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.

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.

Transduction of bacteriocin determinants in group A streptococci.

Determinants of streptococcin A-FF22 (SA) production and host cell immunity have been transduced to three serologically distinct Group A streptococci. Streptomycin resistance markers were not cotransducible with bacteriocin determinants. SA+ transductants of strains unrelated to the parent SA+ strain were unstable but SA+ transductants of a spontaneous SA- derivative of the parent appeared to be stable.

Group A streptococcal bacteriocin. Production, purification, and mode of action.

A bacteriocin, streptocin A, was isolated from the supernatant fluid of tryptic soy broth cultures of Group A streptococcus strain FF-22. Evidence was obtained which supports the view that the failure to recover active streptocin A after growth of the producer strain in certain fluid media is due to the inactivation of the bacteriocin by concomitantly synthesized streptococcal proteinase. The bacteriocin was purified 139-fold and the active product appeared to be of uniform size, having a molecular weight of approximately 8,000. Streptocin A was bactericidal, but not lytic, for a susceptible Group A streptococcus and the lethal effect was markedly temperature dependent. The bacteriocin inhibited the synthesis of DNA, RNA, and protein, and also prevented the uptake and incorporation of glucose by the sensitive cells. Degradation of RNA occurred, but appeared to be less than that produced by a staphylococcal bacteriocin. This effect may be due to differences in the killing potency of the two bacteriocins in preparations having similar inhibitory activity when measured by lawn culture assays.

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.

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.

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.

Cloning and sequence analysis of zooA, a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin.

The nucleotide sequence has been determined for zooA, a gene encoding the bacteriocin-like inhibitory substance zoocin A in Streptococcus zooepidemicus strain 4881. The zooA gene product corresponds to the 285-amino acid (aa) zoocin A pre-peptide from which a leader sequence is cleaved to form the 262-aa biologically active molecule of estimated molecular mass 27,877 Da. Expression of zooA in a Gram-negative host was shown by the extracellular release from Escherichia coli, containing cloned zooA, of a biologically active peptide having an identical range of anti-bacterial activity to that of zoocin A, purified from S. zooepidemicus strain 4881. Data base searches revealed sequences having homologies with known muralytic proteins produced by both Gram-positive and Gram-negative bacteria and indicate a 'mix and match' blending of domain-type structures, the C-terminal putative receptor-recognition region of the molecule being joined by a threonine-proline-rich linker to an N-terminal putative catalytic region having homology with several known endopeptidases, including lysostaphin.

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.

Lantibiotic-mediated anti-lactobacillus activity of a vaginal Staphylococcus aureus isolate.

Staphylococcus aureus strain 26 inhibited the growth of 23 of 26 lactobacilli of endocervical origin, but only two of 17 staphylococci, in deferred antagonism tests. The inhibitory agent, a bacteriocin-like inhibitory substance (BLIS) named staphylococcin Au-26, was obtained from vigorously shaken liquid cultures containing a 0.1% (v/v) supplement of Tween 80 and was purified by chromatographic fractionation on XAD-2, carboxymethyl Sephadex and reversed phase HPLC. The molecular mass of staphylococcin Au-26 was estimated by SDS-PAGE to be approx. 2700. The detection of lanthionine residues in the molecule, the high stability to heating at acidic but not alkaline pH values and inactivation by proteinases indicate that staphylococcin Au-26 is a member of the lantibiotic class of peptide antibiotics--the first reported to be produced by a S. aureus strain. Primary sequence analysis showed that the N-terminus of the molecule is isoleucine, a characteristic also displayed by the lantibiotics nisin, epidermin and gallidermin.

Factors affecting production of the group A streptococcus bacteriocin SA-FF22.

Factors influencing the production of streptococcin A-FF22 (SA-FF22) in liquid media were examined. Despite good growth of the producer strain, no SA-FF22 was detected during incubation at 40 degrees C, at pH 7, in Brain Heart Infusion Broth or in Mg(2+)-supplemented media. Optimal SA-FF22 production occurred at 32 degrees C, at pH 6.7, in cultures in Tryptic Soy Broth supplemented with glucose 2.25% and yeast extract 1%. Under these conditions SA-FF22 remained cell-associated but could be extracted with acid.

"Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors.

A scheme for the "fingerprinting" of streptococci according to their production of (P typing) and sensitivity to (S typing) bacteriocine-like inhibitory substances has been developed. P typing of 450 beta-haemolytic streptococci by their action on a set of nine standard indicator strains revealed that 80% of strains produced one or more detectable inhibitors, and that 17 different P types could be recognised. Production of some inhibitors seemed to be a property of strains of a particular serological group or type. Bacteriocine-like substances were produced by streptococci of serological groups, A, B, C, D, E, F and G. Nine strains were selected as standard producers for S typing. These strains differed in their spectra of inhibition, but all seemed to be active only against gram-positive bacteria. One producer, a group-F streptococcus, specifically inhibited group-A streptococci. The conditions of incubation were critical for demonstration of inhibitor production. A requirement for blood and for incubation at 32 degrees C were important factors. None of the inhibitors was induced by ultraviolet irradiation. The observed inhibitory effects were not attributable to either hydrogen peroxide or low pH, but to the production of a variety of substances having diverse physicochemical properties and production requirements. Most of the inhibitors do not seem to be produced in liquid media. The "fingerprinting" procedure is simple and inexpensive, and provides a reliable means of subdividing streptococcal strains that may find application as a supplement to the existing serological typing schemes.

Proteinase-related broad-spectrum inhibitory activity among group-A streptococci.

Some 10% of group-A streptococci have inhibitory activity against all nine strains (eight of them streptococci) in a set of indicators in an inhibitor-production typing (P-typing) scheme. This activity was associated with the concurrent synthesis of cell-associated proteinase by the streptococcal strain. Inhibitor production was prevented either by incubation of the test strain in conditions inimical to proteinase production, e.g., at low temperature and alkaline pH, or by addition to the medium of substances, such as glucose, iodoacetic acid, lincomycin, Congo red or trypan blue, that had an anti-proteinase effect. Inhibitory activity was not detectable in liquid cultures, but freeze-thaw extracts of cultures of group-A streptococcus strain A1013 on Gibco Columbia Agar Base (Gibco Diagnostics, Madison, WI, USA) had some inhibitory activity. The inhibitor was concentrated and partially purified, and the active agent was shown to be a high-mol.-wt cationic protein which was bactericidal for various bacteria in the logarithmic growth phase, including the homologous producer strain.

Inhibitor production by group-G streptococci of human and of animal origin.

Strains of group-G streptococci were tested by a "fingerprinting" method for the production of (P typing) and sensitivity to (S typing) inhibitory agents, and were biotyped. In the standard P-typing test, 28 of 50 strains of human origin, but none of 30 strains of animal origin, showed inhibitory activity. Of the human strains, 12 formed a bacteriocin that was active on group-A streptococci, including three (strains I2, I5 and I8) of the four streptococci of this group among the indicator strains. Sixteen other human strains inhibited the fourth group-A indicator (strain I7), and to a lesser extent strain I2, by lowering the pH of the typing medium. This acid-mediated inhibition was eliminated by testing on a medium containing calcium carbonate 0.5%; the 16 strains were then completely non-inhibitory, and the bacteriocin-forming strains, the typing pattern of which had originally been I2, I5, I7, I8, showed only inhibition attributable to the action of the bacteriocin. Nearly all group-A streptococci were sensitive to the group-G bacteriocin. The indicator strain I7 and several other members of M-type 28 were exceptions, but their resistance was not associated with the presence of R-antigen 28. Fifteen inhibitor-sensitivity patterns and 12 biotypes were identified among the strains; some of these tended to be associated with either a human or an animal origin. Neither S type nor biotype appeared to correlate with inhibitor production.

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.

Production of a bacteriocine-like substance by group-A streptococci of M-type 4 and T-pattern 4.

