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| 2. |
- Grahn, Niclas, et al.
(författare)
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Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons
- 2003
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Ingår i: FEMS Microbiology Letters. - 0378-1097 .- 1574-6968. ; 219:1, s. 87-91
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Tidskriftsartikel (refereegranskat)abstract
- Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, we have found contaminating bacterial DNA in our reagents used for PCR. Identified bacteria are the water-borne bacterial genera Pseudomonas, Stenotrophomonas, Xanthomonas, Ralstonia and Bacillus. Our results are in concordance with recent reports of contaminated industrial water systems. In light of this conclusion, we believe that there is a need for increased awareness of possible contamination in uncertified widely used molecular biology reagents, including ultra-pure water. Since sequence-based 16S rDNA techniques are used in a variety of settings for bacterial typing and the characterization of microbial communities, we feel that future certification of molecular biology reagents, as free of nucleic acids, would be advantageous. ⌐ 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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| 3. |
- Jonasson, Jon, 1942-, et al.
(författare)
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Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments
- 2002
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 110:3, s. 263-272
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Tidskriftsartikel (refereegranskat)abstract
- The rapid identification of the etiological agent of microbial infections can bring about both clinical and financial benefits. Thus, fast and generally applicable classification methods are needed that will enable us to rapidly distinguish pathogenic bacteria from commensals or saprophytic bacteria found in the same habitat. We here show that provisional classification of bacterial isolates can be performed on a large scale based on 16S rRNA sequence comparisons using PyrosequencingÖ, a recently described real-time DNA sequence analysis technique, and the concept of signature matching. The probes we have developed, together with the new technology, will enable early diagnosis of specific pathogens, which is critical for the rational use of antimicrobial therapy in clinical medicine.
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| 4. |
- Karpati, F, et al.
(författare)
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Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity
- 2001
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - 0903-4641 .- 1600-0463. ; 109:5, s. 389-400
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Tidskriftsartikel (refereegranskat)abstract
- To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.
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| 5. |
- Kissopoulou, Antheia, et al.
(författare)
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Monozygotic twins with myocarditis and a novel likely pathogenic desmoplakin gene variant
- 2020
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Ingår i: ESC Heart Failure. - : Wiley. - 2055-5822. ; 7:3, s. 1210-1216
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Tidskriftsartikel (refereegranskat)abstract
- Myocarditis most often affects otherwise healthy athletes and is one of the leading causes of sudden death in children and young adults. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically determined heart muscle disorder with increased risk for paroxysmal ventricular arrhythmias and sudden cardiac death. The clinical picture of myocarditis and ARVC may overlap during the early stages of cardiomyopathy, which may lead to misdiagnosis. In the literature, we found several cases that presented with episodes of myocarditis and ended up with a diagnosis of arrhythmogenic cardiomyopathy, mostly of the left predominant type. The aim of this case presentation is to shed light upon a possible link between myocarditis, a desmoplakin (DSP) gene variant, and ARVC by describing a case of male monozygotic twins who presented with symptoms and signs of myocarditis at 17 and 18 years of age, respectively. One of them also had a recurrent episode of myocarditis. The twins and their family were extensively examined including electrocardiograms (ECG), biochemistry, multimodal cardiac imaging, myocardial biopsy, genetic analysis, repeated cardiac magnetic resonance (CMR) and echocardiography over time. Both twins presented with chest pain, ECG with slight ST-T elevation, and increased troponin T levels. CMR demonstrated an affected left ventricle with comprehensive inflammatory, subepicardial changes consistent with myocarditis. The right ventricle did not appear to have any abnormalities. Genotype analysis revealed a nonsense heterozygous variant in the desmoplakin (DSP) gene [NM_004415.2:c.2521_2522del (p.Gln841Aspfs*9)] that is considered likely pathogenic and presumably ARVC related. There was no previous family history of heart disease. There might be a common pathophysiology of ARVC, associated with desmosomal dysfunction, and myocarditis. In our case, both twins have an affected left ventricle without any right ventricular involvement, and they are carriers of a novel DSP variant that is likely associated with ARVC. The extensive inflammation of the LV that was apparent in the CMR may or may not be the primary event of ARVC. Nevertheless, our data suggest that irrespective of a possible link here to ARVC, genetic testing for arrhythmogenic cardiomyopathy might be advisable for patients with recurrent myocarditis associated with a family history of myocarditis.
