BACKGROUND
streptococcus pneumoniae  commonly colonises the nasopharynx of healthy humans, especially young children.
carriage is considered a prerequisite for pneumococcal disease and facilitates the transmission of pneumococci throughout communities  <cit> .
dissemination of pneumococci from the nasopharynx to other body sites can give rise to diseases such as meningitis, sepsis, pneumonia, and otitis media.
an estimated  <dig>  children under the age of five die from pneumococcal infections each year, with most deaths occurring in low-income countries where carriage rates are especially high  <cit> .
strategies targeting the reduction of pneumococcal colonisation could potentially reduce this burden of disease.
current pneumococcal conjugate vaccines  induce protection against 10– <dig> of the most common disease-causing serotypes via the induction of anti-capsular antibodies.
although pcvs have successfully reduced carriage and disease caused by vaccine serotypes, they are expensive to produce and have led to an increase in colonisation by non-vaccine serotypes   <cit> .
in recent years, there has been increased interest in the use of probiotics, which are defined as live microorganisms that can confer a health benefit to the host, to reduce pathogen colonisation and respiratory tract infections  <cit> .
proposed mechanisms of probiotic action include inhibition of pathogen colonisation via competition for binding, direct inhibition due to the activity of secreted antimicrobial molecules and the induction of immunomodulatory effects in the host .
streptococcus salivarius is a member of the respiratory tract microbiota and has been commercially available as an oral probiotic for more than a decade  <cit> .
small clinical trials have shown that administration of s. salivarius strains k <dig> and m <dig> can reduce the occurrence of tonsillitis and otitis media  <cit>  and reduce dental plaque levels in children  <cit> , as well as treat halitosis in adults  <cit> .
several in vitro studies have found that s. salivarius can prevent the growth of a range of respiratory pathogens, including the pneumococcus, through production of megaplasmid-encoded bacteriocins and bacteriocin-like inhibitory substances  .
however, the mechanisms by which they inhibit pathogen adherence in vivo are unknown.
we have previously shown that s. salivarius k <dig> can inhibit pneumococcal adherence to a human epithelial cell line   <cit> .
here, we demonstrate that the same phenomenon is observed in detroit  <dig> pharyngeal epithelial cells and investigate the inhibitory mechanisms involved, including the role of the s. salivarius megaplasmid.
our results suggest that s. salivarius k <dig> inhibits pneumococcal adherence by blocking pneumococcal binding sites, although other mechanisms such as direct interference through the action of secreted molecules may also contribute.
methods
bacterial strains, cell lines and culture conditions
bacterial isolates are described in tables  <dig> and  <dig>  pneumococcal isolates were selected to represent a range of serotypes and based on their ability to adhere to human epithelial cells.
s. salivarius isolates were sourced from blis technologies ltd, new zealand.
all isolates were cultured at 37 °c in 5 % co <dig> on horse blood agar  plates, in todd-hewitt broth , or thb supplemented with  <dig>  %  yeast extract .
deferred antagonism testing was carried out on baca  plates supplemented with human blood  and caco <dig>  except where noted.table  <dig>
streptococcus salivarius isolates used in this study
k <dig> mp−
m <dig> mp−
sala: salivaricin a  <cit> ; salb: salivaricin b  <cit> ; sal9: salivaricin  <dig>  <cit> ; salm: salivaricin m  <cit> ; mp: megaplasmid
the detroit  <dig> pharyngeal epithelial carcinoma cell line  was maintained in rpmi  <dig>  supplemented with 10 %  foetal bovine serum .
monolayers were released by incubation with  <dig>  % trypsin/edta  trypsin,  <dig>  mm edta, life technologies) for 10 min at 37 °c in 5 % co <dig> and seeded into 24-well trays at a concentration of  <dig>  ×  <dig> cells/well in rpmi supplemented with 5 % fbs for use in adherence assays.
deferred antagonism testing
testing for deferred antagonism was performed as described previously  <cit> .
briefly, overnight thb cultures of s. salivarius  were used to inoculate a 1-cm-wide line down the middle of a human blood agar plate .
plates were incubated for 18 h at 37 °c in 5 % co <dig>  s. salivarius growth was removed, and the plate inverted over a chloroform-soaked cloth for 30 min to kill any remaining bacteria.
residual chloroform was then evaporated by exposure of the agar surface to air for 30 min.
overnight thb cultures of pneumococcal indicator strains were then streaked at right angles to the previously grown s. salivarius and incubated for a further 12–16 h. plates were examined for growth where s. salivarius had previously grown, and prevention of pneumococcal growth recorded as +  or – .
adherence assays
adherence assays were performed as described previously  <cit>  with several modifications.
d <dig> cells seeded into 24-well trays were washed twice with hanks buffered salt solution  before the addition of 500 μl/well rpmi without serum.
bacteria were grown to log phase in thy  and resuspended in  <dig>  %  nacl  to ~ <dig>  × 109 cfu/ml.
a 10-fold dilution series of s. salivarius was prepared in  <dig>  % nacl and 10 μl aliquots of high , medium , and low  concentrations of bacteria were administered to duplicate wells before centrifugation at  <dig> × g for 3 min to promote bacterial adherence to the cell monolayer.
plates were incubated at 37 °c in 5 % co <dig> for 1 h. pneumococci were prepared as above and added to all wells at a concentration of ~ <dig>  × 106 cfu/well  1 h after s. salivarius.
centrifugation was performed as above and plates were incubated for a further 1 h. cells  were then washed three times with hbss and harvested after the addition of 200 μl  <dig>  % trypsin/edta to each well, incubation for 10 min at 37 °c in 5 % co <dig>  and addition of 800 μl of thy to each well with mechanical disruption.
lysates were stored at −20 °c until tested.
pneumococcal adherence  was determined by quantitative real-time pcr  targeting lyta .
the effect of bacterial infection on d <dig> cells was monitored microscopically and no significant cytopathic effects were observed during the adherence assay conditions.
the effect of s. salivarius on pneumococcal adherence was calculated by normalising all treatments against wells containing pneumococci alone.
