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.

Human Systemic Immune Response to Ingestion of the Oral Probiotic Streptococcus salivarius BLIS K12.

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.

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.

Bacterial survival and adhesion for formulating new oral probiotic foods.

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.

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.

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.

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.

Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.

Alternative mechanisms of action of cationic antimicrobial peptides on bacteria.

Cationic antimicrobial peptides are a novel type of antibiotic offering much potential in the treatment of microbial-related diseases. They offer many advantages for commercial development, including a broad spectrum of action and modest size. However, despite the identification or synthetic production of thousands of such peptides, the mode of action remains elusive, except for a few examples. While the dogma for the mechanism of action of antimicrobial peptides against bacteria is believed to be through pore formation or membrane barrier disruption, some peptides clearly act differently and other intracellular target sites have been identified. This article presents an updated review of how cationic antimicrobial peptides are able to affect bacterial killing, with a focus on internal targets.

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Identification of nlmTE, the locus encoding the ABC transport system required for export of nonlantibiotic mutacins in Streptococcus mutans.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.