Developing Oral Probiotics From Streptococcus salivarius

Philip A Wescombe; John DF Hale; Nicholas CK Heng; John R Tagg

Disclosures

Future Microbiol. 2012;7(12):1355-1371. 

In This Article

Current Contenders

S. salivarius K12

Although S. salivarius K12 was initially selected on the basis of its broad inhibitory activity against S. pyogenes, it has subsequently been demonstrated to provide more diverse health benefits – ranging from the alleviation of halitosis to stimulation of antiviral immune defenses and the reduction of episodes of OM. This broad spectrum of potential health benefits conferred throughout the life of the human host has prompted the adoption of the colloquial moniker for this strain, "BLIS K12 – the probiotic for all ages" (Figure 2).

Figure 2.

Streptococcus salivarius: the probiotic for all ages. Diseases that may be alleviated by Streptococcus salivarius probiotics and the ages at which they generally tend to manifest.
Reproduced with permission from [77].

In 2001, strain K12 became the first S. salivarius to be commercially developed as a probiotic and more than 50 million doses have now been marketed internationally by the New Zealand company BLIS Technologies Ltd (Dunedin, New Zealand). A substantial body of research was undertaken to underpin the safe and efficacious application of the strain to humans and this included a variety of clinical interventions in both animals and humans. Although S. salivarius is not commonly consumed as a naturally occurring food ingredient, it is nevertheless considered a low-risk organism since, in spite of its apparently invariable and plentiful presence in the human oral cavity, it is only very rarely a cause of infection in humans who are immunologically competent.[27] The safety of strain K12 has been specifically supported by a series of studies: affirming the absence of known streptococcal virulence factors and antibiotic resistance determinants; showing its low mutagenicity predisposition; acute and subacute toxicity testing in rats; and a high-dosage trial in humans.[29,35,36] The outcome of these strain-specific studies, together with recognition of the inherent safety of the species, has enabled a self-affirmed 'generally regarded as safe' (or 'GRAS') status to be granted for strain K12 in the USA. Interestingly, the species S. salivarius is still generally classified as a risk group 2 organism in Europe; however, on the basis of its safety profile strain, K12 has been specifically reclassified as a risk group 1 organism in Germany by the Ausschuß für Biologische Arbeitsstoffe (Translation: Committee on Biological Agents).[43]

The original source of S. salivarius K12 was a healthy schoolchild who had maintained a large indigenous oral cavity population of the K12 strain for a period of more than 12 months, during which time no new S. pyogenes infections were experienced. A distinctive (and indeed patentable) feature of strain K12 was its production of two novel lantibiotics (salivaricin A2 and B), both of which were shown in vitro to have inhibitory activity against S. pyogenes, the principal causative agent of streptococcal pharyngitis.[44] Further support, albeit indirect, for the protection offered by S. salivarius BLIS against S. pyogenes infection came from studies showing that children who harbored oral populations of salivaricin A- and/or B-producing S. salivarius had significantly fewer new acquisitions of S. pyogenes than did children who appeared not to have BLIS-producing S. salivarius (17 vs 32%, respectively).[45] Another study showed that children who frequently experienced clinically confirmed sore throats were significantly less likely to have BLIS-producing S. salivarius than children who had not experienced sore throats in the past 3 years.[46] Furthermore, competition experiments between cocultured strain K12 and a bioluminescent S. pyogenes demonstrated that strain K12 binds avidly to human epithelial cell lines and can interfere with the binding of S. pyogenes[28,47] (Figure 3). Oral cavity colonization of humans occurs following its introduction into the mouth and the efficacy of this colonization is enhanced by prior reduction of the levels of the indigenous streptococcal population, as occurs following the use of an antiseptic mouth rinse (e.g., chlorhexidine) or after antibiotic treatment.[15,48,49] Recent, as yet unpublished, studies have also demonstrated that the use of one lozenge a day containing 1 billion viable cfu of strain K12, is sufficient to achieve oral cavity colonization in the majority of subjects [WESCOMBE PA ET AL., UNPUBLISHED DATA]. Further evidence for the protection afforded by strain K12 against streptococcal pharyngitis was gathered during a small preliminary trial in which 24 children with a history of recurrent tonsillitis (0.33 episodes per month) received daily doses of either strain K12 or a placebo. The 18 children receiving strain K12 experienced fewer sore throats (0.10 per month) than did the six children in the placebo group (0.19 per month) [BURTON JP ET AL., UNPUBLISHED DATA].

