Local delivery of nitric oxide prevents endothelial dysfunction in periodontitis.

AIMS: Increased cardiovascular disease risk underlies elevated rates of mortality in individuals with periodontitis. A key characteristic of those with increased cardiovascular risk is endothelial dysfunction, a phenomenon synonymous with deficiencies of bioavailable nitric oxide (NO), and prominently expressed in patients with periodontitis. Also, inorganic nitrate can be reduced to NO in vivo to restore NO levels, leading us to hypothesise that its use may be beneficial in reducing periodontitis-associated endothelial dysfunction. Herein we sought to determine whether inorganic nitrate improves endothelial function in the setting of periodontitis and if so to determine the mechanisms underpinning any responses seen. METHODS AND RESULTS: Periodontitis was induced in mice by placement of a ligature for 14 days around the second molar. Treatment in vivo with potassium nitrate, either prior to or following establishment of experimental periodontitis, attenuated endothelial dysfunction, as determined by assessment of acetylcholine-induced relaxation of aortic rings, compared to control (potassium chloride treatment). These beneficial effects were associated with a suppression of vascular wall inflammatory pathways (assessed by quantitative-PCR), increases in the anti-inflammatory cytokine interleukin (IL)-10 and reduced tissue oxidative stress due to attenuation of xanthine oxidoreductase-dependent superoxide generation. In patients with periodontitis, plasma nitrite levels were not associated with endothelial function indicating dysfunction. CONCLUSION: Our results suggest that inorganic nitrate protects against, and can partially reverse pre-existing, periodontitis-induced endothelial dysfunction through restoration of nitrite and thus NO levels. This research highlights the potential of dietary nitrate as adjunct therapy to target the associated negative cardiovascular outcomes in patients with periodontitis.

Resilience of the oral microbiome.

The human mouth harbors a complex microbiota, the composition of which is potentially influenced by a wide range of factors, including the intake of food and drink, the availability of endogenous nutrients, the host immune system, drug treatments, and systemic diseases. Despite these possible influences, the oral microbiota is remarkably resilient, particularly in comparison with the microbiota of the large intestine. Diet, with the exception of excessive and/or frequent consumption of fermentable carbohydrate or supplementation with nitrate, has minimal impact on the composition of the oral bacterial community. The common oral diseases dental caries and the periodontal diseases is associated with modification of the oral microbiota primarily as a result of the ecological changes induced by excessive acid production and inflammation, respectively. Systemically-administered antimicrobials have only a small effect on the composition of the oral bacterial community, and while locally delivered antimicrobials can have some clinical benefits, the biofilm lifestyle of oral bacteria lends them substantial resistance to the agents used. Saliva plays an important role in oral microbial ecology, by supplying nutrients and providing protection against colonization by nonoral organisms. Dry mouth is one condition that has a major effect on the microbiota, resulting in increased colonization by opportunistic pathogens. Some systemic diseases do affect the oral microbiome, notably diabetes, in which raised levels of glucose in saliva and tissue impact on bacterial nutrition.

Consumer Safety Considerations of Skin and Oral Microbiome Perturbation.

Microbiomes associated with human skin and the oral cavity are uniquely exposed to personal care regimes. Changes in the composition and activities of the microbial communities in these environments can be utilized to promote consumer health benefits, for example, by reducing the numbers, composition, or activities of microbes implicated in conditions such as acne, axillary odor, dandruff, and oral diseases. It is, however, important to ensure that innovative approaches for microbiome manipulation do not unsafely disrupt the microbiome or compromise health, and where major changes in the composition or activities of the microbiome may occur, these require evaluation to ensure that critical biological functions are unaffected. This article is based on a 2-day workshop held at SEAC Unilever, Sharnbrook, United Kingdom, involving 31 specialists in microbial risk assessment, skin and oral microbiome research, microbial ecology, bioinformatics, mathematical modeling, and immunology. The first day focused on understanding the potential implications of skin and oral microbiome perturbation, while approaches to characterize those perturbations were discussed during the second day. This article discusses the factors that the panel recommends be considered for personal care products that target the microbiomes of the skin and the oral cavity.

Tannerella serpentiformis sp. nov., isolated from the human mouth.

