| Extended-spectrum (3-lactamases (ESBLs) which is a large species of P-lactamases can hydrolyze the third generation of cephalosporins and monobactams. ESBLs are mainly produced by Klebsiella pneumoniae and Escherichia coli. In the last decade, the prevalence of ESBLs in clinical isolates is increasing sharply. ESBL-producing strains are becoming one of the most prevalent pathogens of nosocomial infection. The research on ESBLs, especially on ESBLs gene, is becoming one of the hottest research fields in Clinical Microbiology, hi China, the prevalence of ESBL-producing strains is high and the research on ESBLs genes is relatively scarce. We have conducted the following reseaches: collected 362 clinical isolates, Klebsiella pneumoniae and Escherichia coli, from 8 district hospitals in Zhejiang Province and the First Affiliated Hospital, College of Medicine, Zhejiang University; compared and evaluated three phenotypic tests for ESBL-producing strains; identified the genotypes of SHV p-lactamases in ESBL-producing strains; studied some properties of a novel SHY p-lactamase.1. Comparison of ESBL-detecting methods and related gene analysisThere are many phenotypic tests for ESBL-producing strains including double disk test (DDT), E test, three dimensional test, Vitek test, Inhibitor potentiated disk diffusion test and inhibitor potentiated broth dilution test (IPBDT). Although the lasttwo methods are recommended by NCCLS (1999), they cannot be used regularly in most clinical laboratories since their reagents are expensive and both methods are inconvenient to perform. Clinical laboratories still require a convenient, reliable and economic method to detect ESBLs. 68 strains of clinical isolated Escherichia coli and Klebsiella pneumoniae, 10 strains of standard p-lactamase-producing Escherichia coli and a strain of ATCC 25922 were collected. IPBDTs , DDT, E tests and Vitek tests were performed to detect ESBLs in these strains. Comparing with the results of IPBDT, the sensitivity of DDT (96.4%) is higher than that of E test and Vitek test (70.4% and 75.9%, respectively) and the specificities of three tests were all higher than 90%. By gene cloning and sequencing of five strains having different results by IPBDT and DDT, two strains were determined to produce non-TEM, non-SHV and three were determined to produce TEM-1 that is a non-ESBLs. The results suggested that strains producing TEM-1 pMactamases might affect the results of phenotype in ESBL-producing strains.2. The genotypic distribution of SHY p-lactamases in ESBL-producing clinical isolates in Zhejiang Province119 ESBL-producing strains were detected as ESBL-producers by IPBDT from 362 Escherichia coli and Klebsiella pneumoniae strains in Zhejiang Province during September 1998 ~ June 1999. The genotypes of ESBLs were initially determined by polymerase chain reaction (PCR) and determined again by dot blot hybridization. In the ESBLs producers, the prevalence of TEM, non-SHV producers was 55.5%, that of SHV, non-TEM producers was 10.9%, that of both TEM and SHV producers was13.4%, and that of non-TEM, non-SHV producers was 20.2%. The prevalent genotypes of ESBLs were not same in most districts of Zhejiang Province. The purified SHV PCR products were cloned into pGEM-Teasy vectors and sequenced by Sanger's dideoxy chain termination composition method. 29 out of 119 ESBL-producing strains produced SHV p-lactamases. Among these 29 strains: 4 strains produced SHV-1, 1 strain produced SHV-5, 8 strains produced SHV-11, 8 strains produced SHV-12 and 8 strains produced SHV-28. SHV-11, SHV-12 and SHV-28 are the main prevalent genotypes of SHV p-lactamases in ESBL-producing Escherichia coli and Klebsiellapneumoniae in Zhejiang Province.3. Discovery of a novel genotype of P-lactamases named SHV-28 and study of its propertiesThe genes encoding P-lactamases produced by 8 strains of ESBL-producing Klebsiella pneumoniae isolated from Ningbo and Zhoushan District, Zhejiang Province were amplified by PCR. All PCR products had 888 nucleotides without any d...
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