Safety assessment of Streptococcus salivarius M18 a probiotic for oral health.

The development of probiotics targeting non-intestinal body sites continues to generate interest amongst researchers, biotech companies and consumers alike. A key consideration for any bacterial strain to be developed into a probiotic is a robust assessment of its safety profile. Streptococcus salivarius strain M18 was originally isolated from a healthy adult and evaluated for its probiotic capabilities targeted to dental and oral health applications. This publication presents the safety characterisation of strain M18. Application of a diverse range of techniques showed that strain M18 can be specifically distinguished from other S. salivarius using a variety of molecular and phenotypic methodologies and that it lacks any relevant antibiotic resistance or virulence determinants. Direct comparison of the strain M18 safety profile with that of the prototype S. salivarius probiotic, S. salivarius strain K12, supports the proposition that strain M18 is indeed safe for probiotic application in humans.

Bacteriocins of Gram-positive bacteria having activity spectra extending beyond closely-related species.

Bacteriocins are bacterially-produced antimicrobial peptides that have killing activity principally against other relatively closely-related bacteria. Some bacteriocins of the lactic acid bacteria (LAB) have for many years been extensively applied in food biopreservation. However, especially during the last decade, a number of reports have appeared about unanticipated extensions to the generally rather narrow anti-bacterial activity spectrum of some of the LAB bacteriocins and novel applications have been proposed for bacteriocins ranging from controlling the growth of an increasingly-heterogeneous variety of pathogens, including Gram-negative multidrug resistant bacteria, viruses, yeasts, and in particular, difficult to control Mycobacterium spp., to their potential application as anticancer agents. How best can we assess this now rapidly-accumulating stream of reports on potential future applications of bacteriocins? Where is the line between realistic, science-based proposals and highly-speculative fiction and what are the 'critical points' that might help us to draw this line? In this review, we have attempted to analyse a selection of the presently-available data concerning relatively 'unorthodox' (i.e. beyond food preservation) applications of bacteriocins, and, by utilising our set of 'critical points', we endeavour to identify essential or/and missing information that appear crucial for success of the proposed applications.

Beneficial microbes for the oral cavity: time to harness the oral streptococci?

Indigenous microbes are known to influence human health outcomes and various approaches are now being made to positively modulate these microbe-induced outcomes via the administration of probiotics. The application of probiotics that are specific to the oral cavity is a relatively undeveloped field, and their emergence has largely occurred as a reasoned follow-up to initial studies in which probiotics that had already been developed and obtained regulatory approval for intestinal applications were then also evaluated for their putative influence on oral microbiota functionality. These attempts to extend the application of existing probiotics were probably at least in part motivated by recognition of the substantial safety and regulatory hurdles that must be overcome prior to the introduction of a novel probiotic agent. Nevertheless, from an efficacy perspective it appears more logical to develop microbes of oral origin as the specific providers of probiotic solutions for oral diseases, rather than attempting to adapt intestinally-derived strains for this role. Oral bacteria and their bioactive molecules have evolved to operate optimally in this environment and in some cases are known to persist only in oral sites. Amongst the bacteria of more than 700 species now identified within the human oral microbiota, it is the streptococci that are numerically predominant. Although this review highlights the development of the oral cavity bacterium Streptococcus salivarius as an oral probiotic, a number of other streptococcal species have also been shown to have considerable potential as probiotic candidates.

Evaluation of safety and human tolerance of the oral probiotic Streptococcus salivarius K12: a randomized, placebo-controlled, double-blind study.

Streptococcus salivarius is naturally a predominant member of the human oropharynx and the commercial probiotic strain K12 has been consumed for more than a decade. The present study examines the health responses of human volunteers to oral ingestion of high doses of S. salivarius K12. A randomized group of 53 subjects received a dose of 1 × 10(10)cfu S. salivarius K12 (N=25) or placebo (N=28) for 28 days, followed by a 28-day wash out period. Blood, urine and saliva samples were collected at baseline and following treatment and analyzed, while the oral and gastrointestinal tolerance of the subjects to the dosing regimen was determined by use of questionnaires. Adverse events (AE)s were recorded for both groups. No statistically significant differences between the probiotic and placebo treated groups were detected in either the blood clinical chemistry or hematology results (P>0.05). The questionnaire responses of the subjects indicated that both treatments were well tolerated. The frequency and intensity of AEs was similar in the two groups. This data demonstrates that the daily ingestion of S. salivarius K12 over a 28-day period does not adversely affect the human host and supports the safety of its oral delivery in a food-based carrier.

Salivaricin 9, a new lantibiotic produced by Streptococcus salivarius.

