Beneficial modulation of human health in the oral cavity and beyond using bacteriocin-like inhibitory substance-producing streptococcal probiotics.

The human oral cavity contains a diversity of microbial habitats that have been adopted and adapted to as homeland by an amazingly heterogeneous population of microorganisms collectively referred to as the oral microbiota. These microbes generally co-habit in harmonious homeostasis. However, under conditions of imposed stress, as with changes to the host's physiology or nutritional status, or as a response to foreign microbial or antimicrobial incursions, some components of the oral "microbiome" (viz. the in situ microbiota) may enter a dysbiotic state. This microbiome dysbiosis can manifest in a variety of guises including streptococcal sore throats, dental caries, oral thrush, halitosis and periodontal disease. Most of the strategies currently available for the management or treatment of microbial diseases of the oral cavity focus on the repetitive "broad sweep" and short-term culling of oral microbe populations, hopefully including the perceived principal pathogens. Both physical and chemical techniques are used. However, the application of more focused approaches to the harnessing or elimination of key oral cavity pathogens is now feasible through the use of probiotic strains that are naturally adapted for oral cavity colonization and also are equipped to produce anti-competitor molecules such as the bacteriocins and bacteriocin-like inhibitory substances (viz BLIS). Some of these probiotics are capable of suppressing the proliferation of a variety of recognized microbial pathogens of the human mouth, thereby assisting with the restoration of oral microbiome homeostasis. BLIS K12 and BLIS M18, the progenitors of the BLIS-producing oral probiotics, are members of the human oral cavity commensal species Streptococcus salivarius. More recently however, a number of other streptococcal and some non-streptococcal candidate oral probiotics have also been promoted. What is becoming increasingly apparent is that the future for oral probiotic applications will probably extend well beyond the attempted limitation of the direct pathological consequences of oral microbiome dysbiosis to also encompass a plethora of systemic diseases and disorders of the human host. The background to and the evolving prospects for the beneficial modulation of the oral microbiome via the application of BLIS-producing S. salivarius probiotics comprises the principal focus of the present review.

Interferon Gamma Response in Human Saliva Following Exposure to the Oral Probiotic Streptococcus salivarius BLIS K12.

Streptococcus salivarius BLIS K12 is a probiotic strain developed for application to the oral cavity. The strain was originally characterised for its in vitro antibacterial activity against the prominent oral pathogen Streptococcus pyogenes. More recent research has expanded its applications to include reducing halitosis, preventing otitis media and protecting against virus infections of the respiratory tract. A potential mechanism for this anti-viral activity could be the stimulation of salivary interferon gamma (IFN-γ) production in the oral cavity. The aim of this study was to investigate whether the ingestion of and oral cavity colonisation by S. salivarius BLIS K12 is associated with enhancement of IFN-γ levels in saliva. Application of ELISA demonstrated that consumption of S. salivarius BLIS K12 effected an increase in salivary IFN-γ, and this response was more consistent with use of viable cells than following ingestion of heat-killed S. salivarius BLIS K12. Interestingly, those subjects who more successfully colonised with S. salivarius BLIS K12 did not experience a relatively larger increase in their IFN-γ levels, indicating that the observed IFN-γ response occurs independently of colonisation efficacy. In summary, the consumption of S. salivarius BLIS K12 increases salivary levels of IFN-γ, an effect that may contribute to protection of the host against certain virus infections.

Skin Microbiome-The Next Frontier for Probiotic Intervention.

The skin is the largest organ in the human body, and it orchestrates many functions that are fundamentally important for our survival. Although the skin might appear to present a relatively inhospitable or even hostile environment, a multitude of commensals and also some potentially pathogenic microorganisms have successfully adapted to survive and/or thrive within the diverse ecological niches created by the skin's topographical architecture. Dysbiosis within these microbial populations can result in the emergence and pathological progression of skin diseases. Unsurprisingly, this has led to a new focus of research both for the medical dermatology and cosmetic industries that is concerned with modulation of the skin microbiome to help address common microbially mediated or modulated conditions such as acne, body odour, and atopic dermatitis. This review presents an overview of our current understanding of the complex relationship of the skin with its microbiome and then introduces the concept of probiotic intervention for the management of microbial dysbiosis within the skin ecosystem.

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.

Probiotics at War Against Viruses: What Is Missing From the Picture?

