Protective Mechanisms of Respiratory Tract Streptococci againstStreptococcus pyogenes Biofilm Formation and Epithelial Cell Infection

Tomas Fiedler,a Catur Riani,a* Dirk Koczan,b Kerstin Standar,a Bernd Kreikemeyer,a Andreas Podbielskia

University of Rostock, Institute of Medical Microbiology, Virology, and Hygiene, Rostock, Germanya; University of Rostock, Institute of Immunology, Rostock, Germanyb

Streptococcus pyogenes (group A streptococci [GAS]) encounter many streptococcal species of the physiological microbial biomewhen entering the upper respiratory tract of humans, leading to the question how GAS interact with these bacteria in order toestablish themselves at this anatomic site and initiate infection. Here we show that S. oralis and S. salivarius in direct contactassays inhibit growth of GAS in a strain-specific manner and that S. salivarius, most likely via bacteriocin secretion, also exertsthis effect in transwell experiments. Utilizing scanning electron microscopy documentation, we identified the tested strains aspotent biofilm producers except for GAS M49. In mixed-species biofilms, S. salivarius dominated the GAS strains, while S. oralisacted as initial colonizer, building the bottom layer in mixed biofilms and thereby allowing even GAS M49 to form substantialbiofilms on top. With the exception of S. oralis, artificial saliva reduced single-species biofilms and allowed GAS to dominate inmixed biofilms, although the overall two-layer structure was unchanged. When covered by S. oralis and S. salivarius biofilms,epithelial cells were protected from GAS adherence, internalization, and cytotoxic effects. Apparently, these species can haveprobiotic effects. The use of Affymetrix array technology to assess HEp-2 cell transcription levels revealed modest changes afterexposure to S. oralis and S. salivarius biofilms which could explain some of the protective effects against GAS attack. In sum-mary, our study revealed a protection effect of respiratory tract bacteria against an important airway pathogen and allowed afirst in vitro insight into local environmental processes after GAS enter the respiratory tract.

Streptococcus pyogenes (group A streptococci [GAS]) belongs tothe most important bacterial species causing purulent respira-

tory tract and skin infections in humans in more than 720 millionpatients per year (1, 2). Many of the GAS virulence factors in-volved in these infectious processes, as well as the underlying cir-cuits controlling their expression, have been identified in the re-cent years (3–9).

The administration of �-lactams is the treatment of choice tofight purulent GAS infections. Despite this appropriate therapy, 5to 10% of patients suffer from recurrent episodes (10). This phe-nomenon could be associated with the ability of GAS to adhere toand, more importantly, internalize into eukaryotic cells (3, 11).Unfortunately, a high capacity for internalization is apparentlyassociated with resistance to macrolides (12), preventing this in-tracellularly active antibiotic from being a true therapeutic alter-native, especially for recurrent GAS infections. Therefore, newoptions would be welcome to combat such infections, and, in finalconsequence, the spread of the bacteria from such carrier patientsin a nonepidemic setting.

Probiotics, defined as “live microorganisms [that] when ad-ministered in adequate amounts confer a health benefit on thehost” (FAO/WHO, 2002) could serve as a therapeutic alternative(13). Beneficial roles of probiotic bacteria have already been re-ported for treatments in the oral cavity (14). Thus far, only a fewproducts became commercially available, e.g., the Streptococcussalivarius strain K-12, which is sold as BLIS lozenges in New Zea-land (15), and freeze-dried powders of Lactobacillus rhamnosusplus Bifidobacterium casei and Enterococcus faecalis, which are soldas Symbiolact compositum and Symbioflor, respectively (16, 17).Like other probiotics these have three major activity routes. First,using appropriate adhesins, detergents, bacteriocins, or quorum-sensing inhibitors, the probiotic microorganism can outgrow andpermanently replace pathogens. Second, the host defense mecha-nisms are stimulated or modulated in favorable ways to eradicate

persisting pathogens and better resist future pathogen exposure.Third, after the application of antibiotics or antiseptics, voidspaces on human surfaces could be filled by robust harmless bac-teria until the resident microbial biome is reconstituted.

