for this study , our standard 10species  subgingival  biofilm model was used abiding a slightly modified protocol as described previously ( guggenheim et  al . 2009 ; ammann et  al . , biofilms were grown in medium , consisting of 60% of processed whole unstimulated pooled saliva , 30% modified fluid universal medium ( mfum ) ( gmur & guggenheim 1983 ) and 10% heat inactivated human serum . incubation was carried out for 64  h under anaerobic conditions at 37  c . the standard subgingival in  vitro biofilm was composed of actinomyces oris ( omz 745 ; formerly actinomyces naeslundii ) , campylobacter rectus omz 698 , fusobacterium nucleatum subsp . nucleatum omz 598 , porphyromonas gingivalis atcc 33277 ( omz 925 ) , prevotella intermedia atcc 25611 ( omz 278 ) , streptococcus anginosus atcc 9895 ( omz 871 ) , streptococcus oralis sk248 ( omz 607 ) , tannerella forsythia omz 1047 , treponema denticola atcc 35405 ( omz 661 ) and veillonella dispar atcc 17748 ( omz 493 ) . this standard biofilm was supplemented with either s.  epidermidis atcc 14990 ( omz 423 ) ( treatment 1 ) , or s.  aureus atcc 25923 ( omz 1122 ) ( treatment 2 ) or a mixture of the two staphylococci ( treatment 3 ) or a mixture of the latter and an additional boost inoculation of s.  epidermidis after 16  h ( treatment 4 ) . all strains were maintained on columbia blood agar ( cba ) plates , with the exception of t.  forsythia and t.  denticola that were maintained in liquid growth media as described previously ( ammann et  al . biofilms were grown in 24well polystyrene cell culture plates on hydroxyapatite (  9  mm ; clarkson chromatography products , south williamsport , pa , usa ) and titanium discs ( tiunite , nobel biocare , kloten switzerland ) that had been preconditioned ( pelliclecoated ) in 1  ml of pasteurized whole unstimulated saliva , pooled from individual donors , and incubated for 4  h at room temperature . , the pelliclecoated discs were covered with 1  ml of growth medium ( see above ) , and 200  l of a microbial suspension prepared from equal volumes and densities of each strain , corresponding to od550  =  1.0 . the carbohydrate concentration in fum was 0.3% ( w / v ) glucose . after 16  h of incubation , the growth medium was renewed , along with adding 50  l of t.  denticola liquid culture as well as 50  l of s.  epidermidis culture ( od550  =  1.0 ) in treatment 4 ( see above ) . at 16 , 20 , 24 , 40 , 44 and 48  h , biofilms were washed by three consecutive dips in 2  ml of sterile physiological saline . fresh medium was provided after 16 and 40  h. after 64  h , the biofilms were dipwashed again prior to harvesting for quantification by realtime quantitative pcr ( qpcr ) or processing for fluorescence in  situ hybridization ( fish ) staining and confocal laser scanning microscopy ( clsm ) analyses , as described below . after 64  h of biofilm growth , the discs were vortexed vigorously for 1  min in 1  ml of 0.9% nacl and then sonicated at 25w in a sonifier b12 ( branson ultrasonic , urdorf , switzerland ) for 5  s , to harvest the adherent biofilms . the resulting bacterial suspensions were then used for quantification by qpcr as described earlier ( ammann et  al . primer sequences and properties of the standard 10species biofilm are given in table  1 . the staphylococci were quantified using the microbial dna qpcr assays for s.  epidermidis and s.  aureus ( qiagen instruments , hombrechtikon , switzerland ; cat . biofilms were stained by fish using cy3 or famlabelled probes following the protocols described before ( thurnheer et  al . 2004 ; thurnheer & belibasakis 2015 ) . probe sequences and formamide concentrations used for the hybridizations , as well as the nacl concentrations of the washing buffers , are given in table  2 . for counterstaining , biofilms were stained using 3  m syto 59 ( invitrogen , lucerne , switzerland ) ( 20  min , room temperature , in the dark ) , following the fish procedure . after staining , the samples were embedded upsidedown on chamber slides in 100  l of mowiol 488 ( calbiochemnovabiochem , san diego , ca , usa ) ( guggenheim et  al . stained biofilms were examined by clsm at randomly selected positions using a leica tcs sp5 microscope ( leica microsystems , wetzlar , germany ) fitted with an ar laser ( 458  nm/476  nm/488  nm/496  nm/514  nm excitation ) , and a hene laser ( 561  nm/594  nm/633  nm excitation ) . filters were set to 500540  nm to detect fam , to 