Study On Carbapenem Resistance Mechanism Of Clinical Isolates Of Enterobacteriaceae

ObjectiveTo investigate the main mechanisms of Enterobacteriaceae resistant tocarbapenems and the capability of horizontal transfer capability ofdrug-resistant genes. And to provide a scientific basis for rational using ofantibiotics, reducing and controlling the dissemination of drug-resistantstrains.Methods1. Multidrug-resistant Enterobaeteriaceae strains were isolated from ahospital in Sichuan province from January2010to May2011.2.Minimum inhibitory concentration of multidrug-resistantEnterobaeteriaceae to13antibiotics were determined using the agardilution method. Enterobacteriaceae strains resistant to carbapenems werescreened by analyzing the drug-resistant spectrum.3. Modified Hodgetests were used to detect carbapenemase of carbapenems-resistantEnterobacteriaceae.4. EDTA synergistic tests were performed to researchmetallo-beta-lactamases of Enterobacteriaceae resistant to carbapenems.5.Polymerase chain reaction was determined to detect drug-resistant genes. DNA sequencing result of each amplification was compared with thesequence of GenBank database.6. Real-time RT-PCR was carried out toinvestigate the expression level of porin genes.7. Class I integrons ofEnterobacteriaceae resistant to carbapenems were detected by PCR, andtheir sequences were analysed.8. Conjugation experiments were subjectedto reveal the transferability of carbapenem resistant genes.Results1. Sixty strains of multi-drug resistant Enterobaeteriaceae were isolated inthe study.2. Ten strains of Enterobacteriaceae resistant to carbapenemswere screened by analysis the resistance spectrum, inculding5strains ofKlebsiella pneumoniae,4strains of Enterobacter cloacae and1strain ofRaoultella planticola. The rate of isolated Enterobacteriaceae resistant tocarbapenems from multi-drug resistant Enterobacteriaceae was16.67%.The drug-resistant rates of the10isolated strains to ertapenem,meropenem and imipenem were100%,70%,10%, respectively. Tenstrains were all resistant to ceflriarone sodium, ceftazidime andcefoperazone/sulbactam. The rates of Enterobacteriaceae resistant tocefoxitin sodium and cefepime were90%and80%. The rates ofEnterobacteriaceae susceptible to amikacin and polymyxin B were90%,80%, respectively.3. Modified Hodge tests were positive in7of10carbapenems resistance among Enterobacteriaceae strains, including K13,K25, K30, E3, E8, E13and R1.4. EDTA synergistic tests were positive in 4strains resistant to carbapenems, including K13, K17, E3, and R1.5.Among the10strains, the positive rates of carbapenemases related genesIMP, KPC were40%and10%, respectively. The positive rates ofextended spectrum β-lactamases genes CTX, TEM and SHV were100%,50%,80%, respectively. The positive rate of AmpC related gene DHA was20%. After compared with GenBank, IMP genes of4strains wereidentified as IMP-4, KPC gene of1strain was KPC-2, the majority ofextended spectrum β-lactamases were CTX-M3, TEM-1, SHV-12andSHV-28, DHA gene of2strains were DHA-1.6. By using Real-timeRT-PCR technique to analyze the expression level of porin genes, wefound that expression of ompK36gene in2Klebsiella pneumoniae strains,and ompF gene in3Enterobacter cloacae strains was decreased.7. Sevenstrains carried class I integrons. dfrA12-orfF-aadA2was the mostprevalent gene cassette. The most common drug-resistant genes in class Iintegron was aminoglycoside drug-resistant genes(aadA2and aadA16),followed by trimethoprim drug-resistant genes(dfrA12, dfr16and dfrA27).8. Conjugation experiments were performed for5strains. Conjugationexperiments were positive in4IMP-4-producing Enterobacteriaceaestrains. The transconjugants of antimicrobial susceptibility test resultswere similar to the donor’s. Metallo-beta-lactamases were postive in thefour transconjugants by EDTA synergistic test. Plasmid sizes of thetransconjugants were alike. Conclusion1. Ten strains of Enterobacteriaceae resistant to carbapenems werescreened by analyzing the drug resistance spectrum in the research, whichhas strong resistant effect to cephalosperins, and keeps high activity toaminglycosides and polymyxin B.2. Various drug-resistant genes have been found in the strains. The mostcommon drug-resistant genes are carbapenemases related genes andextended spectrum β-lactamases genes.3. Metallo-β-lactamase-producing is one of the main mechanisms ofEnterobacteriaceae strains resistant to carbapenems.4. The decrease of porin expression may be one mechanism ofEnterobacteriaceae strains resistant to carbapenems.5. In the study, we don’t find class I integrons mediating horizontaltransfer of carbapenem-resistant genes.6. Horizontal transfer of metallo-beta-lactamase(IMP-4) gene can bemediated by plasmid.