GAS are transmitted between humans by direct contact or bycontaminated airborne droplets. Therefore, the skin and the up-per respiratory tract are the primary GAS contact sites on newhuman hosts (18). Both anatomic sites are physiologically inhab-ited by several dozen (skin) to several hundred (respiratory tract)different bacterial species (19–23), of which the majority can onlybe demonstrated by molecular techniques (24, 25). Many of thesespecies have probiotic potential that could be exerted by biofilmformation on anatomical sites serving as targets for pathogenicbacteria. Several recent studies have shown that dairy bacteriasuch as Lactobacillus helveticus, L. rhamnosus, L. oris, and L. reuteri,as well as other oral bacterial species, can protect the pharyngealmucosa from GAS attack (26, 27). Thus far, it is unclear whetherthis defense mechanism in vivo involves biofilm growth.

In turn, the pathogens could build their own biofilm or inte-grate into existing ones. GAS form biofilms in vivo and in vitro

Received 6 November 2012 Accepted 6 December 2012

Published ahead of print 14 December 2012

Address correspondence to Bernd Kreikemeyer, bernd.kreikemeyer@med.uni-rostock.de, or Andreas Podbielski, andreas.podbielski@med.uni-rostock.de.

* Present address: Catur Riani, School of Pharmacy, Institut Teknologi Bandung,Bandung, Indonesia.

T.F. and C.R. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03350-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03350-12

February 2013 Volume 79 Number 4 Applied and Environmental Microbiology p. 1265–1276 aem.asm.org 1265

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

(28–30). Several GAS virulence factors have been suggested to beinvolved in biofilm formation, including M protein, antigen I/IIfamily polypeptide AspA, SpeB production, and the GAS pilus(31–36).

Several issues associated with the protective biofilm hypothesishave not been resolved. (i) Can probiotic bacteria interfere withthe biofilm formation capacity of GAS? (ii) Are GAS able to invadeand establish within existing probiotic biofilms. (iii) Can probi-otic bacteria protect epithelial cells from infection with GAS. (iv)Finally, what are the underlying mechanisms of these interactionswith regard to transcription and function levels. Consequently, inthe present study we used the well-studied BLIS S. salivarius K-12strain and two Streptococcus oralis strains (DSMZ reference strainDSM20627 and patient isolate 4087) as representatives of the pre-dominant upper respiratory tract species to test their potentialprotective capacity against GAS biofilm formation and host cellinfection.

MATERIALS AND METHODSStrains and culture conditions. S. pyogenes M49 strain 591 (37) and M6strain 616 (clinical isolate, Centre of Epidemiology and Microbiology,National Institute of Public Health, Prague, Czech Republic), S. salivariusstrain K-12 (BLIS Technologies, Ltd., Dunedin, New Zealand), and S.oralis strains DSM 20627 (German Collection of Microorganisms and CellCultures [DSMZ]) and 4087 (isolate from a healthy person [the presentstudy]) were cultured in brain heart infusion (BHI) or on Columbia bloodagar plates containing 5% sheep erythrocytes at 37°C under a 5% CO2–20% O2 atmosphere. Eukaryotic laryngeal epithelial cell line HEp-2(American Type Culture Collection) was cultured in Dulbecco modifiedEagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) incell culture flasks or 24-well microtiter plates under a 5% CO2–20% O2

atmosphere.Coaggregation experiments. Bacteria were harvested from overnight

cultures by centrifugation and washed twice with coaggregation buffer(tris[hydroxymethyl]aminomethane, 1 mM [pH 8.0]; CaCl2, 0.1 mM;MgCl2, 0.1 mM; NaN3, 0.02%; NaCl, 0.15 M) (38). The suspension ofbacterial cells in coaggregation buffer was adjusted to an optical density at600 nm of 2.0. To assess two-species coaggregation, the respective strainswere mixed 1:1 to a final volume of 4 ml and vortexed for 10 s. After 2 h ofincubation at room temperature coaggregation was determined by visualinspection.