the ability of the pneumococcal isolates to invade cells was measured by incubating cells with media containing 10 μg/ml penicillin and 200 μg/ml gentamicin for 15 min after incubation with pneumococci for 1 h , confirming the validity of this model to investigate adherence.
the adherence of s. salivarius to d <dig> cells at the time of pneumococcal administration was calculated following incubation of ~ <dig>  × 106 cfu/well bacteria for 1 h at 37 °c in 5 % co <dig>  cells were washed three times with hbss to remove non-adherent bacteria and harvested as above.
cfu/well was determined by viable count on hba plates and expressed as mean % adherence ± standard deviation compared to the inoculum added.
we performed preliminary experiments to determine whether heparin, which can inhibit pneumococcal adherence to epithelial cells in vitro by competitively binding to glycosylaminoglycans  <cit> , could also inhibit s. salivarius adherence to d <dig> cells.
cells were incubated with  <dig> u/well of heparin  for 1 h before the addition of s. salivarius as above and results expressed as % adherence compared to s. salivarius without heparin.
s. salivarius adherence to d <dig> cells over time was measured by qpcr targeting dex .
we also investigated whether non-adherent s. salivarius had an effect on pneumococcal adherence.
to do this, adherence assays were performed as described above, except that wells were washed three times with hbss to remove unbound s. salivarius and fresh medium was added after s. salivarius pre-administration and before the addition of pneumococci.
to determine the inhibitory effect of s. salivarius in the absence of cell contact, s. salivarius was pre-administered to d <dig> cells via permeable transwell inserts with a pore size of  <dig>  μm .
to further investigate the mechanisms involved in the inhibition of pneumococcal adherence, preparations of k <dig> were: a) heat-killed by incubating at 70 °c for 30 min with their lack of viability confirmed by the absence of growth on hba; b) enzymatically treated to remove outer surface proteins and carbohydrates of s. salivarius k <dig> by  resuspending k <dig> inocula in a final concentration of 5 mg/ml pronase e  <cit> , or  by the addition of 5 μl of 1 mg/ml sodium periodate  <cit>  to k <dig> inocula prepared in  <dig>  % nacl for 15 min at 37 °c in 5 % co <dig>  and c) reducing k <dig> protein synthesis by resuspending k <dig> inocula in 2 mg/ml spectinomycin and incubating for 30 min at 37 °c in 5 % co <dig>  following all treatments, k <dig> inocula were washed twice in  <dig>  % nacl prior to use in adherence assays.
qpcr
dna extraction and pneumococcal qpcr assays were performed as described previously  <cit> .
primers  and dual-labelled probes  were designed to detect chromosomal  and megaplasmid  target sequences in s. salivarius.
primer/probe sequences and final concentrations were as follows: dexf2: tgaagcagataacttggtggtg ; dexr2: ctctctgctggcacagctt ; dex probe2: hex-agaagtaggtccatcatctgcc-3′-bhq- <dig> ; mpf: aagccttgtgcatcgactct ; mpr: aaccaagacgcgactgttga ; mp probe: fam-tgaccctttttgttggtcgt-3′-bhq- <dig> .
specificity of both assays was confirmed by sequencing the amplified product and testing against a panel of closely related streptococcal species .
qpcr reactions were carried out in duplicate using agilent brilliant iii master mix , containing 1 μl of template dna in 25 μl final volume using a stratagene mx3005p qpcr machine .
the cycling conditions were:  <dig> × 3 min at 95 °c, 40 × 20 s at 95 °c followed by 20 s at 60 °c.
pneumococcal and s. salivarius dna was quantified using standard curves of reference strain isolates  and bacterial loads were calculated as described previously  <cit> , based on 1 pg of genomic dna being equivalent to  <dig>  pneumococcal cells and  <dig>  s. salivarius cells .
statistical analysis
data were analysed using prism  <dig> d  and excel .
student’s t-test was used to compare pneumococcal adherence in all treatment groups.
spearman’s rank test was used to correlate pneumococcal and s. salivarius k <dig> adherence in adherence assays.
a p value of <  <dig>  was considered statistically significant for all assays.
for all experiments n ≥  <dig> unless otherwise stated.
RESULTS
s. salivarius reduces pneumococcal adherence to pharyngeal epithelial cells
previous studies in our laboratory demonstrated that s. salivarius k <dig> inhibits pneumococcal adherence to ccl- <dig> human epithelial cells in vitro, with the strongest effect seen when k <dig> was added before pneumococci .
in this study, we investigated the mechanisms underlying this inhibition using a cell line more relevant to the pneumococcal ecological niche of the nasopharynx, detroit  <dig> human pharyngeal epithelial cells , and two isolates representing serotypes that commonly colonise .
pneumococcal adherence was measured following pre-administration of commercial probiotic s. salivarius strains k <dig> or m <dig> at high, medium and low doses.
all doses of k <dig> significantly inhibited 6a and 19f adherence .
all doses of m <dig> inhibited 6a adherence , but only the high dose inhibited 19f adherence .
k <dig> adherence to d <dig> cells was higher than that of m <dig> .
heparin, which is known to inhibit pneumococcal adherence to epithelial cells by competing for binding to cell surface glycosylaminoglycans  <cit> , did not affect s. salivarius k <dig> adherence to d <dig> cells .fig.
<dig> pneumococcal adherence of serotypes 6a  and 19f  to pharyngeal epithelial cells following pre-administration of s. salivarius.
approximately  <dig>  × 106 cfu pneumococci were added to d <dig> monolayers at an moi of 11: <dig>  pneumococcal adherence was determined when incubated with pneumococci alone , or pre-incubated for 1 h with s. salivarius k <dig> or m <dig> at high , medium , or low  doses.
data are mean + sd; n ≥  <dig>  * indicates p <  <dig> , ** indicates p <  <dig>  when compared to pnc alone
k <dig> adherence showed significant negative correlation with 19f adherence but not with 6a adherence .
significant inhibition of pneumococcal adherence was also observed following removal of non-adherent k <dig> by washing prior to the addition of pneumococci .fig.
