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Developing oral probiotics from Streptococcus salivarius

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Considerable human illness can be linked to the development of oral microbiota disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has emerged as an important source of safe and efficacious probiotics, capable of fostering more balanced, health-associated oral microbiota. Strain K12, the prototype S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes infections, now has an expanded repertoire of health-promoting applications. K12 and several more recently proposed S. salivarius probiotics are now being applied to control diverse bacterial consortia infections including otitis media, halitosis and dental caries. Other potential applications include upregulation of immunological defenses against respiratory viral infections and treatment of oral candidosis. An overview of the key steps required for probiotic development is also presented.
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10.2217/FMB.12.113 © 2012 Future Medicine Ltd ISSN 1746-0913
Future Microbiol. (2012) 7(12), 1355–1371
Future Microbiology
part of
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Consumers seeking health-promoting dietary
supplements have long been conditioned to the
ingestion of yoghurt as a convenient source of
living beneficial microbes (i.e., probiotics). The
definition of the term probiotic has undergone
a series of evolutionary changes [1] and is now
generally accepted to be “live organisms, which
when administered in adequate amounts, confer
a health benefit on the host” [2] . Conventional
probiotics have typically comprised bacteria
of intestinal origin (especially lactobacilli
and bifidobacteria) and their application has
principally been to provide relief for maladies
of the GI tract. However, the realization that
much human illness can be linked either directly
(e.g., dental caries, periodontal disease and
candidosis) or indirectly (e.g., cardiovascular
disease and perhaps even obesity) to the
development of oral microbiota disequilibria has
diverted much contemporary probiotic research
to the development of products that are capable
of fostering a healthy oral microbiota [3] . While
researchers initially tried to establish whether
conventional approved intestinal probiotics
could also influence the oral microbiota, these
bacteria (perhaps unsurprisingly) have no oral
persistence, and any oral cavity health benefits
seem transitory and largely attributable to
immune stimulation [4]. A more logical strategy
is to utilize microbes isolated from their natural
oral habitat in healthy humans as oral probiotics.
The term ‘oral probiotics’ is used here to refer to
beneficial microbes given to the (usually human)
host to help maintain or effect improvements
in their oral health – not intestinally derived
probiotics that are delivered orally!
The scientific origins of oral probiotics can
be traced to the use of mixtures of putative
ora l commensals producing incompletely
characterized inhibitory agents with in vitro
activity against Streptococcus pyogenes [5] or otitis
media (OM) pathogens [6 ]. An alternative but,
for the time being, aborted approach was the
targeting of mutans streptococci using an ultra-
competitive bacteriocin-producing Streptococcus
mutans, genetically modif ied to attenuate
its virulence [7] . Other approaches include
investigations into isolates of Streptococcus
zooepidemicus [8] , Streptococcus oligofermentans
[9] and Veillonella spp. [10 ] .
In this laboratory, we adopted the strategy
of identifying an oral commensal strongly
inhibitory to S. pyogenes one of the principal
pathogens of the human oral cavity. Key criteria
sought for the ideal probiotic candidate were:
nonpathogenic; large populations occurring
naturally within the oral microbiota; and
producer of potent in vitro and in vivo inhibitory
activity against target pathogens to which
resistance does not readily develop. This search
led us to Streptococcus salivarius [11] .
S. salivarius is a pioneer colonizer of the human
oral cavity and persists there as a predominant
member of the native microbiota throughout
the life of its human host [12 –1 4] . In saliva, it is
typically present at levels of up to 1 × 107 colony
forming units (cfu)/ml and this equates to
approximately 1010 cfu ingested daily [15] . In the
healthy (i.e., immunologically competent) host,
it is only extremely rarely a cause of infection.
Many strains are producers of bacteriocin-like
inhibitory substances (BLIS), and in these strains
Developing oral probiotics from
Streptococcus salivarius
Philip A Wescombe1, John DF Hale1, Nicholas CK Heng2 & John R Tagg*1,3
1BLIS Technologies Ltd. Centre for Innovation, PO Box 56, Dunedin, 9054, New Zealand
2Department of Oral Sciences, PO Box 647, University of Otago, Dunedin, 9054, New Zealand
3Department of Microbiology & Immunology, PO Box 56, University of Otago, Dunedin, 9054, New Zealand
*Author for correspondence: Tel.: +64 3 4794953 Fax: +64 3 4798954 john.tagg@otago.ac.nz
Considerable human illness can be linked to the development of oral microbiota
disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has
emerged as an important source of safe and efficacious probiotics, capable of
fostering more balanced, health-associated oral microbiota. Strain K12, the prototype
S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes
infections, now has an expanded repertoire of health-promoting applications. K12
and several more recently proposed S. salivarius probiotics are now being applied to
control diverse bacterial consortia infections including otitis media, halitosis and dental
caries. Other potential applications include upregulation of immunological defenses
against respiratory viral infections and treatment of oral candidosis. An overview of
the key steps required for probiotic development is also presented.
Keywords
bacterial interference
bacteriocin bacteriocin-
like inhibitory substance
immune stimulation
lantibiotic megaplasmid
oral microbiota oral
probiotic Streptoco ccus
saliva rius
Review
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multiple bacteriocin loci are typically present on
transmissible megaplasmids [16 ] . The S. salivarius
BLIS are diverse in their activity spectra and
are thought to play an important role in both
stabilizing the oral microbiota and preventing
overgrowth (or infection) by potential pathogens
[17] . Salivaricin A, the first fully characterized
S. salivarius BLIS, has been detected in the
saliva of subjects harboring salivaricin A-positive
S. salivarius by the application of a highly specific
BLIS auto-induction assay [15]. This provides
direct evidence for the production of bioactive
BLIS in the human oral cavity.
This review updates the reader on some of the
exciting recent research into the development of
S. salivarius probiotics and also outlines critical
steps required in the overall process of bringing
a probiotic to market.
Development of S. salivarius probiotics:
general principles
The Food and Agricultural Organization and
WHO have published a list of recommended
guidelines for the systematic a ssessment
and development of strains that are under
consideration as probiotics [2] . While this
document focuses particularly on intestinal
probiotics, its recommendations can be
considered generally applicable to all probiotics.
Some of the key steps taken in the commercial
development of a probiotic are shown in
FIGURE 1
. It
is important to note that, in practice, the process
does not always follow an orderly pathway
(especially in the developmental stage), and
that some steps may prove especially problematic
and need to be repeated prior to obtaining a
successful and efficacious end-product.
