Production of the lantibiotic salivaricin A and its variants by oral streptococci and use of a specific induction assay to detect their presence in human saliva

Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.     

Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva

Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.     

Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12

The commercial probiotic Streptococcus salivarius strain K12 is the prototype of those S. salivarius strains that are the most strongly inhibitory in a standardized test of streptococcal bacteriocin production and has been shown to produce the 2,368-Da salivaricin A2 (SalA2) and the 2,740-Da salivaricin B (SboB) lantibiotics. The previously uncharacterized SboB belongs to the type AII class of lantibiotic bacteriocins and is encoded by an eight-gene cluster. The genetic loci encoding SalA2 and SboB in strain K12 have been fully characterized and are localized to nearly adjacent sites on pSsal-K12, a 190-kb megaplasmid. Of 61 strongly inhibitory strains of S. salivarius, 19 (31%) were positive for the sboB structural gene. All but one (strain NR) of these 19 strains were also positive for salA2, and in each of these cases of double positivity, the two loci were separated by fewer than 10 kb. This is the first report of a single streptococcus strain producing two distinct lantibiotics.     

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.     

Applications of BLIS typing to studies of the survival on surfaces of salivary streptococci and staphylococci

A typing scheme based on detection of the production of bacteriocin-like inhibitory substances (BLIS) was a useful tool in a series of epidemiological studies of the spread and survival on surfaces of salivary streptococci and staphylococci. The organisms survived for longer periods on glass, plastic, stainless steel and latex surfaces than on unpainted wood or paper. The presence of saliva as a suspending medium prolonged the viability of Staphylococcus aureus C55 and Streptococcus pyogenes FF22 but not of Strep. salivarius Min 5. Young children were shown to carry Strep. salivarius of identical BLIS-types on their fingers and in their saliva. BLIS typing of Strep. salivarius strains obtained from telephone mouthpieces and from the tongue-sealed flap of envelopes was used as a method of tracing the human source of the salivary deposits.     

Relation between natural bacterial colonization and adhesion to human buccal epithelium

As the results of the quantitative study of Streptococcus salivarius adhering to buccal epithelial cells, three levels of their natural colonization were established: low (less than 20 bacteria per epithelial cell), medium (20-50 bacteria), and high (more than 50 bacteria). The characteristics of natural colonization by S. salivarius inversely correlated with the resistance of epithelial cells to the adhesion of Pseudomonas aeruginosa. In the process of interaction with P. aeruginosa highly adhesive strain, S. salivarius, naturally colonizing the cells of the buccal epithelium, decreased in number 2-10 times up to complete desorption. These results may be regarded as the manifestation of one of the mechanisms regulating the microecological balance in the system of mucous membranes.     

Safety assessment of the oral cavity probiotic Streptococcus salivarius K12

Streptococcus salivarius is a prominent member of the oral microbiota and has excellent potential for use as a probiotic targeting the oral cavity. In this report we document safety data relating to S. salivarius K12, including assessment of its antibiogram, metabolic profiles, and virulence determinants, and we examine the microbial composition of saliva following the dosing of subjects with K12.     

Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens

The use of bacteria as probiotics is in continuous development, thanks to their capacity to maintain or restore a host's natural microbiome by interference with and/or inhibition of other microorganisms mediated by antimicrobial peptide production such as bacteriocins. In the oral cavity, Streptococcus salivarius, a non-pathogenic and predominant oral species, is one of the major bacteriocin producers that is able to coexist in this environment and reduce the frequency of colonization of the main pathogens involved in upper respiratory tract infections. The aim of this study was to screen oral bacteria colonizing healthy children for their use as potential oral probiotics. Eighty-one α-hemolytic streptococci isolated from nasal and/or pharyngeal swabs of 31 healthy children aged between two and twelve years were isolated. Among them, 13 α-hemolytic streptococci were selected for their bacteriocin-like inhibitory activity against potential pathogens. These strains were tested for bacteriocin production and assayed for their capacity to adhere to HEp-2 cell lines. Our data showed that 13 bacteriocin producer strains were able to inhibit different gram-positive pathogens. Among them one strain, S. salivarius 24SMB, deposited as DSM 23307, was selected as a potential oral probiotic, thanks to its safety assessment, ability to inhibit Streptococcus pneumoniae and the absence of virulence and antibiotic resistance genes.     

Isolation and some properties of extracellular glucan-producing strains of human oral Streptococcus salivarius

A total of eighteen strains of Streptococcus salivarius, which formed rough gelatinous, rough mucoid or smooth mucoid colonies on sucrose agar media, were isolated from the saliva and tongue dorsum of adults. All of the isolates produced glucans as well as fructans from sucrose. The bulk of the glucans was synthesized by the extracellular enzyme fraction and was water insoluble, whereas most of the fructans were synthesized by the cell-associated enzyme fraction and were water soluble. All strains formed microbial deposits on wire and glass surfaces when cultured in sucrose broth, but their sucrose-dependent adhesion was apparently looser than that produced by a cariogenic S. sobrinus strain. The rough gelatinous colony forming strains possessed a greater ability to synthesize water-insoluble glucans and produced heavier deposits with higher cohesion. Preliminary studies showed that the S. salivarius of such characteristic forms of colony were detected primarily in the saliva and tongue dorsum: the smooth mucoid colony formers appeared to predominate in the tongue coat and the rough mucoid and rough gelatinous colony formers were prominent in saliva. Isolation of these S. salivarius from dental plaques was low.     

Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation

The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30-40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and proline-rich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Applications of BLIS typing to studies of the survival on surfaces of salivary streptococci and staphylococci

J.R. TAGG AND N. L. RAGLAND. 1991. A typing scheme based on detection of the production of bacteriocin-like inhibitory substances (BLIS) was a useful tool in a series of epidemiological studies of the spread and survival on surfaces of salivary streptococci and staphylococci. The organisms survived for longer periods on glass, plastic, stainless steel and latex surfaces than on unpainted wood or paper. The presence of saliva as a suspending medium prolonged the viability of Staphylococcus aureus C55 and Streptococcus pyogenes FF22 but not of Strep. salivarius Min 5. Young children were shown to carry Strep. salivarius of identical BLIS-types on their fingers and in their saliva. BLIS typing of Strep. salivarius strains obtained from telephone mouthpieces and from the tongue-sealed flap of envelopes was used as a method of tracing the human source of the salivary deposits.     

In vitro functional and immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3 association that are relevant to the development of a pharyngeal probiotic product

The use of proper bacterial strains as probiotics for the pharyngeal mucosa is a potential prophylactic strategy for upper respiratory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promising pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by inducing potentially protective effects. In particular, we found that the strains MIMLh5 and ST3 activate U937 human macrophages by significantly inducing the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Nonetheless, the induction of the anti-inflammatory interleukin-10 (IL-10) by MIMLh5 or ST3 was never lower than that of TNF-α, suggesting that these bacteria can potentially exert a regulatory rather than a proinflammatory effect. We also found that the strains MIMLh5 and ST3 induce cyclooxygenase 2 (COX-2) expression and demonstrated that toll-like receptor 2 (TLR-2) participates in the recognition of the strains MIMLh5 and ST3 by U937 cells. Finally, we observed that these microorganisms grow efficiently when cocultured in milk, suggesting that the preparation of a milk-based fermented product containing both MIMLh5 and ST3 can be a practical solution for the administration of these bacteria. In conclusion, we propose the combined use of L. helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyngeal mucosa.     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Lactobacillus salivarius CTC2197 prevents Salmonella enteritidis colonization in chickens.

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10(5) CFU g(-1) was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.