Biofilm culture conditions. For biofilm assays, conditions optimizedfor GAS were used (39). In detail, bacterial overnight cultures in BHIbroth were suspended in fresh BHI broth supplemented with 0.5% glu-cose (BHI-G), adjusted to 104 CFU/ml, and inoculated into 96- or 24-wellmicrotiter plates for quantification of biofilm masses or microscopic anal-yses, respectively. Alternatively, BHI broth mixed 1:3 with artificial saliva(0.1% Lab Lemco Powder, 0.2% yeast extract, 0.5% peptone, 0.25% mu-cin from porcine stomach [type III; Sigma], 6 mM NaCl, 2.7 mM KCl, 3.5mM KH2PO4, 1.5 mM K2HPO4, 0.05% urea [pH 6.7]) was used as culti-vation medium. For one-species biofilms 100 �l of bacterial suspension(104 CFU/ml), and 100 �l of BHI-G were added per well of a 96-well plate.For two-species biofilm cultures, 100 �l of bacterial suspension (104 CFU/ml) of each species were simultaneously inoculated per well. For micro-scopic observations, biofilms were generated in 24-well polystyrene plates(Cellstar Greiner; Bio-One) with inserted round plastic coverslips (Nunc)or in glass-bottom 2/4-chambers (Nunc). Bacteria were inoculated in ra-tios as described above to a final volume of 1 ml. Biofilm analysis wasperformed after incubation for 3 days as standing cultures at 37°C under5% CO2–20% O2 atmosphere. All assays were performed in triplicate(technical replicates) on at least three independent occasions (biologicalreplicates).

Scanning electron microscopy (SEM). Biofilm bacteria were culturedas described above. After the removal of remaining planktonic bacteria,

biofilms on coverslips were washed with phosphate-buffered saline (PBS)and fixed with 2.5% glutaraldehyde overnight. Coverslips were washedtwo to three times with 0.1 M sodium phosphate buffer (pH 7.3), dehy-drated with a series of increasing ethanol concentrations (5 min in 30%, 5min in 50%, 10 min in 70%, 10 min in 90%, and twice for 10 min inethanol absolute), and dried with CO2 by critical point method with aEmitech dryer as outlined by the manufacturer. Dried coverslips werecovered with gold to a 10-nm layer and scanned with a Zeiss DSM 960Aelectron microscope (29).

Confocal laser scanning microscopy (CLSM). The visualization of S.pyogenes in single- and mixed-species biofilms was done by immunoflu-orescence staining using a specific anti-S. pyogenes polyclonal antibodytogether with an Alexa 488-coupled secondary antibody. Hexidine iodinewas used for staining all Gram-positive bacterial species. After the removalof planktonic bacteria, the biofilms were washed once with PBS, fixed with3% paraformaldehyde for 15 min at 4°C, and subsequently blocked with1% FCS at room temperature for 30 min. The biofilm was washed threetimes with PBS and incubated for 1 h at room temperature with a rabbitIgG anti-S. pyogenes antibody (1:5,000 in PBS). After being washed withPBS, the second antibody (goat anti-rabbit-IgG-Alexa Fluor 488) wasadded at a 1:500 dilution in PBS for 45 min at room temperature in thedark. Again, three washing steps with PBS were performed. Hexidineiodine (1 �l in 1 ml of PBS [Invitrogen]) was added for 10 min at roomtemperature in the dark. The biofilm was inspected with a Zeiss invertedmicroscope attached to a Leica TCS SP2 AOBS laser scanning confocalimaging system with an Argon laser at 480- and 488-nm excitation wave-lengths.

Bacteriocin assay. The bacteriocin assay was performed based on amodified deferred antagonism cross streak technique on blood agar (40).Potential bacteriocin producers were streaked on blood agar, incubatedfor 18 h, and subsequently killed with a vapor of chloroform for 4 min.Chloroform vapors were removed by passive ventilation of the agar platefor 10 to 15 min. S. pyogenes strains were streaked across the primarystreak and incubated overnight. Growth inhibition and beta-hemolysisinactivation were observed around the intersection of bacterial streaks.