Our world is now facing a multitude of novel infectious diseases. Bacterial infections are treated with antibiotics, albeit with increasing difficulty as many of the more common causes of infection have now developed broad spectrum antimicrobial resistance. However, there is now an even greater challenge from both old and new viruses capable of causing respiratory, enteric, and urogenital infections. Reports of viruses resistant to frontline therapeutic drugs are steadily increasing and there is an urgent need to develop novel antiviral agents. Although this all makes sense, it seems rather strange that relatively little attention has been given to the antiviral capabilities of probiotics. Over the years, beneficial strains of lactic acid bacteria (LAB) have been successfully used to treat gastrointestinal, oral, and vaginal infections, and some can also effect a reduction in serum cholesterol levels. Some probiotics prevent gastrointestinal dysbiosis and, by doing so, reduce the risk of developing secondary infections. Other probiotics exhibit anti-tumor and immunomodulating properties, and in some studies, antiviral activities have been reported for probiotic bacteria and/or their metabolites. Unfortunately, the mechanistic basis of the observed beneficial effects of probiotics in countering viral infections is sometimes unclear. Interestingly, in COVID-19 patients, a clear decrease has been observed in cell numbers of Lactobacillus and Bifidobacterium spp., both of which are common sources of intestinal probiotics. The present review, specifically motivated by the need to implement effective new counters to SARS-CoV-2, focusses attention on viruses capable of co-infecting humans and other animals and specifically explores the potential of probiotic bacteria and their metabolites to intervene with the process of virus infection. The goal is to help to provide a more informed background for the planning of future probiotic-based antiviral research.

A TaqMan™-based quantitative PCR screening assay for the probiotic Streptococcus salivarius K12 based on the specific detection of its megaplasmid-associated salivaricin B locus.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Investigation of Streptococcus salivarius-mediated inhibition of pneumococcal adherence to pharyngeal epithelial cells.

BACKGROUND: Pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. The probiotic bacterium, Streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. We investigated the mechanism(s) of inhibition using a human pharyngeal epithelial cell line (Detroit 562) following pre-administration of two different strains of S. salivarius. RESULTS: Whilst the bacteriocin-encoding megaplasmids of S. salivarius strains K12 and M18 were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. Experiments testing S. salivarius K12 and two pneumococcal isolates (serotypes 19F and 6A) showed that inhibition of 19F may involve S. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of K12 and 19F, and no inhibition occurred when K12 was prevented from contacting epithelial cells. K12-mediated inhibition of adherence by 6A may involve additional mechanisms, since no correlation was observed between adherence of K12 and 6A, and K12 could inhibit 6A adherence in the absence of cell contact. CONCLUSIONS: These results suggest that S. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. These findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.

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.

Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization.

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy.

Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial.

The prevalence of dental caries continues to increase, and novel strategies to reverse this trend appear necessary. The probiotic Streptococcus salivarius strain M18 offers the potential to confer oral health benefits as it produces bacteriocins targeting the important cariogenic species Streptococcus mutans, as well as the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification, respectively. In a randomized double-blind, placebo-controlled study of 100 dental caries-active children, treatment with M18 was administered for 3 months and the participants were assessed for changes to their plaque score and gingival and soft-tissue health and to their salivary levels of S. salivarius, S. mutans, lactobacilli, ß-haemolytic streptococci and Candida species. At treatment end, the plaque scores were significantly (P = 0.05) lower for children in the M18-treated group, especially in subjects having high initial plaque scores. The absence of any significant adverse events supported the safety of the probiotic treatment. Cell-culture analyses of sequential saliva samples showed no differences between the probiotic and placebo groups in counts of the specifically enumerated oral micro-organisms, with the exception of the subgroup of the M18-treated children who appeared to have been colonized most effectively with M18. This subgroup exhibited reduced S. mutans counts, indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the efficiency of colonization is increased. It was concluded that S. salivarius M18 can provide oral health benefits when taken regularly.

Developing oral probiotics from Streptococcus salivarius.

Considerable human illness can be linked to the development of oral microbiota disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has emerged as an important source of safe and efficacious probiotics, capable of fostering more balanced, health-associated oral microbiota. Strain K12, the prototype S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes infections, now has an expanded repertoire of health-promoting applications. K12 and several more recently proposed S. salivarius probiotics are now being applied to control diverse bacterial consortia infections including otitis media, halitosis and dental caries. Other potential applications include upregulation of immunological defenses against respiratory viral infections and treatment of oral candidosis. An overview of the key steps required for probiotic development is also presented.

Salivaricin G32, a Homolog of the Prototype Streptococcus pyogenes Nisin-Like Lantibiotic SA-FF22, Produced by the Commensal Species Streptococcus salivarius.

Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes-produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.

The spiFEG locus in Streptococcus infantarius subsp. infantarius BAA-102 confers protection against nisin U.

Nisin U is a member of the extended nisin family of lantibiotics. Here we identify the presence of nisin U immunity gene homologues in Streptococcus infantarius subsp. infantarius BAA-102. Heterologous expression of these genes in Lactococcus lactis subsp. cremoris HP confers protection to nisin U and other members of the nisin family, thereby establishing that the recently identified phenomenon of resistance through immune mimicry also occurs with respect to nisin.

Genome sequence of the bacteriocin-producing oral probiotic Streptococcus salivarius strain M18.

Streptococcus salivarius is a Gram-positive bacterial commensal and pioneer colonizer of the human oral cavity. Many strains produce ribosomally synthesized proteinaceous antibiotics (bacteriocins), and some strains have been developed for use as oral probiotics. Here, we present the draft genome sequence of the bacteriocin-producing oral probiotic S. salivarius strain M18.

Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487.

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery.

Metabolism of L-selenomethionine and selenite by probiotic bacteria: in vitro and in vivo studies.

Since selenium supplements have been shown to undergo biotransformation in the gut, probiotic treatment in combination with selenium supplements may change selenium disposition. We investigated the metabolism of L-selenomethionine (SeMet) and selenite by probiotic bacteria in vitro and the disposition of selenium after probiotic treatment followed by oral dosing with SeMet and selenite in rats. When SeMet was incubated anaerobically with individual antibiotic-resistant probiotic strains (Streptococcus salivarius K12, Lactobacillus rhamnosus 67B, Lactobacillus acidophilus L10, and Bifidobacterium lactis LAFTI® B94) at 37°C for 24 h, 11-18% was metabolized with 44-80% of SeMet lost being converted to dimethyldiselenide (DMDSe) and dimethylselenide (DMSe). In similar incubations with selenite, metabolism was more extensive (26-100%) particularly by the lactobacilli with 0-4.8% of selenite lost being converted to DMSe and DMDSe accompanied by the formation of elemental selenium. Four groups of rats (n = 5/group) received a single oral dose of either SeMet or selenite (2 mg selenium/kg) at the time of the last dose of a probiotic mixture or its vehicle (lyoprotectant mixture used to maintain cell viability) administered every 12 h for 3 days. Another three groups of rats (n = 3/group) received a single oral dose of saline or SeMet or selenite at the same dose (untreated rats). Serum selenium concentrations over the subsequent 24 h were not significantly different between probiotic and vehicle treated rats but appeared to be more sustained (SeMet) or higher (selenite) than in the corresponding groups of untreated rats. Probiotic treated rats given SeMet also had selenium concentrations at 24 h that were significantly higher in liver and lower in kidney than untreated rats given SeMet. Thus, treatment with probiotics followed by SeMet significantly affects tissue levels of selenium.

Identification of DysI, the immunity factor of the streptococcal bacteriocin dysgalacticin.

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG-repB-dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

Purification and characterization of a novel delta-lysin variant that inhibits Staphylococcus aureus and has limited hemolytic activity.

Delta-lysins (DL) that are produced by various species of staphylococci are not widely known for their antimicrobial activity. We have purified and characterized a novel DL variant, E229DL and examined its spectrum of inhibitory activity. The biological activity of E229DL, produced by Staphylococcus epidermidis strain E229, shows relatively broad-spectrum activity against Gram-positive pathogens, including representatives of MRSA and epidemic MRSA type 15. E229DL was purified to homogeneity from 95% acidified-methanol extracts of cell cultures by using a series of reversed-phase chromatographic separations. The fully processed form of E229DL is a 25-amino-acid peptide with a predicted mass of 2841.4 Da, but the purified biologically active molecule appears to be N-formylated (mass 2867.33 Da). The DL gene (hld) resembles that of other types of DL, but differs in five codons with hld in Staphylococcus aureus (26 residues) and one codon with the closest homolog, the hld-II in S. warneri (25 residues). The characterization of E229DL showed that its activity is stable in agar exposed to high temperatures (80 degrees C/45 min). In addition, biological testing of the native and synthetic peptides against a range of human and animal erythrocytes and Vero cells indicated that E229DL is an antibacterial agent with no detectable cytopathic effects at concentrations equivalent to the minimum inhibitory concentration for EMRSA15-A208. Initial investigation of the mode of action of E229DL indicated that it is rapidly lytic for target cells. This is the first description of a native form of DL having only limited cytotoxic activity for eukaryotic cells at concentrations that are inhibitory to staphylococci.

Something Old and Something New: An Update on the Amazing Repertoire of Bacteriocins Produced by Streptococcus salivarius.

Streptococcus salivarius has an exclusive and intimate association with humans. We are its sole natural host, and its contribution to the relationship appears overwhelmingly benevolent. Beautifully adapted to its preferred habitat, the human tongue, it only rarely ventures far from this location in the healthy host and indeed appears ill-equipped to become invasive due to a scarcity of virulence attributes. We consider that its strategically advantageous lingual location and numerical predominance allow S. salivarius to carry out a population surveillance and modulation role within the oral microbiota. Some strains are armed with complex arrays of targeted antibiotic weaponry, much of which belongs to the lantibiotic class of bacteriocins and a key to their ability to assemble and utilize this armament is their possession of transmissible multi-bacteriocin-encoding megaplasmid DNA. This review traces the origins of research into S. salivarius bacteriocins and bacteriocin-like inhibitory substances, showcases some of the inhibitory activities that we currently have knowledge of, and speculates about potential directions for ongoing investigation and probiotic application of this previously under-rated human commensal.

Production of the Bsa lantibiotic by community-acquired Staphylococcus aureus strains.

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.