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
Email	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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Project:

Project ID:	PR000984
Project DOI:	doi: 10.21228/M8H68N
Project Title:	Evidence for proline utilisation by oral bacterial biofilms grown in saliva
Project Type:	Research study
Project 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, London, London, SE1 9RT, United Kingdom
Email:	leanne.cleaver@kcl.ac.uk
Phone:	07464626438
Funding Source:	This work was funded by a BBSRC Lido-associated PhD studentship for LC in association with Colgate-Palmolive, USA.

Subject:

Subject ID:	SU001507
Subject Type:	Bacteria
Subject Species:	Multi-species non-defined biofilm consortium

Factors:

Subject type: Bacteria; Subject species: Multi-species non-defined biofilm consortium (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA121810	sample7	Arginine 10mM
SA121811	sample38	Arginine 10mM
SA121812	sample39	Arginine 10mM
SA121813	sample40	Arginine 10mM
SA121814	sample6	Arginine 10mM
SA121815	sample5	Arginine 10mM
SA121816	sample14	Arginine 25mM
SA121817	sample15	Arginine 25mM
SA121818	sample49	Arginine 25mM
SA121819	sample47	Arginine 25mM
SA121820	sample16	Arginine 25mM
SA121821	sample48	Arginine 25mM
SA121822	sample25	Arginine 50mM
SA121823	sample23	Arginine 50mM
SA121824	sample24	Arginine 50mM
SA121825	sample56	Arginine 50mM
SA121826	sample57	Arginine 50mM
SA121827	sample58	Arginine 50mM
SA121828	sample41	Glucose 10mM
SA121829	sample43	Glucose 10mM
SA121830	sample8	Glucose 10mM
SA121831	sample10	Glucose 10mM
SA121832	sample42	Glucose 10mM
SA121833	sample9	Glucose 10mM
SA121834	sample52	Glucose 25mM
SA121835	sample51	Glucose 25mM
SA121836	sample17	Glucose 25mM
SA121837	sample19	Glucose 25mM
SA121838	sample18	Glucose 25mM
SA121839	sample50	Glucose 25mM
SA121840	sample60	Glucose 50mM
SA121841	sample26	Glucose 50mM
SA121842	sample59	Glucose 50mM
SA121843	sample27	Glucose 50mM
SA121844	sample61	Glucose 50mM
SA121845	sample28	Glucose 50mM
SA121846	sample1	Growth Medium
SA121847	sample66	No treatment
SA121848	sample67	No treatment
SA121849	sample65	No treatment
SA121850	sample63	No treatment
SA121851	sample62	No treatment
SA121852	sample64	No treatment
SA121853	sample34	No treatment
SA121854	sample32	No treatment
SA121855	sample30	No treatment
SA121856	sample29	No treatment
SA121857	sample33	No treatment
SA121858	sample31	No treatment
SA121859	sample37	Proline 10mM
SA121860	sample35	Proline 10mM
SA121861	sample3	Proline 10mM
SA121862	sample4	Proline 10mM
SA121863	sample36	Proline 10mM
SA121864	sample2	Proline 10mM
SA121865	sample12	Proline 25mM
SA121866	sample11	Proline 25mM
SA121867	sample13	Proline 25mM
SA121868	sample45	Proline 25mM
SA121869	sample46	Proline 25mM
SA121870	sample44	Proline 25mM
SA121871	sample54	Proline 50mM
SA121872	sample53	Proline 50mM
SA121873	sample55	Proline 50mM
SA121874	sample20	Proline 50mM
SA121875	sample21	Proline 50mM
SA121876	sample22	Proline 50mM

Showing results 1 to 67 of 67

Showing results 1 to 67 of 67

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

Treatment:

Treatment ID:	TR001522
Treatment Summary:	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.

Treatment ID:

TR001522

Treatment Summary:

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.

Sample Preparation:

Sampleprep ID:	SP001515
Sampleprep Summary:	Samples were processed for NMR using a previously published method (6). Briefly, centrifuged spent saliva supernatant and the sterile saliva sample were each mixed with TSP buffer in a 5 mm NMR tube (Bruker, Germany). The tubes were sealed and analysed at the Biomolecular Spectroscopy Centre, King’s College London, UK on a 600 MHz spectrometer (Bruker) for 1H 1D-NMR and 1H-C13 1D- and 2D-NMR. The concentration of metabolites relative to the sterile saliva baseline were collected using Chenomix NMR Suite version 8.5 (Chenomix Ltd, Canada). The 2D C13 spectra were analysed using TopSpin version 3.6.2 (Bruker) and COLMAR (30).

Sampleprep ID:

SP001515

Sampleprep Summary:

Samples were processed for NMR using a previously published method (6). Briefly, centrifuged spent saliva supernatant and the sterile saliva sample were each mixed with TSP buffer in a 5 mm NMR tube (Bruker, Germany). The tubes were sealed and analysed at the Biomolecular Spectroscopy Centre, King’s College London, UK on a 600 MHz spectrometer (Bruker) for 1H 1D-NMR and 1H-C13 1D- and 2D-NMR. The concentration of metabolites relative to the sterile saliva baseline were collected using Chenomix NMR Suite version 8.5 (Chenomix Ltd, Canada). The 2D C13 spectra were analysed using TopSpin version 3.6.2 (Bruker) and COLMAR (30).

Analysis:

Analysis ID:	AN002395
Analysis Type:	NMR
Num Factors:	11
Num Metabolites:	19
Units:	mM

NMR:

NMR ID:	NM000166
Analysis ID:	AN002395
Instrument Name:	Bruker
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	600Hz