Adherence and internalization assays. Adherence to and internaliza-tion into HEp-2 cells was quantified using an antibiotic protection assay(41). Three different seeding strategies were used for this assay: (i) simul-taneous seeding (S. pyogenes and respiratory tract streptococci were addedto the HEp-2 cells at the same time), (ii) S. pyogenes first seeding (S.pyogenes was incubated with HEp-2 cells for 1 h prior to addition of the S.oralis or S. salivarius), and (iii) S. pyogenes last seeding (respiratory tractstreptococci were incubated with HEp-2 cells for 2 h prior to inoculationof S. pyogenes). Moreover, several modifications of the latter method wereperformed. (i) DMEM medium and planktonic respiratory tract bacteriawere removed prior to S. pyogenes infection, and Hep-2 cells were washedthree times with sterile prewarmed PBS preceding the S. pyogenes inocu-lation. (ii) A transwell system was used, preventing direct contact betweenthe respiratory tract bacteria (upper compartment) and HEp-2 cells(lower compartment). In addition, adherent and internalized S. pyogeneschains were visualized using double immune fluorescence as describedpreviously (42). All assays were performed with three technical and at leastthree biological replicates.

Eukaryotic cell viability assay. The live/dead viability/cytotoxicity kitfor animal cells (Molecular Probes; Mobitec) was used to determine via-bility of HEp-2 cells in the presence of S. pyogenes and respiratory tractspecies. Exposure of HEp-2 cells to the respective bacteria was as describedfor adherence and internalization experiments, except that HEp-2 cellswere cultivated in 24-well plates on coverslips. Visualization and inspec-tion of live or dead HEp-2 cells was done under a fluorescence microscopeat 400-fold magnification. In each assay, three randomly chosen micro-scopic fields were documented as a picture. In order to express the resultsas quantitative data, living cells (green fluorescence) were counted andexpressed as the percentage of all cells (live and dead) visible in eachpicture.

Fiedler et al.

1266 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

HEp-2 cell transcriptome analysis. To compare host cell gene expres-sion profile changes caused by S. salivarius K-12 and S. oralis DSMZ,high-density oligonucleotide microarrays were applied. Two biologicalreplicates of total RNA samples from infected HEp-2 cells were hybridizedto Human Genome U133 plus 2.0 array (Affymetrix, St. Clara, CA), in-terrogating 47,000 transcripts with more than 54,000 probe-sets. Arrayhybridization was performed according to the supplier’s instructions us-ing GeneChip expression 3= amplification one-cycle target labeling andcontrol reagents (Affymetrix). Subsequently, washing and staining proto-cols were performed with the Affymetrix Fluidics Station 450. For a signalenhancement, an antibody amplification was carried out with a biotin-ylated anti-streptavidin antibody (Vector Laboratories, United King-dom), which was cross-linked by a goat IgG (Sigma, Germany), followedby a second staining with streptavidin-phycoerythrin conjugate (Molec-ular Probes/Invitrogen). The scanning of the microarray was done with aGeneChip Scanner 3000 (Affymetrix) at a 1.56-�m resolution.

The data analysis was performed using MAS 5.0 (Microarray Suite statis-tical algorithm; Affymetrix) and probe-level analysis using GeneChip Oper-ating Software (GCOS 1.4), and the final data extraction was performed withthe DataMiningTool 3.1 (Affymetrix). Differentially upregulated and down-regulated genes from two independent experiments were clustered manuallyand analyzed for their molecular function using tools in NetAffx analysiscenter (http://www.affymetrix.com/analysis/index.affx).

Statistical analysis. Where applicable, statistical analyses were per-formed using the Student t test. Significant differences were defined whenthe P values were �0.05.