Three strains representing the previously uncultured human oral Tannerella taxon HMT-286 were recently isolated from the subgingival plaque of a patient with chronic periodontitis. The phenotypic and genetic features of strain SP18_26T were compared to those of the type species of Tannerella, Tannerella forsythia. A genome size of 2.97 Mbp (G+C content 56.5 mol%) was previously reported for SP18_26T, compared to a size of 3.28 Mbp (47.1 mol%) in T. forsythia ATCC 43037T. 16S rRNA gene sequence comparisons also revealed 94.3 % sequence identity with T. forsythia ATCC 43037T. Growth was stimulated by supplementation of media with N-acetyl muramic acid, as seen with T. forsythia, but the cells displayed a distinctive snake-like morphology. Fatty acid methyl ester analysis revealed a profile differing from T. forsythia, chiefly in the amount of 3-OH-16 : 0 (four-fold lower in SP18_26T). Overall, metabolic enzyme activity also differed from T. forsythia, with enzyme activity for indole present, but the complement of glycoside hydrolase enzyme activity was smaller than T. forsythia, for example, lacking sialidase and N-acetyl-ß-glucosaminidase - evidence backed up by analysis of its gene content. On the basis of these results, a new species Tannerella serpentiformis is proposed for which the type strain is SP18_26T (=DSM 102894T=JCM 31303T).

The Effect of Influenza Virus on the Human Oropharyngeal Microbiome.

BACKGROUND: Secondary bacterial infections are an important cause of morbidity and mortality associated with influenza infections. As bacterial disease can be caused by a disturbance of the host microbiome, we examined the impact of influenza on the upper respiratory tract microbiome in a human challenge study. METHODS: The dynamics and ecology of the throat microbiome were examined following an experimental influenza challenge of 52 previously-healthy adult volunteers with influenza A/Wisconsin/67/2005 (H3N2) by intranasal inoculation; 35 healthy control subjects were not subjected to the viral challenge. Serial oropharyngeal samples were taken over a 30-day period, and the V1-V3 region of the bacterial 16S ribosomal RNA sequences were amplified and sequenced to determine the composition of the microbiome. The carriage of pathogens was also detected. RESULTS: Of the 52 challenged individuals, 43 developed proven influenza infections, 33 of whom became symptomatic. None of the controls developed influenza, although 22% reported symptoms. The diversity of bacterial communities remained remarkably stable following the acquisition of influenza, with no significant differences over time between individuals with influenza and those in the control group. Influenza infection was not associated with perturbation of the microbiome at the level of phylum or genus. There was no change in colonization rates with Streptococcus pneumoniae or Neisseria meningitidis. CONCLUSIONS: The throat microbiota is resilient to influenza infection, indicating the robustness of the upper-airway microbiome.

World Workshop on Oral Medicine VII: Targeting the oral microbiome Part 2: Current knowledge on malignant and potentially malignant oral disorders.

OBJECTIVE: The World Workshop on Oral Medicine VII chose the oral microbiome as a focus area. Part 1 presents the methodological state of the science for oral microbiome studies. Part 2 was guided by the question: What is currently known about the microbiome associated with oral squamous cell carcinoma and potentially malignant disorders of the oral mucosa? MATERIALS AND METHODS: A scoping review methodology was followed to identify and analyse relevant studies on the composition and potential functions of the oral microbiota using high-throughput sequencing techniques. The authors performed searches in PubMed and EMBASE. After removal of duplicates, a total of 239 potentially studies were identified. RESULTS: Twenty-three studies on oral squamous cell carcinoma, two on oral leukoplakia and four on oral lichen planus were included with substantial differences in diagnostic criteria, sample type, region sequenced and sequencing method utilised. The majority of studies focused on bacterial identification and recorded statistically significant differences in the oral microbiota associated with health and disease. However, even when comparing studies of similar methodology, the microbial differences between health and disease varied considerably. No consensus on the composition of the microbiomes associated with these conditions on genus and species level could be obtained. Six studies on oral squamous cell carcinoma had included in silico predicted microbial functions (genes and/or pathways) and found some similarities between the studies. CONCLUSIONS: Attempts to reveal the microbiome associated with oral mucosal diseases are still in its infancy, and the studies demonstrate significant clinical and methodological heterogeneity across disease categories. The immense richness and diversity of the microbiota clearly illustrate that there is a need for additional methodologically comparable studies utilising deep sequencing approaches in significant cohorts of subjects together with functional analyses. Our hope is that following the recipe as outlined in our preceding companion paper, that is Part 1, will enhance achieving this in the future and elucidate the role of the oral microbiome in oral squamous cell carcinoma and potentially malignant disorders of the oral mucosa.

World Workshop on Oral Medicine VII: Targeting the microbiome for oral medicine specialists-Part 1. A methodological guide.