<dig> correlation of adherence of s. salivarius k <dig> and pneumococcal serotype 6a  and 19f  adherence to pharyngeal epithelial cells.
adherence of s. salivarius and pneumococci to d <dig> monolayers was determined following pre-incubation for 1 h with high , medium , or low  doses of s. salivarius k <dig> and subsequent incubation with ~ <dig>  × 106 cfu pneumococci for 1 h. r = spearman rank correlation coefficient
p valueb
p valueb
p valueb
adirect: pre-administration of k <dig> directly onto d <dig> monolayer; transwells: pre-administration of k <dig> via transwell inserts; direct + wash: pre-administration of k <dig> directly onto d <dig> monolayers followed by a washing step to remove non-adherent k12
b
p values calculated from pneumococcal adherence alone compared to pneumococcal adherence following different methods of k <dig> pre-administration
chigh: ~ <dig>  × 107 cfu/ml; medium: ~ <dig>  × 106 cfu/ml and low: ~ <dig>  × 105 cfu/ml
the k <dig> megaplasmid is essential to prevent pneumococcal growth but not adherence
the megaplasmids of s. salivarius k <dig> and m <dig> encode bacteriocins that prevent the growth of a range of bacterial species on solid media  <cit> , but their roles in inhibition of pneumococcal growth or adherence are unknown.
we compared the ability of k <dig>  m <dig>  their megaplasmid-negative derivatives, and a panel of s. salivarius strains to inhibit the growth of  <dig> pneumococcal isolates by deferred antagonism.
s. salivarius strains k <dig>  m <dig>  a <dig>  nr, t18a and min <dig> inhibited all of the pneumococcal isolates tested .
neither megaplasmid-negative strain  or strain t30a prevented the growth of any of the pneumococcal isolates tested .table  <dig> deferred antagonism of s. salivarius strains against a panel of pneumococcal isolates
pmp843a
“+” indicates inhibition of pneumococcal growth and “−" indicates no inhibition, n = 2
nt not tested
aperformed on horse blood agar plates
since the s. salivarius megaplasmids were essential to prevent the growth of pneumococci, and k <dig> caused a greater reduction in pneumococcal adherence than m <dig> , we next compared the ability of k <dig> and k <dig> mp− strains to inhibit pneumococcal adherence to d <dig> cells.
although the exact copy number of the megaplasmid is unknown, preliminary qpcr analysis confirmed that the ratio of k <dig> chromosomal dna to megaplasmid dna remained stable during bacterial growth  and throughout assay conditions .
all doses of k <dig> mp− significantly inhibited 6a adherence .
when compared to k <dig>  there was some evidence that the k <dig> mp− strain resulted in less inhibition than the k <dig> strain, but this difference was only statistically significant at the high dose .
for the 19f strain, only the high and medium doses of k <dig> mp− significantly inhibited pneumococcal adherence.
we found no differences in the ability of k <dig> and k <dig> mp− to adhere to d <dig> cells  or in their adherence to d <dig> cells over time .fig.
<dig> adherence of pneumococcal serotypes 6a  and 19f  to pharyngeal epithelial cells following pre-administration of megaplasmid positive  and negative  s. salivarius k <dig> strains.
approximately  <dig>  × 106 cfu pneumococci were added to d <dig> monolayers at an moi of 10: <dig>  pneumococcal adherence was determined when incubated with pneumococci alone , or pre-incubated for 1 h with s. salivarius k <dig> or s. salivarius k12mp
- at high ; medium ; or low  doses.
data are mean + sd; n ≥  <dig>  * indicates p <  <dig> , ** indicates p <  <dig>  when compared to pnc alone
fig.
<dig> time course of s. salivarius adherence to d <dig> cells.
cells were inoculated with either ~ <dig>  × 105 cfu of s. salivarius k <dig> or ~ <dig>  × 105 cfu of s. salivarius k12mp- and the number of adherent bacteria measured over three hours.
median ± iqr for both s. salivarius isolates are depicted
cell contact is required for k <dig> to inhibit adherence by serotype 19f, but not serotype 6a
to determine whether contact of s. salivarius with d <dig> cells was required to inhibit pneumococcal adherence, we performed adherence assays in which k <dig> was administered using transwell inserts, preventing direct contact with epithelial cells but allowing the passage of secreted molecules.
using this transwell system, no inhibition of 19f adherence was observed at any dose, while 6a adherence was inhibited at the medium  and low  doses .
preliminary experiments showed that culture supernatants did not inhibit pneumococcal growth in the deferred antagonism test.
protein synthesis may be required for k <dig> to inhibit 6a adherence
the above experiments indicated the involvement of multiple mechanisms for s. salivarius inhibiting pneumococcal adherence.
we further investigated the molecular mechanism of s. salivarius k <dig> inhibition of 6a adherence by performing different treatments on the k <dig> inoculum prior to its use in adherence assays.
denaturing proteins and killing k <dig> cells , interrupting protein synthesis , and removing outer surface carbohydrates  and proteins  all reduced inhibition of 6a adherence compared with untreated k <dig> .
however, only spectinomycin-treated k <dig> could no longer significantly inhibit 6a adherence .fig.
<dig> adherence of pneumococcal serotype 6a to pharyngeal epithelial cells following pre-administration of treated s. salivarius k <dig>  approximately  <dig>  × 106 cfu pneumococci were added to d <dig> monolayers at an moi of 10: <dig>  pneumococcal adherence was determined when incubated with pneumococci alone , or pre-incubated for 1 h with approximately  <dig>  × 107 cfu s. salivarius k <dig> , or treated with sodium periodate, pronase e, heat-killed , or spectinomycin.
data are mean + sd; n ≥  <dig>  * indicates p <  <dig> , ** indicates p <  <dig>  when compared to pnc alone
discussion
in this study, we found that various bacteriocin-producing strains of s. salivarius can prevent pneumococcal growth on solid media and that the commercial probiotic strains, k <dig> and m <dig>  inhibit pneumococcal 6a and 19f adherence to a pharyngeal epithelial cell line.
studies investigating the probiotic mechanisms of s. salivarius have largely focused on the role of bacteriocins and blis.
the ~190 kb megaplasmid of s. salivarius k <dig> harbours genes encoding the bacteriocins salivaricin a <dig> and b, which prevent the growth of some bacterial pathogens, including streptococcus pyogenes, in vitro  <cit> .