Figure 3.

Electron microscope image demonstrating the attachment of Streptococcus salivarius K12 to HEp-2 cells.
Image courtesy of M Rohde.

S. salivarius, Rothia mucilaginosa and an uncharacterized species of Eubacterium were identified as being present in either relatively reduced numbers or absent in tongue dorsum populations of subjects suffering from halitosis.[50] Prompted by this observation, a trial of 23 subjects with halitosis (having breath scores for volatile sulfur compound [VSC] levels of greater than 200 ppb) undertook a 3-day regimen of chlorhexidine mouth rinsing, followed, at intervals, by the use of lozenges containing either S. salivarius K12 or placebo.[49] Assessment of the subjects' VSC levels 1 week after treatment initiation demonstrated that 85% of the K12-treated group and 30% of the placebo group had substantial (>100 ppb) VSC level reductions. While the majority of the subjects tested had a favorable outcome, the mechanism(s) of VSC reduction was not clearly established. In vitro tests showed that the inhibitory spectrum of strain K12 encompasses some of the key Gram-negative anaerobes (including Prevotella spp.) that have been implicated in halitosis.[49] Other mechanisms of competition (e.g., saturation of attachment sites by the newly introduced K12 cells) may also have been influential, particularly as facilitated by the chlorhexidine pretreatment step, which may have reduced populations of some critical adjunct members of the halitosis-associated consortia. Subsequent colonization of the microbe-depleted site by the incoming K12 could also limit anaerobe proliferation through specific BLIS-mediated inhibition of key members of the halitosis-associated microbiota.

OM is the most common bacterial infection in young children and the predominant etiological agents are Streptococcus pneumoniae, S. pyogenes, Moraxella catarrhalis and Haemophilus influenzae. As a preliminary experiment to evaluate the efficacy of probiotic interventions for the control of OM, it was shown that S. salivarius K12, when given to 19 young OM-susceptible children following a 3-day course of amoxicillin, led to colonization of the nasopharynx and/or the adenoid tissue of some subjects.[51] Interestingly, in that study, only 33% of the subjects achieved oral colonization with strain K12. This lower-than-anticipated level of colonization was attributed to the failure of the amoxicillin pretreatment to effect a substantial reduction in the level of the indigenous oral streptococcal populations, since most of these subjects had been preconditioned to regular amoxicillin exposure during the course of their OM therapy.[51] To determine whether delivery of the S. salivarius K12 probiotic to the oral cavity would have any effect on the rate of recurrence of OM, a small study was undertaken at Dunedin Hospital BURTON JP ET AL., UNPUBLISHED DATA. The 13 children enrolled in the study were from the surgical waiting list for grommet implants and all had a history of recurrent acute OM (AOM). The subjects were offered a three-month treatment course of either strain K12 or placebo and nine completed the study. The children receiving the K12 probiotic (n = 6) had far fewer ear infections (0.22 per month) than they did prior to entering the study (0.50 per month, n = 13) and also by comparison with the smaller placebo group (0.55 occurences per month, n = 3) BURTON JP ET AL., UNPUBLISHED DATA. The encouraging results of this study (although only preliminary) indicate that S. salivarius K12 dosing could potentially reduce the occurrence of OM.