Candidate screening & selection
Laboratory-based analyses can help provide
preliminary indications of the potential beneficial
attributes of a probiotic and of its safety for use in
humans. The repertoire, activity spectrum and
potency of bacteriocin production, as detected
in deferred and simultaneous antagonism
tests, sometimes provide a major criterion for
probiotic strain selection. Other desirable traits
include an ability of the candidate strain to
adhere avidly to human epithelial cell lines and,
in cobinding assays, to outcompete pathogens
such as S. pyogenes [1 8, 19 ] or Candida albicans
[20] . For S. salivarius probiotics, the production
of putatively beneficial enzymes, such as urease
[21, 22] and dextranase [23, 24] , have also been
evaluated. Following the strain screening (or
‘auditioning’) phase and prior to commitment of
major developmental (or ‘grooming’) resources
for the provisionally selected probiotic candidate,
an intensive strainbackground’ and ‘identity
check’ must be performed. The Food and
Agricultural Organization/WHO guidelines
state that probiotics should have an official generic
and species designation [2] . Strain-specific genetic
and physiological characterization is important
since interstrain differences within a microbial
species commonly occur for characteristics
that may prove critical for probiotic efficacy;
for example, their bacteriocin and exoenzyme
repertoires. Strain-specific characterization aids
subsequent assessments of probiotic performance
and also allows for accurate epidemiological
surveillance of the strain following its seeding
within complex indigenous ecosytems in its
newly adopted human host [25 ,2 6] .
Following this predevelopmental screening
phase, the probiotic front-runner candidate should
be lodged in an internationally recognized strain
repository, such as the American Type Culture
Collection (ATCC) or the Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH.
Safety evaluation
Ensuring that a probiotic is safe for human
consumption should be of paramount
consideration. The WHO guidelines stipulate
that any bacterial species, including those
having a history of human consumption, has
the potential to cause disease – especially in
immunocompromised individuals – and that
it is the role of the manufacturer to assess the
potential risk of the strain being developed
[2]. While safety should be verified prior to
commercial release, in practice it is an ongoing
process and requires continual in vitro and
in vivo ana lysis.
An important first step in safety evaluation is
a thorough search of the literature. Identification
of the history of use and reports of infection
resulting from the chosen species/strain should
be noted. For S. salivarius, although there have
been several reports of infection, the majority
of these have followed iatrogenic or traumatic
cerebrospinal fluid contamination [2 7].
In vitro safety checks should form an integral
part of the early strain selection process. These
tests include: metabolic profiling to assess the
production of deleterious byproducts (e.g.,
d-lactate); antibiogram determination to
accepted standards, such as those established by
the Clinical and Laboratory Standards Institute,
to indicate antibiotic resistance and preferred
options should there be a need to treat probiotic
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infection; toxicity to cell lines and blood
(hemolysis) [28] ; and the presence of virulence
factors [29 ]. The mutagenic ability of a strain can
be assessed by the Ames test.
Many genetic techniques are available to
assess the presence/absence of virulence factors
and to aid strain identification. Although
PCR and pulsed-field gel electrophoresis have
traditionally been applied [29], the current speed
and relative cost-efficiency of entire genome
sequencing now allows researchers to easily
obtain genome ‘snapshots’ of a probiotic strain.
Full genome sequencing allows for rapid and
accurate identification of known virulence genes,
antibiotic resistance determinants, colonization
factors and genetic transfer mechanisms. At the
time of writing, the genomes of six S. salivarius
strains, namely, M18 [30], PS4 [31], JIM8777
[32] , CCHSS3 [33] , 57.I [34 ] and K12 (BioProject
Accession No. ALIF01000000) have been
sequenced.
Following in vitro testing, trials in animals
(typically rats) allow for an in situ safety
assessment of the probiotic and help predict
potential toxicity for the human host. Typically,
researchers will study the effect of the probiotic
by analyzing changes to total body weight,
individual organ weight, key biochemistry
markers (e.g., enzyme fluctuations), urine and
blood [35] .
Human trials should take the form of large
double-blind placebo-controlled studies to
reveal statistically significant outcomes. Such
trials should be carried out using the anticipated
commercial formulation and dosage levels to
ensure its safety is evaluated in a ‘real world
situation [36 ].
Stability & shelf life
Probiotics are biological products whose
viability is influenced by a variety of complex
physiological and chemical factors. To ensure
that the correct dose (number of viable cfu)
is delivered upon consumption, knowledge
of factors influencing the stability of the final
product is critical. ‘Stability’ and ‘shelf life’ are
closely related concerns. Stability refers to the
survivability/viability of the probiotic strain in
a particular format, while shelf life determines
how long the product can be sold while retaining
stability. Stability is affected by many different
factors including manufacturing conditions
(e.g., exposure to temperature and pressure,
growth media, fermentation times, product
blending and handling systems), auxiliary
ingredients (e.g., pH and ionic strength) and
composition of the final product (i.e., liquid,
solid and water availability), as well as storage
temperature and packaging. It may take several
production ‘runs’ to derive the final successful
product format.
Product format
The manner of delivery is an important
consideration. Factors such as palatability and
effectiveness of delivery need to be optimized
and, in particular, adequate contact time with
host tissues needs to be achieved to foster
attachment and colonization of the strain. A
variety of product formats for S. salivarius have
been considered, with current formulations
including lozenges, ice cream, chewing gum,
mouthwashes and yoghurt.
Dosing schedule
In contrast with the apparent modus operandi of
most intestinal probiotics, it is generally believed
that for oral probiotics, persistent colonization
is required in order to achieve optimal health
benefits. Therefore, trials need to be carried out
to determine the dosage regimens required to
Selection stage
Strain screening
Repository deposition
Safety evaluation
Developmental stage
Cell production
Stability and shelf life
Product format
Dosing schedule
Efficacy
Clinical trials
Production stage
In-house vs outsourced
Quality assurance
Figure 1. Steps required for the
development of a probiotic.
Developing oral probiotics from Streptococcus salivarius Review
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effect oral colonization. It seems that the best
opportunity for successful implantation of an
oral probiotic is following either rinsing with oral
antiseptics, such as chlorhexidine, or a course
of antibiotic therapy (i.e., when the indigenous
microbiota has been reduced in numbers).
Although dosing levels to determine colonization
efficacy and toxicity are ideally best assessed in
humans, some relevant data can also be gained
from trials in experimental animals [35].
Efficacy
By definition, a probiotic should confer a health
benefit, typically measured as a reduction in
symptoms or prevention of disease. Although
BLIS-producing S. salivarius may show strong
inhibitory activity in in vitro assays, this activity,
importantly, also needs to be translated to an
in vivo environment. Ideally, double-blind,
placebo-controlled studies of the individual
strain in its final product format are required to
determine benefits [2]. This requirement has now
attracted the attention of the major regulatory
authorities such as the US FDA and European
Food Safety Authority. Many current probiotics,
however, have not adhered to this requirement,
probably largely due to the expense and logistics
involved in carrying out large clinical trials. The
large regulatory hurdle of proving efficacy for a
particular probiotic, while useful for ensuring
products are eff icacious, may also lead to
consumers missing out on benefits from some
newly developed products. This issue could
be addressed by regulatory bodies through
adoption of a staggered claim system, where low-
level claims (e.g., for preventative rather than
therapeutic benefits) could be made for products
that have been proven to be safe, but which
have not yet been fully assessed for efficacy.
