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Molecular analysis of the oral microbiota of dental diseases
Umeå University, Faculty of Medicine, Odontology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.

Place, publisher, year, edition, pages
Umeå: Odontologi , 2008. , 61 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 105
Keyword [en]
early childhood caries, early periodontitis, 16S rDNA cloning and sequencing, whole genomic DNA probes, oligonucleotide DNA probes, PCR, diversity, molecular, aggregatibacter actinomycetemcomitans, serotypes
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-1911ISBN: 978-91-7264-657-5 (print)OAI: oai:DiVA.org:umu-1911DiVA: diva2:142399
Public defence
2008-11-28, 933, 1A, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-11-10 Created: 2008-11-10 Last updated: 2009-05-04Bibliographically approved
List of papers
1. Microbial Risk Markers for Childhood Caries in Pediatricians’ Offices
Open this publication in new window or tab >>Microbial Risk Markers for Childhood Caries in Pediatricians’ Offices
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2010 (English)In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 89, no 4, 378-383 p.Article in journal (Refereed) Published
Abstract [en]

Dental caries in pre-school children has significant public health and health disparity implications. To determine microbial risk markers for this infection, this study aimed to compare the microbiota of children with early childhood caries with that of caries-free children. Plaque samples from incisors, molars, and the tongue from 195 children attending pediatricians’ offices were assayed by 74 DNA probes and by PCR to Streptococcus mutans. Caries-associated factors included visible plaque, child age, race, and snacking habits. Species were detected more frequently from tooth than tongue samples. Lactobacillus gasseri (p < 0.01), Lactobacillus fermentum, Lactobacillus vaginalis, and S. mutans with Streptococcus sobrinus (all p < 0.05) were positively associated with caries. By multifactorial analysis, the probiotic Lactobacillus acidophilus was negatively associated with caries. Prevotella nigrescens was the only species (p < 0.05) significantly associated with caries by the ‘false discovery’ rate. Analysis of the data suggests that selected Lactobacillus species, in addition to mutans streptococci, are risk markers for early childhood caries.

Place, publisher, year, edition, pages
Sage Publications, 2010
Keyword
early childhood caries, S. mutans, Lactobacillus
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-3611 (URN)10.1177/0022034509360010 (DOI)000275566800010 ()
Note
Working title: Oral Microbiota of Pre-School Children in Pediatrician’s OfficesAvailable from: 2008-11-10 Created: 2008-11-10 Last updated: 2017-12-14Bibliographically approved
2. Molecular analysis of the oral microbiota of severe early childhood caries
Open this publication in new window or tab >>Molecular analysis of the oral microbiota of severe early childhood caries
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3612 (URN)
Available from: 2008-11-10 Created: 2008-11-10 Last updated: 2010-01-13Bibliographically approved
3. Subgingival and tongue microbiota during early periodontitis.
Open this publication in new window or tab >>Subgingival and tongue microbiota during early periodontitis.
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2006 (English)In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 85, no 4, 318-323 p.Article in journal (Refereed) Published
Abstract [en]

Periodontal infections have a microbial etiology. Association of species with early disease would be useful in determining which microbes initiate periodontitis. We hypothesized that the microbiota of subgingival and tongue samples would differ between early periodontitis and health. A cross-sectional evaluation of 141 healthy and early periodontitis adults was performed with the use of oligonucleotide probes and PCR. Most species differed in associations with sample sites; most subgingival species were associated with subgingival samples. Few species were detected more frequently in early periodontitis by DNA probes. Porphyromonas gingivalis and Tannerella forsythia (Tannerella forsythensis) were associated with early periodontitis by direct PCR. In conclusion, the microbiota of tongue samples was less sensitive than that of subgingival samples in detecting periodontal species, and there was overlap in species detected in health and early periodontitis. Detection of periodontal pathogens in early periodontitis suggests an etiology similar to that of more advanced disease.

Identifiers
urn:nbn:se:umu:diva-22257 (URN)10.1177/154405910608500407 (DOI)16567551 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13
4. Clinical characteristics and microbiota of progressing slight chronic periodontitis in adults.
Open this publication in new window or tab >>Clinical characteristics and microbiota of progressing slight chronic periodontitis in adults.
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2007 (English)In: Journal of Clinical Periodontology, ISSN 0303-6979, E-ISSN 1600-051X, Vol. 34, no 11, 917-930 p.Article in journal (Refereed) Published
Abstract [en]

AIM: This study sought clinical and microbial risk indicators for progressing slight periodontitis. MATERIAL AND METHODS: One hundred and seventeen periodontally healthy or slight periodontitis adults (20-40 years) were monitored clinically at 6-month intervals followed by supragingival cleaning. Inter-proximal sites with >1.5 mm increase in clinical attachment over 18 months were considered disease active. Subgingival plaque was analysed by 78 16S rDNA and 38 whole-genomic DNA probes and by PCR to Porphyromonas gingivalis and Tannerella forsythia. Characteristics were compared between active and inactive subjects. RESULTS: Twenty-two subjects showed disease activity principally at molars. Mean baseline gingival and plaque indices, bleeding on probing, probing depth and clinical attachment level (CAL) were higher in active subjects. DNA probes detected species and not-yet-cultivated phylotypes from chronic periodontitis, although few species were associated with active subjects. By PCR P. gingivalis (p=0.007) and T. forsythia (p=0.075) were detected more frequently during monitoring in active subjects. Stepwise logistic analysis associated baseline levels of gingival index, clinical attachment and bleeding with subsequent clinical attachment loss. CONCLUSIONS: Gingivitis and CAL were significantly associated with progressing slight periodontitis in 20--40-year-old adults. Species associated with moderate and advanced chronic periodontitis were detected in slight periodontitis.

Identifiers
urn:nbn:se:umu:diva-22259 (URN)10.1111/j.1600-051X.2007.01126.x (DOI)17877747 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13
5. Lack of serotype antigen expression in aggregatibacter actinomycetemcomitans
Open this publication in new window or tab >>Lack of serotype antigen expression in aggregatibacter actinomycetemcomitans
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3615 (URN)
Available from: 2008-11-10 Created: 2008-11-10 Last updated: 2010-01-13Bibliographically approved

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