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Appl. Environ. Microbiol. doi:10.1128/AEM.01419-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Molecular Analysis of the Subgingival Microbiota in Health and Disease

School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, U.K, Plant, Animal and Microbial Science Dept. IGER (Institute of Grassland and Environmental Research), Plas Gogerddan, Aberystwyth, SY23 3EB, UK; School of Dentistry, University of Manchester, Higher Cambridge Street, Manchester M15 6FH

^* To whom correspondence should be addressed. Email: andrew.mcbain{at}manchester.ac.uk.

	Abstract

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterised by PCR-DGGE using primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analysed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis using multiplex PCR. Associations of patient age, gender and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed using logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 - 40 distinct DGGE bands) with substantial individual variation in community profile. Species diversity in health and disease was determined using the Shannon-Weaver index of diversity, and compared using the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many non-typical oral species, and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR however, detected T. forsythensis, A. actinomycetemcomitans and P. gingivalis in 9% 16%, 29% and of patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P<0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. were significant predictors of disease (P<0.05) and that there was no significant difference (P>0.05) in terms of eubacterial species diversity between health and disease.

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