The oral microbiome, a complex ecosystem of microbes, is crucial for oral health. Imbalances in this ecosystem can lead to various oral diseases. Probiotics, live beneficial bacteria, offer a potential solution by strengthening oral defences. This study aimed to develop and evaluate a novel toothpaste containing Streptococcus salivarius M18, a probiotic strain. After ensuring compatibility with toothpaste ingredients, a stable formulation with desirable properties was created. The toothpaste demonstrated cleaning efficacy and antimicrobial activity against oral pathogens in vitro. A clinical trial involving healthy adults showed that all doses of the probiotic toothpaste significantly increased S. salivarius M18 levels in saliva, with the effect persisting even after discontinuation. These findings suggest that the toothpaste effectively delivers the probiotic to the oral cavity and promotes colonisation. Further research is needed to optimise the formulation and assess its long-term impact on oral health.

The discovery and application of bacteriocin-producing probiotics, such as Streptococcus salivarius K12 (BLIS K12), represent significant advances in the prevention and management of bacterial infections, particularly in the oral cavity and upper respiratory tract. Originally developed for its bacteriocin-mediated inhibition of the important bacterial pathogen Streptococcus pyogenes, BLIS K12 has more recently also demonstrated potential in the modulation and prevention of viral infections, including COVID-19. Emerging evidence also suggests a broader role for BLIS K12 in immune regulation, with implications for controlling hyperinflammatory responses and enhancing mucosal immunity. Of particular interest is recent work indicating that BLIS K12 can modulate antibody responses against viral antigens, such as the SARS-CoV-2 spike protein, positioning it as a unique adjunct in managing viral infections. This review chronicles the pathway of BLIS K12’s probiotic development, emphasizing its relevant bacteriocin mechanisms, oral health applications, emerging antiviral properties, and potential broader health benefits through immune modulation, all of which position it as a significant non-pharmacological adjunct in managing respiratory and immune health

Probiotics in cosmetics represent a new and burgeoning area of research, attracting attention from cosmetic manufacturers and customers looking for natural or additional alternatives in their skincare regimens. Probiotics not only promote beneficial microbiomes to improve skin health but can also modulate the visual, topographical, and mechanical attributes to improve skin quality. This study was conducted in healthy adults to determine the effect of direct skin application of a topical serum formulation containing the live probiotic Micrococcus luteus Q24 on several key cosmetic skin quality parameters and its beneficial impact on the modulation of the skin microbiome. Quantitative changes in various skin parameters were measured using a skin analyser device and skin swabs were analysed using whole-genome sequencing to monitor microbial diversity and levels of probiotic colonization. Compared to the baseline, significant reductions in the pores, spots, wrinkles, and impurities scores, and an increase of 101% in the hydration score were observed. Additionally, 45–80% of participants showed a decrease in pores, spots, wrinkles, and impurities and 90% of participants showed an increase in hydration after 25 days of probiotic serum application. The WGS analysis of the skin swab samples showed a significant increase in the relative abundance of M. luteus Q24. The results from this study indicate that topical application of a serum containing the probiotic M. luteus Q24 offers the benefit of improving skin health quality.

Synbiotics are mixtures of prebiotics and probiotics that enhance the activity of probiotic bacteria when co-administered to provide greater benefits to the host. Traditionally, the synbiotics that have been discovered enhance gut probiotic strains and are nutritionally complex molecules that survive digestive breakdown until they reach the later stages of the intestinal tract. Here, we screened and identified sugars or sugar substitutes as synbiotics for the oral probiotic strains Streptococcus salivarius BLIS K12 and BLIS M18. Using a modified deferred antagonism assay, we found that 0.5% (w/v) galactose and 2.5% (w/v) raffinose were the best candidates for use as synbiotics with BLIS K12 and M18, as they trigger enhanced antimicrobial activity against a range of bacteria representing species from the mouth, gut, and skin. Using reverse transcriptase quantitative PCR, we found that this enhanced antimicrobial activity was caused by the upregulation of the lantibiotic genes salA, salB, and sal9 in either K12 or M18. This led to the conclusion that either 2.5% (w/v) raffinose or 0.5% (w/v) galactose, respectively, are suitable synbiotics for use in conjunction with BLIS K12 and M18 to enhance probiotic performance.

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.

Probiotics developed for topical applications in humans have the potential to beneficially modulate microbial imbalances on the skin surface and thereby improve skin health. This study was conducted to determine whether topical formulations containing the human skin commensal Micrococcus luteus strain Q24 (BLIS Q24) are safe, tolerable and efficacious when used by healthy human subjects. M. luteus Q24 was assessed in vitro for haemolytic activity and its antibiotic susceptibility profile. Formulations of strain Q24 were evaluated for the preliminary safety and tolerability in healthy human participants. Forty-seven adults were randomly assigned to four single-site, single-blind randomised placebo or baseline controlled or active-controlled trials. Skin swab samples were collected for differential viable counts to monitor levels of probiotic colonisation. M. luteus Q24 was found to be non-haemolytic and susceptible to commonly used antibiotics. The M. luteus Q24 formulations were safe and tolerable and >90% of the participants reported improvements from baseline in the appearance (e.g., radiance and hydration) of their treated skin. Additionally, participants observed a reduction in pore size, skin clarity and enhanced skin softness. No adverse effects were reported. A dose-related significant increase was observed in the levels of M. luteus Q24 isolated from skin swabs of the probiotic-treated subjects. Placebo-controlled trials in human subjects involving the topical application of different doses of M. luteus Q24 formulations were supportive of the safety, tolerability and efficacy of probiotic M. luteus Q24. Self-reported skin health assessments by the subjects indicated that M. luteus Q24 has good potential as a probiotic for improving skin health quality.

