Persistence of the Oral Probiotic Streptococcus salivarius M18 Is Dose Dependent and Megaplasmid Transfer Can Augment Their Bacteriocin Production and Adhesion Characteristics

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18’s persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.     

Anti-Inflammatory Properties of Streptococcus salivarius,a Commensal Bacterium of the Oral Cavity and Digestive Tract

Streptococcus salivarius is one of the first colonizers of the human oral cavity and gut after birth and therefore may contribute to the establishment of immune homeostasis and regulation of host inflammatory responses. The anti-inflammatory potential of S. salivarius was first evaluated in vitro on human intestinal epithelial cells and human peripheral blood mononuclear cells. We show that live S. salivarius strains inhibited in vitro the activation of the NF-κB pathway on intestinal epithelial cells. We also demonstrate that the live S. salivarius JIM8772 strain significantly inhibited inflammation in severe and moderate colitis mouse models. These in vitro and in vivo anti-inflammatory properties were not found with heat-killed S. salivarius, suggesting a protective response exclusively with metabolically active bacteria.     

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118α are identical, their companion peptides (Sln2 and ABP-118β, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment

Staphylococcus aureus is an opportunistic pathogen that can colonize human and animal intestinal tracts, causing certain gastrointestinal diseases. The adherence of enteric pathogens to host intestinal epithelial cells is important for their pathogenesis. In the present study, Lactobacillus salivarius and Lactobacillus plantarum were investigated in vitro to examine their ability to competitively exclude S. aureus. Various factors involved in attachment, including bacterial status and cell concentration, growth phase, competition patterns, and surface-layer protein extracts, were also investigated. Live lactobacilli in the mid-log growth phase exhibited maximum inhibitory activity when lactobacilli were pre- or co-incubated with S. aureus. However, the inhibitory activity was significantly reduced when the lactobacilli were inactivated by heating or treated with LiCl. Furthermore, both lactobacilli possessed certain cell surface properties, such as hydrophobicity, autoaggregation, and coaggregation ability. L. salivarius and L. plantarum strongly inhibited S. aureus adherence to Caco-2 cells and their inhibition activity was significantly influenced by several factors that affect adhesion inhibition.     

Streptococcal Bacteriocin-Like Inhibitory Substances: Some Personal Insights into the Bacteriocin-Like Activities Produced by Streptococci Good and Bad

The background to the discovery and commercial development of the first Streptococcus salivarius probiotic is documented. A 40-year search of the genus Streptococcus for a harmless natural antagonist of Streptococcus pyogenes had as its operational basis a simple deferred antagonism “fingerprinting” procedure, the application of which results in each tested strain being accorded an inhibitor production (P)-type and inhibitor sensitivity (S)-type profile. Systematic application of this schema has opened a “Pandora’s Box” of novel streptococcal bacteriocin-like inhibitory substances (BLIS). The numerically prominent commensal S. salivarius is proposed to have a pivotal population-modulating role within the oral microbiota of humans. The probiotic strain S. salivarius K12 produces several megaplasmid-encoded BLIS including the lantibiotics salivaricin A and salivaricin B. Strain K12 and other BLIS-producing S. salivarius are currently in use or under development for application to the control of a variety of common maladies and infections of the human oral cavity.     

Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva

Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.     

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

Extended Safety Data for the Oral Cavity Probiotic Streptococcus salivarius K12

Previous studies of the bacteriocin-producing Streptococcus salivarius K12 monitored a variety of intrinsic strain characteristics of potential relevance to its application as an oral probiotic in humans. These included the content of antibiotic resistance and virulence determinants, the production of deleterious metabolic by-products and its genetic stability. In the present study, we examined additional safety factors including the responses of rats to either short- or long-term oral dosing with strain K12 preparations. In addition, the potential genotoxicity of strain K12 was tested using a bacterial reverse mutation assay. To determine the occurrence and concentrations in human saliva of S. salivarius having the same bacteriocin phenotype as strain K12, saliva samples from 780 children were evaluated. The level of dosing with strain K12 required to achieve oral cavity colonization levels similar to those occurring naturally for this type of bacteriocin-producing S. salivarius was established using 100 human subjects. Following the oral instillation of lyophilized S. salivarius K12 cells in these subjects, its persistence was not at levels higher than those found naturally for this type of bacterium. The various sets of data obtained in this study showed no evidence of genotoxicity and no acute or subacute toxicity effects associated with strain K12. Based on the previously published data, the long history of use by humans and the information presented here, it is concluded that S. salivarius K12 is safe for human consumption.     