An unanticipated application of S. salivarius K12 could be to ameliorate the development of oral candidosis. A number of early studies indirectly demonstrated that S. salivarius may inhibit oral candida,[52–55] but more recently Ishijima et al.[20] found a direct protective effect against Candida albicans after oral dosing with strain K12. In this latest study, K12 was shown to bind preferentially to the hyphae of C. albicans and to prevent its attachment to a plastic substratum. Interestingly, K12 was not able to directly inhibit C. albicans in a deferred antagonism assay, indicating that the bacteriocins encoded for by strain K12 do not target yeast and further supporting other observations that mechanisms other than the ability to target pathogens with antimicrobial molecules can also contribute to the health benefits of probiotics. When tested using an in vivo mouse model for oral candidosis, a dose-dependent improvement in symptom score was observed for mice dosed with K12 at 24 and 3 h before and at 3, 24 and 27 h after C. albicans inoculation, when compared with mice in a saline-treated group. Follow-up clinical evaluation of the efficacy of K12 in candidosis control in humans now seems imperative.

Although it is now well established that exposure to probiotic bacteria can impact upon the host's immune system, the outcome of these interactions can be quite strain-specific. Several in vitro cell culture experiments have indicated that strain K12 can help to maintain cell homeostasis. In one microarray-based study, it was demonstrated that co-culture with either strain K12 or certain bacterial pathogens differentially influenced the expression levels of 1530 genes in human bronchial epithelial cells.[56]S. salivarius K12 altered the expression of 660 genes (572 of which were specific to K12) and, in particular, those involved in innate immune defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. In this same study, Staphylococcus aureus influenced the expression of 323 genes. The ratio of upregulated to downregulated genes was 5:2 for K12, but this ratio was reversed for S. aureus, further illustrating the different signaling roles of strain K12 and bacterial pathogens. Closer analysis of the affected gene pathways indicated that K12 potentially contributes to the maintainance of homeostasis between human and bacterial cells by reducing proinflammatory responses. In particular, K12 was shown, by enzyme-linked immunosorbent assay, to reduce the levels (from 318 to 5.1 pg/ml) of the cytokine IL-8 produced by the bronchial cell line in response to the presence of Pseudomonas aeruginosa.[56] IL-8 has been demonstrated to have a major involvement in the pathogenesis of gingivitis and so dosing with strain K12 may potentially help ameliorate some of the inflammatory manifestations of this disease. The secretion of Gro-α, an inducible neutrophil chemotactic factor synthesized in epithelial tissues during inflammation, was also inhibited by the presence of strain K12 when the epithelial cells were exposed to flagellin (a known inducer of IL-8 secretion by epithelial cells), further emphasizing the protective role strain K12 can play for the host. The mechanism of immunosuppression by strain K12 appeared to be at least partially explained through the inhibition of activation of the NF-κB pathway (a family of transcription factors that function as dimers and regulate genes involved in immunity, inflammation and cell survival). Interestingly, the most significantly over-represented pathway in the array studies was the unified interferon signaling pathway. In this pathway, type I and II interferons signal through their specific receptors to upregulate the expression of a large number of genes responsible for innate immunity against viral infection, antitumor activity, priming of the LPS response and anti-inflammatory effects. This indicates that, while K12 cells can act to reduce inflammation, they may also 'prime' the epithelial cells through tonic signaling to respond rapidly and appropriately to the detection of viral or bacterial exposure in order to limit the spread of infection – a role that has recently been ascribed, in general, to commensal bacteria.[57]

Other preliminary studies have demonstrated that high-level oral dosing with S. salivarius K12 elicits increased salivary levels of IFN-γ.[58] These observations were further supported by investigations with mouse splenocytes, in which IFN-γ levels, but not the pro-inflammatory cytokines IL-1β or TNF-α, were increased in response to co-culturing with strain K12 [WALES J ET AL., UNPUBLISHED DATA]. Interestingly, it seems that not all S. salivarius elicit similar immune responses, since S. salivarius strain ATCC 25975 was reported to upregulate IL-6, IL-8 and TNF-α gene expression.[59] Indeed, in that study it seemed that strain ATCC 25975 was even more efficient at inducing the release of proinflammatory mediators than was C. albicans. These apparently contradictory findings emphasize the importance of not extrapolating the specific findings for one probiotic candidate strain to all members of that same species. The initial findings of induction by strain K12 of an anti-inflammatory response have subsequently been independently corroborated by Guglielmetti et al.,[47] who showed that IL-6, IL-8 and TNF-α levels were significantly reduced when FaDu cells were co-cultured with K12. These findings will be discussed below in relationship to the probiotic candidate strain S. salivarius ST3.

