Jørn A. Aas

Defining the Normal Bacterial Flora of the Oral Cavity

ABSTRACT More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. Our purposes were (i) to utilize culture-independent molecular techniques to extend our knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and (ii) to determine the site and subject specificity of bacterial colonization. Nine sites from five clinically healthy subjects were analyzed. Sites included tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils. 16S rRNA genes from sample DNA were amplified, cloned, and transformed into Escherichia coli. Sequences of 16S rRNA genes were used to determine species identity or closest relatives. In 2,589 clones, 141 predominant species were detected, of which over 60% have not been cultivated. Thirteen new phylotypes were identified. Species common to all sites belonged to the genera Gemella, Granulicatella, Streptococcus, and Veillonella. While some species were subject specific and detected in most sites, other species were site specific. Most sites possessed 20 to 30 different predominant species, and the number of predominant species from all nine sites per individual ranged from 34 to 72. Species typically associated with periodontitis and caries were not detected. There is a distinctive predominant bacterial flora of the healthy oral cavity that is highly diverse and site and subject specific. It is important to fully define the human microflora of the healthy oral cavity before we can understand the role of bacteria in oral disease.

Bacteria of Dental Caries in Primary and Permanent Teeth in Children and Young Adults

ABSTRACT Although Streptococcus mutans has been implicated as a major etiological agent of dental caries, our cross-sectional preliminary study indicated that 10% of subjects with rampant caries in permanent teeth do not have detectable levels of S. mutans. Our aims were to use molecular methods to detect all bacterial species associated with caries in primary and permanent teeth and to determine the bacterial profiles associated with different disease states. Plaque was collected from 39 healthy controls and from intact enamel and white-spot lesions, dentin lesions, and deep-dentin lesions in each of 51 subjects with severe caries. 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identities. In a reverse-capture checkerboard assay, 243 samples were analyzed for 110 prevalent bacterial species. A sequencing analysis of 1,285 16S rRNA clones detected 197 bacterial species/phylotypes, of which 50% were not cultivable. Twenty-two new phylotypes were identified. PROC MIXED tests revealed health- and disease-associated species. In subjects with S. mutans, additional species, e.g., species of the genera Atopobium, Propionibacterium, and Lactobacillus, were present at significantly higher levels than those of S. mutans. Lactobacillus spp., Bifidobacterium dentium, and low-pH non-S. mutans streptococci were predominant in subjects with no detectable S. mutans. Actinomyces spp. and non-S. mutans streptococci were predominant in white-spot lesions, while known acid producers were found at their highest levels later in disease. Bacterial profiles change with disease states and differ between primary and secondary dentitions. Bacterial species other than S. mutans, e.g., species of the genera Veillonella, Lactobacillus, Bifidobacterium, and Propionibacterium, low-pH non-S. mutans streptococci, Actinomyces spp., and Atopobium spp., likely play important roles in caries progression.

Microbial Risk Indicators of Early Childhood Caries

ABSTRACT The aim of this study was to use molecular identification methods, such as 16S RNA gene sequence and reverse-capture checkerboard hybridization, for identification of the bacteria associated with dental caries and with dental health in a subset of 204 twins aged 1.5 to 7 years old. A total of 448 plaque samples (118 collected from caries-free subjects and 330 from caries-active subjects) were used for analysis. We compared the bacteria found in biofilms of children exhibiting severe dental caries, with different degrees of lesion severity, with those found in biofilms of caries-free children. A panel of 82 bacterial species was selected, and a PCR-based reverse-capture checkerboard method was used for detection. A simple univariate test was used to determine the overabundance and underabundance of bacterial species in the diseased and in the healthy groups. Features identified with this univariate test were used to construct a probabilistic disease prediction model. Furthermore, a method for the analysis of global patterns of gene expression was performed to permit simultaneous analysis of the abundance of significant species by allowing cross-bacterial comparisons of abundance profiles between caries-active and caries-free subjects. Our results suggested that global patterns of microbial abundance in this population are very distinctive. The top bacterial species found to be overabundant in the caries-active group were Actinomyces sp. strain B19SC, Streptococcus mutans, and Lactobacillus spp., which exhibited an inverse relationship to beneficial bacterial species, such as Streptococcus parasanguinis, Abiotrophia defectiva, Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis.