High-level health claims (e.g., replacements
for current treatments) should be reserved for
products that have satisfied the requirements of
successful outcomes from multiple double-blind
placebo-controlled trials.
Probiotic production
The production of probiotics can be achieved
either in-house for companies with large
infrastructure or, alternatively, be outsourced to
facilities that have the appropriate fermentation
capabilities and expertise. Regardless of the
production process chosen, it is essential for the
successful delivery of probiotics that a stringent
quality assurance program is in place ensuring
that the probiotic is delivered in a safe and
efficacious manner. Such a program should
have control over the ingredients used in the
fermentation and subsequent formulation of
the delivery format for the probiotic. It should
also closely monitor the product for safety and
ensure that throughout the stated shelf life of the
probiotic an efficacious dose is delivered.
Profiles of proposed S. salivarius
probiotics: past, present & potential
Early entries
S. salivarius strains TOVE-R and K58 were given
preliminary consideration in the pioneering days
of applied bacterial interference research for their
potential to control the major Streptococcus-
associated infections of the human oral cavity
– dental caries and streptococcal pharyngitis.
S. salivariu s TOVE-R
The ability of certain S. salivarius to interfere
with the proliferation of the streptococci most
commonly implicated in the etiology of dental
caries was demonstrated by Tanzer et al. [37] .
Initial oral colonization of rats with S. salivarius
strain TOVE-R (R for rough colony morphology)
prevented the subsequent establishment by
fecal transmission of S. mutans 10449S and
Streptococcus sobrinus 6715–13WT. TOVE-R
itself did not contribute to caries development
and its transmission to rats already infected
by 10449S or colonization of rats prior to
10449S exposure inhibited caries induction. In
a further study, TOVE-R was demonstrated to
colonize rat dental plaque already containing
S. mutans or S. sobrinus and to persist as a
prominent member of the plaque microbiota
[38] . Moreover, colonization by TOVE-R
effected an approximately 50% reduction in
the total recoverable S. mutans and S. sobrinus
populations on the teeth. The authors suggested
that TOVE-R colonization may have clinical
therapeutic utility for suppressing existing
infection of humans by the mutans streptococci,
but no follow-up clinical studies have been
reported. Interestingly, the only follow-up study
of TOVE-R seems to be its apparently successful
application to the alleviation of periodontal
disease in a beagle model [39] .
S. salivariu s K58
The first S. salivarius specifically selected for
its potential to interfere with the colonization
of the upper respiratory tract by S. pyogenes was
strain K58 [5]. The bioactive agent inhibitory to
the growth of S. pyogenes was partially purified
from culture supernatants and named enocin
(based on the names the investigators, Eugene
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and Christine) [5] . Enocin was characterized
as a low-molecular-weight heat-labile molecule
and, since its mode of action appeared to involve
interference with pantothenate utilization, it
inhibited the growth of organisms requiring
exogenous pantothenate such as S. pyogenes [5] .
Interestingly, of 29 S. salivarius strains isolated
during a series of clinical studies [40 –4 2], nine
produced enocin-like activity against S. pyogenes,
indicating that this agent may be frequently
expressed by S. salivarius. Unfortunately, no
further studies of this strain or of enocin have
been reported. In our experience, S. salivarius K58
inhibits the growth of S. pyogenes in simultaneous
antagonism tests, but not in deferred antagonism
BLIS P-typing tests, indicating that the agent is
produced early in the growth phase and rapidly
lost/degraded [Tagg JRetal., Unpublished Data].
Current contenders
S. salivariu s 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)
.
In 2001, strain K12 became the f irst
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 [2 9,35 ,3 6] . 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)[4 3].
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 [4 4] . 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
Group B
streptococcus
Otitis media
Tonsillitis
Dental caries
Periodontal disease
HalitosisCandidosis
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].
Developing oral probiotics from Streptococcus salivarius Review
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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 ,4 8 , 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 PAet 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J P et al., Unpublished Data].
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
5 µm
Figure 3. Electron microscope image demonstrating the attachment of
Streptococcus salivarius K12 to HEp-2 cells.
Image courtesy of M Rohde.
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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 a nd 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 J Pet 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 ana lysis 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 t o 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-, an inducible
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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-B 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 [5 7] .
Other preliminary studies have demonstrated
that high-level oral dosing with S. salivarius
K12 elicits increased salivary levels of IFN- [58] .
These observations were further supported by
investigations with mouse splenocytes, in which
IFN- levels, but not the pro-inflammatory
cytokines IL-1 or TNF-, were increased
in response to co-culturing with strain K12
[WalesJet a l., 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- 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- 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 inf lammation a nd
apoptosis.
S. salivariu s 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 [6 0], MPS [3 0] and M [30] . Salivaricin A2
and 9 are well-characterized bacteriocins with
broad activity against S. pyogenes as well as
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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 ,6 4] . 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.
New nominations
S. salivariu s ST3
Using strain K12 as a model oral probiotic for
comparative purposes, Guglielmetti et al. [18]
obtained 56 probiotic candidate strains from
pharyngeal sites of four healthy donors. From
this initial group, 11 S. salivarius, established to
be of separate lineages, were further investigated
for their potential as probiotics targeting
prevention of S. pyogenes pharyngitis. Assessment
of their probiotic potential included tests of their
binding efficacy to the human epithelial cell lines
FaDu and HaCaT – an attempt to model the
primary adhesion target for invading S. pyogenes
[66] . The strains having the highest adhesion
indices to FaDu cells were ST3 and K12. As
another efficacy measure, the bioluminescent
indicator strain S. pyogenes C11LucFF was used
to monitor the exclusion of S. pyogenes affected
by preincubation of either FaDu or HaCaT
cells with each candidate probiotic. Exclusion
was strain-specific, with S. salivarius strains
ST1 and RS1 observed to be significantly more
effective at excluding the S. pyogenes than strain
K12 in this assay. Interestingly, however, strain
K12 was at least as effective as strain ST1 in
competition assays, in which combinations of
the bioluminescent S. pyogenes and S. salivarius
probiotic were added together to the cell
lines. One possible reason for the apparently
superior ability of strain K12 over strain ST3 to
antagonize the binding of S. pyogenes may be its
production of the bacteriocins salivaricin A and
B, both of which are known to inhibit the growth
of S. pyogenes in vitro. By contrast, strain ST3 did
not appear to produce any bacteriocin activity
under the test conditions used for this study.
