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Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

ABSTRACT: Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas chromatography-mass spectrometry GC-MS and revealed nine compounds representing 99.6% of fatty acids, where unsaturated and saturated fatty acids formed 93.8% and 5.8 % respectively. Oleic and linoleic acids were the major unsaturated fatty acids. Noteworthy, the fatty acids at the concentration of 31.3 mg L-1 completely inhibited Streptococcus mutans biofilm, and water insoluble extracellular polysaccharide production in both polystyrene plates, and tooth model assay using saliva-coated hydroxyapatite discs. Inhibition of biofilm correlated significantly and positively with the inhibition of water insoluble extracellular polysaccharide (R=1, p <0.0001). Furthermore, Arthrographis kalrae fatty acids at a concentration of 7.8 mg L-1 exhibited acidogenesis-mitigation activity. They did not show bactericidal activity against Streptococcus mutans and cytotoxic activity against human oral fibroblast cells at the concentration used. On the other hand, saliva-coated hydroxyapatite discs treated with sub-minimum biofilm inhibitory concentration of fatty acids showed disturbed biofilm architecture with a few unequally distributed clumped matrices using fluorescence microscopy. Our findings revealed that the intracellular fatty acid arrays derived from endophytic Arthrographis kalrae could contribute to the biofilm-preventing alternatives, specifically Streptococcus mutans biofilms.

SUBMITTER: Abdel-Aziz MM

PROVIDER: S-EPMC7277960 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

Abdel-Aziz Marwa M MM Emam Tamer M TM Raafat Marwa M MM

Biomolecules 20200525 5

Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas ...[more]

PMID: 32466324

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Project description:Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 μg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 μg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2',7'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 μg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.

Project description:BackgroundQuorum sensing (QS) system can regulate the expression of virulence factors and biofilm formation in Streptococcus mutans. Antimicrobial photodynamic therapy (aPDT) inhibits quorum quenching (QQ), and can be used to prevent microbial biofilm. We thereby aimed to evaluate the anti-biofilm potency and anti-metabolic activity of nano-quercetin (N-QCT)-mediated aPDT against S. mutans. Also, in silico evaluation of the inhibitory effect of N-QCT on the competence-stimulating peptide (CSP) of S. mutans was performed to elucidate the impact of aPDT on various QS-regulated genes.MethodsCytotoxicity and intracellular reactive oxygen species (ROS) generation were assessed following synthesis and confirmation of N-QCT. Subsequently, the minimum biofilm inhibitory concentration (MBIC) of N-QCT against S. mutans and anti-biofilm effects of aPDT were assessed using colorimetric assay and plate counting. Molecular modeling and docking analysis were performed to confirm the connection of QCT to CSP. The metabolic activity of S. mutans and the expression level of various genes involved in QS were evaluated by flow cytometry and reverse transcription quantitative real-time PCR, respectively.ResultsSuccessful synthesis of non-toxic N-QCT was confirmed through several characterization tests. The MBIC value of N-QCT against S. mutans was 128 μg/mL. Similar to the crystal violet staining, the results log10 CFU/mL showed a significant degradation of preformed biofilms in the group treated with aPDT compared to the control group (P < 0.05). Following aPDT, metabolic activity of S. mutans also decreased by 85.7% (1/2 × MBIC of N-QCT) and 77.3% (1/4 × MBIC of N-QCT), as compared to the control values (P < 0.05). In silico analysis showed that the QCT molecule was located in the site formed by polypeptide helices of CSP. The relative expression levels of the virulence genes were significantly decreased in the presence of N-QCT-mediated aPDT (P < 0.05).ConclusionsThe combination of N-QCT with blue laser as a QQ-strategy leads to maximum ROS generation, disrupts the microbial biofilm of S. mutans, reduces metabolic activity, and downregulates the expression of genes involved in the QS pathway by targeting genes of the QS signaling system of S. mutans.

Dataset Information

Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

Publications

Hindering of Cariogenic <i>Streptococcus mutans</i> Biofilm by Fatty Acid Array Derived from an Endophytic <i>Arthrographis kalrae</i> Strain.

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