Salivaricin 9 (Sal9) is a 2560 Da lantibiotic having just 46 % amino acid identity with its closest known homologue, the Streptococcus pyogenes lantibiotic SA-FF22. The Sal9 locus (designated siv) in Streptococcus salivarius strain 9 was partially sequenced and localized to an approximately 170 kb megaplasmid, which also harbours the locus for the lantibiotic salivaricin A4. The entire locus was fully characterized in the draft genome sequence of S. salivarius strain JIM8780 and shown to consist of eight genes, having the following putative functions: sivK, sensor kinase; sivR, response regulator; sivA, Sal9 precursor peptide; sivM, lantibiotic modification enzyme; sivT, ABC transporter involved in the export of Sal9 and concomitant cleavage of its leader peptide; and sivFEG, encoding lantibiotic self-immunity. Intriguingly, in contrast to strain 9, the siv locus was chromosomally located in strain JIM8780--the first lantibiotic locus shown not to be exclusively plasmid-associated in S. salivarius. Sal9-containing extracts specifically induced lantibiotic production in both strain 9 and strain JIM8780, indicating that Sal9 functions as a signal peptide for upregulation of its own biosynthesis. Screening representative strains of three streptococcal species (S. salivarius, S. pyogenes and S. mitis) for sivA indicated that it was present only in S. salivarius, with 12 of 28 tested S. salivarius positive. Since Sal9 was inhibitory to all tested S. pyogenes strains it appears to have potential as an important component of the bacteriocin armoury of S. salivarius probiotics intended to control S. pyogenes infections of the human oral cavity.

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei.

Commercially prepared lamb was stored at -1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.

Extended Safety Data for the Oral Cavity Probiotic Streptococcus salivarius K12.

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

Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12.

A powder preparation of the oral probiotic Streptococcus salivarius K12 has been given to 19 young otitis media-prone children following a 3-day course of amoxicillin administered as a preliminary to ventilation tube placement. In two subjects, the use of strain K12 appeared to effect the expansion of an indigenous population of inhibitory S. salivarius. In other children, strain K12 colonisation extended beyond the oral cavity to also include the nasopharynx or adenoid tissue. The relatively low proportion (33%) of subjects that colonised was attributed to failure of the amoxicillin pre-treatment to sufficiently reduce the indigenous S. salivarius populations prior to dosing with strain K12 powder.

Commensal bacilli inhibitory to mastitis pathogens isolated from the udder microbiota of healthy cows.

AIMS: To isolate from the microbiota of the healthy cow udder commensal bacteria having antimicrobial activity against bovine mastitis pathogens, with a long-term view to their potential application as antimastitis probiotics. METHODS AND RESULTS: Bacterial isolates from four healthy cow udders were tested for inhibitory activity against three Gram-positive indicator bacteria. This led to the selection of nine broadly inhibitory strains. All were of the Bacillus genus and their antimicrobial activities, which appeared heterogeneous on the basis of their antibacterial spectra and heat susceptibilities, enabled grouping of the inhibitory bacilli into six different inhibitory profiles. All displayed strong in vitro activity against Gram-positive mastitis pathogens. Inhibitory bacilli were recovered from each of the 11 udder samples collected over 7 months from one of these cows and the isolates included representatives of all six inhibitory profiles. CONCLUSIONS: Bacilli present in the udder microbiota of healthy cows can produce a variety of broadly active inhibitors of Gram-positive bacteria, including potential mastitis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibitor-producing strains of commensal Bacillus species have been identified, which may have the potential for use as possible antimastitis probiotics.

A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters.

AIMS: To determine whether dosing with bacteriocin-producing Streptococcus salivarius following an antimicrobial mouthwash effects a change in oral malodour parameters and in the composition of the oral microbiota of subjects with halitosis. MATERIALS AND RESULTS: Twenty-three subjects with halitosis undertook a 3-day regimen of chlorhexidine (CHX) mouth rinsing, followed at intervals by the use of lozenges containing either S. salivarius K12 or placebo. Assessment of the subjects' volatile sulphur compound (VSC) levels 1 week after treatment initiation showed that 85% of the K12-treated group and 30% of the placebo group had substantial (>100 ppb) reductions. The bacterial composition of the saliva was monitored by culture and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Changes in the PCR-DGGE profiles occurred in most subjects following K12 treatment. In vitro testing showed that S. salivarius K12 suppressed the growth of black-pigmented bacteria in saliva samples and also in various reference strains of bacteria implicated in halitosis. CONCLUSIONS: Administration of bacteriocin-producing S. salivarius after an oral antimicrobial mouthwash reduces oral VSC levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this preliminary study indicates that the replacement of bacteria implicated in halitosis by colonization with competitive bacteria such as S. salivarius K12 may provide an effective strategy to reduce the severity of halitosis.

The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics.

