α-d-(1 → 3)-graft-(1 → 6)-glucan: Comb-like polysaccharide synthesized in vitro with α-1,3/1,6-glucosyltransferase L from Streptococcus salivarius.

We demonstrated that a unique polysaccharide with extremely high molecular weight can be easily obtained via a low-cost, mild reaction in a water medium from sucrose, a photosynthetic product. α-1,3/1,6-Glucosyltransferase L (GtfL) from Streptococcus salivarius produced water-insoluble α-d-glucan from sucrose at 37 °C. Gel permeation chromatography revealed the molecular weight was extremely high; the weight-average molecular weight values were more than 1,000,000 irrespective of the substrate concentration. The Smith degradation of neat glucan and NMR spectroscopic analyses of the acetyl derivative revealed a structure similar to that of a comb-type graft copolymer, α-d-(1 â†’ 3)-graft-(1 â†’ 6)-glucan. The anhydroglucose units (AGUs) in the main-chain backbone are linked by (1 â†’ 3)-glycosidic bonds, whereas a side chain consisting of four AGUs via (1 â†’ 6)-glycosidic bonds alternately extends from C6 of the main chain.

Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk reduce chemokine IL-8 production in response to TNF-α in Caco-2 cell line, an exploratory study.

Streptococci are a predominant genera of the human milk microbiome. Among different lactic acid bacteria (LAB) a few Streptococcal strains are also considered as probiotics. Probiotic bacteria are reported to modulate immunity when consumed in adequate amount and bacterial hydrophobicity can be considered as a preliminary experiment for the adhesive capability of probiotic bacteria to the epithelial cells. The present study aimed to investigate the probiotic, hydrophobic and immune modulation property of Streptococcus lactarius MB622 and Streptococcus salivarius MB620, isolated from human milk. S. lactarius MB622 and S. salivarius MB620 displayed higher hydrophobicity (78 % and 59 % respectively) in addition to intrinsic probiotic properties such as gram positive classification, catalase negative activity, resistance to artificially stimulated gastric juice and gastrointestinal bile salt concentration. In conclusion Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk when administered in sufficient amount and for certain duration could be used to reduce inflammation inside the colon by reducing the production of inflammatory booster (IL-8) in diseased state.

Galacto-Oligosaccharide Supplementation Modulates Pathogen-Commensal Competition between Streptococcus agalactiae and Streptococcus salivarius.

The members of the infant microbiome are governed by feeding method (breastmilk vs. formula). Regardless of the source of nutrition, a competitive growth advantage can be provided to commensals through prebiotics - either human milk oligosaccharides (HMOs) or plant oligosaccharides that are supplemented into formula. To characterize how prebiotics modulate commensal - pathogen interactions, we have designed and studied a minimal microbiome where a pathogen, Streptococcus agalactiae engages with a commensal, Streptococcus salivarius. We discovered that while S. agalactiae suppresses the growth of S. salivarius via increased lactic acid production, galacto-oligosaccharides (GOS) supplementation reverses the effect. This result has major implications in characterizing how single species survive in the gut, what niche they occupy, and how they engage with other community members.

Safety assessment of Streptococcus salivarius DB-B5 as a probiotic candidate for oral health.

Streptococcus salivarius DB-B5 was previously isolated from the supragingival plaque of a healthy female adult and selected for development as a probiotic candidate for oral health. Probiotics are an important emerging therapeutic method for preventing, treating, and maintaining oral health. Although S. salivarius is a predominant member of the commensal oral microbiota and generally regarded as a safe species, it is recognized that each strain needs to be comprehensively assessed for safety. This study describes the in silico, in vitro, and clinical testing that were conducted to evaluate the safety of S. salivarius DB-B5. Both 16S rRNA and multi-gene phylogenetic reconstruction was used to confirm the taxonomic identity of this strain. Bioinformatic analysis of the genome demonstrated the absence of transmissible antibiotic resistance genes or virulence factors. Phenotypic testing further showed S. salivarius DB-B5 to be susceptible to clinically relevant antibiotics. S. salivarius DB-B5 displayed weak alpha-hemolysis, and does not produce biogenic amines. In a randomized, double-blind, placebo-controlled clinical study, consumption of S. salivarius DB-B5 at 10 billion CFU/day for 4 weeks by healthy adults was safe and well-tolerated (ClinicalTrials.gov registry number NCT04492631). This work has indicated that S. salivarius DB-B5 is a safe probiotic candidate.

Evolution of Lantibiotic Salivaricins: New Weapons to Fight Infectious Diseases.

