ABSTRACT
Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the muta- tions were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krusei Both the sensitivity and the specificity of the microarray were 100%. Conclu- sion DNA microarray is a quite reliable method to identify Candida spp. and fluconazole resistance-associ- ated mutations in the ERG11 gene of C. albicans.
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Objective To investigate the genotype distribution of CTX-M-type extended-spectrum β-1actamase(ESBLs) by denaturing high-performance liquid chromatography(DHPLC) in Hunan Province and the accuracy of DHPLC assay. Methods The blaCTX-M genes of standard strains and clinical ESBLs-producing Enterobacteriaceae were amplified by multiplex PCR followed by DHPLC and genotype determination. 25 isolates randomly selected were sequenced to assess the accuracy of DHPLC method. Results Among 142 ESBLs-producing isolates, 109 isolates carried blaCTX-M gene (76. 8% ). Four different CTX-M genotypes were detected by DHPLC, including CTX-M-3 (33 isolates), CTX-M-15 (19 isolates), CTX-M-14 (52 isolates) and CTX-M-9 (5 isolates). The DHPLC typing of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing, but one CTX-M-15 isolate typed by DHPLC was shown to be CTX-M-82 by sequencing. Conclusion DHPLC is a powerful tool for genotyping of the resistance gene and is worth being applied in the clinical and scientific research with accurate, rapid and economic advantages.
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Objeetive To detect ERG11 gene mutations in clinical isolares of Candida albicans resistant to fluconazole.and discuss their relationship with formation of drug resistance.Methods Clinical specimens were collected.CHROMagar mediuln and amplification of the fragment spanning the conserved sequence of 25S rDNA including some transposable introns.were used to identify subtype Candida albicans isolates.FIuconazole sensitivity was detected in vitro through microdilution and Rosco tablets.The other three fragment of ERG11 gene were amplified and followed by sequencing with resistant type strain ATCC 76615-19 and Candida albicans Darlington strain with two sensitive isolates as controh.Results Fifteen resistant isolates of Candida albicans were found,all of which were type A.Sixteen silent mutations and 11 missense mutations were detected.Mutations in ATCC 76615-19 and Darlington strain were same with what had been reported.In the 2 sensitive strains.G640A(E165K),A945C(E266D)and G1609A/G(V488I)occurred,as well as the other 9 silent mutations.Only G487T(A114S)and T916C(Y257H)existed in each of 14 resistant isolates.In the other one resistant isolate,T541C(Y132H),T495A(D116E),A530C (K128T)and T1493A(F449Y)occurred Mong with 8 silent mutations.Conclusions The occurrence of G487T(A114S)and 1916C(Y257H)in 14 isolates from different sources suggested they may involve in fluconazole resistance.The novel mutation T1493A(F449Y)can appear in resistant isolves of Candida albicans.
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OBJECTIVE To invesigate the prevalence of SHV-producing Enterobacteriaceae isolates in Hunan Province and identify the subtype of SHV encoding gene and the epidemiological aspect of SHV-producing isolates. METHODS Isolates were collected and identified as well as subjected to ESBLs detection.PCR and DNA sequencing were used to determine the genotype of SHV enzymes.The homology of SHV-producing strains were detected by RAPD. RESULTS Twenty-six of 171 Enterobacteriaceae isolates were confirmed to produce blaSHV genes,and 5 subtypes of SHV-type ?-lactamases were determined,including 9 strains of SHV-28,7 strains of SHV-12,7 strains of SHV-1,2 strains of SHV-11 and 1 strain of SHV-5.There were 6 RAPD types in 19 isolates of SHV-producing Klebsiella pneumoniae,5 RAPD types in 5 isolates of SHV-producing Enterobacter cloacae. CONCLUSIONS SHV-12 is the predominant genotype of SHV ESBLs producing Enterobacteriaceae in Hunan Province,and clone spread has played a certain role in SHV-type ?-lactamase producing K.pneumoniae.SHV ESBLs are not found in Escherichia coli.