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.

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.

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.

The bitterness of vegetables is a leading reason why they are avoided by children and some adults. Bitterness is perceived via TAS2R receptors located on the tongue. In contrast, astringency is a mouthfeel rather than a taste, and is perceived as a dry, puckering sensation. To date few reports have suggested any interactions between the two processes even though they often occur simultaneously in many real foods. In this study, we have used Brussels sprouts as an exemplar bitter vegetable and examined the influence of a number of different interventions on perceived intensity. Subjects rated the intensity of Brussels sprouts before and after three interventions: gravy, red wine, and water. Only red wine caused a significant (p < .0001) decrease in VAS scale, from 5.5 to 3.5 on a 10‐point labeled magnitude scale. The results suggest the astringency of the red wine affected the perception of bitter in the Brussels sprout. Some possible mechanisms are discussed. Practical applications This report reveals a possible insight into how bitterness is perceived in humans. By using astringency to affect salivary proteins, we suggest they may play a role in the detection of bitterness. This may be by helping to transport bitterness compounds to the taste bud receptors or a separate mechanism. Potentially this also opens up new ways to block bitterness.