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.

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.

Streptococcus salivarius K12 is an oral probiotic known to contribute to protection against oral pathogenic bacteria in humans. Studies of immune responses to S. salivarius K12 have focused on the oral cavity, and systemic immune responses have not yet been reported. The aim of this study was to identify acute systemic immune responses to the commercial product, S. salivarius BLIS K12, in a double-blinded, placebo-controlled human clinical trial. It was hypothesised that consumption of S. salivarius BLIS K12 would induce an anti-inflammatory response and a decrease in pro-inflammatory cytokines. Blood samples were obtained from participants prior to a single dose of S. salivarius BLIS K12 or a placebo and then secondary blood samples were obtained 24 h and 7 days post-consumption. Samples were analysed using multi-parametric flow cytometry, to quantify immune cell frequency changes, and by a LEGENDplex assay of human inflammatory cytokines. Consumption of S. salivarius BLIS K12 was associated with increased levels of IL-8 at 24 h. The frequency of Tregs increased in samples taken 7 days after probiotic consumption, and IL-10 concentrations were higher at 7 days than 24 h after consumption. There was no difference in the frequency and/or activation of CD4+ T cells, CD8+ T cells, B cells and NK cells. Interestingly, there was an increase in IL-12, 7 days after the consumption of S. salivarius BLIS K12. Collectively, this research demonstrates that ingestion of the probiotic S. salivarius K12 can induce changes in the systemic immune response. The implications of the generation and type of immune response warrant further study to determine potential health benefits.

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 are defined as live microorganisms, which, when administered in adequate amounts, confer health benefits to the host. Traditionally, probiotic food research has heavily focused on the genera Bifidobacteria and Lactobacilli, along with their benefits for gut health. Recently with the identification of new probiotic strains specifically intended for oral health applications, the development of probiotic foods for oral health benefits has garnered interest, with a renewed focus on identifying new food formats for delivering probiotics. The development of novel oral probiotic foods is highly complex, as the composition of a food matrix dictates: (1) bacterial viability during production and shelf life and (2) how bacteria partition with components within a food matrix and subsequently adhere to oral cavity surfaces. At present, virtually no information is available on oral probiotic strains such as Streptococcus salivarius; specifically, how orally-derived strains survive under different food parameters. Furthermore, limited information exists on the partition behavior of probiotics with food components, governed by physico-chemical interactions and adhesion phenomena. This review aspires to examine this framework by providing a foundation with existing literature related to the common probiotic genera, in order to inform and drive future attempts of designing new oral probiotic food formats.

The genus Streptococcus is one of the most diverse of the bacterial genera and presently comprises 70 defined species inhabiting a wide variety of ecological habitats. Certain species are used in the production of food products but the majority are commensal colonizers or pathogens of humans and other animals. Many are producers of bacteriocins, especially of the lantibiotic class; the tongue-dwelling Streptococcus salivarius one of the more prolific bacteriocinogenic species. Some strains of S. salivarius harbour especially large (> 100kb) megaplasmids, otherwise unreported amongst other oral bacteria. These properties, together with its extremely low pathogenic potential, favour S. salivarius as a source of oral probiotics to target infections of humans caused by other streptococci including pharyngitis (Streptococcus pyogenes) and dental caries (Streptococcus mutans). In this chapter, we provide current information on both the lantibiotic and heat-labile salivaricins produced by S. salivarius. We also present a section on the potential applications of bacteriocin-producing streptococci as oral probiotics, including a profile of S. salivarius probiotic products already available and a summary of some of the steps required to commercialize new strains.

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.

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.

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, β-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.

Consumers seeking health-promoting dietary supplements have long been conditioned to the regular ingestion of yoghurt as a convenient source of living beneficial microbes (viz. probiotics). Conventional probiotics have typically been bacteria of intestinal origin (especially lactobacilli and bifidobacteria) and their application has principally been to provide relief for maladies of the gastrointestinal tract. However, the realisation that much human illness can be linked either directly (dental caries, periodontal disease and candidosis) or indirectly (cardiovascular disease and perhaps even obesity) to the development of oral microbiota disequilibria has diverted much of the thrust of contemporary probiotic research towards the establishment and maintenance of a healthy oral microbiota. Step one was to determine whether conventional intestinal probiotics could influence the oral microbiota, but these (perhaps unsurprisingly) have no oral persistence and any oral cavity health benefits are transitory and largely attributable to immune stimulation. Enter, Streptococcus salivarius K12 - the world's first purposely-selected oral probiotic, a bacterium derived from the oral microbiota of a healthy human and shown to colonise the oral cavity and to express a wide variety of anti-competitor molecules, termed BLIS (bacteriocin-like inhibitory substances) capable of targeting oral pathogens and also thought to have a role in the stabilisation of a healthy oral microbiota.

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.