our results showed that the k <dig> megaplasmid is required to prevent pneumococcal growth in vitro, but is not essential to inhibit pneumococcal adherence.
therefore the primary mechanism of inhibition of pneumococcal adherence in this model does not appear to be mediated by megaplasmid-encoded bacteriocins; although we note that lack of inhibition of 19f adherence by a low dose of k12mp- indicates a possible role for the megaplasmid at this concentration.
instead, our findings indicated that s. salivarius k <dig> can inhibit pneumococcal adherence by blocking pneumococcal binding sites on d <dig> cells.
we observed a correlation between s. salivarius k <dig> adherence to d <dig> cells and inhibition of adherence by pneumococcal isolate pmp <dig> .
we also found that k <dig> contact with cells was essential for this inhibition.
k <dig> binding to d <dig> cells did not require megaplasmid-encoded molecules, since a megaplasmid-negative strain of k <dig> displayed equal adherence.
we observed differences in the inhibition of pneumococcal adherence depending on both the strain of s. salivarius used and the pneumococcal isolate tested.
this was not unexpected, given that s. salivarius strains vary in their capacity to adhere to epithelial cells and inhibit other pathogens  <cit> .
the pneumococcus is a diverse pathogen, with different strains and serotypes known to behave differently in vitro and in vivo  <cit> .
overall, s. salivarius k <dig> displayed stronger inhibition of pneumococcal adherence than the m <dig> strain, possibly due to its increased capacity to adhere to d <dig> cells.
interestingly, k <dig> adherence to d <dig> cells did not correlate with 6a adherence, suggesting that mechanisms other than s. salivarius-mediated blocking of pneumococcal binding sites on epithelial cells may be involved in the inhibition of 6a adherence.
this was further supported by the inhibition of 6a adherence by all doses of m <dig> despite its low adherence capability.
although a high dose of megaplasmid-negative k <dig> could still inhibit 6a adherence, the effect was significantly less than that observed with megaplasmid-positive k <dig>  indicating a possible contribution to inhibition by the k <dig> megaplasmid in this assay.
transwell experiments demonstrated that for 19f, contact of s. salivarius k <dig> with d <dig> cells was required to inhibit pneumococcal adherence, consistent with a previous report showing that contact with epithelial cells was needed for s. salivarius k <dig> to inhibit adherence by s. pyogenes to hep- <dig> cells  <cit> .
however, low doses of k <dig> administered in the absence of cell contact could inhibit adherence of 6a.
this could be due to bacteriocins or other secreted products, which may not necessarily be encoded on the megaplasmid.
the differences observed using varying doses of probiotic suggest that quorum sensing may play a role in the expression of s. salivarius genes relevant to pneumococcal inhibition.
further transwell experiments that test the k <dig> megaplasmid-negative strain may determine whether the molecules involved are chromosomally or plasmid encoded.
preliminary attempts to detect if bacteriocins were secreted through transwell membranes by deferred antagonism were unsuccessful, however this approach may have been insufficiently sensitive.
overall, the data obtained in this study suggest that the primary mechanism by which s. salivarius k <dig> inhibits pneumococcal adherence to d <dig> cells is by blocking pneumococcal attachment to the epithelial cell surface.
a number of pneumococcal surface molecules have been shown to play a role in adherence, such as the adhesion molecule psaa, which binds to cell surface carbohydrates  <cit> .
for s. salivarius, various cell surface molecules including fibrils and fimbriae have been implicated in adherence  <cit> .
as heparin does not block s. salivarius adherence to d <dig> cells, glycosylaminoglycans do not seem to be a shared cell surface receptor.
since airway epithelial cells derived from carcinomas, such as d <dig> cells, can display altered expression of surface molecules compared to primary human epithelial cells  <cit> , further investigation of common receptors used by these species should include testing of primary cells.
although inhibition of pneumococcal adherence could be due to competition for receptor-mediated binding, it could also result through a less specific mechanism, such as steric hindrance, or the ability of s. salivarius to mask pneumococcal binding sites.
other potential mechanisms by which s. salivarius may inhibit pneumococcal adherence, such as immune modulation of epithelial cells  <cit> , were not examined in this study.
additionally, the in vitro model lacks the complexity of the nasopharynx, which can harbour a wide range of colonising bacterial species that interact with each other and with epithelial cells.
whilst megaplasmid-encoded bacteriocins are required to inhibit the growth of pneumococci on solid media, our data suggest that it is the adherence of s. salivarius to epithelial cells that primarily blocks pneumococcal adherence in vitro.
therefore, strategies to use s. salivarius as a probiotic in the respiratory tract should optimise the ability of this species to colonise the target tissue.
in vitro studies performed in our laboratory, and by others, have shown that the addition of probiotic bacteria to epithelial cells prior to pathogen administration is more effective than attempting to disrupt established pathogen colonisation  <cit> .
in clinical studies, high doses of m <dig> achieve greater colonisation rates and density in the oral cavity compared to lower doses  <cit> .
clinical studies of s. salivarius k <dig> as an oral probiotic have involved daily administration of high doses of the bacterium in an effort to achieve long-term colonisation of the oral cavity .
s. salivarius may be better suited as a probiotic for the oral cavity, where it predominates, than for the nasopharynx, the preferred niche of the pneumococcus.
strategies that facilitate s. salivarius adherence to the nasopharyngeal epithelium, such as the nasal spray recently employed by santagati and colleagues  <cit> , would likely be needed for this probiotic to inhibit pneumococcal colonisation in vivo.