Advances in high-throughput sequencing technologies have allowed for a rapid increase in knowledge about the human microbiome in both healthy and diseased states, which is expected to increase our understanding of multifactorial diseases. The World Workshop on Oral Medicine VII chose the microbiome as one of its topics of focus. Part 1 of this review provides updated knowledge in the field of microbiome research, describes the advantages and disadvantages of currently available sequencing technologies, and proposes a seven-step "recipe" for designing and performing studies that is supported by contemporary evidence. Part 2 of this review in a companion paper discusses the results of high-throughput sequencing studies published to date on the microbiota associated with oral mucosal diseases. The goal of this collective enterprise is to encourage more oral medicine specialists to become engaged in multidisciplinary collaborations to investigate the role of the microbiome in relation to oral diseases, which could potentially lead to enhanced diagnosis, risk assessment and treatment of these patients.

Actinomyces and related organisms in human infections.

Actinomyces israelii has long been recognized as a causative agent of actinomycosis. During the past 3 decades, a large number of novel Actinomyces species have been described. Their detection and identification in clinical microbiology laboratories and recognition as pathogens in clinical settings can be challenging. With the introduction of advanced molecular methods, knowledge about their clinical relevance is gradually increasing, and the spectrum of diseases associated with Actinomyces and Actinomyces-like organisms is widening accordingly; for example, Actinomyces meyeri, Actinomyces neuii, and Actinomyces turicensis as well as Actinotignum (formerly Actinobaculum) schaalii are emerging as important causes of specific infections at various body sites. In the present review, we have gathered this information to provide a comprehensive and microbiologically consistent overview of the significance of Actinomyces and some closely related taxa in human infections.

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.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BACKGROUND: Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. RESULTS: Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. CONCLUSIONS: Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.

The oral microbiome in health and disease.

The human mouth harbours one of the most diverse microbiomes in the human body, including viruses, fungi, protozoa, archaea and bacteria. The bacteria are responsible for the two commonest bacterial diseases of man: dental caries (tooth decay) and the periodontal (gum) diseases. Archaea are restricted to a small number of species of methanogens while around 1000 bacterial species have been found, with representatives from the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, Synergistetes and Tenericutes and the uncultured divisions GN02, SR1 and TM7. Around half of oral bacteria are as yet uncultured and culture-independent methods have been successfully used to comprehensively describe the oral bacterial community. The human oral microbiome database (HOMD, www.homd.org) provides a comprehensive resource consisting of descriptions of oral bacterial taxa, a 16S rRNA identification tool and a repository of oral bacterial genome sequences. Individuals' oral microbiomes are highly specific at the species level, although overall the human oral microbiome shows few geographical differences. Although caries and periodontitis are clearly bacterial diseases, they are not infectious diseases in the classical sense because they result from a complex interaction between the commensal microbiota, host susceptibility and environmental factors such as diet and smoking. Periodontitis, in particular, appears to result from an inappropriate inflammatory reaction to the normal microbiota, exacerbated by the presence of some disease-associated bacterial species. In functional terms, there appears to considerable redundancy among the oral microbiota and a focus on functional rather than phylogenetic diversity may be required in order to fully understand host-microbiome interactions.

Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity.

SGP1(T), a strain belonging to a lineage of the phylum Synergistetes with no previously cultivated representatives was subjected to a comprehensive range of phenotypic and genotypic tests. For good growth the strain was dependent on co-culture with, or extracts from, selected other oral bacteria. Cells of strain SGP1(T) were asaccharolytic and major amounts of acetic acid and moderate amounts of propionic acid were produced as end products of metabolism in peptone-yeast extract-glucose broth supplemented with a filtered cell sonicate of Fusobacterium nucleatum subsp. nucleatum ATCC 25586(T) (25 %, v/v). Hydrogen sulphide was produced and gelatin was weakly hydrolysed. The major cellular fatty acids were C(14 : 0), C(18 : 0) and C(16 : 0). The DNA G+C content of strain SGP1(T) was 63 mol%. Phylogenetic analysis of the full-length 16S rRNA gene showed that strain SGP1(T) represented a novel group within the phylum Synergistetes. A novel species in a new genus, Fretibacterium fastidiosum gen. nov., sp. nov., is proposed. The type strain of Fretibacterium fastidiosum is SGP1(T) ( = DSM 25557(T) = JCM 16858(T)).

Description of Alloprevotella rava gen. nov., sp. nov., isolated from the human oral cavity, and reclassification of Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov.