Guglielmetti et al. further characterized strains
ST3, RS1 and K12 according to their ability to
modulate the immune responses of FaDu cell
lines [1 8] . Interestingly, all three strains were well
tolerated on co-culture with the epithelial cells,
with no strains stimulating a pro-inflammatory
response a finding that, on reflection, might
indeed be anticipated for an oral cavity commensal
species [18] . Co-culture with strain K12 was
demonstrated to reduce baseline IL-6, IL-8 and
TNF- levels, with IL-8 and IL-6 well below
the levels obtained for the other two S. salivarius
strains (ST3 and RS1). All three S. salivarius
were effective at reducing the levels of IL-6 and
IL-8 following stimulation of the FaDu cells by
IL-1 (a prototypical pro-inflammatory cytokine
that plays a central role in the inflammation
amplification cascade) illustrating the general
anti-inflammatory activity of S. salivarius. IL-6,
IL-8 and TNF- are known to be mediators
of the pro-inflammatory response that causes
most of the tissue damage in periodontal
disease and results from the host response to
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bacterial infection. Downregulation of the pro-
inflammatory response is now a major aim of
treatment regimes for periodontal disease [6 7],
highlighting a potential further application for
probiotic therapy with S. salivarius strains such
as K12 and ST3. A further observation was the
apparent upregulation of GM-CSF and IFN-
levels in IL-1-stimulated FaDu cells co-cultured
with K12 [18] . GM-CSF stimulates stem cells
to produce granulocytes, a process crucial for
fighting infection, while IFN- is a cytokine
essential for innate and adaptive immunity
against viral and intracellular bacterial infections
and for tumor control. This stimulation of
GM-CSF and IFN- by strain K12 indicates that
another beneficial effect of probiotic treatment
with this strain may be to enhance the body’s
natural defenses against virus infection. Immune
enhancement of this nature (also described as
low-level tonic signaling), enabling the body to
respond more rapidly to virus infection, has also
been described for some other probiotics [57]. For
example, prophylactic probiotic administration
has been demonstrated to limit the duration and
severity of virus infections of the respiratory tract
in human subjects, indicating that the effects of
probiotics can extend beyond the GI tract [6 8].
Strains ST3 and RS1 appeared to drive a slightly
different immune response to that of strain K12
in FaDu cells, in that, while they shared the
ability to reduce IL-6, IL-8 and TNF- levels,
they upregulated the levels of MIP and MCP-1
– both of which are proinflammatory cytokines
[18] . The authors suggest that this observed
upregulation may benefit the host through
upregulation of their immune defenses against
pathogenic bacteria, a comment that is supported
by similar observations made about the Gram-
negative probiotic strain Escherichia coli Nissle
[69] . Interestingly, strain RS1 was shown to
reduce the levels of IFN- produced by IL-1-
stimulated FaDu cells, perhaps indicating that
exposure to this strain could potentially render
the host more susceptible to viral infection.
Ability to produce urease was another
characteristic assessed for each of the candidate
probiotic strains. Strains K12 and RS1 were
demonstrated both to be strongly ureolytic, a trait
considered beneficial due to its effect in reducing
the acidity of dental plaque and, thereby, possibly
delaying the onset and progression of dental
caries [70, 71]. By contrast, strain ST3 appeared
non-ureolytic and, on further examination, was
found to lack ureC, which encodes the main
subunit of the urease complex [18 ] . The authors
suggested that the inability to hydrolyze urea
could be considered beneficial, in that it could
result in there being less damage to the host’s
mucosal cells from exposure to ammonia.
These observations highlight the potential for
different strains to fulfill different roles in the
oral cavity and, perhaps, for them to be targeted
to applications in individuals with specif ic
health needs.
Recently, strain ST3 was examined for its
possible use as an oropharyngeal probiotic in
combination with a second putative probiotic
bacterium, Lactobacillus helveticus strain
MIMlh5 [19] . The two strains were investigated
for their ability to coordinately modulate the host
innate immune system in a manner beneficial
to the host. Although exposure to this strain
combination significantly induced expression
of the proinflammatory cytokine TNF-, the
levels achieved were never above the co-induced
levels of the anti-inf lammator y molecule
IL-10, indicating that the effect was probably
regulatory rather than pro-inf lammatory.
However, a further observation that did support
the potential immune benefits of prophylaxis
with this strain combination was the induced
expression of COX-2, the gene encoding the
isoform of prostaglandin synthase H, which is
known to be induced by several stimuli including
bacterial components. Prostaglandin synthase H
synthesizes prostaglandins, which are known to
contribute to the protection of the gastrointestinal
mucosa and are also involved in both the
induction of oral tolerance, by guiding T cells
towards an immunosuppressive phenotype [72] ,
and the resolution of inflammation [73]. A rapid
upregulation of COX-2 expression in response
to injury or inflammation has, furthermore,
been reported to restore mucosal integrity, thus
reducing the time available for pathogens to
penetrate the innate defenses of the intact mucosa
[74] . In addition to these immunological effects,
the probiotic strain combination was shown to
interfere with the binding of S. pyogenes to FaDu
cells, while not interfering with the binding
characteristics of each probiotic partner strain,
indicating that they were compatible for use in
a combined probiotic product. It is of interest to
note that L. helveticus strain MIMlh5 was able
to antagonize S. pyogenes better than strain ST3
in the luminescence assay deployed. However,
since S. salivarius is more commonly located in
the oral cavity, it is to be expected that it may
perform better in vivo, emphasizing the need for
clinical studies on human subjects to confirm
the benefits of each probiotic strain. Both strains
were also found to grow efficiently in milk,
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indicating that fermented milk products may be
suitable delivery vehicles for the probiotics [19 ] .
S. salivariu s 24SMB
Santagati et al. recent ly screened 81
-hemolytic streptococci isolated from nasal
and/or pharyngeal swabs of healthy children,
with the intention of identifying commensal
bacteriocin-producing bacteria for use in the
prevention of upper respiratory tract infections
[28] . The principal selection criteria were: safety
for the host, strong adhesion to HEp-2 cells
and inhibitory activity against S. pneumoniae.
S. salivarius strain 24SMB had an inhibitory
spectrum apparently specific for S. pneumoniae
when tested on Columbia agar base supplemented
with 5% horse blood and 0.1% CaCO3. When
tested on TSYCa, the activity spectrum increased
to also include three S. pyogenes strains. The
authors concluded that, since strain 24SMB
appeared to contain no known bacteriocin loci,
it may express one or more as yet uncharacterized
bacteriocins. Interestingly, one of the main
criteria for the selection of 24SMB was its ability
to bind to HEp-2 cells, which were originally
thought to have been derived from an epidermoid
carcinoma of the larynx but, subsequently, have
been established to have arisen from HeLa
(derived from cervical cancer) cell contamination
[75]. Since HEp-2 cells are positive for keratin
by immunoperoxidase staining and therefore do
share this characteristic of human tissue surfaces,
they may have some relevance for evaluation of
oral probiotic attachment. However, for oral
probiotic selection, it seems that cell lines that
are established to have oral cavity origins should
preferably be used. In any case, although strain
SMB24 was shown to bind well to HEp-2 cells
(50–57%), strain K12 was also found to bind
to a similar extent (50 60%), indicating no
benefit in this regard for strain SMB24 over
the currently established K12 probiotic. The
safety of strain SMB24 was evaluated through a
screen for potential streptococcal virulence genes
and testing for sensitivity to antibiotics. Strain
SMB24 was negative for all potential virulence
genes tested and, additionally, was sensitive to all
antibiotics used in the screen. Overall, although
the investigations are still preliminary, strain
SMB24 appears to have good potential for use
as a probiotic to prevent upper respiratory tract
infections.