CONCLUSIONS
bacteriocin-encoding megaplasmids of s. salivarius strains k <dig> and m <dig> were essential to prevent pneumococcal growth on solid media but were not required to inhibit pneumococcal adherence to pharyngeal epithelial cells.
our results suggest that s. salivarius k <dig> employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells.
further research is needed to identify the specific molecules involved.
these findings contribute to our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.
additional files
additional file 1: supporting information s <dig>  raw data used to generate figures and tables.
additional file 2: table s <dig>  specificity of dex and mp gene targets to detect streptococcus salivarius.
dna was extracted from pure cultures grown on hba agar and  <dig>  ng dna of each isolate listed was used as a template for the dex and mp qpcr reactions, as described in the methods section.
mean ct from duplicate wells is shown.
abbreviations
atccamerican type culture collection
blisbacteriocin-like inhibitory substance
cfucolony forming unit
dnadeoxyribonucleic acid
edtaethylenediaminetetraacetic acid
fbsfoetal bovine serum
hbahorse blood agar
hbsshanks buffered salt solution
moimultiplicity of infection
pcrpolymerase chain reaction
thbtodd hewitt broth
thytodd hewitt broth supplemented with yeast extract

 streptococcus pneumoniae commonly colonises the nasopharynx of healthy humans, especially young children. carriage is considered a prerequisite for pneumococcal disease and facilitates the transmission of pneumococci throughout communities. dissemination of pneumococci from the nasopharynx to other body sites can give rise to diseases such as meningitis, sepsis, pneumonia, and otitis media. an estimated children under the age of five die from pneumococcal infections each year, with most deaths occurring in low-income countries where carriage rates are especially high. strategies targeting the reduction of pneumococcal colonisation could potentially reduce this burden of disease. current pneumococcal conjugate vaccines induce protection against 10– of the most common disease-causing serotypes via the induction of anti-capsular antibodies. although pcvs have successfully reduced carriage and disease caused by vaccine serotypes, they are expensive to produce and have led to an increase in colonisation by non-vaccine serotypes. in recent years, there has been increased interest in the use of probiotics, which are defined as live microorganisms that can confer a health benefit to the host, to reduce pathogen colonisation and respiratory tract infections. proposed mechanisms of probiotic action include inhibition of pathogen colonisation via competition for binding, direct inhibition due to the activity of secreted antimicrobial molecules and the induction of immunomodulatory effects in the host. streptococcus salivarius is a member of the respiratory tract microbiota and has been commercially available as an oral probiotic for more than a decade. small clinical trials have shown that administration of s. salivarius strains k and m can reduce the occurrence of tonsillitis and otitis media and reduce dental plaque levels in children, as well as treat halitosis in adults. several in vitro studies have found that s. salivarius can prevent the growth of a range of respiratory pathogens, including the pneumococcus, through production of megaplasmid-encoded bacteriocins and bacteriocin-like inhibitory substances. however, the mechanisms by which they inhibit pathogen adherence in vivo are unknown. we have previously shown that s. salivarius k can inhibit pneumococcal adherence to a human epithelial cell line. here, we demonstrate that the same phenomenon is observed in detroit pharyngeal epithelial cells and investigate the inhibitory mechanisms involved, including the role of the s. salivarius megaplasmid. our results suggest that s. salivarius k inhibits pneumococcal adherence by blocking pneumococcal binding sites, although other mechanisms such as direct interference through the action of secreted molecules may also contribute. methods bacterial strains, cell lines and culture conditions bacterial isolates are described in tables  and pneumococcal isolates were selected to represent a range of serotypes and based on their ability to adhere to human epithelial cells. s. salivarius isolates were sourced from blis technologies ltd, new zealand. all isolates were cultured at 37 °c in 5 % co on horse blood agar plates, in todd-hewitt broth, or thb supplemented with  % yeast extract. deferred antagonism testing was carried out on baca plates supplemented with human blood and caco except where noted.table streptococcus salivarius isolates used in this study k mp− m mp− sala: salivaricin a; salb: salivaricin b; sal9: salivaricin; salm: salivaricin m; mp: megaplasmid the detroit pharyngeal epithelial carcinoma cell line was maintained in rpmi supplemented with 10 % foetal bovine serum. monolayers were released by incubation with  % trypsin/edta trypsin,  mm edta, life technologies) for 10 min at 37 °c in 5 % co and seeded into 24-well trays at a concentration of × cells/well in rpmi supplemented with 5 % fbs for use in adherence assays. deferred antagonism testing testing for deferred antagonism was performed as described previously. briefly, overnight thb cultures of s. salivarius were used to inoculate a 1-cm-wide line down the middle of a human blood agar plate. plates were incubated for 18 h at 37 °c in 5 % co s. salivarius growth was removed, and the plate inverted over a chloroform-soaked cloth for 30 min to kill any remaining bacteria. residual chloroform was then evaporated by exposure of the agar surface to air for 30 min. overnight thb cultures of pneumococcal indicator strains were then streaked at right angles to the previously grown s. salivarius and incubated for a further 12–16 h. plates were examined for growth where s. salivarius had previously grown, and prevention of pneumococcal growth recorded as +  or –. adherence assays adherence assays were performed as described previously with several modifications. d cells seeded into 24-well trays were washed twice with hanks buffered salt solution before the addition of 500 μl/well rpmi without serum. bacteria were grown to log phase in thy and resuspended in  % nacl to ~ × 109 cfu/ml. a 10-fold dilution series of s. salivarius was prepared in  % nacl and 10 μl aliquots of high, medium, and low concentrations of bacteria were administered to duplicate wells before centrifugation at × g for 3 min to promote bacterial adherence to the cell monolayer. plates were incubated at 37 °c in 5 % co for 1 h. pneumococci were prepared as above and added to all wells at a concentration of ~ × 106 cfu/well 1 h after s. salivarius. centrifugation was performed as above and plates were incubated for a further 1 h. cells were then washed three times with hbss and harvested after the addition of 200 μl  % trypsin/edta to each well, incubation for 10 min at 37 °c in 5 % co and addition of 800 μl of thy to each well with mechanical disruption. lysates were stored at −20 °c until tested. pneumococcal adherence was determined by quantitative real-time pcr targeting lyta. the effect of bacterial infection on d cells was monitored microscopically and no significant cytopathic effects were observed during the adherence assay conditions. the effect