Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques

Background

Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.

Methodology/Principal Findings

Supragingival plaque samples from caries-free children incubated with ¹³C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by Lactobacillus and Propionibacterium species, both of which have been previously found within carious lesions from children.

Conclusions/Significance

Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.     

In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.     

Early Canine Plaque Biofilms: Characterization of Key Bacterial Interactions Involved in Initial Colonization of Enamel

Periodontal disease (PD) is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops.     

A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva,Gingivitis or Mild Periodontitis

Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.     

Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity

Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva.     

Oral microflora in betel-chewing adults of the Karen tribe in Thailand

ObjectiveTo study a possible influence of betel chewing on the composition of the oral microflora in plaque and saliva and on oral health parameters as well as a possible betel effect on oral bacteria in vitro.Material and methodsThirty-two adults (16 betel chewers and 16 non-betel-chewing controls) of the Karen Hill tribe in Thailand were investigated. Saliva samples and 2 pooled supragingival plaque samples were taken from each individual for microbial analysis with culture and 4 subgingival samples for analysis with the DNA–DNA hybridization method against 12 periodontitis associated bacterial species. Caries (DMFS), plaque (PlI%) and bleeding on probing (% BoP) was registered as well as number of sites with >5 mm probing pocket depth (PPD). Water extract of the betel (areca chatechu) nut was tested for its antimicrobial effect in vitro against 10 oral bacterial species with the agar diffusion method.ResultsAn antimicrobial effect of betel nut water extract was found on the oral microorganisms in vitro. The levels of mutans streptococci and lactobacilli in saliva were low or absent in both chewers and controls. The prevalence of the periodontitis associated bacteria was >90%. Betel chewers had significantly lower levels of some bacteria in subgingival plaque (Prevotella intermedia p < 0.001) than non-chewers. This study population was low in missing teeth (mean 0.7 and 0.3), caries decay (DS 2.1 vs 1.6), and number of deep pockets (mean 1.9 vs 1.3). Great variation in oral hygiene (PlI and BoP) between the subjects was seen.ConclusionsAn antimicrobial effect of the betel nut was found in vitro and with a possible effect also in vivo however it did not seem to influence clinical parameters such as plaque index, caries prevalence (DMFS), bleeding on probing and number of deep pockets.     

Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples

The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H₂O₂) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H₂O₂ production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

L-Arginine Destabilizes Oral Multi-Species Biofilm Communities Developed in Human Saliva

The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37^oC. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm³/μm²) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi-species oral biofilm development and community composition and enhances the activity of CPC. The incorporation of LAHCl into oral healthcare products may be useful for enhanced biofilm control.     

Mechanisms underlying interactions between two abundant oral commensal bacteria

Complex polymicrobial biofilm communities are abundant in nature particularly in the human oral cavity where their composition and fitness can affect health. While the study of these communities during disease is essential and prevalent, little is known about interactions within the healthy plaque community. Here we describe interactions between two of the most abundant species in this healthy microbiome, Haemophilus parainfluenzae and Streptococcus mitis. We discovered that H. parainfluenzae typically exists adjacent to mitis group streptococci in vivo with which it is also positively correlated based on microbiome data. By comparing in vitro coculture data to ex vivo microscopy we revealed that this co-occurrence is density dependent and further influenced by H₂O₂ production. We discovered that H. parainfluenzae utilizes a more redundant, multifactorial response to H₂O₂ than related microorganisms and that this system’s integrity enhances streptococcal fitness. Our results indicate that mitis group streptococci are likely the in vivo source of NAD for H. parainfluenzae and also evoke patterns of carbon utilization in vitro for H. parainfluenzae similar to those observed in vivo. Our findings describe mechanistic interactions between two of the most abundant and prevalent members of healthy supragingival plaque that contribute to their in vivo survival.Subject terms: Microbiome, Bacterial physiology, Bacteriology, Biofilms, Microbial ecology