S. salivariu s T30
An important target for S. salivarius probiotics
is AOM, the most common bacterial infection
in growing children – the key causative agents
of which are S. pneumoniae, S. pyogenes,
M. catarrhalis and H. influenzae. It is
presumed that the infection originates from
the nasopharynx with the bacteria entering the
middle ear via the Eustachian tube. Current
treatments include the use of broad-spectrum
antibiotics and insertion of tympanostomy tubes.
Antibiotic dosing fosters resistance development
and also weakens the natural defenses through a
reduction in the numbers of the normal healthy
microbiota. Tympanostomy tube insertion is
costly and carries with it the associated risks of
general anesthesia (in some cases) and possible
membrane damage. Some probiotic-based
strategies to reduce the incidence of OM have
been explored, including the successful use of
inhibitory -hemolytic streptococci (a mixture
of Streptococcus mitis, Streptococcus sanguinis
and Streptococcus oralis) as a nasal spray [6 ]. A
complication of this is that the three species
utilized in the formulation are all recognized
human pathogens, with S. mitis associated
with lung infection and abscess formation, and
both S. oralis and S. sanguinis implicated in
endocarditis. Therefore, a S. salivarius probiotic,
if also shown to be clinically efficacious, may
be viewed more favorably by regulatory bodies
for use in young children. With this in mind
Walls et al. [76] looked at the nasopharyngeal
microflora of 20 children with recurrent AOM
(having more than six episodes of AOM in the
previous year) and 15 healthy controls (having
no more than one AOM episode). While no
significant correlation was observed between
the groups for the presence of BLIS-producing
streptococci, three S. salivarius isolates (two
from the control group) were inhibitory to
representative strains of at least three of the four
major species of AOM pathogens in vitro [76] .
Interestingly, for some subjects, salivaricin A-
and B-producing S. salivarius were isolated from
the nasopharynx, but not the saliva, indicating
that S. salivarius producing these bacteriocins
may be particularly well-adapted for growth in
the nasopharynx. One isolate (strain T30) was
characterized further and demonstrated to have
broad inhibitory activity against all four major
AOM pathogens when grown on TSYE agar
under anaerobic conditions, but not on BaCa
medium or when incubated with a 5% CO2 in
air atmosphere [76] . This strain was patented
for use in the prevention or treatment of OM
but was withdrawn in 2006 [102] . However,
the concept of using a strongly competitive
nasopharyngeal-localized S. salivarius for the
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probiotic-mediated control of AOM certainly
seems meritorious.
Future perspective
With the recent rapid expansion in the variety
of available probiotics and in their delivery
vehicles, consumers are developing a growing
enthusiasm for the health benefits to be derived
from their consumption. While the majority of
probiotics have been designed for use in the GI
tract, it is clear that there is now an impetus to
progress the field to encompass other regions of
the body, including the oral cavity. As this review
has shown, there are now a number of candidate
probiotic strains from the species S. salivarius
that have been proposed for application to the
control of microbial diseases of the oral cavity.
While the safety of S. salivarius for application
to humans appears to have been well established,
there is still only relatively limited clinical
evidence to support claims of health benefit.
Most current supporting evidence has been
based on either in vitro studies or the results
of clinical trials that have been limited in size.
There is no doubt that, in the next few years,
the benefits to be gained from these probiotics
(both in terms of health and commercial gain)
will provide the incentive for clinical studies
of sufficient magnitude to clearly establish the
roles that S. salivarius probiotics can play in the
human oral cavity, the upper respiratory tract
and beyond
(FIGURE 4)
. It is also apparent that, due
to strain variation (even in the immunological
responses that they evoke), individual strains
will be selected for their specific health benefits
which could include the prevention of: dental
caries; OM; streptococcal sore throat; halitosis;
oral thrush; general immune priming and
potentially many more
(TABLE 1)
. This promises
to be a rapidly evolving and rewarding research
area to observe and to participate in over the
next decade.
Gum
Tooth
Saliva
Pharynx
Tongue
Plaque-localized
S. salivarius
Competitive
exclusion and
inhibition of
S. mutans
Urease – pH
maintenance
Dextranase – plaque
biolm disruption?
S. mutans
C. albicans
Indigenous
S. salivarius
Megaplasmid
transfer
Direct inhibition
and exclusion
Antimicrobial
production
Malodorous
bacteria
S. salivarius
probiotic
Reduction in
gingivitis-induced
inammation
Direct inhibition
and exclusion of
S. pyogenes
S. pyogenes
Epithelial
cell
Immunological
modulation
S. salivarius
cell wall
fragments
IL-8, IL-6,
TNF-α
IFN-γ
IL-8, IL-6,
TNF-α
Future Microbiol. © Future Science Group (2012)
Figure 4. Influence of Streptococcus salivarius probiotics in the oral cavity. Health benefits can occur through the direct inhibition
and exclusion of pathogens, modulation of the human immune system to reduce pathogen-induced inflammation or by ‘priming’ the
immune system to respond rapidly to viral or bacterial infection.
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Table 1. Streptococcus salivarius strains investigated for their potential as probiotics.