Potential probiotic properties of phytase-producing Lactobacillus salivarius FC113

Probiotics have known efficacy as dietary supplements. Here, Lactobacillus strain F113 was characterized for probiotic use. Strain FC113 was selected as having the highest phytase activity (403.6 U) among tested strains showing acid tolerance and nitrite production. FC113 was tentatively identified as Lactobacillus salivarius based on an API 50 CHL assay and 16S rRNA gene analysis. The production of interleukin (IL)-1α and tumor necrosis factor (TNF)-α was measured in in vitro culture experiments. Cytokine production by L. salivarius FC113 at 1?×?10⁷ CFU/ml was approximately 175.5?±?36.40 pg/mL IL-1α and 353.5?±?61.79 pg/mL TNF-α. L. salivarius FC113 was profoundly resistant to artificial gastric juice (pH 2.5, 1 % pepsin), and persisted for 24 h in artificial bile acid. According to results obtained with an API ZYM kit, L. salivarius FC113 did not generate carcinogenic enzymes. L. salivarius F113 had an inhibitory effect on food-borne pathogens, and adhered strongly to HT-29 human intestinal epithelial cell lines. These results show that L. salivarius FC113 has probiotic characteristics, and exhibits high feed bioavailability in the host animal, in addition to an immune-stimulating effect.     

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.     

Chlamydomonas reinhardtii-expressed multimer of Bacteriocin LS2 potently inhibits the growth of bacteria

Bacteriocin LS2 was isolated from the Lactobacillus salivarius BGHO1 strain in 2012. Since then, its antibacterial activity has not been examined. Here, Chlamydomonas reinhardtii was used to express a C-terminal hemagglutinin (HA) and 6×His double tagged three repeats of Bacteriocin LS2 (3×Bacteriocin LS2). 3×Bacteriocin LS2 expression was stable following passaging in C. reinhardtii cells for six months and its yield accounted for 0.28% of total soluble proteins of the host cells. C. reinhardtii-derived 3×Bacteriocin LS2 inhibited the growth of four tested bacteria of both gram-positive and gram-negative with minimum inhibitory concentration (MIC) values between 75 and 90 μg/mL, indicating that this peptide is more potent than other bacteriocins like nesin and bacteriocin MA047A which have a MIC beyond 165 μg/mL in general. The recombinant 3×Bacteriocin LS2 maintained high stability over a wide range of temperature and pHs, showed tolerance to proteases, exhibited low hemolytic activity against rabbit erythrocytes and low cytotoxicity to human embryonic kidney 293 T (HEK 293 T) cells. In addition, C. reinhardtii-derived 3×Bacteriocin LS2 penetrated cell membranes and destroyed the morphology of targeted bacterial cells to different extents. In summary, our study shows that C. reinhardtii can be used as a platform for the production of active Bacteriocin LS2.     

Potential of Lactic Streptococci to Produce Bacteriocin

A survey was made on the bacteriocin-producing potential of lactic streptococci. Bacteriocin-like activities were isolated and partially purified from about 5% of the 280 strains investigated. The frequency of production varied from about 1% in Streptococcus lactis subsp. diacetylactis to 9 and 7.5% in S. lactis and Streptococcus cremoris, respectively. Eight strains of S. cremoris produced bacteriocins which, on the basis of heat stability at different pH values and inhibitory spectrum, could be divided into four types. From 54 S. lactis strains, 5 strains produced inhibitory substances, namely, three nisin-like antibiotics and two different bacteriocins. Only 1 of 93 S. lactis subsp. diacetylactis strains produced a bacteriocin which was very similar to bacteriocins of type I in S. cremoris. All of the bacteriocins that were partially purified by ammonium sulfate precipitation showed very limited inhibitory spectra. Most of the lactic streptococci and a few members of the genera Clostridium, Leuconostoc, and Pediococcus were inhibited. None of the bacteriocins acted on gram-negative bacteria. The bacteriocinogenic strains were also characterized on the basis of plasmid content. All strains possessed between one and nine plasmids ranging from 1 to 50 megadaltons.     

Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.     

Influence of Oral Probiotic <Emphasis Type="Italic">Streptococcus salivarius</Emphasis> K12 on Ear and Oral Cavity Health in Humans: Systematic Review

Traditionally, probiotics are linked to the good health of the intestine and most clinical studies focus on that field. Evidence of oral probiotic use for ear and oral cavity disease prevention with impact on human health is limited. This work reviews existing studies and literature on Streptococcus salivarius K12 as an oral probiotic and effects of S. salivarius K12 on human ear and oral cavity human health. The studies were accessed via database searches: MEDLINE, PubMed, and Elsevier. The search included/focused on/encompassed publications from 2003 to 2016 with keywords related to K12 Streptococcus salivarius, bacteriocin-like inhibitory substances (BLIS) K12, probiotic K12 salivarius, and K12 probiotic health effects. Only a small amount of studies was identified: the total of 68 studies was identified, 35 of which were relevant after screening, and 9 were included in the final analysis. Very little literature is available about the association/correlation between/connection/interrelation of S. salivarius K12 with/and human ear and oral cavity health. S. salivarius K12 may have a role in reducing the occurrence and/or severity of secretory otitis media (SOM) and also in prevention of streptococcal and viral pharyngotonsillitis in children. Research highlights that S. salivarius K12 has shown promising results in treatment of halitosis, but data are still deficient. Further studies need to be initiated to improve understanding of the association of oral probiotic S. salivarius K12 with human ear and oral cavity health.     

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.Subject terms: Bacteriology, Biofilms, Microbiome, Clinical microbiology     

In vitro Inhibition of Clinical Isolates of Otitis Media Pathogens by the Probiotic Streptococcus salivarius BLIS K12

Otitis media is a common childhood infection, frequently requiring antibiotics. With high rates of antibiotic prescribing and increasing antibiotic resistance, new strategies in otitis media prevention and treatment are needed. The aim of this study was to assess the in vitro inhibitory activity Streptococcus salivarius BLIS K12 against otitis media pathogens. Efficacy of the bacteriocin activity of S. salivarius BLIS K12 against the otitis media isolates was assessed using the deferred antagonism test. Overall, 48% of pathogenic isolates exhibited some growth inhibition by S. salivarius BLIS K12. S. salivarius BLIS K12 can inhibit the in vitro growth of the most common pathogens.

     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

Three Distinct Two-Component Systems Are Involved in Resistance to the Class I Bacteriocins,Nukacin ISK-1 and Nisin A,in Staphylococcus aureus

Staphylococcus aureus uses two-component systems (TCSs) to adapt to stressful environmental conditions. To colonize a host, S. aureus must resist bacteriocins produced by commensal bacteria. In a comprehensive analysis using individual TCS inactivation mutants, the inactivation of two TCSs, graRS and braRS, significantly increased the susceptibility to the class I bacteriocins, nukacin ISK-1 and nisin A, and inactivation of vraSR slightly increased the susceptibility to nukacin ISK-1. In addition, two ABC transporters (BraAB and VraDE) regulated by BraRS and one transporter (VraFG) regulated by GraRS were associated with resistance to nukacin ISK-1 and nisin A. We investigated the role of these three TCSs of S. aureus in co-culture with S. warneri, which produces nukacin ISK-1, and Lactococcus lactis, which produces nisin A. When co-cultured with S. warneri or L. lactis, the braRS mutant showed a significant decrease in its population compared with the wild-type, whereas the graRS and vraSR mutants showed slight decreases. Expression of vraDE was elevated significantly in S. aureus co-cultured with nisin A/nukacin ISK-1-producing strains. These results suggest that three distinct TCSs are involved in the resistance to nisin A and nukacin ISK-1. Additionally, braRS and its related transporters played a central role in S. aureus survival in co-culture with the strains producing nisin A and nukacin ISK-1.