RESULTSCocultures of S. pyogenes and respiratory tract streptococci inliquid culture media. Only scarce information exists regardinghow S. salivarius and S. oralis could influence cultures of GAS.Thus, coculture experiments were performed by growing GASserotypes M49 and M6 in direct (tubes) or indirect (transwellsystem) contact with the two respiratory tract streptococci in sev-eral combinations. In initial coaggregation experiments, coincu-bation of the different species was shown to increase the bacterialdispersion in combinations of GAS with S. salivarius and S. oralisDSMZ (see Fig. S1 in the supplemental material), indicating alower degree of coaggregation in planktonic mixtures of thesespecies. Serial combinations of high to low bacterial CFU countswere used to test whether bacterial numbers play a role in thisinteraction. From the outcome of these experiments, only mean-ingful results are shown. As shown in Fig. 1, the direct contactexperiments revealed that a high initial CFU count of S. salivarius

FIG 1 Cocultivation experiments with S. pyogenes and respiratory tract streptococci. The figure shows CFU of S. pyogenes M49 (A to C) and M6 (D to F) after 24 h ofeither coincubation in direct contact (light gray bars) or in transwell assays (dark gray bars) with S. salivarius (Ss) and S. oralis (So) DSMZ strain or strain 4087,respectively. Numbers below the bars indicate the initially inoculated number of bacteria (in CFU/ml). Error bars resulted from three independent experiments.

Oral Streptococci and GAS Biofilm Formation

February 2013 Volume 79 Number 4 aem.asm.org 1267

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

and S. oralis mixed with a low GAS M49 and M6 CFU countcaused the repression of GAS growth. In contrast, low numbers ofS. salivarius CFU inhibited the growth of low initial GAS M49 andM6 CFU (Fig. 1A and D).

In experiments using the transwell system, secreted substancesfrom most bacteria did not decrease GAS CFU, except for S. sali-varius (Fig. 1). The initial mixture of 106 CFU of S. salivarius/mlplus 103 CFU of GAS/ml led to a 7-log decrease in GAS CFUcompared to untreated controls. Furthermore, the S. oralis strainsreduced GAS M49 CFU by 1 log (Fig. 1B and C). No other straincombination or CFU variation revealed significant effects. Theeffect of S. salivarius on GAS M49 was more prominent than theeffect on GAS M6. Apparently, S. salivarius secreted a diffusiblesubstance that exerted a growth inhibition effect on GAS, and theGAS M6 strain is more resistant against its action.

The fitness of the tested respiratory tract species in the presenceof GAS strains was also monitored in the direct contact system (seeFig. S2 in the supplemental material). Even mixtures of initiallyhigh GAS CFU with low S. salivarius and S. oralis CFU did notprevent the growth of the respiratory tract streptococci in themixed cultures.

Bacteriocin production. One possible explanation for some ofthe results of the coculture experiments could be the secretion ofdiffusible substances such as bacteriocins. Thus, we tested bacte-riocin production by the respiratory tract species using a solidblood agar test system. The potential producers were grown in afirst streak and inactivated by chloroform vapor. Hence, any ob-served effect was caused by chloroform-stable, diffusible sub-stances that were produced by the first streaked strain. The resultsshown in Fig. 2 revealed that exclusively S. salivarius could killboth GAS strains by bacteriocin production.

Combined species biofilm phenotypes. GAS are able to formbiofilms in vitro. However, the biofilm phenotype is serotypedependent (29, 39). Here, biofilm formation of GAS M6(strong biofilm producer) and M49 (poor to moderate biofilmproducer) was investigated in the presence of S. salivarius andS. oralis strains to observe whether GAS can integrate intorespiratory tract biofilms or GAS biofilms can be disturbed byrespiratory tract bacteria.

Initial tests revealed that BHI supplemented with 0.5% (wt/vol) glucose best supported growth of all species (data not shown).Prior to mixed-species experiments, the biofilm-forming ability ofall strains was assessed using monospecies cultures. From thesafranin assay results and SEM pictures (Fig. 3), it is apparent thatall species developed significant biofilm masses except for the GASM49 strain in accordance with published results (29). Bacterialcoaggregation was previously shown to play an important role in

biofilm development and bacterial interaction with host cells (43).Therefore, coaggregation capacity of BHI-grown single- andmixed-species was examined. Only the GAS strains revealed anaggregation effect in single-species experiments. In addition, S.oralis 4087 coaggregates with both of the GAS strains tested (seeFig. S1A-C in the supplemental material).

