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Identification and characterisation of bacteria based on 16S rDNA techniques with special reference to Helicobacter pylori in the gastro-intestinal tract
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The overall aim of this study was to establish molecular techniques for the detection and identification of "difficult to grow" - bacteria in mixed bacterial populations in clinical samples without the need for culturing procedures.

Material and Methods: Thirty-nine strains of Mobiluncus were used as a model system for phylogenetic classification of fastidious bacteria based on 16S ribosomal DNA (rDNA) sequences. To test the application of the 16S rDNA broad range PCR concept for detecting bacteria clinically, urine samples spiked with Chlamydia trachomatis elementary bodies and real urine test samples from 12 C. trachomatis positive and negative male volunteers were tested in a semiblind manner against routine procedures. Furthermore, gastric biopsy samples from 22 individuals (13 defined as having Helicobacter pylori-associated gastritis, and 9 defined as normal controls) were evaluated for the presence of Helicobacter 16S rDNA and the virulence genes cagA, vacA, and ureA. PCR products were also applied to temporal temperature gel electrophoresis (TTGE) gels for profiling the microbial flora. Finally, intestinal biopsies from 22 patients (11 diagnosed as Crohn's disease (CD), and 11 non-CD patients) were investigated using probes targeting potential pathogens that have been suggested to be involved in CD.

Results: We were able to confirm the current species designation of Mobiluncus, although the results did not support the division of M. curtisii into subspecies. For the detection of C. trachomatis in urine, the in-house system was shown to be as sensitive as a commercially available PCR system. The search for Helicobacter pylori in gastric biopsies revealed the presence of Helicobacter DNA in 20 of 22 individuals. The molecular techniques were apparently too sensitive compared with routinely used techniques. TTGE revealed a complex microbial flora both in the normal control group and in the gastritis group, with dominance of Helicobacter in the gastritis group. The present results might lend support to the hypothesis that Helicobacter are indigenous biota of the human stomach. cagA was amplified in all Helicobacter positive specimens. None of the specimens in the control group carried a H. pylori type strain identical vacA genotype. ureA negative mutants were also found in this group. The 16S rDNA sequence data might also indicate phylogenetic heterogeneity. The mixed bacterial flora found in CD inflammatory lesions is consistent with the idea that the enteric microflora enters primary lesions, where secondary invaders may elicit chronic inflannnatory response.

Conclusion: This thesis has demonstrated the usefulness of 16S rDNA based techniques for detection, identification, and characterisation of individual bacterial pathogens as well as for profiling of mixed bacterial flora in clinical specimens.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 112 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 622
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28646Local ID: 13802ISBN: 91-7219-579-7 (print)OAI: oai:DiVA.org:liu-28646DiVA: diva2:249457
Public defence
2000-04-28, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-01Bibliographically approved
List of papers
1. Broad-range PCR amplification and DNA sequence analysis reveals variable motifs in 16S rRNA genes of Mobiluncus species
Open this publication in new window or tab >>Broad-range PCR amplification and DNA sequence analysis reveals variable motifs in 16S rRNA genes of Mobiluncus species
1995 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 103, no 7-8, 755-763 p.Article in journal (Refereed) Published
Abstract [en]

Using DNA primers based on highly conserved regions of bacterial 16S ribosomal RNA genes, a technique was established for detection of Mobiluncus species by polymerase chain reaction (PCR) and hybridization analysis. Part of the 16S rRNA genes of Mobiluncus mulieris, Mobiluncus curtisii and uncharacterized Mobiluncus strains were analyzed by broad-range PCR amplification and direct DNA sequencing analysis. Sequence comparison of the partial 16S rRNA genes of Mobiluncus curtisii, Mobiluncus mulieris and atypical Mobiluncus strains studied indicated genus and species-specific motifs within the variable regions V3, V4 and V9 of 16S ribosomal DNAs. A Mobiluncus curtisii-specific primer, located within the variable region V3 of the 16S rRNA gene, was designed for Southern blot hybridization analysis of broad-range PCR products. Broad-range amplification combined with a M. curtisii-specific hybridization probe, Mob V3, distinguished between Mobiluncus curtisii, Mobiluncus mulieris, and atypical Mobiluncus strains.

Keyword
16S rRNA genes, PCR, DNA hybridization, bacterial vaginosis, Mobiluncus
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79458 (URN)10.1111/j.1699-0463.1995.tb01434.x (DOI)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
2. Classification of the genus Mobiluncus based on comparative partial 16S rRNA gene analysis
Open this publication in new window or tab >>Classification of the genus Mobiluncus based on comparative partial 16S rRNA gene analysis
1996 (English)In: International Journal of Systematic Bacteriology, ISSN 0020-7713, Vol. 46, no 1, 332-336 p.Article in journal (Refereed) Published
Abstract [en]

On the basis of partial 16S rRNA gene sequences and the results of Southern blot analyses, we confirmed the division of the genus Mobiluncus into the species Mobiluncus curtisii and Mobiluncus mulieris. Division of M. curtisii into M. curtisii subsp. curtisii and M. curtisii subsp. holmesii was not supported by our data.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79459 (URN)10.1099/00207713-46-1-332 (DOI)8573515 (PubMedID)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
3. Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes
Open this publication in new window or tab >>Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes
1996 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 104, no 1-6, 451-458 p.Article in journal (Refereed) Published
Abstract [en]

Broad range PCR amplification and genus-specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1–10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.

Keyword
16S rRNA genes, diagnostic PCR, Chlamydia trachomatis, Helicobacter pylori, Mobiluncus curtisii
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79460 (URN)10.1111/j.1699-0463.1996.tb00741.x (DOI)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
4. Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?
Open this publication in new window or tab >>Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?
Show others...
1998 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 47, no 8, 695-704 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79462 (URN)10.1099/00222615-47-8-695 (DOI)9877190 (PubMedID)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
5. 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
Open this publication in new window or tab >>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
Show others...
2000 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, Vol. 49, no 9, 817-822 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24957 (URN)10966230 (PubMedID)9368 (Local ID)9368 (Archive number)9368 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-08-01Bibliographically approved
6. Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR
Open this publication in new window or tab >>Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR
Show others...
1999 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, Vol. 48, no 3, 263-268 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25305 (URN)10.1099/00222615-48-3-263 (DOI)10334593 (PubMedID)9746 (Local ID)9746 (Archive number)9746 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-08-01

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