Five strains of anaerobic, gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that these strains represented a novel group within the family Prevotellaceae, and the most closely related species was Prevotella tannerae. P. tannerae and the novel taxon are deeply branched from the genus Prevotella, with sequence identities to the type strain of the type species of Prevotella, Prevotella melaninogenica, of 82.2 and 85.6 %, respectively. The novel genus Alloprevotella gen. nov. is proposed to accommodate the novel species Alloprevotella rava gen. nov., sp. nov. and the previously named Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov. The type species is Alloprevotella tannerae. The type strain of Alloprevotella rava is 81/4-12(T) ( = DSM 22548(T)  = CCUG 58091(T)) and the type strain of Alloprevotella tannerae is ATCC 51259(T)  = CCUG 34292(T)  = CIP 104476(T)  = NCTC 13073(T). Alloprevotella rava is weakly to moderately saccharolytic and produces moderate amounts of acetic acid and major amounts of succinic acid as end products of fermentation. Strains are sensitive to 20 % bile and hydrolyse gelatin. The principal cellular long-chain fatty acids are anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C17 : 0 and iso-C17 : 0 3-OH. The G+C content of the DNA of the type strain is 47 mol%.

Design and evaluation of novel primers for the detection of genes encoding diverse enzymes of methylotrophy and autotrophy.

The phylogenetic significance of the diversity of key enzymes of methylotrophic and autotrophic metabolism is discussed. Primers for these key enzymes were designed using gene sequences encoding methanol dehydrogenase (mxaF; using subsets from database sequences for 22 Bacteria), hydroxypyruvate reductase (hpr; 36 sequences), methylamine dehydrogenase (mauA; 12 sequences), methanesulfonate monooxygenase (msmA; four sequences), and the ccbL and cbbM genes of ribulose bisphosphate carboxylase (26 and 23 sequences). These were effective in amplifying the correct gene products for the target genes in reference organisms and in test organisms not previously shown to contain the genes, as well as in some methylotrophic Proteobacteria isolated from the human mouth. The availability of the new primers increases the probability of detecting diverse examples of the genes encoding these key enzymes both in natural populations and in isolated bacterial strains.

The microbiota and radiotherapy for head and neck cancer: What should clinical oncologists know?

Radiotherapy is a linchpin in head and neck squamous cell carcinoma (HN-SCC) treatment. Modulating tumour and/or normal tissue biology offers opportunities to further develop HN-SCC radiotherapy. The microbiota, which can exhibit homeostatic properties and be a modulator of immunity, has recently received considerable interest from the Oncology community. Microbiota research in head and neck oncology has also flourished. However, available data are difficult to interpret for clinical and radiation oncologists. In this review, we focus on how microbiota research can contribute to the improvement of radiotherapy for HN-SCC, focusing on how current and future research can be translated back to the clinic. We include in-depth discussions about the microbiota, its multiple habitats and relevance to human physiology, mechanistic interactions with HN-SCC, available evidence on microbiota and HNC oncogenesis, efficacy and toxicity of treatment. We discuss clinically-relevant areas such as the role of the microbiota as a predictive and prognostic biomarker, as well as the potential of leveraging the microbiota and its interactions with immunity to improve treatment results. Importantly, we draw parallels with other cancers where research is more mature. We map out future directions of research and explain clinical implications in detail.

Facultative methylotrophs from the human oral cavity and methylotrophy in strains of Gordonia, Leifsonia, and Microbacterium.

We show that bacteria with methylotrophic potential are ubiquitous in the human mouth microbiota. Numerous strains of Actinobacteria (Brevibacterium, Gordonia, Leifsonia, Microbacterium, Micrococcus, Rhodococcus) and Proteobacteria (Achromobacter, Klebsiella, Methylobacterium, Pseudomonas, Ralstonia) were isolated, and one strain of each of the eleven genera was studied in detail. These strains expressed enzymes associated with methylotrophic metabolism (methanol, methylamine, and formate dehydrogenases), and the assimilation of one-carbon compounds by the serine pathway (hydroxypyruvate reductase). Methylotrophic growth of the strains was enhanced by the addition of glass beads to cultures, suggesting that they may naturally occur in biofilms in the mouth. This is the first report of Gordonia, Leifsonia, and Rhodococcus being present in the mouth and of the unequivocal demonstration for the first time of the methylotrophic potential of strains of Gordonia, Leifsonia, and Microbacterium.

Prevotella fusca sp. nov. and Prevotella scopos sp. nov., isolated from the human oral cavity.