Lantibiotic salivaricins are polycyclic peptides containing lanthionine and/or ß-methyllanthionine residues produced by certain strains of Streptococcus salivarius, which almost exclusively reside in the human oral cavity. The importance of these molecules stems from their antimicrobial activity towards relevant oral pathogens which has so far been applied through the development of salivaricin-producing probiotic strains. However, salivaricins may also prove to be of great value in the development of new and novel antibacterial therapies in this era of emerging antibiotic resistance. In this review, we describe the biosynthesis, antimicrobial activity, structure, and mode of action of the lantibiotic salivaricins characterized to date. Moreover, we also provide an expert opinion and suggestions for future development of this important field of microbiology.

Inhibitory effect of Streptococcus salivarius K12 and M18 on halitosis in vitro.

BACKGROUND: The aim of the study was to observe the antimicrobial activity of Porphyromonas gingivalis and Treponema denticola as well as the effect on reducing volatile sulfur compounds (VSCs). MATERIALS AND METHODS: After P. gingivalis and T. denticola were cultured with or without Streptococcus salivarius K12 and M18, VSCs were measured by Oral Chroma. In order to analyze the mechanism for malodor control, the antimicrobial activity of S. salivarius K12 and M18 against P. gingivalis and T. denticola was assessed. SPSS 21.0 was used for data analysis with the Kruskal-Wallis and Jonckheere-Terpstra tests. Mann-Whitney test was applied for post hoc analysis. RESULTS: P. gingivalis and T. denticola VSC levels were reduced by high concentrations of S. salivarius K12 and M18 during coculture. The concentrations were lower than those of single culture (p < .05). An antimicrobial effect was detected on P. gingivalis, and T. denticola by 50% S. salivarius K12 and M18. The spent culture medium and whole bacteria of S. salivarius K12 and M18 reduced the levels of VSCs below the amount in a single culture of P. gingivalis and T. denticola (p < .05). CONCLUSION: S. salivarius K12 and M18 decreased the levels of VSCs originating from P. gingivalis and T. denticola.

Subtle selectivity in a pheromone sensor triumvirate desynchronizes competence and predation in a human gut commensal.

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal Streptococcus salivarius, the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens.

Antagonistic effects of Streptococcus and Lactobacillus probiotics in pharyngeal biofilms.

Direct antagonism towards pathogens including Streptococcus pyogenes is a proposed mechanism of pharyngeal probiosis but off-target effects on the symbiotic microbiota of the throat are possible and may be beneficial, harmful or neutral. We have assessed the bacteriological effects of two candidate Lactobacillus probiotics and the established pharyngeal probiotic Streptococcus salivarius K12. Antagonism towards S. pyogenes and potential off-target effects were determined using sessile monospecies biofilms and pharyngeal microcosms, respectively. The candidate probiotics were antagonistic towards S. pyogenes (rank order of increasing potency, Lactobacillus acidophilus < Lactobacillus plantarum < Streptococcus salivarius) in the absence of significant acidification or cell-cell contact. Streptococcus salivarius and L. plantarum caused significant reductions in viable counts of streptococci in pharyngeal microbiotas, whilst S. salivarius also caused reductions in staphylococci. In contrast, changes in pharyngeal eubacterial DNA profiles were limited overall. In summary, the three candidate probiotics suppressed axenic Streptococcus pyogenes biofilms by mechanisms that did not depend on cell-cell contact or acidification and did not markedly destabilize complex pharyngeal microbiotas derived from healthy individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: Candidate probiotic bacteria deployed to prevent or treat bacterial pharyngitis will interact with the target bacteria such as Streptococcus pyogenes as well as with the microbiota of the throat, where off-target effects are possible. Three candidate probiotics Lactobacillus acidophilus, Lactobacillus plantarum and Streptococcus salivarius reduced viability within extant S. pyogenes biofilms through the elaboration of diffusible factors other than fermentation acids but did not markedly disrupt ex situ pharyngeal microcosms. This work demonstrates the application of in vitro pharyngeal models in the preclinical testing of the safety and efficacy of candidate pharyngeal probiotics.

Biology of Oral Streptococci.

Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity, which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this article, we discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus, because this genus is now divided into eight distinct groups, and oral species are found in six of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation, thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali, displaying an important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans. Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

Impact of growth pH and glucose concentrations on the CodY regulatory network in Streptococcus salivarius.

