Summary of Study ST001433
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000984. The data can be accessed directly via it's Project DOI: 10.21228/M8H68N This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001433 |
| Study Title | Evidence for proline utilisation by oral bacterial biofilms grown in saliva |
| Study Type | Research study |
| Study Summary | 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 whole mouth saliva and then grown for 7 days using sterilised whole mouth saliva supplemented with proline, arginine and 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 metabolised proline to 5-aminopentanoate, butyrate and propionate, and actively utilised 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. |
| Institute | King's College London |
| Department | Centre for Host Microbiome Interactions |
| Last Name | Cleaver |
| First Name | Leanne |
| Address | Floor 17, Tower Wing, Guy's Hospital, Great Maze Pond |
| leanne.cleaver@kcl.ac.uk | |
| Phone | 07464626438 |
| Submit Date | 2020-07-23 |
| Num Groups | 11 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(NMR) |
| Analysis Type Detail | NMR |
| Release Date | 2020-08-03 |
| Release Version | 1 |
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Collection:
| Collection ID: | CO001502 |
| Collection Summary: | Saliva was processed and biofilms were grown using a previously published methodology with modifications (Cleaver, Moazzez and Carpenter, 2019). Saliva samples were centrifuged (5000g for 5 mins), and the supernatant was collected, pooled, boiled in sealed tubes for 20 minutes to sterilise, and left to cool to room temperature; hereafter referred to as sterile saliva. The loose pellet from the spun saliva samples was pooled and combined with enough sterile saliva to facilitate inoculation of the hydroxyapatite (HA) discs. Samples were vortexed vigorously to resuspend the pellet; this constituted the pooled saliva inoculum. The remaining sterile saliva was split into 11 aliquots. D-proline, L-arginine, and glucose were added to the aliquots to achieve concentrations of 10mM, 25mM and 50mM respectively for each (based on concentrations of proline obtained from previous publication (6)). One aliquot was also supplemented with 10mM carbon 13 labelled (C13) D-proline. Two aliquots were left unsupplemented to act as a positive and negative control. Six HA discs in a sterile microtitre plate were inoculated per treatment condition with 1 ml pooled saliva inoculum (negative control discs were inoculated with sterile saliva only to check for sterility) and incubated for 24 hours aerobically in a 40 L aerobic incubator (GenLab Ltd, UK) at 37°C. After this time the discs were washed three times with sterile phosphate buffered saline (PBS) and 1 ml of supplemented/unsupplemented saliva per condition was added to the discs. The discs were then incubated anaerobically in a 3.5 litre anaerobic jar with an Anaerogen anaerobic generator pack (Oxoid, UK) at 37°C for 72 hours. After this spent saliva from the biofilms was removed and stored at -20°C for further analysis, discs were washed three times with PBS, and refreshed with supplemented/unsupplemented saliva and further incubated anaerobically at 37°C for 72 hours. After this final incubation the spent saliva was removed and stored at -20°C for further analysis. |
| Sample Type: | Bacterial cells |