Bacterial Profiles of Root Caries in Elderly Patients

ABSTRACT Culture-based studies have shown that Streptococcus mutans and lactobacilli are associated with root caries (RC). The purpose of the present study was to assess the bacterial diversity of RC in elderly patients by use of culture-independent molecular techniques and to determine the associations of specific bacterial species or bacterial communities with healthy and carious roots. Plaque was collected from root surfaces of 10 control subjects with no RC and from 11 subjects with RC. The bacterial 16S rRNA genes from extracted DNA were PCR amplified, cloned, and sequenced to determine species identity. From a total of 3,544 clones, 245 predominant species or phylotypes were observed, representing eight bacterial phyla. The majority (54%) of the species detected have not yet been cultivated. Species of Selenomonas and Veillonella were common in all samples. The healthy microbiota included Fusobacterium nucleatum subsp. polymorphum, Leptotrichia spp., Selenomonas noxia, Streptococcus cristatus, and Kingella oralis. Lactobacilli were absent, S. mutans was present in one, and Actinomyces spp. were present in 50% of the controls. In contrast, the microbiota of the RC subjects was dominated by Actinomyces spp., lactobacilli, S. mutans, Enterococcus faecalis, Selenomonas sp. clone CS002, Atopobium and Olsenella spp., Prevotella multisaccharivorax, Pseudoramibacter alactolyticus, and Propionibacterium sp. strain FMA5. The bacterial profiles of RC showed considerable subject-to-subject variation, indicating that the microbial communities are more complex than previously presumed. The data suggest that putative etiological agents of RC include not only S. mutans, lactobacilli, and Actinomyces but also species of Atopobium, Olsenella, Pseudoramibacter, Propionibacterium, and Selenomonas.

Osteoradionecrosis contains a wide variety of cultivable and non-cultivable bacteria

Abstract Background: Direct microscopy, 1 1The chief editor, Ingar Olsen, has had no part in the review and decision process of this paper.anaerobic culture and DNA–DNA hybridization have previously demonstrated an association between microorganisms and osteoradionecrosis (ORN). The purpose of our study was to use culture independent molecular techniques to detect bacteria in necrotic bone lesions of the mandible after radiation therapy. Design: Bacterial DNA was extracted from eight deep medullar specimens from resected mandibles (six cases), including one patient with relapse. 16S rRNA genes were PCR amplified, cloned, transformed into Escherichia coli and sequenced to determine species identity and closest relatives. Results: From the analysis of 438 clones, 59 predominant species were detected, 27% of which have not been cultivated. The predominant species detected from radionecrotic mandibles were Campylobacter gracilis, Streptococcus intermedius, Peptostreptococcus sp. oral clone FG014, uncultured bacterium clone RL178, Fusobacterium nucleatum, and Prevotella spp. The study demonstrated intersubject variability of the bacteria present in ORN. In contrast to the diverse bacterial profile detected in primary infection, only a few members of the oral indigenous flora were identified from the relapse case. Conclusions: Diverse bacterial profiles in specimens of ORN in marrow spaces of the mandible were detected by culture independent molecular techniques. To better understand the pathogenesis and to improve the therapy of the infection, detection of all members of the complex bacterial flora associated with ORN is necessary.

Cultivated and not-yet-cultivated bacteria in oral biofilms.

This review gives an overview of the bacterial diversity of cultivated and not-yet-cultivated bacterial species in oral biofilms. Examples are given from the healthy oral cavity of youngsters, adults, and the elderly; caries in primary and permanent teeth; root caries in the elderly; subgingival plaque; aggressive periodontitis; chronic periodontitis; necrotizing ulcerative periodontitis; halitosis; noma; endodontic infections; and spreading odontogenic infections. Transfer of biofilm bacteria to the blood is also discussed. Techniques used for identifying these organisms are mainly PCR, cloning, and 16S rRNA gene sequencing, as well as microarrays. As much as 50% or more of the microbiota in oral biofilms cannot yet be cultured. This may have significant implications for our knowledge of the pathogens in major biofilm infections in the oral cavity such as caries, periodontitis, peri-implantitis, and mucositis. Furthermore, several bacterial species not traditionally believed to be oral pathogens have also been shown to be associated with disease.