Strain Target area (s) Commercialized Antibacterial
agents
Safety Efficacy
K12 Oral health
Halitosis
Otitis media
Strep sore throat
Oral thrush
Anti-inflammatory
Yes Salivaricin A
Salivaricin B
GRAS
High-dose human trials
Absence of streptococcal
virulence genes
Antibiotic sensitivity
Low mutagenicity
Acute and subacute studies in
rats
More than 50 million doses
sold over 10 years
Genome sequence
Clinical:
Colonization trials in humans
Small-scale clinical trials
Otitis media, recurrent tonsillitis
Clinical studies on halitosis
Bacteriocin detection in human oral cavity
Increased salivary levels of IFN-
In vitro:
Anti-inflammatory effect that reduces inflammation by a range of pathogenic
bacteria
Anti-Candida exclusion studies
Good adhesion to FaDu and HEp-2 epithelial cell lines
Inhibition of many pathogenic bacteria including S. pneumoniae, M. catarrhalis
and S. pyogenes
S. pyogenes exclusion assays
M18 Dental caries
Gingivitis
Periodontal disease
Anti-inflammatory
Strep sore throat
Yes Salivaricin A
Salivaricin M
Salivaricin MPS
Salivaricin 9
Human trials
Antibiotic sensitivity
Genome sequence
Clinical:
S. mutans reduction for colonized individuals
In vitro:
Inhibition of S. mutans and S. pyogenes
Immunological studies – anti-inflammatory effect – protection against periodontal
pathogen-induced inflammation
Dextranase production
Urease production
ST3 Strep sore throat
Anti-inflammatory
No Not known Antibiotic sensitivity
Inability to hydrolyze urea
In vitro:
Good adhesion to FaDu epithelial cell line
Inhibition of S. pyogenes
S. pyogenes exclusion assays
Immunological studies – anti-inflammatory effect
24SMB Otitis media
Strep sore throat
No Not known Antibiotic sensitivity
Absence of streptococcal
virulence genes
No harmful enzymatic activity
In vitro:
Inhibition of S. pneumoniae and S. pyogenes
Adhesion to HEp-2 cells
T30 Otitis media No Not known No specific testing In vitro:
Inhibition of S. pneumoniae, M. catarrhalis, H. influenzae and S. pyogenes
Isolated from nasopharynx
Tove- R Dental caries No Not known No specific testing Clinical:
Rat model demonstration of efficacy to prevent S. mutans colonization and
reduce existing numbers
Has capability to colonize dental plaque
K58 Strep sore throat No Enocin No specific testing Inhibitor capable of interfering with pantothenate utilization – active against
S. pyogenes
Isolated from a child resistant to S. pyogenes colonization
GRAS: Generally regarded as safe; H.: Haemophilus species; M.: Moraxella species; S.: Streptococcus species.
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References
Papers of special note have been highlighted as:
of interest
 of considerable interest
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Quirynen M. Probiotics and oral healthcare.
Periodontol 48, 111–147 (2008).
2. Food and Agricultural Organization/W HO.
Guidelines for the eva luation of probiotics in
food. In: Report of a Joint FAO/WHO Working
Group on Drafting Guidelines for the
Evaluation of Probiotics in Food. Food and
Agricultural Organization, Rome, Italy
(2002).
 Guidance paper on the steps required in
the assessment and development of
potential probiotics.
3. Zarco MF, Vess TJ, Ginsburg GS.
The oral microbiome in health and disease
and the potential impact on personalized
dental medicine. Oral Dis. 18, 109–120
(2012).
4. Hansen JN. Nisin as a model food
preservative. Crit. Rev. Food Sci. Nutr.
34, 69–93 (1994).
5. Sanders CC, Sanders WE. Enocin: an
antibiotic produced by Streptococcus
salivarius that may contribute to protection
against infections due to Group A
Streptococci. J. Infect. Dis. 146, 683–690
(1982).
6. Roos K, Hakansson EG, Holm S. Effect of
recolonisation with “interfering” a lpha
streptococci on recurrences of acute and
secretory otitis media in children :
randomised placebo controlled trial. BMJ
322, 210–212 (20 01).
7. Hillman JD, Mo J, McDonell E,
Cvitkovitch D, Hillman CH. Modification
of an effector strain for replacement therapy
of dental caries to enable clinical safety
trials. J. Appl. Microbiol. 102, 1209–1219
(2007).
8. Balakrishnan M, Simmonds RS, Tagg JR.
Diverse activity spectra of bacteriocin-like
inhibitory substances having activity against
mutans streptococci. Caries Res. 35, 75– 80
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Financial & competing interests
disclosure
JR Tagg is Professor Emeritus at the University of
Otago and is the founding scientist of BLIS
Tec hn ol o gi es Lt d , w hi ch is a co mp an y t ha t s pe c ia l-
izes in the development and production of probiotic
microorganisms including Streptococcus salivarius
strains K12 and M18. PA Wescombe and JDF
Hale are employed by BLIS Technologies Ltd.
NCK Heng is employed by the University of Otago.
The authors have no other relevant affiliations or
financial involvement with any organization or
entity with a financial interest in or financial con-
flict with the subject matter or materials discussed
in the manuscript apart from those disclosed.
No writing assistance was utilized in the
production of this manuscript.
Executive summary
Probiotics
Live cells, the use of which benefits the health of the consumer.
Some probiotics effect beneficial modulation of the indigenous microbiota via:
Colonization to occupy space or to effect increased anticompetitor activity within the native biofilm;
Megaplasmid transmission to modify the genetic repertoire of the existing microbiota;
Immune stimulation – a transient effect not requiring probiotic colonization, but potentially helping to control virus infection by
upregulating the host’s immune defenses.
Streptococcus salivarius
Pioneer oral cavity commensal that remains a predominant component of the oral microbiota throughout life.
Rarely disease-associated in healthy (immunologically competent) humans.
Can harbor transmissible megaplasmids.
Some strains produce multiple bacteriocins, sometimes referred to as anticompetitor molecules or bacteriocin-like inhibitory
substances (BLIS).
S. salivarius K12
The prototype oral probiotic, originally selected for its strong anticompetitor activity against S. pyogenes.
Shown to have a role in the control of consortia bacterial infections, such as otitis media and halitosis, and also possibly in the reduction
of oral candidosis and infections by certain upper respiratory tract viruses.
Other candidate S. salivarius probiotics: past & present
Early contenders, TOVE-R and K58, had nonbacteriocin-mediated anticompetitor activities.
Strain M18 selected because of its uncommon BLIS activity against mutans streptococci.
New contenders ST3 and 24SMB were selected for their binding efficacy to epithelial cells and in vitro antipathogen activity, but neither
produce characterized bacteriocins.
The anti-AOM candidate strain T30 was selected for its BLIS activity against otitis media pathogens and was originally isolated from the
nasopharynx of a healthy child.
Bringing an oral probiotic from benchtop to marketplace
Producing a safe product is of paramount importance.
Significant investigations of the potential benefits of a strain need to be conducted before commencing efficacy trials.
Oral cavity probiotics require different considerations than intestinal probiotic products.
Clinical trials evaluating the efficacy of the final product are becoming increasingly important.
The road to market is not linear and may require several attempts to create an efficacious formulation.
Review Wescombe, Hale, Heng & Tagg
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Developing oral probiotics from Streptococcus salivarius Review
... Штамм K12 пробиотика S. salivarius, первоначально введенный для борьбы с инфекциями, вызванными Streptococcus pyogenes, в настоящее время имеет доказанную эффективность и в отношении Streptococcus pneumoniae, Haemophilus influenzaе и Moraxella catarrhalis, являющихся одними из основных этиологических факторов бактериальных инфекций респираторного тракта у детей и взрослых [63]. ...