570630  nm for cy3 and to 660710  nm for syto 59 . zseries were generated by vertical optical sectioning with the slice thickness set at 1.02  m . scans were recombined and processed using imaris 7.6.5 software ( bitplane , zrich , switzerland ) , without any qualitative changes to the raw images . three independent experiments were performed , and within each experiment every group was represented in triplicate biofilm cultures . hence , statistics were performed on nine individual data points , deriving from the nine individual biofilm cultures per experimental group . the statistical significance of the differences in microbial numbers between the control group ( standard 10species  subgingival  biofilm ) and the four treatments was evaluated by twoway analysis of variance ( anova ) , corrected by tukey 's multiple comparisons test ( significance level p  <  0.05 ) . undetectable values were ascribed the lowest detection limit value of the assay to allow for logarithmic transformation . comparisons were performed between the control group and each experimental group , for each individual species . the data were analysed using the prism version 6 , statistical analysis software ( graphpad , la jolla , ca , usa ) . for this study , our standard 10species  subgingival  biofilm model was used abiding a slightly modified protocol as described previously ( guggenheim et  al . 2009 ; ammann et  al . , biofilms were grown in medium , consisting of 60% of processed whole unstimulated pooled saliva , 30% modified fluid universal medium ( mfum ) ( gmur & guggenheim 1983 ) and 10% heat inactivated human serum . incubation was carried out for 64  h under anaerobic conditions at 37  c . the standard subgingival in  vitro biofilm was composed of actinomyces oris ( omz 745 ; formerly actinomyces naeslundii ) , campylobacter rectus omz 698 , fusobacterium nucleatum subsp . nucleatum omz 598 , porphyromonas gingivalis atcc 33277 ( omz 925 ) , prevotella intermedia atcc 25611 ( omz 278 ) , streptococcus anginosus atcc 9895 ( omz 871 ) , streptococcus oralis sk248 ( omz 607 ) , tannerella forsythia omz 1047 , treponema denticola atcc 35405 ( omz 661 ) and veillonella dispar atcc 17748 ( omz 493 ) . this standard biofilm was supplemented with either s.  epidermidis atcc 14990 ( omz 423 ) ( treatment 1 ) , or s.  aureus atcc 25923 ( omz 1122 ) ( treatment 2 ) or a mixture of the two staphylococci ( treatment 3 ) or a mixture of the latter and an additional boost inoculation of s.  epidermidis after 16  h ( treatment 4 ) . all strains were maintained on columbia blood agar ( cba ) plates , with the exception of t.  forsythia and t.  denticola that were maintained in liquid growth media as described previously ( ammann et  al . biofilms were grown in 24well polystyrene cell culture plates on hydroxyapatite (  9  mm ; clarkson chromatography products , south williamsport , pa , usa ) and titanium discs ( tiunite , nobel biocare , kloten switzerland ) that had been preconditioned ( pelliclecoated ) in 1  ml of pasteurized whole unstimulated saliva , pooled from individual donors , and incubated for 4  h at room temperature . , the pelliclecoated discs were covered with 1  ml of growth medium ( see above ) , and 200  l of a microbial suspension prepared from equal volumes and densities of each strain , corresponding to od550  =  1.0 . the carbohydrate concentration in fum was 0.3% ( w / v ) glucose . after 16  h of incubation , the growth medium was renewed , along with adding 50  l of t.  denticola liquid culture as well as 50  l of s.  epidermidis culture ( od550  =  1.0 ) in treatment 4 ( see above ) . at 16 , 20 , 24 , 40 , 44 and 48  h , biofilms were washed by three consecutive dips in 2  ml of sterile physiological saline . fresh medium was provided after 16 and 40  h. after 64  h , the biofilms were dipwashed again prior to harvesting for quantification by realtime quantitative pcr ( qpcr ) or processing for fluorescence in  situ hybridization ( fish ) staining and confocal laser scanning microscopy ( clsm ) analyses , as described below . after 64  h of biofilm growth , the discs were vortexed vigorously for 1  min in 1  ml of 0.9% nacl and then sonicated at 25w in a sonifier b12 ( branson ultrasonic , urdorf , switzerland ) for 5  s , to harvest the adherent biofilms . the resulting bacterial suspensions were then used for quantification by qpcr as described earlier ( ammann et  al . primer sequences and properties of the standard 10species biofilm are given in table  1 . the staphylococci were quantified using the microbial dna qpcr assays for s.  epidermidis and s.  aureus ( qiagen instruments , hombrechtikon , switzerland ; cat . biofilms were stained by fish using cy3 or famlabelled probes following the protocols described before ( thurnheer et  al . 