resistance rate,and individual drug resistance rate of different spp of CRE.Results A total of 124 enterobacteriaceae with reduced susceptibility to carbapenems were collected,including 84 Klebsiella pneumoniae(67.7%),24 Enterobacter cloacae(19.4%),10 Escherichia coli(8.1%)and 6 Klebsiella oxytoca(4.8%).All CRE strains showed resistance to ceftriaxone,ceftazidime,cefepime and other cephalosporin antibiotics.More than 80.0%of the isolates were not sensitive(moderate or resistant)to tobramycin and cotrimoxazole.The insensitivity of CRE to gentamicin,piperacillin/tazobactam,ciprofloxacin and tigecycline were 56.5%,46.8%,38.7%and 20.2%,respectively.There are some differences between the resistance of different strains.Klebsiella pneumoniae had a relatively low resistance rate,the insensitivity to piperacillin/tazobactam,ciprofloxacin,and tigecycline is less than 30%.Enterobacter cloacae was less susceptible to imipenem and meropenem(both were 25.0%),insensitive to gentamicin,tobramycin and piperacillin/tazobactam were 75.0%,to ciprofloxacin and tigecycline the insensitivity rate were more than 40.0%.The insensitivity rates of Escherichia coli to piperacillin/tazobactam and ciprofloxacin were 90.0%and 60.0%respectively.The drug resistance of Klebsiella oxytoca was more serious,the test antibacterials all showed insensitivity except tigecycline(16.7%).Conclusions Multiple strains of CRE infection occurred in this area and showed multiple drug resistance,of which K.pneumoniae was the main strain.There was a certain difference in resistance among different strains.K.pneumoniae showed low resistance rate,and E.cloacae,E.coli and K.oxytoca were relatively high.Analysis of drug resistance is conducive to the discovery of drug-resistant strains of bacteria,and for the subsequent treatment of opportunistic infections with pathogenic bacteria.Part II Study on Drug Resistant Phenotypes and Genotypes of Carbapenem-resistant EnterobacteriaceaeObjective To study the resistant phenotypes and genotypes of carbapenem-resistant enterobacteriaceae,and to investigate the application of Vitek 2 applied expert systems(AES)and MET for the detection of carbapenemase from enterobacteriaceae.Methods The phenotype of carbapenemase was determined by the results reported of Vitek 2 instrument AES system and MHT.The blaKPC,blaVIM,blaIMP,blaNDM-1 and blaOXA-48 gene were amplified by PCR,then positive results were sequenced and blasted to determine genotype.The information of positive strains were registered for a Genebank number.Since the results of gene sequencing as the gold standard,the performance of Vitek 2 compact AES and MHT for the detection of carbapenemase in enterobacteriaceae was calculated based on sequencing result,including specificity,sensitivity,test efficacy,negative predictive value and positive predictive value.Results The results showed that 102 isolates was positive by MHT(82.3%),the proportion of K.pneumoniae,E.cloacae,E.coli and K.oxytoca were 83.3%,75.0%,80.0%and 100.0%respectively.The positive strains were detected 116 strains by AES,the proportion of carbapenemase-producing K.pneumoniae,E.cloacae,,E.coli and K.oxytoca in the corresponding strains was 95.2%,91.7%,80.0%and 100.0%,respectively.The 54 IMP-4 carbapenemase producing isolates were confirmed by PCR amplification and sequencing,including 42 K.pneumoniae,8 E.cloacae and 4 K oxytoca.The 40 IMP-8 carbapenemase producing isolates were contained by 20 K.pneumoniae,12 E.cloacae,6 E.coli and 2 K.oxytoca,the other 30 isolates did not detect carbapenemase.The specificity,sensitivity,test power,negative predictive value and positive predictive value of AES was 26.7%,100.0%,82.3%,100.0%and 81.0%,for the detection of carbapenemase in enterobacteriaceae.The specificity,sensitivity,test power,negative predictive value and positive predictive value of MHT was 66.7%,97.9%,90.3%,90.9%and 90.2%for the detection of carbapenemase in enterobacteriaceae.Conclusions The main resistance mechanism of CRE strains in this area is the production of IMP carbapenemase.IMP-4 type and IMP-8 type MBLs are the main types of enzyme production.The AES of Vitek 2 systems and MHT showed high sensitivity to the detection of IMP-type enzyme phenotypes produced by enterobacteriaceae.Both of the above methods can be used for the enzyme-producing information of early-prompt strains in order to take isolation measures in time.Part ? Study on Molecular Epidemiology of Enterobacteriaceae Producing IMP-type MetalloenzymeObjective To study the molecular epidemiology of IMP-type CRE infected