Novel bacterial proteolytic and metabolic activity associated with dental erosion-induced oral dysbiosis.

BACKGROUND: Dental erosion is a disease of the oral cavity where acids cause a loss of tooth enamel and is defined as having no bacterial involvement. The tooth surface is protected from acid attack by salivary proteins that make up the acquired enamel pellicle (AEP). Bacteria have been shown to readily degrade salivary proteins, and some of which are present in the AEP. This study aimed to explore the role of bacteria in dental erosion using a multi-omics approach by comparing saliva collected from participants with dental erosion and healthy controls. RESULTS: Salivary proteomics was assessed by liquid-chromatography mass spectrometry (LC-MS) and demonstrated two altered AEP proteins in erosion, prolactin inducible protein (PIP), and zinc-alpha-2 glycoprotein (ZAG). Immunoblotting further suggested that degradation of PIP and ZAG is associated with erosion. Salivary microbiome analysis was performed by sequencing the bacterial 16S rRNA gene (V1-V2 region, Illumina) and showed that participants with dental erosion had a significantly (p < 0.05) less diverse microbiome than healthy controls (observed and Shannon diversity). Sequencing of bacterial mRNA for gene expression (Illumina sequencing) demonstrated that genes over-expressed in saliva from erosion participants included H + proton transporter genes, and three protease genes (msrAB, vanY, and ppdC). Salivary metabolomics was assessed using nuclear magnetic resonance spectrometry (NMR). Metabolite concentrations correlated with gene expression, demonstrating that the dental erosion group had strong correlations between metabolites associated with protein degradation and amino acid fermentation. CONCLUSIONS: We conclude that microbial proteolysis of salivary proteins found in the protective acquired enamel pellicle strongly correlates with dental erosion, and we propose four novel microbial genes implicated in this process. Video Abstract.

Salivary bacterial community profile in normal-weight and obese adolescent patients prior to orthodontic treatment with fixed appliances.

OBJECTIVES: The aim of this study was to compare the intra-oral bacterial profile of normal-weight and obese adolescents prior to orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Nineteen adolescent patients were recruited into two groups based upon body mass index (BMI) and classified as normal-weight or obese. Unstimulated whole mouth saliva was obtained for 5 minutes. Bacterial DNA extraction was performed from saliva, and 16S rRNA gene sequencing of the V1-2 variable regions was undertaken followed by analysis using the mothur pipeline. RESULTS: Saliva from a total of 19 adolescent patients with mean (SD) age 15.6 (1.8) years were divided into 10 normal-weight with mean BMI of 19.4 (2.2) kg/m2 and 9 obese with mean BMI of 30.2 (3.5) kg/m2 . A total of 156 783 sequences were obtained from the 19 samples with no significant differences in richness or diversity between sample groups by obesity status or gender (AMOVA). The bacterial community in both groups was dominated by bacterial genera characteristic of the human mouth, which included Streptococcus, Porphyromonas, Veillonella, Gemella, Prevotella, Fusobacterium and Rothia. CONCLUSION: There were no differences in alpha or beta diversity of oral bacterial communities between normal-weight and obese orthodontic patients. Obese adolescents attending for orthodontic treatment had a similar microflora to their normal-weight counterparts.

Evidence for Proline Utilization by Oral Bacterial Biofilms Grown in Saliva.

Within the mouth bacteria are starved of saccharides as their main nutrient source between meals and it is unclear what drives their metabolism. Previously oral in vitro biofilms grown in saliva have shown proteolytic degradation of salivary proteins and increased extracellular proline. Although arginine and glucose have been shown before to have an effect on oral biofilm growth and activity, there is limited evidence for proline. Nuclear magnetic resonance (NMR) spectroscopy was used to identify extracellular metabolites produced by bacteria in oral biofilms grown on hydroxyapatite discs. Biofilms were inoculated with stimulated whole mouth saliva and then grown for 7 days using sterilized stimulated whole mouth saliva supplemented with proline, arginine or glucose as a growth-medium. Overall proline had a beneficial effect on biofilm growth-with significantly fewer dead bacteria present by biomass and surface area of the biofilms (p < 0.05). Where arginine and glucose significantly increased and decreased pH, respectively, the pH of proline supplemented biofilms remained neutral at pH 7.3-7.5. SDS-polyacrylamide gel electrophoresis of the spent saliva from proline and arginine supplemented biofilms showed inhibition of salivary protein degradation of immature biofilms. NMR analysis of the spent saliva revealed that proline supplemented biofilms were metabolically similar to unsupplemented biofilms, but these biofilms actively metabolized proline to 5-aminopentanoate, butyrate and propionate, and actively utilized glycine. This study shows that in a nutrient limited environment, proline has a beneficial effect on in vitro oral biofilms grown from a saliva inoculum.

Mixed aerobic-anaerobic incubation conditions induce proteolytic activity from in vitro salivary biofilms.

Oral biofilms have not been studied using both metabolome and protein profiling concurrently. Bacteria produce proteases that lead to degradation of functional salivary proteins. The novel protocol described here allows for complete characterisation of in vitro oral biofilms, including proteolytic, metabolic, and microbiome analysis. Biofilms were grown on hydroxyapatite discs from whole mouth saliva, using sterilised saliva as a growth-medium, in different growth environments. Salivary protein degradation was assessed from spent saliva growth-medium using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and metabolic activity by nuclear magnetic resonance (NMR). Discs were assessed for depth and coverage of biofilms by confocal laser scanning microscopy (CLSM), and biofilms were collected at the end of the experiment for 16S rRNA gene sequence analysis. There was a significant difference in biofilm viability, salivary protein degradation, and metabolites identified between biofilms grown aerobically and biofilms exposed to an anaerobic environment. Bacterial 16S rRNA gene sequencing showed the predominant genus in the 7-day aerobic biofilms was Streptococcus, in aerobic-anaerobic and anaerobic 7-day biofilms Porphyromonas, and in aerobic-anaerobic and anaerobic 13-day biofilms Fusobacterium. This data suggests new growth requirements and capabilities for analysing salivary biofilms in vitro, which can be used to benefit future research into oral bacterial biofilms.

A case of polymicrobial anaerobic spondylodiscitis due to Parvimonas micra and Fusobacterium nucleatum.

Introduction. Here, we present a case of polymicrobial anaerobic spondylodiscitis. Case Presentation. A forty-five year-old female patient was referred to a specialist orthopaedic hospital with an eight week history of back pain without fevers. X-ray imaging and magnetic resonance imaging showed acute osteomyelitis of the twelfth thoracic and first lumbar vertebrae. Prolonged enrichment cultures grew Parvimonas micra and Fusobacterium nucleatum, identified by matrix-assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry (MS). The patient was successfully treated with six weeks of intravenous ertapenem and oral clindamycin. Conclusion. Anaerobic discitis is rare, and polymicrobial discitis is rarer still. A PubMed literature review revealed only seven cases of F. nucleatum discitis and only twelve cases of P. micra discitis; this includes only one other reported case of a polymicrobial discitis due to infection with both anaerobes. We emphasise the importance of prolonging enrichment culture and the use of fast yet accurate identification of anaerobes using MALDI-ToF MS in these infections.