PCR-based restriction pattern typing of the vacA gene provides evidence for a homogeneous group among Helicobacter pylori strains associated with peptic ulcer disease.
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The results of PCR-based molecular typing of Helicobacter pylori strains by restriction fragment length polymorphism analysis of a 1, 161-bp nucleotide sequence of the midregion of the vacA gene are reported. A total of 48 H. pylori strains isolated from gastric biopsy specimens obtained from 18 patients with peptic ulcer dyspepsia, 15 patients with nonulcer dyspepsia, and 15 asymptomatic H. pylori-infected subjects were studied. Highly heterogeneous restriction patterns were obtained by digestion of PCR products with SauII, BglII, and HhaI, whereas HaeIII digestion resulted in a strictly homogeneous profile for H. pylori strains isolated from 14 of 18 (77.7%) patients with peptic ulcer dyspepsia, but a strictly homogeneous profile was found for strains from only 8 of 15 (53.3%) patients with nonulcer dyspepsia (P = 0.163) and 5 of 15 (33.3%) asymptomatic H. pylori-infected subjects (P = 0.014). A potentially important aspect of the results obtained is the clinical relevance, since a single restriction pattern seems to be able to identify the majority of H. pylori strains associated with peptic ulcer disease. (+info)
Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions.
(26/8395)
Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi. (+info)
Molecular markers demonstrate that the first described multidrug-resistant Mycobacterium bovis outbreak was due to Mycobacterium tuberculosis.
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We genetically characterized multidrug-resistant Mycobacterium tuberculosis complex strains which caused a nosocomial outbreak of tuberculosis affecting six human immunodeficiency virus (HIV)-positive patients and one HIV-negative staff member (E. Bouvet, E. Casalino, G. Mendoza-Sassi, S. Lariven, E. Vallee, M. Pernet, S. Gottot, and F. Vachon, AIDS 7:1453-1460, 1993). The strains showed all the phenotypic characteristics of Mycobacterium bovis. They presented a high copy number of IS6110, the spacers 40 to 43 in the direct repeat locus, and the mtp40 fragment. They lacked the G-A mutation at position 285 in the oxyR gene and the C-G mutation at position 169 in the pncA gene. These genetic characteristics revealed that these were dysgonic, slow-growing M. tuberculosis strains mimicking the M. bovis phenotype, probably as a consequence of cellular alterations associated with the multidrug resistance. Spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis confirmed that the outbreak was due to a single strain. However, the IS6110 RFLP pattern of the strain isolated from the last patient, diagnosed three years after the index case, differed slightly from the patterns of the other six strains. A model of a possible genetic event is presented to explain this divergence. This study stresses the value of using several independent molecular markers to identify multidrug-resistant tubercle bacilli. (+info)
Molecular evidence for heterogeneity of the multiple-drug-resistant Mycobacterium tuberculosis population in Scotland (1990 to 1997).
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Multiple-drug-resistant Mycobacterium tuberculosis (MDR-MTB) has been well studied in hospitals or health care institutions and in human immunodeficiency virus-infected populations. However, the characteristics of MDR-MTB in the community have not been well investigated. An understanding of its prevalence and circulation within the community will help to estimate the problem and optimize the strategies for control and prevention of its development and transmission. In this study, MDR-MTB isolates from Scotland collected between 1990 and 1997 were characterized, along with non-drug-resistant isolates. The results showed that they were genetically diverse, suggesting they were unrelated to each other and had probably evolved independently. Several new alleles of rpoB, katG, and ahpC were identified: rpoB codon 525 (ACC-->AAC; Thr525Asn); katG codon 128 (CGG-->CAG; Arg128Gln) and codon 291 (GCT-->CCT; Ala291Pro); and the ahpC synonymous substitution at codon 6 (ATT-->ATC). One of the MDR-MTB isolates from an Asian patient had an IS6110 restriction fragment length polymorphism pattern very similar to that of the MDR-MTB W strain and had the same drug resistance-related alleles but did not have any epidemiological connection with the W strains. Additionally, a cluster of M. tuberculosis isolates was identified in our collection of 715 clinical isolates; the isolates in this cluster had genetic backgrounds very similar to those of the W strains, one of which had already developed multiple drug resistances. The diverse population of MDR-MTB in Scotland, along with a low incidence of drug-resistant M. tuberculosis, has implications for the control of the organism and prevention of its spread. (+info)
Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water.
