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  • 1
    In: International Journal of Nephrology and Renovascular Disease, Informa UK Limited, Vol. Volume 14 ( 2021-02), p. 23-31
    Type of Medium: Online Resource
    ISSN: 1178-7058
    Language: English
    Publisher: Informa UK Limited
    Publication Date: 2021
    detail.hit.zdb_id: 2508160-3
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  • 2
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 43, No. 8 ( 2005-08), p. 3985-3994
    Abstract: Molecular techniques such as spa typing and multilocus sequence typing use DNA sequence data for differentiating Staphylococcus aureus isolates. Although spa typing is capable of detecting both genetic micro- and macrovariation, it has less discriminatory power than the more labor-intensive pulsed-field gel electrophoresis (PFGE) and costly genomic DNA microarray analyses. This limitation hinders strain interrogation for newly emerging clones and outbreak investigations in hospital or community settings where robust clones are endemic. To overcome this constraint, we developed a typing system using DNA sequence analysis of the serine-aspartate (SD) repeat-encoding region within the gene encoding the keratin- and fibrinogen-binding clumping factor B ( clfB typing) and tested whether it is capable of discriminating within clonal groups. We analyzed 116 S. aureus strains, and the repeat region was present in all isolates, varying in sequence and in length from 420 to 804 bp. In a sample of 36 well-characterized genetically diverse isolates, clfB typing subdivided identical spa and PFGE clusters which had been discriminated by whole-genome DNA microarray mapping. The combination of spa typing and clfB typing resulted in a discriminatory power (99.5%) substantially higher than that of spa typing alone and closely approached that of the whole-genome microarray (100.0%). clfB typing also successfully resolved genetic differences among isolates differentiated by PFGE that had been collected over short periods of time from single hospitals and that belonged to the most prevalent S. aureus clone in the United States. clfB typing demonstrated in vivo, in vitro, and interpatient transmission stability yet revealed that this locus may be recombinogenic in a primarily clonal population structure. Taken together, these data show that the SD repeat-encoding region of clfB is a highly stable marker of microvariation, that in conjunction with spa typing it may serve as a DNA sequence-based alternative to PFGE for investigating genetically similar strains, and that it is useful for analyzing collections of isolates in both long-term population-based and local epidemiologic studies.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 3
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 44, No. 2 ( 2000-02), p. 326-336
    Abstract: Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptible Mycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMB-resistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB , a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 4
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 42, No. 2 ( 2004-02), p. 792-799
    Abstract: Strain typing of microbial pathogens has two major aims: (i) to index genetic microvariation for use in outbreak investigations and (ii) to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region ( spa typing) provides a rapid and accurate method to discriminate Staphylococcus aureus outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether spa typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within S. aureus. Using spa and coa typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that S. aureus likely has a primarily clonal population structure and that spa typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages. spa typing performed better than MLEE, PFGE, and coa typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of S. aureus .
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 5
    In: Infection and Immunity, American Society for Microbiology, Vol. 71, No. 12 ( 2003-12), p. 7099-7108
    Abstract: Protective immunity against pulmonary tuberculosis (TB) is characterized by the formation in the lungs of granulomas consisting of macrophages and activated T cells producing tumor necrosis factor alpha and gamma interferon, both required for the activation of the phagocytes. In 90% of immunocompetent humans, this response controls the infection. To understand why immunity fails in the other 10%, we studied the lungs of six patients who underwent surgery for incurable TB. Histologic examination of different lung lesions revealed heterogeneous morphology and distribution of acid-fast bacilli; only at the surface of cavities, i.e., in granulomas with a patent connection to the airways, were there numerous bacilli. The mutation profile of the isolates suggested that a single founder strain of Mycobacterium tuberculosis may undergo genetic changes during treatment, leading to acquisition of additional drug resistance independently in discrete physical locales. Additional drug resistance was preferentially observed at the cavity surface. Cytokine gene expression revealed that failure to control the bacilli was not associated with a generalized suppression of cellular immunity, since cytokine mRNA was up regulated in all lesions tested. Rather, a selective absence of CD4 + and CD8 + T cells was noted at the luminal surface of the cavity, preventing direct T-cell-macrophage interactions at this site, probably allowing luminal phagocytes to remain permissive for bacillary growth. In contrast, in the perinecrotic zone of the granulomas, the two cell types colocalized and bacillary numbers were substantially lower, suggesting that in this microenvironment an efficient bacteriostatic or bactericidal phagocyte population was generated.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 1483247-1