2004 ; thurnheer & belibasakis 2015 ) . probe sequences and formamide concentrations used for the hybridizations , as well as the nacl concentrations of the washing buffers , are given in table  2 . for counterstaining , biofilms were stained using 3  m syto 59 ( invitrogen , lucerne , switzerland ) ( 20  min , room temperature , in the dark ) , following the fish procedure . after staining , the samples were embedded upsidedown on chamber slides in 100  l of mowiol 488 ( calbiochemnovabiochem , san diego , ca , usa ) ( guggenheim et  al . stained biofilms were examined by clsm at randomly selected positions using a leica tcs sp5 microscope ( leica microsystems , wetzlar , germany ) fitted with an ar laser ( 458  nm/476  nm/488  nm/496  nm/514  nm excitation ) , and a hene laser ( 561  nm/594  nm/633  nm excitation ) . filters were set to 500540  nm to detect fam , to 570630  nm for cy3 and to 660710  nm for syto 59 . zseries were generated by vertical optical sectioning with the slice thickness set at 1.02  m . scans were recombined and processed using imaris 7.6.5 software ( bitplane , zrich , switzerland ) , without any qualitative changes to the raw images . three independent experiments were performed , and within each experiment every group was represented in triplicate biofilm cultures . hence , statistics were performed on nine individual data points , deriving from the nine individual biofilm cultures per experimental group . the statistical significance of the differences in microbial numbers between the control group ( standard 10species  subgingival  biofilm ) and the four treatments was evaluated by twoway analysis of variance ( anova ) , corrected by tukey 's multiple comparisons test ( significance level p  <  0.05 ) . undetectable values were ascribed the lowest detection limit value of the assay to allow for logarithmic transformation . comparisons were performed between the control group and each experimental group , for each individual species . the data were analysed using the prism version 6 , statistical analysis software ( graphpad , la jolla , ca , usa ) . a standard 10species  subgingival  biofilm was used as the ground model for this study , grown either on ha or titanium discs . when s.  aureus or s.  epidermidis was included in the initial inoculum , either individually or together , all of the remaining original 10 species were grown unimpeded in the biofilm . significant ( p  <  0.05 ) changes in c.  rectus , p.  gingivalis and t.  forsythia numbers were observed only when s.  epidermidis was reinoculated ( i.e.  booster  ) after 16  h following the initiation of biofilm formation . the numbers of p.  gingivalis and t.  forsythia were increased by 2.9fold and 3.2fold , respectively , when the two staphylococci were present in the biofilm , whereas c.  rectus decreased by 6.1fold . yet , these changes in numbers remained within one step of the log10 scale . importantly , s.  aureus was successfully incorporated and grown in the biofilm under any of the tested conditions . this was also the case for s.  epidermidis when inoculated together with the 10  subgingival  species . however , when s.  epidermidis and s.  aureus were inoculated together along with the 10 other species , the growth of the former was significantly inhibited by approximately 3 steps of the log10 scale ( fig . cells / biofilm of the standard 10species subgingival biofilm grown on hydroxyapatite ( a ) or titanium discs ( b ) ( control group ; blue ) , containing additionally staphylococcus epidermidis ( treatment 1 ; red ) , or staphyoccous aureus ( treatment 2 ; green ) , or s.  epidermidis  +  s.  aureus ( treatment 3 ; purple ) or s.  epidermidis  +  s.  aureus  +  a boost inoculation of s.  epidermidis after 16  h of growth ( treatment 4 ; orange ) . statistically significant differences compared with the control group are indicated with asterisks ( * p  <  0.05 ; * * p  <  0.01 ; * * * p  <  0.001 ) . biofilm formation on titanium surfaces was thereafter investigated in a similar manner ( fig . it was found that presence of s.  aureus or s.  epidermidis in the inoculum ( together or individually ) did not cause any changes in the numbers of the 10 original species after 64  h of biofilm growth . accordingly , s.  aureus and s.  epidermidis were able to successfully grow as part of the biofilm , along with the other species . however , when these two staphylococci were inoculated together , only s.  aureus was able to grow in the biofilm , whereas the growth of s.  epidermidis was suppressed . this newly established biofilm model whereby s.  epidermidis or s.  aureus was able to integrate among the 10  subgingival  species , was also analysed structurally by clsm ( fig .  