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| 6. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing
- 2000
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - 0903-4641 .- 1600-0463. ; 108:1, s. 67-73
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Tidskriftsartikel (refereegranskat)abstract
- A multiplex PCR assay for the detection of vancomycin resistance (van) genes in enterococci was established. Primers targeting the 16S rRNA gene were included in the reaction mixture. Multiple-primer DNA sequencing of the PCR products provided species identification through partial nucleotide sequences of 16S rRNA genes, as well as confirmation of the correct identification of vanA, vanB, vanC-1, and vanC-2/3 genotypes. Thirty-nine enterococcal clinical isolates and type strains were examined for the presence of vancomycin resistance determinants. Twelve other isolates from a clinical reference collection (some of them having vanA, vanB, vanC-1, or vanC-2/3 genotypes) were used as controls. Hybridization and partial DNA sequence analysis of multiplex PCR products revealed that none of the clinical isolates had a vanA genotype and only one had a vanB genotype, vanC- 1 was found in three clinical isolates, and vanC-2/3 in one. Results obtained with the reference and type strains were in agreement with earlier results.
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| 7. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Differential virulence-gene mRNA expression in coccoid forms of Helicobacter pylori
- 2001
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 285:2, s. 530-536
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Tidskriftsartikel (refereegranskat)abstract
- Controversy exists whether coccoid forms of Helicobacter pylori maintain transcriptional and translational processes. The aim of the present study was to investigate mRNA levels in coccoid H. pylori and, if possible, to establish a correlation with the state of nonrandom fragmentation of rRNA in those cells. For that purpose, UreA, UreI, CagA, VacA, SodB, and Hsp60 mRNA levels in bacillary and coccoid forms of H. pylori CCUG 17874T, H. pylori 26695, and H. pylori J99, respectively, were studied by means of a multiplex reverse-transcription PCR assay and Southern blot analysis of the RT-PCR-amplified products. Nonrandom fragmentation of 23S rRNA was assessed by a recently described assay. Virulence-gene-derived mRNA transcripts were visualized in DNase I-treated RNA preparations. All three strains revealed the presence of different mRNA patterns in bacillary and coccoid forms. Putative promoter sequences similar to the consensus Escherichia coli -10 hexamer TATAAA box were present in all six virulence genes analyzed. Moreover, the decrease seen in mRNA levels during conversion into the coccoid form appeared to correlate with the 23S rRNA nonrandom fragmentation pattern. The present data indicate that modulation of virulence-gene expression is differently regulated in bacillary and coccoid H. pylori.
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| 8. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions
- 2001
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - 0903-4641 .- 1600-0463. ; 109:3, s. 209-216
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Tidskriftsartikel (refereegranskat)abstract
- Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.
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| 9. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Probing 23S ribosomal RNA cleavage sites in coccoid Helicobater pylori.
- 2001
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Ingår i: Helicobacter. - : Wiley. - 1083-4389 .- 1523-5378. ; 6:2, s. 100-109
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Tidskriftsartikel (refereegranskat)abstract
- Background. Previous studies have revealed that extensive nonrandom fragmentation of ribosomal RNA occurs during conversion of Helicobacter pylori to the coccoid form. The 16S rRNA fragmentation has been characterised in some detail. The aim of the present study was to define corresponding cleavage-sites in the 3'-half of the 23S rRNA molecule. Materials and Methods. Northern blot analysis using 23S rRNA specific antisense riboprobes and a 5'-end- labelled oligonucleotide probe was used to analyse the 23S rRNA fragmentation pattern in coccoid H. pylori type strain CCUG 17874T and H. pylori 26695, for which the genome has been sequenced. A double- stranded cDNA-dependent (ds-cDNA) primer- extension analysis technique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the peptidyl-transferase centre was used to determine cleavage sites at the nucleotide level. Results. We report here the mapping of putative cleavage sites within domains IV and V, enclosing the peptidyl transferase centre, in the 3'-half of the 23S rRNA molecule. Three cleavage sites were located in domain IV. Two other cleavage sites were located in the peptidyl transferase centre, and one presumptive multiple-break site between helices 77 and 78 in domain V. The DNA motifs were different from the postulated A + U rich single-strand cleavage sites recognised by RNase E, which has been implicated in rRNA degradation in Escherichia coli. Conclusions. The present analysis suggests that a hitherto unknown mechanism is responsible for the nonrandom fragmentation of rRNA in coccoid H. pylori, which may have important consequences for the growth, and survival of the bacterium.
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| 10. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis
- 2000
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Ingår i: Journal of Medical Microbiology. - 0022-2615 .- 1473-5644. ; 49:9, s. 817-822
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.
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