BACKGROUND: Streptococcus salivarius is an abundant isolate of the human oral microbiota. Since both pH and glucose availability fluctuate frequently in the oral cavity, the goal of this study was to investigate regulation by CodY, a conserved pleiotropic regulator of Gram positive bacteria, in response to these two signals. The chemostat culture system was employed to precisely control the growth parameters, and the transcriptomes of wild-type S. salivarius 57.I and its CodY-null derivative (ΔcodY) grown at pH 7 and 5.5, with limited and excessive glucose supply were determined. RESULTS: The transcriptomic analysis revealed that CodY was most active at pH 7 under conditions of glucose limitation. Based on whether a CodY binding consensus could be located in the 5' flanking region of the identified target, the transcriptomic analysis also found that CodY shaped the transcriptome via both direct and indirect regulation. Inactivation of codY reduced the glycolytic capacity and the viability of S. salivarius at pH 5.5 or in the presence of H2O2. Studies using the Galleria mellonella larva model showed that CodY was essential for the toxicity generated from S. salivarius infection, suggesting that CodY regulation was critical for immune evasion and systemic infections. Furthermore, the CodY-null mutant strain exhibited a clumping phenotype and reduced attachment in biofilm assays, suggesting that CodY also modulates cell wall metabolism. Finally, the expression of genes belonging to the CovR regulon was affected by codY inactivation, but CodY and CovR regulated these genes in opposite directions. CONCLUSIONS: Metabolic adaptation in response to nutrient availability and growth pH is tightly linked to stress responses and virulence expression in S. salivarius. The regulation of metabolism by CodY allows for the maximal utilization of available nutrients and ATP production. The counteractive regulation of the CovR regulon could fine tune the transcriptomes in response to environmental changes.

Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.

Comparing the impact of ultrafine particles from petrodiesel and biodiesel combustion to bacterial metabolism by targeted HPLC-MS/MS metabolic profiling.

Alterations of gut bacterial metabolism play an important role in their host metabolism, and can result in diseases such as obesity and diabetes. While many factors were discovered influencing the gut bacterial metabolism, exposure to ultrafine particles (UFPs) from engine combustions were recently proposed to be a potential risk factor for the perturbation of gut bacterial metabolism, and consequentially to obesity and diabetes development. This study focused on evaluation of how UFPs from diesel engine combustions impact gut bacterial metabolism. We hypothesize that UFPs from different type of diesel (petrodiesel vs. biodiesel) will both impact bacterial metabolism, and the degree of impact is also diesel type-dependent. Targeted metabolic profiling of 221 metabolites were applied to three model gut bacteria in vitro, Streptococcus salivarius, Lactobacillus acidophilus and Lactobacillus fermentum. UFPs from two types of fuels, petrodiesel (B0) and a biodiesel blend (B20: 20% soy biodiesel/80% B0 by volume), were exposed to the bacteria and their metabolic changes were compared. For each bacterial strain, metabolites with significantly changed abundance were observed in both perturbations, and all three strains have increased number of altered metabolites detected from B20 UFPs perturbation in comparison to B0 UFPs. Multivariate statistical analysis further confirmed that the metabolic profiles were clearly different between testing groups. Metabolic pathway analyses also demonstrated several important metabolic pathways, including pathways involves amino acids biosynthesis and sugar metabolism, were significantly impacted by UFPs exposure.

Comparison of Tunable Diode Laser Absorption Spectroscopy and Isothermal Micro-calorimetry for Non-invasive Detection of Microbial Growth in Media Fills.

Two methods were investigated for non-invasive microbial growth-detection in intact glass vials as possible techniques for automated inspection of media-filled units. Tunable diode laser absorption spectroscopy (TDLAS) was used to determine microbially induced changes in O2 and CO2 concentrations within the vial headspaces. Isothermal microcalorimetry (IMC) allowed the detection of metabolic heat production. Bacillus subtilis and Streptococcus salivarius were chosen as test organisms. Parameters as robustness, sensitivity, comparability and time to detection (TtD) were evaluated to assess method adequacy. Both methods robustly detected growth of the tested microorganisms within less than 76 hours using an initial inoculum of <10CFU. TDLA turned out to be less sensitive than TDLA and IMC, as some false negative results were observed. Compared to the visual media-fill examination of spiked samples, the investigated techniques were slightly slower regarding TtD. Although IMC showed shorter TtD than TDLAS the latter is proposed for automating the media-fill inspection, as larger throughput can be achieved. For routine use either TDLA or a combination of TDLA and TDLA should be considered. IMC may be helpful for replacing the sterility assessment of commercial drug products before release.

Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.