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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mode of action of dysgalacticin: a large heat-labile bacteriocin.

OBJECTIVES: The mode of action of dysgalacticin, a large (21.5 kDa), heat-labile bacteriocin that is active against the human pathogen Streptococcus pyogenes, was investigated. METHODS: We used recombinant dysgalacticin to determine its mode of action against S. pyogenes. Antimicrobial activity of dysgalacticin was determined by MIC assays and viability counts. The extracellular pH of glucose-energized S. pyogenes cell suspensions was measured to determine the influence of dysgalacticin on glucose fermentation. To examine the effect of dysgalacticin on glucose transport, uptake of [14C]glucose and the non-metabolizable analogue [3H]2-deoxyglucose (2DG) was measured. Furthermore, the effect of dysgalacticin on membrane integrity, intracellular potassium concentration, membrane potential and [14C]serine uptake was determined. RESULTS: Dysgalacticin was bactericidal towards S. pyogenes and inhibited glucose fermentation by non-growing cell suspensions. Dysgalacticin blocked transport of both glucose and 2DG, indicating that dysgalacticin targets the phosphoenolpyruvate-dependent glucose- and mannose-phosphotransferase system (PTS) of S. pyogenes. This inhibitory activity was voltage-independent, and in addition to the inhibition of glucose transport, dysgalacticin increased the permeability of the cytoplasmic membrane mediating leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [14C]serine uptake, a membrane potential-dependent process in S. pyogenes. CONCLUSIONS: Taken together, these data indicate that dysgalacticin targets the glucose- and/or mannose-PTS as a receptor leading to inhibition of sugar uptake. As a result of this interaction, dysgalacticin perturbs membrane integrity leading to loss of intracellular K+ ions and dissipation of the membrane potential, ultimately leading to cell death.

Inhibition by Lactobacillus sakei of other species in the flora of vacuum packaged raw meats during prolonged storage.

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm(-2) of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.

Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat.

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.

Ubericin A, a class IIa bacteriocin produced by Streptococcus uberis.

Streptococcus uberis, a causal agent of bovine mastitis, produces ubericin A, a 5.3-kDa class IIa (pediocin-like) bacteriocin, which was purified and characterized. The uba locus comprises two overlapping genes: ubaA (ubericin A precursor peptide) and ubaI (putative immunity protein). Ubericin A is the first streptococcal class IIa bacteriocin to be characterized.

Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity.

BACKGROUND: Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55alpha) and 55% (LtnA2 and C55beta) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems. RESULTS: So close is this relatedness that the hybrid peptide pairs LtnA1:C55beta and C55alpha:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55alpha, but not C55beta. In order to investigate in closer detail the significance of the differences between LtnA1 and C55alpha, three residues in LtnA1 were replaced with the equivalent residues in C55alpha. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop. CONCLUSION: It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system.

Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners.

BACKGROUND: Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats for Lactobacillus crispatus, L. gasseri and Gardnerella vaginalis and developed new formats for Atopobium vaginae, L. iners and L. jensenii to obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years. RESULTS: The 71 vaginal microflora samples of these women were categorized, using the Ison and Hay criteria, as refined by Verhelst et al. (2005), as follows: grade Ia: 8 samples, grade Iab: 10, grade Ib: 13, grade I-like: 10, grade II: 11, grade III: 12 and grade IV: 7.L. crispatus was found in all but 5 samples and was the most frequent Lactobacillus species detected. A significantly lower concentration of L. crispatus was found in grades II (p < 0.0001) and III (p = 0.002) compared to grade I. L. jensenii was found in all grades but showed higher concentration in grade Iab than in grade Ia (p = 0.024). A. vaginae and G. vaginalis were present in high concentrations in grade III, with log10 median concentrations (log10 MC), respectively of 9.0 and 9.2 cells/ml. Twenty (38.5%) of the 52 G. vaginalis positive samples were also positive for A. vaginae. In grade II we found almost no L. iners (log10 MC: 0/ml) but a high concentration of L. gasseri (log10 MC: 8.7/ml). By contrast, in grade III we found a high concentration of L. iners (log10 MC: 8.3/ml) and a low concentration of L. gasseri (log10 MC: 0/ml). These results show a negative association between L. gasseri and L. iners (r = -0.397, p = 0.001) and between L. gasseri and A. vaginae (r = -0.408, p < 0.0001). CONCLUSION: In our study we found a clear negative association between L. iners and L. gasseri and between A. vaginae and L. gasseri. Our results do not provide support for the generally held proposition that grade II is an intermediate stage between grades I and III, because L. gasseri, abundant in grade II is not predominant in grade III, whereas L. iners, abundant in grade III is present only in low numbers in grade II samples.

The effect of ingestion of milk supplemented with salivaricin A-producing Streptococcus salivarius on the bacteriocin-like inhibitory activity of streptococcal populations on the tongue.

The colonization efficacies of salivaricin A (SalA)-producing Streptococcus salivarius strains 20P3 and 5 were compared when given in milk to 219 children, using either 2-day or 9-day dosing regimens. Colonization levels overall were superior for strain 5, and the 9-day dosing schedule resulted in higher levels of both initial colonization and strain persistence. The indigenous streptococcal tongue populations of 20 (10.9%) of the 189 children in the 2-day trial showed markedly increased SalA-like inhibitory activity following use of the S. salivarius-supplemented milk. All 20 of these children were found to have had relatively small (<5% of total S. salivarius) indigenous tongue populations of SalA-producing S. salivarius, and the relative proportions and/or inhibitory activity of these SalA producers on the childrens' tongues increased following ingestion of the S. salivarius-supplemented milk. Because SalA is known to be strongly inhibitory to Streptococcus pyogenes, an important implication of this study is that the consumption of SalA-producing probiotic S. salivarius could potentially help to effect a sustained increase in SalA-mediated protection against S. pyogenes infection.