In summary, it appears that strain K12 is well suited for use as an oral cavity and upper respiratory tract probiotic due to its natural propensity to inhabit the human oral cavity and be strongly competitive with a number of potential oral pathogens that have adapted to the same ecological niche. In addition, the immune responses of cell lines to co-incubation with S. salivarius K12 indicate that it elicits no proinflammatory response but rather an anti-inflammatory response, as well as modulating genes associated with adhesion to the epithelial layer and homeostasis. By these strategies, S. salivarius K12 appears to be well-tolerated on the epithelial surface, while also actively protecting the host by BLIS-mediated inhibition of pathogen replication and stimulation of cytokine-mediated reduction of virus replication and pathogen-induced inflammation and apoptosis.

S. salivarius M18

Some early reports indicated that certain S. salivarius strains (especially TOVE-R as aforementioned) may have a role in the limitation of dental caries. Following the successful discovery and introduction of the probiotic strain K12, BLIS Technologies Ltd. conducted extensive follow-up deferred antagonism testing of candidate BLIS-producing S. salivarius to identify strains having inhibitory spectra that included bacterial species putatively associated with the development of dental caries. In this screen, S. salivarius strain M18 (formerly known as Mia) was found to inhibit all tested S. mutans and S. sobrinus (collectively referred to as the mutans streptococci). Other species inhibited by strain M18 included: Actinomyces viscosus, Actinomyces naeslundii, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Listeria monocytogenes, H. influenzae, Staphylococcus saprophyticus and Staphylococcus cohnii.[101] This unusually broad spectrum of inhibition indicated that strain M18, in addition to potentially reducing the risk of dental caries, may also have additional benefits for the host in helping to limit the growth of a variety of common bacterial pathogens of the upper respiratory tract.

To date, four bacteriocin loci have been identified in the M18 genome: salivaricin A2,[101] 9,[60] MPS[30] and M.[30] Salivaricin A2 and 9 are well-characterized bacteriocins with broad activity against S. pyogenes as well as other upper respiratory tract pathogens, but not against mutans streptococci. Salivaricin MPS is less well characterized, but is known to be a large 60 kDa bacteriocin with specific activity against S. pyogenes.[61] Salivaricins A2, 9 and MPS have been found to be megaplasmid-encoded in strain M18.[16,30] By contrast, salivaricin M appears to be chromosomally encoded and, recently, has not only been shown to be a lantibiotic, but also to be the molecule responsible for the observed activity of strain M18 against mutans streptococci.[30] Interestingly, unlike most other S. salivarius bacteriocins, salivaricin M appears to be optimally produced in vitro on TSYCa agar (trypticase soy broth supplemented with 2% yeast extract, 0.1% CaCO3 and 1.5% agar), and less effectively on BaCa (blood-containing) agar in deferred antagonism assays, an observation indicating that there is strict regulation of its locus expression.

Preliminary colonization trials have indicated that, in children who colonize well with strain M18, the salivary levels of mutans streptococci are maintained at reduced levels for significant periods (at least 27 days) by comparison with placebo-dosed control subjects, in whom the mutans streptococci levels returned to pretreatment levels within 4–6 days.[101,62]

A variety of pathogens have been implicated in the development of gingivitis and periodontitis and it has also been shown that the etiology of these diseases is strongly linked to the inflammatory response of the host cells to the bacterial pathogens.[63,64] To determine whether strain M18 can potentially impact on pathogen-induced pro-inflammatory cytokine expression in gingival fibroblasts, strains M18 and K12 were coincubated with gingival fibroblasts both prior to and concommitantly with exposure to periodontal pathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. Strains M18 and K12 both significantly inhibited the expression of the pro-inflammatory cytokines IL-6 and -8, commonly associated with gingivitis – indicating that dosing with these probiotics may potentially be useful in the treatment of gingivitis.[65] Appropriately controlled large-scale clinical trials further investigating the potential for M18 probiotic interventions in the control of dental caries and gingivitis now appear warranted.

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