A unique and characteristic bacteriocine-like inhibitor elaborated by M-type 4, T-pattern 4, group-A streptococci was isolated and partially purified. This inhibitor was found to be produced optimally in Todd-Hewitt broth; after extraction and concentration, was shown to be protein in nature, and to have a m.w. of c. 8000. It was extremely heat stable and acid tolerant, but was quickly inactivated in alkaline conditions. It could be demonstrated in cell-bound form, but 99.5% was found in culture supernates. It was specifically adsorbed by viable sensitive cells, and its mode of action was bacteristatic.

Microbially-produced peptides having potential application to the prevention of dental caries.

Strategies advanced to decrease the occurrence of dental caries have in the past typically focussed upon attempting to reduce plaque accumulation by application of broad-spectrum antibacterial agents. In recent years however there has been growing interest in the application of a more targeted approach to the selective elimination from plaque of those bacterial species that are specifically implicated as the aetiological agents of this disease. This review focuses upon a number of the small bacterially-produced peptide antibiotics known as bacteriocins that are currently being explored for their potential role in the treatment and prevention of dental caries.

Bacteriocin-like inhibitory activity associated with beta-hemolytic strains of Streptococcus salivarius.

Seven beta-hemolytic Streptococcus salivarius isolates produced bacteriocin-like inhibitory activity in deferred antagonism tests using a set of nine indicator bacteria (I1-I9). Five of these S. salivarius strains (KWF, TOVE-R, K17, K21, and K26) were inhibitory to indicators I2, I5, I6, and I7. Mutated non-hemolytic derivatives showed concomitant loss of inhibitory activity against I2, I5, and I6, but retained activity against I7. Inhibitory activity against I2, I5, and I6 was restored in beta-hemolytic revertants of such mutants. Strain 3638 was inhibitory to all of the indicator organisms except I3, and this pattern of inhibitory activity was retained by non-hemolytic derivatives. It appeared that strain 3638 produced an additional broadly-active inhibitory agent, since a mutant (strain 3638A), which was apparently defective in the production of this inhibitor, retained both the beta-hemolytic and I2-, I5-, I6-, and I7-inhibitory activities. Non-hemolytic derivatives of strain 3638A were inhibitory only to I7. Strain 3638, therefore, appeared to produce at least three inhibitory agents: one active only on I7; another acting on I2, I5, and I6 (and associated with beta-hemolytic activity); and a third apparently active on all of the indicators other than I3. S. salivarius strain JH inhibited all nine indicator strains and possessed a beta-hemolytic character which differed from that of the other strains in being readily eliminated on treatment with the plasmid-curing agent novobiocin. Non-hemolytic derivatives of JH retained inhibitory activity against the complete set of indicators.(ABSTRACT TRUNCATED AT 250 WORDS)

Incidence and characterization of anti-microbial effects produced by Actinomyces viscosus and Actinomyces naeslundii.

Sixty-two facultative Actinomyces strains isolated from dental plaque were tested for the production of bacteriocin-like inhibitory effects by a deferred antagonism method. When incubated anaerobically, all isolates produced identical inhibitory patterns against 15 indicator organisms, but under microaerophilic conditions, little inhibitory activity was observed. Activity was not evident after anaerobic incubation on a medium buffered by 0.5% (w/v) calcium carbonate. Gas-liquid chromatographic analyses of agar blocks removed from the inhibitory zones indicated that, compared with microaerophilic conditions, anaerobic incubation encouraged production of high concentrations of lactic and succinic acids, and the concomitant fall in the pH was probably responsible for the inhibitory effects.

The production of bacteriocin-like substances by the oral bacterium Streptococcus salivarius.

Inhibitory substances produced by six strains of Streptococcus salivarius were isolated and partially characterized. The six prototype producer strains were selected initially on the basis of their differing spectra of inhibitory activity when tested against a set of nine standard indicator strains. Optimal production conditions were defined for each producer strain and inhibitor-containing extracts were obtained for characterization studies. All of the inhibitors appeared to be proteinaceous substances of molecular weight greater than 3500. When tested against a Streptococcus pyogenes indicator strain, one of the inhibitors was bactericidal, but the other five appeared to be bacteriostatic. Some differences between the inhibitors were observed with respect to heat and enzyme sensitivities.