patients and analyze the application value of Vitek MS mass spectrometry in the homology analysis of CRE strains.Methods The molecular epidemiology of the strain was studied by pulsed field gel electrophoresis(PFGE).The results were interpreted according to the Tenover standard.Simultaneously,the cluster analysis of protein peak was using the SuperSpectrum function of the Vitek MS mass spectrometer.The results were compared with those of PFGE homology.Results According to the Tenover standard,84 strains of carbapenem-resistant K.pneumoniae were divided into 11 different PFGE-DNA profiles(A-K).Among the 42 strains of IMP-4 type metalloenzymes,19 strains were the same type(type A),14 strains were B type,6 strains were C type,the other three strains were independent genotypes(D type,E type,F type).In 20 strains producing IMP-8 type metalloenzymes,12 isolates were the same G type and 8 isolates were the same H type.Another 22 strains without carbapenemase were divided into three genotypes(type ?,type J,type K).Twenty-four strains of carbapenem-resistant E.cloacae were divided into eight types of PFGE-DNA(A-H).Among 8 strains of IMP-4 type metalloenzyme strains,6 strains were type A,the other strains were type B.In 12 strains of IMP-8 type belong to different genotypes(C type,D type,E type,F type).The other four strains did not produce carbapenemase belong to the G type and H type.Ten strains of carbapenem-resistant E.coli belong to different PFGE-DNA profiles(A-E).Six strains of carbapenem-resistant K.oxytoca were classified into different PFGE-DNA profiles(A-C).The SuperSpectrum functional protein peak comparison results of the Vitek MS mass spectrometer have some similarities with PFGE.The error rates of K.pneumoniae,E.cloacae,E.coli and K.oxytoca were 9.5%(8 cases),12.5%(3 cases),10%(1 case)and 16.7%(1 case).Conclusions There are certain homology between IMP-type enzyme CRE strains appearing in different hospitals and different departments in Liaocheng region,indicating that the strain has produced a partial regional epidemic.The Vitek MS mass spectrometry technology can be initially applied to homology analysis of CRE strains,but accurate homology typing still requires PFGE detection.Part ? Analysis of Clinical Pathogenic and Treatment of Carbapenem-resistant Enterobacteriaceae InfectionObjective To investigate the data of clinical infections in patients infected with CRE,retrospectively analyze the risk factors and treatment options for infection,and explore the rational prevention and treatment of IMP-producing CRE strains.Methods To collect clinical datas of patients infected with CRE strains,including basic information,treatment,and prognosis.The type of infection is divided into ventilator-associated bacterial pneumonia(VABP),bacteremia,complicated urinary tract infection(cUTI)/acute pyelonephritis(AP),hospital-acquired bacterial pneumonia(HABP),superficial wound infection(SWI),biliary tract infection(BTI),deep wound infection(DWI)and sterile body fluids infection(SBFI)due to CRE.The clinical data of patients infected with CRE strains were compared with the patients infected with carbapenem-sensitive stains in the same period.The basic information such as sex,age,disease,hospitalization,operation,replacement of bed and use of antimicrobial were investigated.The data were analyzed with SPSS 18.0.Results According to the type of infection CRE cases,31 VABP,22 bacteremia,18 cUTI/AP,16 HABP,16 SWI,9 BTI,7 DWI and 5 SBFI were identified.The patient population had significant immunocompromised(33 of 124,26.6%)and severe sepsise(43 of 124,34.7%).Most patients with single-agent empirical treatment,and the purpose of treatment with more anti-bacterial drugs.CRE infection with poor prognosis(28-day mortality was 22.6%,28/124),some patients infected using fluoroquinolone therapy based on susceptibility to achieve good results.The control studies showed significant differences in the number of days of hospitalization,invasive operation and bed replacement between the two groups(P<0.01).The use of carbapenem antibiotics before detected strains were different in the two groups(P<0.05).Conclusions The presence of IMP-type carbapenemase-producing CRE strains in this region has caused multiple site infections.Infected patients often have poor conditions,severe complications and high mortality rates.Long-term hospitalization,invasive procedures and use of carbapenems are high risk factors for CRE infection.The quinolone antibacterial agents can effectively treat part of the CRE infections producing IMP enzyme.