of s. salivarius on pneumococcal adherence was calculated by normalising all treatments against wells containing pneumococci alone. the ability of the pneumococcal isolates to invade cells was measured by incubating cells with media containing 10 μg/ml penicillin and 200 μg/ml gentamicin for 15 min after incubation with pneumococci for 1 h, confirming the validity of this model to investigate adherence. the adherence of s. salivarius to d cells at the time of pneumococcal administration was calculated following incubation of ~ × 106 cfu/well bacteria for 1 h at 37 °c in 5 % co cells were washed three times with hbss to remove non-adherent bacteria and harvested as above. cfu/well was determined by viable count on hba plates and expressed as mean % adherence ± standard deviation compared to the inoculum added. we performed preliminary experiments to determine whether heparin, which can inhibit pneumococcal adherence to epithelial cells in vitro by competitively binding to glycosylaminoglycans, could also inhibit s. salivarius adherence to d cells. cells were incubated with u/well of heparin for 1 h before the addition of s. salivarius as above and results expressed as % adherence compared to s. salivarius without heparin. s. salivarius adherence to d cells over time was measured by qpcr targeting dex. we also investigated whether non-adherent s. salivarius had an effect on pneumococcal adherence. to do this, adherence assays were performed as described above, except that wells were washed three times with hbss to remove unbound s. salivarius and fresh medium was added after s. salivarius pre-administration and before the addition of pneumococci. to determine the inhibitory effect of s. salivarius in the absence of cell contact, s. salivarius was pre-administered to d cells via permeable transwell inserts with a pore size of  μm. to further investigate the mechanisms involved in the inhibition of pneumococcal adherence, preparations of k were: a) heat-killed by incubating at 70 °c for 30 min with their lack of viability confirmed by the absence of growth on hba; b) enzymatically treated to remove outer surface proteins and carbohydrates of s. salivarius k by resuspending k inocula in a final concentration of 5 mg/ml pronase e, or by the addition of 5 μl of 1 mg/ml sodium periodate to k inocula prepared in  % nacl for 15 min at 37 °c in 5 % co and c) reducing k protein synthesis by resuspending k inocula in 2 mg/ml spectinomycin and incubating for 30 min at 37 °c in 5 % co following all treatments, k inocula were washed twice in  % nacl prior to use in adherence assays. qpcr dna extraction and pneumococcal qpcr assays were performed as described previously. primers and dual-labelled probes were designed to detect chromosomal and megaplasmid target sequences in s. salivarius. primer/probe sequences and final concentrations were as follows: dexf2: tgaagcagataacttggtggtg; dexr2: ctctctgctggcacagctt; dex probe2: hex-agaagtaggtccatcatctgcc-3′-bhq-; mpf: aagccttgtgcatcgactct; mpr: aaccaagacgcgactgttga; mp probe: fam-tgaccctttttgttggtcgt-3′-bhq-. specificity of both assays was confirmed by sequencing the amplified product and testing against a panel of closely related streptococcal species. qpcr reactions were carried out in duplicate using agilent brilliant iii master mix, containing 1 μl of template dna in 25 μl final volume using a stratagene mx3005p qpcr machine. the cycling conditions were: × 3 min at 95 °c, 40 × 20 s at 95 °c followed by 20 s at 60 °c. pneumococcal and s. salivarius dna was quantified using standard curves of reference strain isolates and bacterial loads were calculated as described previously, based on 1 pg of genomic dna being equivalent to pneumococcal cells and  s. salivarius cells. statistical analysis data were analysed using prism d and excel. student’s t-test was used to compare pneumococcal adherence in all treatment groups. spearman’s rank test was used to correlate pneumococcal and s. salivarius k adherence in adherence assays. a p value of <  was considered statistically significant for all assays. for all experiments n ≥  unless otherwise stated.  s. salivarius reduces pneumococcal adherence to pharyngeal epithelial cells previous studies in our laboratory demonstrated that s. salivarius k inhibits pneumococcal adherence to ccl- human epithelial cells in vitro, with the strongest effect seen when k was added before pneumococci. in this study, we investigated the mechanisms underlying this inhibition using a cell line more relevant to the pneumococcal ecological niche of the nasopharynx, detroit human pharyngeal epithelial cells, and two isolates representing serotypes that commonly colonise. pneumococcal adherence was measured following pre-administration of commercial probiotic s. salivarius strains k or m at high, medium and low doses. all doses of k significantly inhibited 6a and 19f adherence. all doses of m inhibited 6a adherence, but only the high dose inhibited 19f adherence. k adherence to d cells was higher than that of m. heparin, which is known to inhibit pneumococcal adherence to epithelial cells by competing for binding to cell surface glycosylaminoglycans, did not affect s. salivarius k adherence to d cells.fig. pneumococcal adherence of serotypes 6a and 19f to pharyngeal epithelial cells following pre-administration of s. salivarius. approximately  × 106 cfu pneumococci were added to d monolayers at an moi of 11: pneumococcal adherence was determined when incubated with pneumococci alone, or pre-incubated for 1 h with s. salivarius k or m at high, medium, or low doses. data are mean + sd; n ≥  * indicates p < , ** indicates p <  when compared to pnc alone k adherence showed significant negative correlation with 19f adherence but not with 6a adherence. significant inhibition of pneumococcal adherence was also observed following removal of non-adherent k by washing prior to the addition of pneumococci.fig. correlation of adherence of s. salivarius k and pneumococcal serotype 6a and 19f adherence to pharyngeal epithelial cells. adherence of s. salivarius and pneumococci to d monolayers was determined following pre-incubation for 1 h with high, medium, or low doses of s. salivarius k and subsequent incubation with ~ × 106 cfu pneumococci for 1 h. r = spearman rank correlation coefficient p valueb p valueb p valueb adirect: pre-administration of k directly onto d monolayer; transwells: pre-administration of k via transwell inserts; direct + wash: pre-administration of k directly onto d monolayers followed by a washing step to remove non-adherent k12 b p values calculated from pneumococcal adherence alone compared to pneumococcal adherence following different methods of k pre-administration chigh: ~ × 107 cfu/ml; medium: ~ × 106 cfu/ml and low: ~ × 105 cfu/ml the k megaplasmid is essential to prevent pneumococcal growth but not adherence the megaplasmids of s. salivarius k and m encode bacteriocins that prevent the growth of a range of bacterial species on solid media, but their roles in inhibition of pneumococcal growth or adherence are unknown. we compared the ability of k m their megaplasmid-negative derivatives, and a panel of s. salivarius strains to inhibit the growth of pneumococcal isolates by deferred antagonism. s. salivarius strains k m a nr, t18a and min inhibited all of the pneumococcal isolates tested. neither megaplasmid-negative strain or strain