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We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections. (+info)
Analysis with a combination of macrorestriction endonucleases reveals a high degree of polymorphism among Bordetella pertussis isolates in eastern France.
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From 1990 to 1996, routine screening for whooping cough identified 399 patients with a calmodulin-dependent adenylate cyclase-positive test result and yielded 69 Bordetella pertussis isolates. None of the patients were fully vaccinated, and most were less than 6 months old. Analysis of total DNA by pulsed-field gel electrophoresis (PFGE) after XbaI, SpeI, or DraI macrorestriction yielded 19, 15, and 5 different patterns, respectively, whereas ribotyping failed to demonstrate any strain polymorphism. Discrimination among the isolates was improved by combining the PFGE profiles. Some patterns were more frequent, but the corresponding patients were not clearly epidemiologically related. The patterns for two strains obtained during a 3-month period from patients who were neighbors differed by the length of a single DNA fragment. These data strongly suggest that one type of isolate is widely spread throughout the world and is carried by individuals other than patients who develop a true illness. (+info)
Bartonella koehlerae sp. nov., isolated from cats.
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Two of the 25 Bartonella isolates recovered during a prevalence study of Bartonella henselae bacteremia in domestic cats from the greater San Francisco Bay region were found to differ phenotypically and genotypically from all prior B. henselae isolates. These isolates, C-29 and C-30, which were recovered from the blood of two pet cats belonging to the same household, grew on chocolate agar as pinpoint colonies following 14 days of incubation at 35 degrees C in a candle jar but failed to grow on heart infusion agar supplemented with 5% rabbit blood. Additional phenotypic characteristics distinguished the isolates C-29 and C-30 from other feline B. henselae isolates. The restriction patterns obtained for C-29 and C-30 by citrate synthase PCR-restriction fragment length polymorphism (RFLP) analysis as well as by genomic RFLP could not be distinguished from each other but were distinctly different from that of the B. henselae type strain. In reciprocal reactions, DNAs from strains C-29 and C-30 were 97 to 100% related under optimal and stringent DNA reassociation conditions, with 0 to 0.5% divergence within related sequences. Labeled DNA from the type strain of B. henselae was 61 to 65% related to unlabeled DNAs from strains C-29 and C-30 in 55 degrees C reactions, with 5.0 to 5.5% divergence within the related sequences, and 31 to 41% related in stringent, 70 degrees C reactions. In reciprocal reactions, labeled DNAs from strains C-29 and C-30 were 68 to 92% related to those of the B. henselae type strain and other B. henselae strains, with 5 to 7% divergence. The 16S rRNA gene sequence of strain C-29 was 99.54% homologous to that of the type strain of B. henselae. On the basis of these findings, the two isolates C-29 and C-30 are designated a new species of Bartonella, for which we propose the name Bartonella koehlerae. The type strain of Bartonella koehlerae is strain C-29 (ATCC 700693). (+info)
Specificity of IS6110-based DNA fingerprinting and diagnostic techniques for Mycobacterium tuberculosis complex.
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Restriction fragment length polymorphism and hybridization of DNA extracted from Mycobacterium tuberculosis, nontuberculous mycobacteria, and nonmycobacterial species with a probe derived from IS6110 confirmed that IS6110 was specific to M. tuberculosis complex. In addition, DNA amplification with IS6110-specific primers yielded a 181-bp fragment only in DNA from M. tuberculosis complex isolates. (+info)