2 ) technically , it was not possible to perform clsm on the biofilm grown on titanium discs , as the biofilm displayed increased detachment from this surface during the execution of the fishstaining protocol . the structure and bacterial distribution of the 10species ( control ) biofilm is visualized in fig .  2a , whereas the presence of s.  epidermidis ( fig . 2c ) was individually confirmed by fish staining , using fluorescencelabelled 16s rrna oligonucleotide probes ( table  2 ) . in both staphylococci groups , there were small but distinctive bacterial cell clusters of the associated species ( red colour ) , which were scattered across the biofilm mass ( green colour ) . in the case where s.  aureus and s.  epidermidis were simultaneously coinoculated ( fig . 2d ) , or when s.  epidermidis was reinoculated after 16  h ( fig . 2e ) , only s.  aureus was identified in the biofilm , whereas s.  epidermidis was not detectable . the distribution and clustering of s.  aureus when coinoculated with s. epidermidis did not differ from the biofilm group where s.  aureus was inoculated alone . these findings corroborate the low detection levels of s.  epidermidis by qpcr in the corresponding biofilm groups ( fig .  1 ) . clsm images of the standard 10species subgingival biofilm after 64  h of growth on hydroxyapatite discs ( control group ; a ) containing additionally staphylococcus epidermidis ( treatment 1 ; b ) , or staphyoccous aureus ( treatment 2 ; c ) , or s.  epidermidis  +  s.  aureus ( treatment 3 ; d ) or s.  epidermidis  +  s.  aureus  +  a boost inoculation of s.  epidermidis after 16  h of growth ( treatment 4 ; e ) . due to fish staining with 16s rrna probes , sepi229 and saur229 s.  epidermidis and s.  aureus appear blue and red , respectively . the present study established and characterized an in  vitro multispecies  submucosal  biofilm model , which is of relevance to periimplantitis . it is based on the advancement of the original 10species  subgingival  biofilm grown anaerobically on ha discs ( guggenheim et  al . the novelty lies in the growth of the biofilm on titanium discs , as well as the incorporation of s.  aureus or s.  epidermidis individually in its structure . staphylococci have allegedly been more associated with periimplantitis than periimplant health , or periodontitis ( heitzmayfield & lang 2010 ; belibasakis 2014 ) . studies have also shown that s.  aureus dna counts are greater on dental implants than on natural teeth , as evaluated by dna however , such methods should be considered with more caution due to potential crossreactivity between taxa that could lead to overinterpretation of the findings ( charalampakis & belibasakis 2015 ) . yet , culturedependent methods have confirmed the presence of staphylococci in periimplantitis , albeit rather infrequency ( leonhardt et  al . hence , there is sufficient reasoning to incorporate further staphylococci into our 10species experimental biofilm model . formation of monospecies biofilms of s.  epidermidis has previously been investigated in relation to titanium surfaces ( burgers et  al . 2012 ) . both s.  aureus and s.  epidermidis were able to grow as part of the biofilm , at numbers comparable to the other constituent  subgingival  species . moreover , it was possible for the biofilms to grow on both ha and titanium surfaces , denoting that there is no selective advantage of the growth of this biofilm on one surface over the other . these results are in line with the recent observation that s.  aureus can efficiently grow within a biofilm consisting of another six  supragingival species , without affecting their numeric composition ( thurnheer & belibasakis 2015 ) . within that biofilm , s.  aureus appeared to localize in small and rather secluded clusters of its own species . this observation is very similar to the localization pattern of either s.  aureus or s.  epidermidis observed in the present  submucosal  biofilm . this may denote that staphylococci can grow in a sparse , yet distinctive , pattern as part of oral biofilms , without outcompeting in growth and spatial arrangement with the remaining constituent species , as has been shown in the case of escherichia coli ( thurnheer & belibasakis 2015 ) . a competition trend between the newly introduced staphylococci that is , when s.  epidermidis was coinoculated with s.  aureus , the former failed to grow in the biofilm . clearly , this denotes an ecological advantage of s.  aureus over s.  epidermidis under the present microenvironmental conditions . this may explain the more frequent detection of s.  aureus than s.  epidermidis in biofilms from sites with periimplantitis ( mombelli & decaillet 2011 ) . otherwise , there is also contrary evidence that the monospecies competition between s.  aureus and s.  epidermidis by means of quorumsensing may generally be in favour of s.  epidermidis , which might explain its predominance on skin and infections on indwelling medical devices ( otto et  al . yet , one has to consider that every microenvironmental niche of the human body is ecologically different and may therefore provide selective conditions for the growth of different bacteria , or their interactions with each other . hence , within a  submucosal  oral biofilm , such as the one developed in this study , the behavioural interaction between s.  aureus and s.  epidermidis can be different than on skin . it is worth noting at this stage that , with the present experimental approach , it is not possible to gauge whether this effect was due to direct suppression of s.  epidermidis by s.  aureus , or a community effect of s.  aureus on this polymicrobial species biofilm . in conclusion , the present study has established and characterized  submucosal  biofilm model for periimplantitis grown on titanium surfaces , by individually integrating s.  aureus or s.  epidermidis . the model can be used for testing potential modalities for the prevention or treatment of periimplantitis , before being applied into the clinics . moreover , this model also revealed a competitive interaction between s.  aureus and s.  epidermidis in oral biofilms , whereby the former outcompetes the growth of the latter . this is an interesting microecological observation that may explain the more frequent detection of s.  aureus than s.  epidermidis , in periimplantitis biofilms .
abstractobjectives 
 staphylococcus spp . are postulated to play a role in periimplantitis . 
 this study aimed to develop a  submucosal  in  vitro biofilm model , by integrating two staphylococci into its composition.materials and methodsthe standard  subgingival  biofilm contained actinomyces oris , fusobacterium nucleatum , streptococcus oralis , veillonella dispar , campylobacter rectus , prevotella intermedia , streptococcus anginosus , porphyromonas gingivalis , tannerella forsythia and treponema denticola , and was further supplemented with staphyoccous aureus and/or staphylococcus epidermidis . 
 biofilms were grown anaerobically on hydroxyapatite or titanium discs and harvested after 64  h for realtime polymerase chain reaction , to determine their composition . 
 confocal laser scanning microscopy and fluorescence in  situ hybridization were used for identifying the two staphylococci within the biofilm.resultsboth staphylococci established within the biofilms when added separately . 
 however , when added together , only s.  aureus grew in high numbers , whereas s.  epidermidis was reduced almost to the detection limit . 
 compared to the standard subgingival biofilm , addition of the two staphylococci had no impact on the qualitative or quantitative composition of the biofilm . when grown individually in the biofilm , s.  epidermidis and s.  aureus formed small distinctive clusters and 
 it was confirmed that s.  epidermidis was not able to grow in presence of s.  aureus.conclusions 
 staphyoccous aureus and s.  epidermidis can be individually integrated into an oral biofilm grown on titanium , hence establishing a 
 submucosal  biofilm model for periimplantitis . 
 this model also revealed that s.  aureus outcompetes s.  epidermidis when grown together in the biofilm , which may explain the more frequent association of the former with periimplantitis .