... К настоящему времени проведено значительное количество клинических исследований по безопасности и эффективности применения штамма S. salivarius К12. Так, в ряде клинических исследований показана эффективность и безопасность S. salivarius К12 в профилактике [63,[72][73][74][75] и лечении [76] острых респираторных инфекций у детей и взрослых пациентов. Применение S. salivarius К12 оказалось эффективным и в профилактике острых, рекуррентных/ рецидивирующих и хронических заболеваний лор-органов: тонзиллита [65,[75][76][77], фарингита [78,79], тонзиллофарингитов [70,72,80,81], среднего отита [63,78,82]. ...
... Так, в ряде клинических исследований показана эффективность и безопасность S. salivarius К12 в профилактике [63,[72][73][74][75] и лечении [76] острых респираторных инфекций у детей и взрослых пациентов. Применение S. salivarius К12 оказалось эффективным и в профилактике острых, рекуррентных/ рецидивирующих и хронических заболеваний лор-органов: тонзиллита [65,[75][76][77], фарингита [78,79], тонзиллофарингитов [70,72,80,81], среднего отита [63,78,82]. ...
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SARS-CoV-2 infection can cause changes in the organs and tissues of the oral cavity, which is associated with a wide distribution of angiotensin-converting enzyme type 2 in the oral cavity, mainly epithelial cells of the oral mucosa, gums and fibroblasts of the periodontal ligament. Thus, the oral mucosa is susceptible to SARS-CoV-2 infection and may act as a gateway for the virus, as well as a reservoir for SARS-CoV-2. We searched the literature for the period from the beginning of the pandemic until May 30, 2022, devoted to the study of changes in the organs and tissues of the oral cavity with a new coronavirus infection (COVID-19) in the electronic search engines PubMed/MEDLINE and Scopus. A special place in the study of changes in the organs and tissues of the oral cavity with a new coronavirus infection (COVID-19) is occupied by periodontal pathology. A number of reviews and clinical studies conclude the importance of good oral hygiene and periodontal health as an important aspect of COVID-19 prevention and management. Oral probiotics can be considered as a promising direction for correcting changes in organs and tissues of the oral cavity in COVID-19.
... Oral probiotics strains, developed for dental health, have been investigated in clinical studies. Some of these include the application of Streptococcus salivarius strains M18 and K12 as reported by Wescombe et al. [27] and He et al. [29]. It was demonstrated that Str. ...
... salivarius M18 is effective in inhibiting the oral pathogen Str. mutans, thereby reducing the risk of dental caries [27,28]. In the other study, Str. ...
... thermophilus, and Streptococcus thermophilus) are considered as safe. Although Str. salivarius has a disputable reputation, MacDonald et al. [55] and Wescombe et al. [27] suggested that strains belonging to this species can be applied as beneficial strains. On the contrary, Wilson et al. [56] reported that Str. ...
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This study aimed to select beneficial strains from the oral cavity of healthy volunteers and to evaluate these as potential oral probiotic candidates. The selection process was based on the isolation, differentiation, identification, and safety assessment of LAB strains, followed by a series of experiments for the selection of appropriate candidates with beneficial properties. In the screening procedure, 8 isolates from the oral cavity of a Caucasian volunteers were identified as Streptococcus (Str.) salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lactiplantibacillus plantarum (Lb.) (Lactobacillus plantarum) ST63HK and ST66HK; Latilactobacillus sakei (Lb.) (Lactobacillus sakei) ST69HK; and Lactobacillus (Lb.) gasseri ST16HK based on 16S rRNA sequencing. Physiological and phenotypic tests did not show hemolytic, proteinase, or gelatinase activities, as well as production of biogenic amines. In addition, screening for the presence of efaA, cyt, IS16, esp, asa1, and hyl virulence genes and vancomycin-resistant genes confirmed safety of the studied strains. Moreover, cell-to-cell antagonism indicated that the strains were able to inhibit the growth of tested representatives from the genera Bacillus, Enterococcus, Streptococcus, and Staphylococcus in a strain-specific manner. Various beneficial genes were detected including gad gene, which codes for GABA production. Furthermore, cell surface hydrophobicity levels ranging between 1.58% and 85% were determined. The studied strains have also demonstrated high survivability in a broad range of pH (4.0–8.0). The interaction of the 8 putative probiotic candidates with drugs from different groups and oral hygiene products were evaluated for their MICs. This is to determine if the application of these drugs and hygiene products can negatively affect the oral probiotic candidates. Overall, antagonistic properties, safety assessment, and high rates of survival in the presence of these commonly used drugs and oral hygiene products indicate Str. salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lb. plantarum ST63HK and ST66HK; Lb. sakei ST69HK; and Lb. gasseri ST16HK as promising oral cavity probiotic candidates.
... The use of the S. salivarius K12 strain has been shown to increase the host's ability to produce a prompt interferon response not accompanied by a parallel inflammatory response based on TNF-α, IL-1β, and IL-6 [18]. While the ability to produce an interferon response in the host would seem horizontally shared by other S. salivarius strains [47], the inability to determine the concomitant presence of an inflammatory response would instead seem to concern the S. salivarius K12 strain in a peculiar way [48]. A prompt and rapid interferon response, notoriously anti-viral, could, at least theoretically, explain the observations made in our pilot study. ...
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Anatomical and physiological considerations indicate that the oral cavity is a primary source of the lung microbiota community, and recent studies have shown that the microbiota in the lungs contributes to immunological homeostasis, potentially altering the organ’s susceptibility to viral infection, including SARS-CoV-2. It has been proposed that, in the case of viral infection, lung Gram-negative bacteria could promote the cytokine cascade with a better performance than a microbiota mainly constituted by Gram-positive bacteria. Recent observations also suggest that Prevotella-rich oral microbiotas would dominate the oral cavity of SARS-CoV-2-infected patients. In comparison, Streptococcus-rich microbiotas would dominate the oral cavity of healthy people. To verify if the modulation of the oral microbiota could have an impact on the current coronavirus disease, we administered for 14 days a well-recognized and oral-colonizing probiotic (S. salivarius K12) to hospitalized COVID-19 patients. The preliminary results of our randomized and controlled trial seem to prove the potential role of this oral strain in improving the course of the main markers of pathology, as well as its ability to apparently reduce the death rate from COVID-19. Although in a preliminary and only circumstantial way, our results seem to confirm the hypothesis of a direct involvement of the oral microbiota in the construction of a lung microbiota whose taxonomic structure could modulate the inflammatory processes generated at the pulmonary and systemic level by a viral infection.
... Salivaricin A2 and B are two bacteriocins produced by this bacterium. Salivaricins have been reported to suppress Streptococcus pyogenes, a bacterium that can cause pharyngitis [61].Bacteriocins that produced from S. salivarius K12 were capable to stop all the bacteria that cause halitosis from growing. The establishing of novel formulas that contain salivaricin and K12 strain or both of them is worth thinking [62]. ...
