GNPS human saliva derived in vitro biofilms
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ FT
ORGANISM(S): Oral Bacterial Flora
SUBMITTER: Anna Edlund Jeffrey McLean
PROVIDER: MSV000078583 | GNPS | Thu Apr 10 16:18:00 BST 2014
REPOSITORIES: GNPS
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Project description:Streptococcus mutans were grown in rich SHI-medium as biofilms anaerobically. Biofilms were starved in minimal medium followed by glucose amendment (at pH 7). Replicate samples for MS/MS-small molecule and transcription analyses were collected at pH 7 and at pH 3.5 (20 hrs after glucose spiking). Only secreted molecules were analyzed with MS/MS (no cell lysis protocol was employed).
2014-04-10 | MSV000078581 | GNPS
Project description:These biofilms were established from saliva samples as described in Edlund et al. 2013 (Microbiome, doi:10.1186/2049-2618-1-25). After starving the biofilms in minimal medium anaerobiaclly glucose was added to the cultures. Samples for metatranscriptomics and metabolomics were collected in parallel at pH 7 (prior to glucose amendment), pH 4.2 (6 hours after glucose amendment) and pH 5.2 (9 hours after pH amendment). Secreted small molecules were extracted from biofilms (cells were not lysed) at the differnet pH stages.
2014-04-10 | MSV000078579 | GNPS
Project description: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.
2020-07-23 | ST001433 | MetabolomicsWorkbench
Project description:In this experiment we collected small molecule data that represent excreted molecules by Streptococcus mutans growing as a biofilm. The S. mutans biofilms were established and incubated in anaerobic conditions. Samples were collected before and after a drastic pH drop due to glucose amendments. Control samples are included in this folder that represent molecules that were extracted from sterilized growth media only. These peaks should be subtracted from the biofilm samples prior to analyses.
2014-04-10 | MSV000078578 | MassIVE
Project description:Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation pH is a major environmental factor that regulates gene expression in many bacteria. Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and the pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we used DNA microarray to perform genome-wide transcriptional analysis of S. mutans in response to acidic pH. The results showed that adaptation of S. mutans to pH 5.5 for 2 hrs induced differential expression of nearly 14% of genes in the genome, including 169 up-regulated genes and 108 down-regulated genes, largely categorized into six groups. Especially, we found that the genes encoding multiple two-component systems, including CiaHR, LevSR, LiaSR, ScnKR, HK/RR07 and ComDE, were up-regulated during acid adaptation. These findings were further confirmed by real time qRT-PCR and phenotypic assays of the gene deletion mutants. The results support that the multiple two-component systems are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.
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Project description:Transcriptional analysis of glucose shock vs. steady-state growth in the parent strain and an acid sensitive mutant strain of S. mutans RNA was extracted from 4 replicate samples of S. mutans UA159 and UR117 (fabM mutant strain) grown in continuous culture to a steady-state pH value of 7. The cultures were exposed to a glucose shock (200mM) and samples were collected upon achieving culture pH value of 5.5. pH control was re-established and cultures were allowed to grow to a steady-state pH value of 5 (for UA159) and 5.5 (for fabM mutant). RNA was labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from S. mutans UA159 cultures grown to mid-log.
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Project description:Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation pH is a major environmental factor that regulates gene expression in many bacteria. Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and the pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we used DNA microarray to perform genome-wide transcriptional analysis of S. mutans in response to acidic pH. The results showed that adaptation of S. mutans to pH 5.5 for 2 hrs induced differential expression of nearly 14% of genes in the genome, including 169 up-regulated genes and 108 down-regulated genes, largely categorized into six groups. Especially, we found that the genes encoding multiple two-component systems, including CiaHR, LevSR, LiaSR, ScnKR, HK/RR07 and ComDE, were up-regulated during acid adaptation. These findings were further confirmed by real time qRT-PCR and phenotypic assays of the gene deletion mutants. The results support that the multiple two-component systems are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH. Total RNAs were isolated from S. mutans UA159 cells (0.6 at OD600) grown in a TYG broth (3% tryptone, 0.3% yeast extract and 20 mM glucose) at either pH 5.5 or pH 7.5. The RNAs were treated with RNase-free DNase 1 and purified by Qiagen RNeasy mini columns. The purified RNAs were used to generate cDNA probes by an indirect labeling method based on the protocol from TIGR. The cDNAs were coupled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen). The labeled cDNA probes from three different cultures of UA159 were hybridized to the S. mutans microarray slides obtained from PFGRC (http://pfgrc.tigr.org). Array hybridization was conducted using a protocol from the PFGRC with minor modification. After hybridization, washes and dried, the array slides were scanned by ScanArray 5000XL Reader (Perkin Elmer, Boston, MA). After the array slides were scanned, the resulting images were loaded into TIGR Spotfinder software (http://www.tigr.org/software/) and overlaid. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, and the intensity values of each spot were determined. Signal intensities of individual channels from an array slide were averaged and normalized using an array data analysis software (MIDAS) by using LOWESS and iterative log mean centering with default settings, followed by in-slide replicate analysis. A t-test was used to determine the consistency of ratios across replicate hybridizations. Only genes whose ratios were ⥠2-fold changes (either increase or decrease) with 99% confidence interval (P ⤠0.01) were considered statistically significant.
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Project description:The formation of biofilms is closely associated with persistent and chronic infections, and physiological heterogeneity such as pH and oxygen gradients renders biofilms highly resistant to conventional antibiotics. To date, effectively treating biofilm infections remains a significant challenge. Herein, we report the fabrication of micellar nanoparticles adapted to heterogeneous biofilm microenvironments, enabling nitric oxide (NO) release through two distinct photoredox catalysis mechanisms. The key design feature involves the use of tertiary amine (TA) moieties, which function as sacrificial agents to avoid the quenching of photocatalysts under normoxic and neutral pH conditions and proton acceptors at acidic pH to allow deep biofilm penetration. This biofilm-adaptive NO-releasing platform shows excellent antibiofilm activity against ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) biofilms both in vitro and in a mouse skin infection model, providing a strategy for combating biofilm heterogeneity and biofilm-related infections.
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