t30a prevented the growth of any of the pneumococcal isolates tested.table deferred antagonism of s. salivarius strains against a panel of pneumococcal isolates pmp843a “+” indicates inhibition of pneumococcal growth and “−" indicates no inhibition, n = 2 nt not tested aperformed on horse blood agar plates since the s. salivarius megaplasmids were essential to prevent the growth of pneumococci, and k caused a greater reduction in pneumococcal adherence than m, we next compared the ability of k and k mp− strains to inhibit pneumococcal adherence to d cells. although the exact copy number of the megaplasmid is unknown, preliminary qpcr analysis confirmed that the ratio of k chromosomal dna to megaplasmid dna remained stable during bacterial growth and throughout assay conditions. all doses of k mp− significantly inhibited 6a adherence. when compared to k there was some evidence that the k mp− strain resulted in less inhibition than the k strain, but this difference was only statistically significant at the high dose. for the 19f strain, only the high and medium doses of k mp− significantly inhibited pneumococcal adherence. we found no differences in the ability of k and k mp− to adhere to d cells or in their adherence to d cells over time.fig. adherence of pneumococcal serotypes 6a and 19f to pharyngeal epithelial cells following pre-administration of megaplasmid positive and negative s. salivarius k strains. approximately × 106 cfu pneumococci were added to d monolayers at an moi of 10: pneumococcal adherence was determined when incubated with pneumococci alone, or pre-incubated for 1 h with s. salivarius k or s. salivarius k12mp - at high; medium; or low doses. data are mean + sd; n ≥  * indicates p < , ** indicates p <  when compared to pnc alone fig. time course of s. salivarius adherence to d cells. cells were inoculated with either ~  × 105 cfu of s. salivarius k or ~ × 105 cfu of s. salivarius k12mp- and the number of adherent bacteria measured over three hours. median ± iqr for both s. salivarius isolates are depicted cell contact is required for k to inhibit adherence by serotype 19f, but not serotype 6a to determine whether contact of s. salivarius with d cells was required to inhibit pneumococcal adherence, we performed adherence assays in which k was administered using transwell inserts, preventing direct contact with epithelial cells but allowing the passage of secreted molecules. using this transwell system, no inhibition of 19f adherence was observed at any dose, while 6a adherence was inhibited at the medium and low doses. preliminary experiments showed that culture supernatants did not inhibit pneumococcal growth in the deferred antagonism test. protein synthesis may be required for k to inhibit 6a adherence the above experiments indicated the involvement of multiple mechanisms for s. salivarius inhibiting pneumococcal adherence. we further investigated the molecular mechanism of s. salivarius k inhibition of 6a adherence by performing different treatments on the k inoculum prior to its use in adherence assays. denaturing proteins and killing k cells, interrupting protein synthesis, and removing outer surface carbohydrates and proteins all reduced inhibition of 6a adherence compared with untreated k. however, only spectinomycin-treated k could no longer significantly inhibit 6a adherence.fig. adherence of pneumococcal serotype 6a to pharyngeal epithelial cells following pre-administration of treated s. salivarius k approximately  × 106 cfu pneumococci were added to d monolayers at an moi of 10: pneumococcal adherence was determined when incubated with pneumococci alone, or pre-incubated for 1 h with approximately  × 107 cfu s. salivarius k, or treated with sodium periodate, pronase e, heat-killed, or spectinomycin. data are mean + sd; n ≥  * indicates p < , ** indicates p <  when compared to pnc alone discussion in this study, we found that various bacteriocin-producing strains of s. salivarius can prevent pneumococcal growth on solid media and that the commercial probiotic strains, k and m inhibit pneumococcal 6a and 19f adherence to a pharyngeal epithelial cell line. studies investigating the probiotic mechanisms of s. salivarius have largely focused on the role of bacteriocins and blis. the ~190 kb megaplasmid of s. salivarius k harbours genes encoding the bacteriocins salivaricin a and b, which prevent the growth of some bacterial pathogens, including streptococcus pyogenes, in vitro. our results showed that the k megaplasmid is required to prevent pneumococcal growth in vitro, but is not essential to inhibit pneumococcal adherence. therefore the primary mechanism of inhibition of pneumococcal adherence in this model does not appear to be mediated by megaplasmid-encoded bacteriocins; although we note that lack of inhibition of 19f adherence by a low dose of k12mp- indicates a possible role for the megaplasmid at this concentration. instead, our findings indicated that s. salivarius k can inhibit pneumococcal adherence by blocking pneumococcal binding sites on d cells. we observed a correlation between s. salivarius k adherence to d cells and inhibition of adherence by pneumococcal isolate pmp. we also found that k contact with cells was essential for this inhibition. k binding to d cells did not require megaplasmid-encoded molecules, since a megaplasmid-negative strain of k displayed equal adherence. we observed differences in the inhibition of pneumococcal adherence depending on both the strain of s. salivarius used and the pneumococcal isolate tested. this was not unexpected, given that s. salivarius strains vary in their capacity to adhere to epithelial cells and inhibit other pathogens. the pneumococcus is a diverse pathogen, with different strains and serotypes known to behave differently in vitro and in vivo. overall, s. salivarius k displayed stronger inhibition of pneumococcal adherence than the m strain, possibly due to its increased capacity to adhere to d cells. interestingly, k adherence to d cells did not correlate with 6a adherence, suggesting that mechanisms other than s. salivarius-mediated blocking of pneumococcal binding sites on epithelial cells may be involved in the inhibition of 6a adherence. this was further supported by the inhibition of 6a adherence by all doses of m despite its low adherence capability. although a high dose of megaplasmid-negative k could still inhibit 6a adherence, the effect was significantly less than that observed with megaplasmid-positive k indicating a possible contribution to inhibition by the k megaplasmid in this assay. transwell experiments demonstrated that for 19f, contact of s. salivarius k with d cells was required to inhibit pneumococcal adherence, consistent with a previous report showing that contact with epithelial cells was needed for s. salivarius k to inhibit adherence by s. pyogenes to hep- cells. however, low doses of k administered in the absence of cell contact could inhibit adherence of 6a. this could be due to bacteriocins or other secreted products, which may not necessarily be encoded on the megaplasmid. the differences observed using varying doses of probiotic suggest that quorum sensing may play a role in the expression of s. salivarius genes relevant to pneumococcal inhibition. further transwell experiments that test the k megaplasmid-negative strain may determine whether the molecules involved are