Disruption of the adh (acetoin dehydrogenase) operon has wide-ranging effects on Streptococcus mutans growth and stress response

The agent largely responsible for initiating dental caries, Streptococcus mutans produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2-7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii , did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex , encoding an NAD + /NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans . IMPORTANCE Dental caries is often initiated by Streptococcus mutans , which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

Exploratory Study on Application of MALDI-TOF-MS to Detect SARS-CoV-2 Infection in Human Saliva

SARS-CoV-2 has caused a large outbreak since its emergence in December 2019. COVID-19 diagnosis became a priority so as to isolate and treat infected individuals in order to break the contamination chain. Currently, the reference test for COVID-19 diagnosis is the molecular detection (RT-qPCR) of the virus from nasopharyngeal swab (NPS) samples. Although this sensitive and specific test remains the gold standard, it has several limitations, such as the invasive collection method, the relative high cost and the duration of the test. Moreover, the material shortage to perform tests due to the discrepancy between the high demand for tests and the production capacities puts additional constraints on RT-qPCR. Here, we propose a PCR-free method for diagnosing SARS-CoV-2 based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling and machine learning (ML) models from salivary samples. Kinetic saliva samples were collected at enrollment and ten and thirty days later (D0, D10 and D30), to assess the classification performance of the ML models compared to the molecular tests performed on NPS specimens. Spectra were generated using an optimized protocol of saliva collection and successive quality control steps were developed to ensure the reliability of spectra. A total of 360 averaged spectra were included in the study. At D0, the comparison of MS spectra from SARS-CoV-2 positive patients (n = 105) with healthy healthcare controls (n = 51) revealed nine peaks that significantly distinguished the two groups. Among the five ML models tested, support vector machine with linear kernel (SVM-LK) provided the best performance on the training dataset (accuracy = 85.2%, sensitivity = 85.1%, specificity = 85.3%, F1-Score = 85.1%). The application of the SVM-LK model on independent datasets confirmed its performances with 88.9% and 80.8% of correct classification for samples collected at D0 and D30, respectively. Conversely, at D10, the proportion of correct classification had fallen to 64.3%. The analysis of saliva samples by MALDI-TOF MS and ML appears as an interesting supplementary tool for COVID-19 diagnosis, despite the mitigated results obtained for convalescent patients (D10).

Excess Iodine Exposure Acutely Increases Salivary Iodide And Antimicrobial Hypoiodous Acid Concentrations In Humans

The lactoperoxidase (LPO)-hydrogen peroxide-halides reaction (LPO system) converts iodide and thiocyanate (SCN-) into hypoiodous acid (HOI) and hypothiocyanite (OSCN-), respectively. Since this system has been implicated in defense of the airways and oropharynx from microbial invasion, we measured the concentrations of these analytes in human saliva before and after iodine administration to test the hypothesis that an iodide load increases salivary iodide and HOI concentrations. Salivary iodide, SCN-, HOI and OSCN- were measured using standard methodology. Salivary iodide and HOI levels significantly increased after iodinated contrast injection compared with baseline levels, whereas there was no significant change in salivary SCN- and OSCN- levels. The contrast dye iodine load and changes of salivary iodide and HOI levels were positively correlated, suggesting that higher iodide in the circulation increases iodide output and salivary HOI production. Excess iodine exposure in humans increases the salivary output of iodide, increasing salivary HOI concentrations with no effect on SCN-/OSCN- levels. This first of its kind study suggests that a sufficient but safe iodide supplementation may augment the generation of antimicrobial HOI by the salivary LPO system against airborne viral pathogens, including coronaviruses and influenza viruses, a possible inexpensive means of effectively curbing viral pandemics.

Azelaic Acid Esters as Pluripotent Immunomodulatory Molecules: Nutritional Supplements or Drugs

Azelaic acid and its esters, the azelates, occur naturally in organisms ranging from plants to humans. We have shown that diethyl azelate (DEA) exhibits a broad range of immunomodulatory activities in vitro and in vivo, and mitigates insulin resistance. To further investigate the therapeutic utility of DEA, we evaluated its mutagenicity in Salmonella typhimurium strains, examined metabolism of DEA in rat, dog, monkey and human primary hepatocytes and in human saliva, determined pharmacokinetics of DEA after an oral dose in rats, and queried its physicochemical properties for drug-like characteristics. DEA was not mutagenic in bacterial strains ± rat liver metabolic activation system S-9. It was chemically unstable in hepatocyte culture medium with a half-life of <1 h and was depleted by the hepatocytes in <5 min, suggesting rapid hepatic metabolism. DEA was also quickly degraded by human saliva in vitro. After an oral administration of DEA to rats, the di- and monoester were undetectable in plasma while the levels of azelaic acid increased over time, reached maximum at <2 h, and declined rapidly thereafter. The observed pharmacological properties of DEA suggest that it has value both as a drug or a nutritional supplement.

A Novel 3D Titanium Surface Produced by Selective Laser Sintering to Counteract Streptococcus oralis Biofilm Formation

The topography of implant surfaces influences the interaction relationship between material and bacteria. The aim of this work was to characterize a novel 3D titanium surface, produced using Selective Laser Sintering (SLS), and to compare the bacterial interaction with machined and double acid etching (DAE) discs. The surface was characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), and Energy Dispersive X-ray Spectrometry (EDX). The wettability was measured using the sessile method. The microbiological investigation consisted in the cultivation of a bacterial pioneer, Streptococcus oralis, on titanium surfaces, previously covered by human saliva in order to form the acquired pellicle. Then, colony forming units (CFUs), biofilm biomass quantification, analyses of viable and dead cells, and SEM observation were determined after 24 h of S. oralis biofilm formation on the different discs. A significantly higher nano-roughness with respect to the other two groups characterized the novel 3D surface, but the wettability was similar to that of machined samples. The microbiological assays demonstrated that the 3D discs reported significantly lower values of CFUs and biofilm biomass with respect to machined surfaces; however, no significant differences were found with the DAE surfaces. The live/dead staining confirmed the lower percentage of living cells on DAE and 3D surfaces compared with the machined. This novel 3D surface produced by SLS presented a high antiadhesive and antibiofilm activity.

A core salivary microbiome shows the high prevalence of bacterial members yet variability across human populations

Human saliva contains diverse bacterial communities, reflecting human health status, dietary patterns and contributing to variability in the sensory perception of food. Many descriptions of salivary microbiome diversity compare commonalities and differences with reference to a diseased state, but the composition of healthy saliva has not been described. Here, we use a meta-analysis approach to define and explore the core membership of the human salivary microbial community by collecting and re-analysing raw 16S rRNA amplicon sequencing data from 47 studies with 2206 saliva samples. We found 68 core bacterial taxa that were consistently detected. Differences induced by various host intrinsic and behaviour factors, including gender, age, geographic location, tobacco usage, and alcohol consumption, were evident. The core of the salivary microbiome was verified by collecting and analysing saliva in an independent study, and this analysis showed that the impact of geographic variation is likely due to diet.