Two strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to belong to two separate taxa. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that the strains were both related to, but distinct from, the type strain of Prevotella melaninogenica. Two novel species, Prevotella fusca sp. nov. and Prevotella scopos sp. nov., are proposed to accommodate these strains. Both strains were saccharolytic and produced acetic and succinic acids, with lesser amounts of lactic and isovaleric acids, as end products of fermentation, and both were sensitive to 20 % bile. The principal cellular long-chain fatty acids of both strains were ai-C(15 : 0), 3-OH i-C(17 : 0), 3-OH C(16 : 0), i-C(15 : 0) and C(16 : 0). The DNA G+C contents of the type strains of Prevotella fusca (W1435(T)  = DSM 22504(T)  = CCUG 57946(T)) and Prevotella scopos (W2052(T)  = DSM 22613(T ) = CCUG 57945(T)) were 43 and 41 mol%, respectively. The two species could be differentiated by gelatin hydrolysis, cellobiose and ribose fermentation, and production of ß-glucosidase.

Scardovia wiggsiae sp. nov., isolated from the human oral cavity and clinical material, and emended descriptions of the genus Scardovia and Scardovia inopinata.

Six strains of anaerobic, pleomorphic Gram-positive bacilli, isolated from the human oral cavity and an infected arm wound, were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed that the isolates were most closely related to Scardovia inopinata CCUG 35729(T) (94.8-94.9 % 16S rRNA gene sequence similarity). The isolates were saccharolytic and produced acetic and lactic acids as end products of fermentation. The major fatty acids were C(16 : 0) (49.8 %) and C(18 : 1)ω9c (35.8 %). Polar lipid analysis revealed a variety of glycolipids, diphosphatidylglycerol, an unidentified phospholipid and an unidentified phosphoglycolipid. No respiratory quinones were detected. The peptidoglycan was of the type A4α L-Lys-Thr-Glu, with L-lysine partially replaced by L-ornithine. The DNA G+C content of one of the strains, C1A_55(T)(,) was 55 mol%. A novel species, Scardovia wiggsiae sp. nov., is proposed to accommodate the six isolates, with the type strain C1A_55(T) (=DSM 22547(T)=CCUG 58090(T)).

Effect of rinsing with ethanol-containing mouthrinses on the production of salivary acetaldehyde.

It has been suggested that the use of alcohol-containing mouthrinses could lead to the presence of acetaldehyde in saliva. In this cross-over study, salivary acetaldehyde levels and microbial profiles were determined before and after rinsing with ethanol-containing mouthrinses with essential oils (EO) and cetyl pyridinium chloride (CPC) as the active ingredients, and with 21.6% ethanol and water controls. After rinsing with all ethanol-containing rinses, acetaldehyde was detected in saliva after 30 s but declined to low levels after 5 min. The highest peak levels were seen with the ethanol control (median = 82.9 µM at 2 min) and were significantly higher than those seen at the same time after rinsing with the EO rinse (43.1 µM). There was no correlation between microbial counts or plaque scores and acetaldehyde levels, although dividing the subjects on the basis of a peak acetaldehyde salivary concentration of > 90.8 µM after the ethanol rinse revealed that the high responders were highly significantly more likely to harbour salivary yeasts than were the low responders. Rinsing with ethanol-containing mouthrinses causes a rapid, but transient, increase in salivary acetaldehyde levels.

Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque.

Metagenomic techniques are used to analyse bacterial communities allowing both culturable and unculturable species to be represented. However, the screening of oral metagenomic samples can be hindered by high animal host DNA content. This study evaluated methods for the reduction of human DNA concentrations within oral metagenomic samples. Plaque samples were collected from 27 patients presenting with periodontal disease and treated to remove human DNA using either selective lysis of eukaryotic cells at several buffer concentrations or differential centrifugation after treatment with trypsin and/or detergents. Human and bacterial DNA levels were determined by quantitative polymerase chain reaction (qPCR). The human DNA content of plaque extracts was significantly reduced by all treatments compared with an untreated control (P < 0.05). However, differential centrifugation simultaneously reduced the bacterial DNA content unless samples were pretreated with a detergent. Observations of Gram stained samples that were processed using differential centrifugation without detergent suggest that many bacteria remain adhered to human cells. An approach that uses differential centrifugation in parallel with selective lysis is recommended to fully represent the oral microbiota in metagenomic samples, including those tightly adhered to human cells and more delicate bacteria such as Mycoplasma.