Determination of arginine catabolism by salivary pellet

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

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“…The concentration of ammonium produced was measured using an enzymatic assay, described by Hoogenkamp and ten Cate [27].…”

Section: Enzymatic Ammonium Assaymentioning

confidence: 99%

Stability and Resilience of Oral Microcosms Toward Acidification and Candida Outgrowth by Arginine Supplementation

Koopman

Röling²,

Buijs

et al. 2014

Microb Ecol

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“…Nevertheless, fractions of salivary pellet are components of human saliva and secreted to the oral cavity via the physiological salivary flow. Clinically, the salivary pellet is used to prepare DNA for diagnostic purposes in oral cancer and arginine catabolism [ 23 , 24 ]. The overall question about the possible contribution of the salivary endotoxins and those within the salivary pellet to oral homeostasis has not yet been answered.…”

Section: Discussionmentioning

confidence: 99%

Salivary pellets induce a pro-inflammatory response involving the TLR4–NF-kB pathway in gingival fibroblasts

et al. 2016

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BackgroundWhole saliva provokes a substantial pro-inflammatory response in gingival fibroblasts. This raises the question whether the salivary pellet, which is used for diagnostic purposes, also has a pro-inflammatory capacity and, if yes, what the underlying mechanisms at the molecular level are.MethodsWe examined the ability of extensively washed salivary pellets to provoke the expression of chemokines in gingival fibroblasts by real-time polymerase chain reaction and immunoassays. Protein composition was determined with proteomic analysis. Endotoxins were analyzed by a Limulus assay and removed by affinity chromatography. The inhibitors TAK-242 and BAY11-7082 were used to determine the involvement of the TLR4 and NF-kB signaling, respectively. Western blot was performed to detect phosphorylated p65.ResultsThe experiments show that salivary pellets and the corresponding washing solution contain pro-inflammatory activity without impairing cell viability. Proteomic analysis revealed proteins with a binding capacity for lipopolysaccharides, and the Limulus assay indicated the presence of endotoxin in the salivary pellets. Blocking TLR4 with TAK-242 and depletion of endotoxins both lowered the capacity of salivary pellets to increase chemokine expression and phosphorylation of p65. BAY11-7082 suppressed chemokine expression in response to the salivary pellets. Autoclaving salivary pellets also reduced their pro-inflammatory activity.ConclusionsThe data support the molecular mechanism of a TLR4-NF-kB-dependent pro-inflammatory response of the gingival fibroblasts exposed to preparations of washed salivary pellets. Together, the data indicate that the salivary pellet is rich in endotoxin but it is mainly a heat labile fraction that accounts for the chemokine expression in the bioassay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12903-016-0229-5) contains supplementary material, which is available to authorized users.

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“…To quantify the amount of protein (biomass) present in the biofilm, 400‐ μ l aliquots of each biofilm suspension were processed according to Hoogenkamp et al . . In short, biofilm samples were diluted in 800 μ l MilliQ water and bead beated twice for 30 s using Ø = 0.1 mm glass beads.…”

Section: Methodsmentioning

confidence: 99%

Effects of three γ‐alkylidene‐γ‐lactams on the formation of multispecies biofilms

Almeida

Pimenta

Pereira

et al. 2018

European J Oral Sciences

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This study evaluated the inhibitory effects of lactams on Streptococcus mutans, Enterococcus faecalis, and Candida glabrata multispecies biofilm formation. γ-Alkylidene-γ-lactams 1, 2, and 3 [solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Glass coverslips were conditioned with either the lactams or 3.5% DMSO (control) for 1 h, inoculated with microbial cultures, and incubated for 48 h. To assess the effect of the lactams on biofilm formation, the following parameters were determined: the biofilm biomass (by both crystal violet staining and protein determination); the amount of insoluble polysaccharides of the extracellular matrix; and the number of viable and total cells [by both colony-forming unit counting and quantitative real-time PCR (qPCR)]. Data were analysed using one-way anova and post-hoc Tukey tests. Lactams 1, 2, and 3 promoted a statistically significant reduction in the amount of biofilm biomass, but only lactam 3 resulted in a statistically significant reduction in the number of attached viable E. faecalis. Both total protein content and the amount of extracellular polysaccharides decreased significantly. The effects of γ-alkylidene-γ-lactams 1, 2, and 3 on the inhibition of multispecies biofilm formation were evident by their ability to reduce the amount of protein and extracellular polysaccharides.

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Determination of arginine catabolism by salivary pellet

Abstract: Graphical abstract

Cited by 8 publications

References 3 publications

Stability and Resilience of Oral Microcosms Toward Acidification and Candida Outgrowth by Arginine Supplementation

Stability and Resilience of Oral Microcosms Toward Acidification and Candida Outgrowth by Arginine Supplementation

Salivary pellets induce a pro-inflammatory response involving the TLR4–NF-kB pathway in gingival fibroblasts

Effects of three γ‐alkylidene‐γ‐lactams on the formation of multispecies biofilms

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