The primary treatment for oral malodour is the reduction of bacterial populations, especially those present on the tongue, by use of a variety of antimicrobial agents or mechanical devices. However, shortly after treatment the problematic bacteria quickly repopulate the tongue and the malodour returns. In our studies, we have used a broadly-active antimicrobial (chlorhexidine) to effect temporary depletion of the oral microbiota and then have attempted to repopulate the tongue surface with Streptococcus salivarius K12, a benign commensal probiotic. The objective of this is to prevent re-establishment of non-desirable bacterial populations and thus help limit the re-occurrence of oral malodour over a prolonged period. In this paper, we discuss why contemporary probiotics are inadequate for treatment of oral malodour and examine the rationale for selection of particular bacterial species for future use in the treatment of this condition. In our preliminary trials of the use of a chlorhexidine rinse followed by strain K12 lozenges, the majority (8/13) of subjects with confirmed halitosis maintained reduced breath levels of volatile sulphur compounds for at least 2 weeks. We conclude that probiotic bacterial strains originally sourced from the indigenous oral microbiotas of healthy humans may have potential application as adjuncts for the prevention and treatment of halitosis.

Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius.

BACKGROUND & OBJECTIVES: Streptococcus salivarius is a numerically prominent member of the human oral microbiota that produces a variety of bacteriocin-like inhibitory substances (BLIS) having in vitro inhibitory activity against S. pyogenes. Our previous studies of S. salivarius isolates from children using a deferred antagonism BLIS production (P)-typing scheme showed that the 9 per cent of children having large populations of P-type 677 S. salivarius experienced fewer S. pyogenes acquisitions than either the 11 per cent of children having predominant P-type 226 populations or the 60 per cent of children with largely non-inhibitory (P-type 000) S. salivarius. Amongst the other BLIS P-types detected were a number of strongly-inhibitory (P-type 777) S. salivarius. In the present study the inhibitory agents produced by prototype strains of P-types 226, 677 and 777 S. salivarius are compared. METHODS: The prototype BLIS-producing S. salivarius strains SN, 20P3, and K12 were isolated from tongue swabbings. BLIS P-typing was done using standard procedures. The BLIS molecules were purified and characterized. RESULTS: S. salivarius SN (P-type 226) produces a heat-labile muramidase. S. salivarius 20P3 (P-type 677) produces the 2315 Da lantibiotic salivaricin A and S. salivarius K12 (P-type 777) produces two lantibiotics; salivaricin A2 (2368 Da) and salivaricin B (2733 Da). INTERPRETATION & CONCLUSION: The P-type 777 S. salivarius strain produced salivaricin A2 and salivaricin B. The combined production of two anti-S. pyogenes BLIS activities by this strain indicates that it could be adopted as a colonizing strain in bacterial interference trials.

Genetic basis for mutacin N and of its relationship to mutacin I.

BACKGROUND & OBJECTIVES: The mutans streptococci (MS) are a group of 7 species of dental cariesassociated bacteria of which Streptococcus mutans and Streptococcus sobrinus are the most important in humans. Many MS produce bacteriocin-like inhibitory substances (BLIS), some of which have been characterised as small peptides capable of inhibiting the growth of closely-related species. These peptides have most commonly been referred to as mutacins. S. mutans strains N and UA140 appear to have closely similar BLIS activities. Both produce mutacins that seem to target the same species of bacteria. On closer analysis however, these two strains have been shown to produce distinctly different mutacins, known as mutacin N and mutacin I respectively. In the present study the mutacin N structural gene (mutN) was cloned and compared with the mutacin I structural gene (mutA). METHODS: Cloning and sequencing of S. mutans N was done. The distribution of mutN using DNA from 216 streptococcal strains was determined by dot blotting. RESULTS: Mut N was cloned and sequenced from an 1800 bp Bam HI/Eco RI fragment. PCR with the mutN primers mutNF and mutNR on the four mutN-positive strains identified identical bands to S. mutans N. The location of mutN differs significantly from that of mutA in that it is directly upstream of comC, a gene encoding a putative competence stimulating factor. INTERPRETATION & CONCLUSION: The close upstream proximity of mutN to comC suggests a link between mutacin N production and competence development. Further studies need to be done to detect competence-related genes in S. mutans strain N.

Different bacteriocin activities of Streptococcus mutans reflect distinct phylogenetic lineages.

