ABSTRACT
Objective To evaluate the applicationof the double-disk synergy test(DDST) and combined disk test(CDT) in clini cal metal enzyme phenotype deteetion.To evaluate the value of multiplex PCR in detecting the metallo-β-1actamase in clinical.Methods 56 strains of metallo-β-1actamase-positive strains were identified [NDM-1(n=9),VIM(n=32),IMP(n=15)]for appraise the two methods.By optimizing the design of PCR primers,3 pairs of primers were designed and detected (IMP,VIM,NDM-1) in one tube for evaluate the method.Results The DDST was 80.36％(45/56),and 100.00％(56/56) in the CDT.The accuracy of multiplex PCR was 100.00 ％ (56/56),and the size of the amplified fragment was used to distinguish three types of metallo-β-lactamase.Conclusion The CDT is more suitable for clinical application than DDST.Multiplex PCR has the characteristics of simple,rapid and accurate in detection of metallo-β-1actamase.It is suitable for daily use of clinical microbiological laboratory,which will help the clinical timely and effective administration.
ABSTRACT
Objective To evaluate the applicationof the double-disk synergy test(DDST) and combined disk test(CDT) in clini cal metal enzyme phenotype deteetion.To evaluate the value of multiplex PCR in detecting the metallo-β-1actamase in clinical.Methods 56 strains of metallo-β-1actamase-positive strains were identified [NDM-1(n=9),VIM(n=32),IMP(n=15)]for appraise the two methods.By optimizing the design of PCR primers,3 pairs of primers were designed and detected (IMP,VIM,NDM-1) in one tube for evaluate the method.Results The DDST was 80.36％(45/56),and 100.00％(56/56) in the CDT.The accuracy of multiplex PCR was 100.00 ％ (56/56),and the size of the amplified fragment was used to distinguish three types of metallo-β-lactamase.Conclusion The CDT is more suitable for clinical application than DDST.Multiplex PCR has the characteristics of simple,rapid and accurate in detection of metallo-β-1actamase.It is suitable for daily use of clinical microbiological laboratory,which will help the clinical timely and effective administration.
ABSTRACT
Objective To investigate the resistance-mechanism of the carbapenems-resistant Klebsiella pneumoniae isolated from clinical.Methods The clinical isolates of carbapenems-resistant Klebsiella pneumoniae from top three comprehensive hospitals of Nanjing area were examined by 40 beta-lactamase,porin-coding genes and linkage of KPC-ISKpn6 using PCR method,the PCR positive results were picked out for sequencing and sequencing BLAST search for comparison analysis.Results Twenty-four strains of carbapenems-resistant Klebsiella pneumoniae were detected,the positive rate of A beta-lactamase TEM-1 and SHV was 100％ (24/24),KPC-2 and LAP-2 was 95.8％ (23/24),45.8％ (11/24) respectively,and C beta-lactamase DHA was 4.2％ (1/24).Meanwhile,the positive detection rates of KPC-ISKpn6 linkage was 95.8％ (23/24),and the mutation rate of porin-coding genes ompK35 and ompK36 were up to 95.8％ (23/24) and 100％ (24/24).Conclusion High incidence of beta-lactamase TEM-1,SHV,KPC-2 and LAP-2 was found in the group of Klebsiella pneumoniae isolates,and carbapenems-resistant of which was primarily due to the high carrying rate of KPC-2 and the high mutation rate of porin-coding genes ompK35 and ompK36.The Insertion sequence ISKpn6 may be involved in the KPC-2 gene mediated-expression.
ABSTRACT
Objective To investigate the drug resistance and its distribution induced by β-lactamases and class Ⅰ integrons with the deficiency of Omp genes in Enterobacter cloacaes.Methods Totally 112 strains of Enterobacter cloacaes were isolated during January 2008 and May 2011.The identification of strains was performed by using Vitek-2 Compact automatic system; and antibiotic susceptibility was determined by K-B method.Isolates of E.cloacae were screened for carbapenemases by modified Hodge test,improved three dimensional test and EDTA-meropenem synergy test.Genes encoding AmpC β-lactamase,metallo-β-1actamases (MBLs) and OXA-like β-1actamases were screened by multiple PCR.Single PCR was used to detect ISEcpl,OmpK35/36,NDM-1 and OXA-48.The variable regions of class Ⅰ integrons were amplified and sequenced.Results Among 112 isolates,6 (5.4％) demonstrated positive in the modified Hodge test and 14 ( 12.5％ ) were positive in the improved three-dimensional test.No carbapenemases gene was found.There were 29(25.9％ ) strains positive for ESBLs genes,ISEcpl was found in the upstream of all the CTX-M-type ESBLs; OXA-1 ESBLs were detected in 2 isolates.AmpC β-lactamase genes were positive in 45 (40.2％) strains,and 82.2％ (37/45) were MIR-3 type.Twenty two isolates carried class Ⅰ integrons,and four different cassettes arrangements were identified within 16 strains:9 isolates harbored aadB-aadA2 ( 1 000 bp),5 isolates with dfrAl5 (700 bp),2 isolates with aadAl ( 1 000 bp).One isolate harbored all the above gene cassettes.The deficiency of OmpK35/36 was found in all strains.Conclusion ESBL,AmpC β-lactamase and the deficiency of OmpK35/36 are correlated with the resistance to carbapenems in Enteobacter clocace,and class Ⅰ integrons may also partly account for the multidrug-resistance.