chromosomally or plasmid encoded. preliminary attempts to detect if bacteriocins were secreted through transwell membranes by deferred antagonism were unsuccessful, however this approach may have been insufficiently sensitive. overall, the data obtained in this study suggest that the primary mechanism by which s. salivarius k inhibits pneumococcal adherence to d cells is by blocking pneumococcal attachment to the epithelial cell surface. a number of pneumococcal surface molecules have been shown to play a role in adherence, such as the adhesion molecule psaa, which binds to cell surface carbohydrates. for s. salivarius, various cell surface molecules including fibrils and fimbriae have been implicated in adherence. as heparin does not block s. salivarius adherence to d cells, glycosylaminoglycans do not seem to be a shared cell surface receptor. since airway epithelial cells derived from carcinomas, such as d cells, can display altered expression of surface molecules compared to primary human epithelial cells, further investigation of common receptors used by these species should include testing of primary cells. although inhibition of pneumococcal adherence could be due to competition for receptor-mediated binding, it could also result through a less specific mechanism, such as steric hindrance, or the ability of s. salivarius to mask pneumococcal binding sites. other potential mechanisms by which s. salivarius may inhibit pneumococcal adherence, such as immune modulation of epithelial cells, were not examined in this study. additionally, the in vitro model lacks the complexity of the nasopharynx, which can harbour a wide range of colonising bacterial species that interact with each other and with epithelial cells. whilst megaplasmid-encoded bacteriocins are required to inhibit the growth of pneumococci on solid media, our data suggest that it is the adherence of s. salivarius to epithelial cells that primarily blocks pneumococcal adherence in vitro. therefore, strategies to use s. salivarius as a probiotic in the respiratory tract should optimise the ability of this species to colonise the target tissue. in vitro studies performed in our laboratory, and by others, have shown that the addition of probiotic bacteria to epithelial cells prior to pathogen administration is more effective than attempting to disrupt established pathogen colonisation. in clinical studies, high doses of m achieve greater colonisation rates and density in the oral cavity compared to lower doses. clinical studies of s. salivarius k as an oral probiotic have involved daily administration of high doses of the bacterium in an effort to achieve long-term colonisation of the oral cavity. s. salivarius may be better suited as a probiotic for the oral cavity, where it predominates, than for the nasopharynx, the preferred niche of the pneumococcus. strategies that facilitate s. salivarius adherence to the nasopharyngeal epithelium, such as the nasal spray recently employed by santagati and colleagues, would likely be needed for this probiotic to inhibit pneumococcal colonisation in vivo.  bacteriocin-encoding megaplasmids of s. salivarius strains k and m were essential to prevent pneumococcal growth on solid media but were not required to inhibit pneumococcal adherence to pharyngeal epithelial cells. our results suggest that s. salivarius k employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. further research is needed to identify the specific molecules involved. these findings contribute to our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future. additional files additional file 1: supporting information s raw data used to generate figures and tables. additional file 2: table s specificity of dex and mp gene targets to detect streptococcus salivarius. dna was extracted from pure cultures grown on hba agar and  ng dna of each isolate listed was used as a template for the dex and mp qpcr reactions, as described in the methods section. mean ct from duplicate wells is shown. abbreviations atccamerican type culture collection blisbacteriocin-like inhibitory substance cfucolony forming unit dnadeoxyribonucleic acid edtaethylenediaminetetraacetic acid fbsfoetal bovine serum hbahorse blood agar hbsshanks buffered salt solution moimultiplicity of infection pcrpolymerase chain reaction thbtodd hewitt broth thytodd hewitt broth supplemented with yeast extract 
these results suggest that s. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. experiments testing s. salivarius k and two pneumococcal isolates showed that inhibition of 19f may involve s. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of k and 19f, and no inhibition occurred when k was prevented from contacting epithelial cells. the probiotic bacterium, streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. probioticsprobiotic mechanismsrespiratory tractstreptococcus salivariusstreptococcus pneumoniaepneumococcuscolonisationadherencemurdoch childrens research instituten/avictorian government's operational infrastructure support programn/aaustralian postgraduate award, australian governmentn/amanning jayne http://dx.doi.org/ /501100000925national health and medical research councilapp1087957satzke catherine issue-copyright-statement© the author 2016 k12-mediated inhibition of adherence by 6a may involve additional mechanisms, since no correlation was observed between adherence of k and 6a, and k could inhibit 6a adherence in the absence of cell contact. we investigated the mechanism of inhibition using a human pharyngeal epithelial cell line following pre-administration of two different strains of s. salivarius. whilst the bacteriocin-encoding megaplasmids of s. salivarius strains k and m were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. these findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future. pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease.
 pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. the probiotic bacterium, streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. we investigated the mechanism of inhibition using a human pharyngeal epithelial cell line following pre-administration of two different strains of s. salivarius.  whilst the bacteriocin-encoding megaplasmids of s. salivarius strains k and m were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. experiments testing s. salivarius k and two pneumococcal isolates showed that inhibition of 19f may involve s. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of k and 19f, and no inhibition occurred when k was prevented from contacting epithelial cells. k12-mediated inhibition of adherence by 6a may involve additional mechanisms, since no correlation was observed between adherence of k and 6a, and k could inhibit 6a adherence in the absence of cell contact.  these results suggest that s. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. these findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords probioticsprobiotic mechanismsrespiratory tractstreptococcus salivariusstreptococcus pneumoniaepneumococcuscolonisationadherencemurdoch childrens research instituten/avictorian government's operational infrastructure support programn/aaustralian postgraduate award, australian governmentn/amanning jayne http://dx.doi.org/ /501100000925national health and medical research councilapp1087957satzke catherine issue-copyright-statement© the author 2016 