Bacteriocins produced by mutans streptococci are known as mutacins. In this study 16 broadly active mutacin-producing Streptococcus mutans strains from New Zealand, North America and Europe were classified into four groups (A-D) on the basis of differences in their activity in deferred antagonism tests against either the homologous producer strain (to test for presence of self-immunity) or indicator strains Staphylococcus aureus 46 and Enterococcus faecium TE1. Two of the strains included in the study (UA140 and UA96) were representatives of the group I and II mutacin producer strains previously described by Caufield and co-workers. One of the New Zealand isolates of group A (S. mutans strain N) appeared to produce inhibitory activity similar to that of the group I prototype strain UA140. Four other New Zealand isolates of group B (S. mutans strains M19, M34, B34 and D14) had mutacin II-like activity. The group B mutacin producers differed from the group A mutacin producers in their additional activity against Staph. aureus 46. Seven S. mutans strains (M46, B46, B57, M12, M28, B28 and 13M) were distinguished from the group A and group B mutacin producers in that they inhibited E. faecium TE1. These were called group C mutacin producers. Strains H7 and H23 resembled the group C strains in their action on both indicator strains TE1 and 46. However, these two strains failed to exhibit immunity to their own inhibitory products in the deferred antagonism test and were separately classified as group D mutacin producers. Phylogenetic analysis of the strains by several genotypic and phenotypic characteristics revealed that the mutacin groups were associated with distinct evolutionary lineages of S. mutans.

Intra- and interspecies signaling between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1 lantibiotic peptides.

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

Diverse activity spectra of bacteriocin-like inhibitory substances having activity against mutans streptococci.

Mutans streptococci (MS) are known to be causative agents of dental caries. It has been suggested that these cariogenic bacteria could be eliminated from dental plaque by application of bacteriocins or bacteriocin-like inhibitory substances (BLIS). In the present study 272 bacterial strains of the genera Streptococcus, Enterococcus and Staphylococcus were tested for their production of BLIS activity against MS by use of a deferred antagonism test on agar media supplemented with either whole blood or yeast extract. Although only 14.3% of the test strains displayed anti-MS activity, the inhibitory agents produced by these strains were characterised by considerable diversity in the range of their inhibitory action against both MS and other common oral streptococcal species. It is suggested that combinations of relatively specifically targeted anti-MS BLIS may have potential application to the prevention of dental caries.

A new alkaline pH-adjusted medium enhances detection of beta-hemolytic streptococci by minimizing bacterial interference due to Streptococcus salivarius.

A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing Streptococcus salivarius against beta-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of beta-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of S. salivarius bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of beta-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The beta-hemolytic streptococcal isolates recovered from the samples included 314 group A (S. pyogenes), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded S. pyogenes, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of S. pyogenes isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of S. pyogenes isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07; P < 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient S. pyogenes strains which might otherwise go undetected on SBA and other isolation media.

Streptococcus mutans strain N produces a novel low molecular mass non-lantibiotic bacteriocin.

Streptococcus mutans strain N was shown to have bacteriocin production and immunity characteristics consistent with those of Group I mutacin-producing strains of S. mutans. The bacteriocin mutacin N was purified from agar cultures of S. mutans strain N using XAD andp6 reversed phase chromatography. The molecular mass of mutacin N was 4806 Da and the entire 49 amino acid sequence was determined by N-terminal sequencing. Database searches indicate that mutacin N is a novel bacteriocin, but with some homology to the protein IIC domain of a hypothetical sugar-phosphotransferase enzyme from Acholeplasma florum.

High pharyngeal carriage rates of Streptococcus pyogenes in Dunedin school children with a low incidence of rheumatic fever.

AIM: To document the incidence and type distribution of Streptococcus pyogenes in a group of Dunedin children throughout the 1997 school year. METHODS: The 780 children recruited from ten primary schools had their throats swabbed on each reporting of pharyngitis. Additional pharyngeal swabbings were obtained monthly from a representative subset of these children, referred to as group 1. All swab samples were plated on CNA-P, a blood agar medium that facilitates detection of haemolytic streptococci. S. pyogenes isolates were classified according to the RFLP patterns of PCR products of their emm genes (ERP typing). Representative isolates of each ERP pattern were also emm-typed, a sequence typing method that correlates with serological M-typing. RESULTS: 28% of the group 1 children were found to carry S. pyogenes for more than two months. This carriage rate is similar to that previously detected in Dunedin and Waikato schoolchildren, but is higher than that generally reported in other countries. Although the predominant S. pyogenes types detected in Dunedin are similar to those in North Island populations, some of the types frequently associated with North Island cases of rheumatic fever and glomerulonephritis were absent or isolated infrequently from the Dunedin children. CONCLUSION: The high pharyngeal carriage rates of S. pyogenes in Dunedin schoolchildren, without the concomitant increased occurrence of post-streptococcal sequelae observed in North Island populations may, in part, be due to a relatively lower occurrence of the M-types most commonly implicated in these diseases.

Dental caries is a preventable infectious disease.

Dental caries is the most common infectious disease affecting humans. The principal causative agents are a group of streptococcal species collectively referred to as the mutans streptococci of which Streptococcus mutans and Streptococcus sobrinus are the most important agents of human caries. This review outlines what is currently known about these ubiquitous pathogens and discusses novel methods for elimination of these bacteria from dental plaque.