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Bacteriocins are proteinaceous multifunctional compounds, generated by ribosomes and have strong antibacterial activity at specific quantities. some members of archaea and bacteria manufacture bacteriocins which inhibit the nourishment of closely related or similar bacterial strains. Bacteriocins are sorted into three main classes depending on their physiochemical and structural characteristics: bacteriocin class I, bacteriocin class II, and bacteriocin class III. the infections caused by bacteria which is resistant to antibiotic are considered as global health problem. Because of their broad-or narrow-spectrum efficacy towards antibiotic-resistant bacteria, bacteriocins may be a potential solution to this global problem. Bacteriocins prevent the nourishment of aim organisms via affect mainly on the envelope of the cell and via affecting expression of gene as well as production of protein inside the cell. the majority of bacteriocin producers are lactic acid bacteria (LAB), a type of bacterium that may be found normally in nutriment and possess important role since ancient times in the manufacture of dairy product. Bacteriocins regarded antimicrobial peptide which considered safe and nontoxic but When tested using cell culture-based techniques, several bacteriocins have been found to have some cytotoxicity. Bacteriocins have been used for food preservation, a variety of therapeutic applications including peptic ulcer treatment, spermicidal effect, woman care, anticancerous element, use in veterinary, skin and oral care, as well as promotion of plant growth in agriculture.
... 15.480493 doi: bioRxiv preprint Several strains of S. salivarius produce the lantibiotic salivaricin A, with five variants having been identified to date 17 . Notably, S. salivarius strain K12, a salivaricin B and salivaricin A2 co-producer with antagonistic activity against the pathogen Streptococcus pyogenes, has been developed as a commercial probiotic and has passed rigorous safety assessment for human use 18,19 . As strains of S. salivarius have been shown to benefit human health in numerous clinical trials [20][21][22] , there is considerable merit in screening other strains for traits with a view to the further development of novel probiotics with antimicrobial activity 23 . ...
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... В нашем исследовании наблюдалось отсутствие нежелательных побочных эффектов у всех пациентов и отмечена их хорошая приверженность к лечению. Безопасность штамма K12 ранее подтверждена серией исследований: показаны отсутствие известных факторов вирулентности стрептококков и детерминант устойчивости к антибиотикам, а также его низкая предрасположенность к мутагенности [20]. Доказано, что прием пробиотика БактоБЛИС снижает частоту эпизодов стрептококковой инфекции глотки примерно на 80% как у взрослых, так и у детей [21,22]. ...
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Chapter
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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.
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Streptococcus salivarius is a commensal species commonly found in the human oropharyngeal tract. Some strains of this species have been developed for use as oral probiotics, while others have been associated with a variety of opportunistic human infections. Here, we report the complete sequence of strain PS4, which was isolated from breast milk of a healthy woman.
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Conference Paper
Objective: Numerous pathogenic microorganisms are associated with oral inflammatory diseases such as gingivitis and periodontitis. Modifying the oral microflora via probiotic application may therefore be a useful mechanism for reducing these ailments. The objective of this study was to determine the effects of two commercially-available oral probiotics on pathogen-induced pro-inflammatory cytokine expression in gingival fibroblasts (GFs). Methods: Human primary GFs were cultivated as adherent monolayers in 24 well plates. The monolayers were first challenged separately for 4 and 8 hours with periodontitis-associated Porphyromonas gingivalis 33277, Aggregatibacter actinomycetemcomitans Y4 and Fusobacterium nucleatum 10593, and oral probiotic strains Streptococcus salivarius K12 and M18 at a 25:1 multiplicity of infection (MOI). GFs were then challenged simultaneously with equal amounts of the pathogens and probiotics at the same MOI, with and without an additional probiotic pre-incubation step. Culture supernatants were collected and assessed for expression of IL-6 and IL-8 using Luminex bead-based technology. Analysis of variance with Bonferroni's post test was used to determine significance, assessed at p<0.05 (N=3). Results: When applied separately or in combination to GF monolayers, all three pathogens significantly increased both IL-6 and IL-8 expression over control levels at 4 and 8 hours. In contrast, neither probiotic strain alone affected expression of either cytokine at either timepoint. When incubated alongside the pathogens, both probiotic strains were found to significantly inhibit the majority of this pathogen-induced cytokine upregulation, regardless of whether the monolayers were pre-incubated with the probiotics or not. The effects were far greater for IL-8, where all probiotic-pathogen combinations significantly reduced expression at both timepoints. Conclusions: Commercially-available oral probiotic strains S. salivarius K12 and M18 were both able to downregulate the expression of cytokines associated with inflammatory conditions like gingivitis and periodontitis. Therefore, these strains may offer novel preventive and therapeutic options for patients suffering from both diseases.
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A total of eight bacterial isolates belonging to six species, and a select group of 12 oral Candida albicans isolates, were used to study the effect of bacteria on germ-tube formation. Briefly, each bacterial suspension (105–6 cells/ml) was mixed with a C. albicans suspension (107 cells/ml) and incubated at 37 °C for 90 min with bovine serum, and the percentage germ-tube-positive Candida cells was quantified using a haemocytometer, under light microscopy. In general, out of eight bacteria, Streptococcus sanguis SK21A, Streptococcus salivarius SK56, Escherichia coli ATCC 25922, and S. salivarius OBU3 suppressed germ-tube formation to varying degrees, with different C. albicans isolates. Porphyromonas gingivalis Pg 50, Lactobacillus casei ATCC 7469 and Prevotella intermedia OBU4 elicited significant enhancement of germ-tube formation, whereas S. sanguis OBU 2 had no effect. E. coli ATCC 25922 was the only organism to show statistically significant suppression of germ-tube formation (p=0.0312). A significant increase in the germ tube production of C. albicans isolated from HIV-infected compared with HIV-free individuals was also noted. The current results tend to suggest that commensal and transient oral bacterial populations may selectively influence the differential expression of germ-tube-forming ability of C. albicans isolates.
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Signals from commensal bacteria can influence immune cell development and susceptibility to infectious or inflammatory diseases. However, the mechanisms by which commensal bacteria regulate protective immunity after exposure to systemic pathogens remain poorly understood. Here, we demonstrate that antibiotic-treated (ABX) mice exhibit impaired innate and adaptive antiviral immune responses and substantially delayed viral clearance after exposure to systemic LCMV or mucosal influenza virus. Furthermore, ABX mice exhibited severe bronchiole epithelial degeneration and increased host mortality after influenza virus infection. Genome-wide transcriptional profiling of macrophages isolated from ABX mice revealed decreased expression of genes associated with antiviral immunity. Moreover, macrophages from ABX mice exhibited defective responses to type I and type II IFNs and impaired capacity to limit viral replication. Collectively, these data indicate that commensal-derived signals provide tonic immune stimulation that establishes the activation threshold of the innate immune system required for optimal antiviral immunity.
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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. Keywords Streptococcus salivarius -Safety-BLIS-Bacteriocins-Probiotics