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  • 6
    Online Resource
    Online Resource
    American Society for Microbiology ; 2006
    In:  Journal of Clinical Microbiology Vol. 44, No. 7 ( 2006-07), p. 2398-2403
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 44, No. 7 ( 2006-07), p. 2398-2403
    Abstract: Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene ( bca , bac , rib , alp1 , or alp3 ), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1498353-9
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  • 7
    Online Resource
    Online Resource
    Microbiology Society ; 2004
    In:  Journal of Medical Microbiology Vol. 53, No. 2 ( 2004-02-01), p. 107-113
    In: Journal of Medical Microbiology, Microbiology Society, Vol. 53, No. 2 ( 2004-02-01), p. 107-113
    Abstract: Thirty-seven multidrug-resistant and 13 pan-susceptible isolates of Mycobacterium tuberculosis were analysed for the diversity of genotypes associated with known drug-resistance mechanisms. The isolates were obtained from patients attending a university tuberculosis clinic in Monterrey, Mexico. A total of 25 IS 6110 -RFLP patterns were obtained from the multidrug-resistant tuberculosis (MDR-TB) isolates. Approximately 65 % of the MDR-TB isolates were attributed to secondary resistance. Different drug-susceptibility patterns were seen with the clustered isolates. The percentage of isolates resistant to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) was 100, 97.3, 48.7 and 67.6, respectively. The most common resistance-associated polymorphisms for the four drugs were as follows: INH, Ser315Thr (67.6 %) in katG ; RIF, Ser450Leu (41.7 %) in rpoB ; EMB, Met306Ile/Val/Leu (66.7 %) in embB ; and STR, Lys43Arg (24 %) in rpsL . Drug-resistance-associated mutations were similar to changes occurring in isolates from other areas of the world, but unique, previously unreported, mutations in katG ( n = 5), rpoB ( n = 1) and rrs ( n = 3) were also identified.
    Type of Medium: Online Resource
    ISSN: 0022-2615 , 1473-5644
    RVK:
    Language: English
    Publisher: Microbiology Society
    Publication Date: 2004
    detail.hit.zdb_id: 2083944-3
    SSG: 12
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  • 8
    Online Resource
    Online Resource
    Microbiology Society ; 2006
    In:  Journal of Medical Microbiology Vol. 55, No. 6 ( 2006-06-01), p. 775-783
    In: Journal of Medical Microbiology, Microbiology Society, Vol. 55, No. 6 ( 2006-06-01), p. 775-783
    Abstract: Group B Streptococcus (GBS) is an important pathogen responsible for a variety of diseases in newborns and the elderly. A clinical GBS isolate is considered nontypable (NT) when serological methods fail to identify it as one of nine known GBS serotypes. Eight clinical isolates (designated A1–A4, B1–B4) showed PFGE profiles similar to that of a GBS serotype V strain expressing R1, R4 surface proteins. These unique isolates were further characterized by immunologic and genetic methods. Rabbit sera to isolates A1 and A2 reacted weakly with concentrated HCl extracts of A1–A4 isolates, but not with those of B1–B4 isolates. In addition, a type V capsular polysaccharide (CPS) inhibition ELISA revealed that cell wall extracts from isolates A1–A4, but not from B1–B4, expressed low but measurable amounts of type V CPS. Molecular serotyping with PCR analysis showed that all eight isolates contained a type V-specific CPS gene ( cpsO ) and harboured the gene encoding the surface protein Alp3. Multilocus sequence typing identified isolate A1 as belonging to a new sequence type (ST) designated ST-173, whereas the other seven isolates keyed to ST-1. Sequencing of the 18 genes (17 736 bp) in the cps locus showed that each NT isolate harboured one to three unique polymorphisms, and also identified an IS 1381 element in cpsE of the B4 isolate. Collectively, genetic and immunologic analyses revealed that these NT isolates expressing R1, R4 proteins have a genetic profile consistent with that of type V, an emergent, antigenically diverse and increasingly prevalent GBS serotype.
    Type of Medium: Online Resource
    ISSN: 0022-2615 , 1473-5644
    RVK:
    Language: English
    Publisher: Microbiology Society
    Publication Date: 2006
    detail.hit.zdb_id: 2083944-3
    SSG: 12
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  • 9
    In: The Journal of Infectious Diseases, Oxford University Press (OUP), Vol. 190, No. 1 ( 2004-07), p. 99-106
    Type of Medium: Online Resource
    ISSN: 0022-1899 , 1537-6613
    URL: Issue
    RVK:
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2004
    detail.hit.zdb_id: 1473843-0
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  • 10
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 49, No. 6 ( 2005-06), p. 2210-2217
    Abstract: Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis . DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis .
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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