After we established the optimal conditions for monospeciesbiofilms, we investigated the isolates’ behavior in mixed-speciessettings. As documented by SEM and CLSM, biofilms of both GASserotype strains plus S. salivarius were dominated by S. salivariusbacteria, since GAS were hardly visible in this combination (Fig. 4;see also Fig. S3 in the supplemental material). This finding wassupported by the biofilm masses as quantified by safranin staining.The amounts of the S. salivarius/GAS two-species biofilms werealmost identical to those of S. salivarius monospecies biofilms butwere significantly higher than GAS M6 and M49 monospeciesbiofilm masses (Fig. 4). Obviously, the observed effects were in-dependent of the biofilm-forming capacity of the individual GASstrain.

A different picture emerged in the case of the mixed GAS/S.oralis biofilms. Both short-chain growing S. oralis strains predom-inated the bottom layer and long-chain growing GAS strains in thetop layer of the two-species biofilm (Fig. 4; see also Fig. S3 in thesupplemental material), indicating that S. oralis acted as a support

FIG 2 Bacteriocin production. S. salivarius K-12 (A), S. oralis DSMZ (B), andS. oralis strain 4087 (C) were grown on Columbia agar plates overnight andkilled by chloroform exposure. Subsequently, S. pyogenes M6 and M49 wereperpendicularly streaked across the primary streak and incubated for another24 h. Growth inhibition zones are visible close to the S. salivarius streak. He-molysin production is obviously not selectively suppressed in growing S. pyo-genes bacteria.

FIG 3 Single-species biofilms. The biofilm-forming capacity of M6 and M49S. pyogenes strains, as well as of S. salivarius K-12, S. oralis DSMZ 20627, and S.oralis 4087 strains, was assessed after 72 h of incubation by safranin stainingand absorption measurements at 492 nm. Error bars resulted from three in-dependent experiments. The panel below the graph shows SEM images of thebiofilms of the respective strains after 72 h of incubation.

Fiedler et al.

1268 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

for GAS biofilm development. Obviously, the biofilm was not asexclusively formed by S. oralis, as was the case for S. salivarius, butthis time was somewhat affected by the biofilm-forming capacityof the individual GAS strains. In the case of GAS M49, biofilmmasses in combination with the S. oralis strains were significantlyhigher than the biofilm masses of GAS M49 or each S. oralis strainalone (Fig. 4A). In contrast, biofilm masses of GAS M6 combinedwith either S. oralis strain did not significantly differ from that ofsingle-species biofilms (Fig. 4B).

Artificial saliva effects on biofilms. Copious amounts of salivaare present in parts of the human upper respiratory tract. Besidesphysiological effects on host surfaces, it has an important role inmicrobial homoeostasis on these surfaces (44). Therefore, the ef-fect of artificial saliva was determined for monospecies andmixed-species biofilms.

Artificial saliva reduced all monospecies biofilm masses exceptfor S. oralis DSMZ (see Fig. S4 in the supplemental material). Theaddition of artificial saliva also reduced the masses of the mixed-species biofilms involving S. salivarius (Fig. 5). The predominanceof S. salivarius in combinations with GAS disappeared upon salivaaddition. Now, both GAS strains prevailed in the SEM pictures,the M6 strain more than the M49 strain (compare Fig. 4 to 5).

The results for the GAS/S. oralis combinations in the presenceof artificial saliva diverged in a strain-dependent manner. Combi-nations of the two GAS strains with S. oralis DSMZ did not lead toa statistically significant change in biofilm masses (Fig. 5). Theeffect of artificial saliva on the GAS/S. oralis 4087 combinationsdiffered for the M49 and M6 strains. In combination with the M49strain, the biofilm mass was drastically decreased, whereas thebiofilm mass was unaffected by saliva in the S. oralis 4087/GAS M6

FIG 4 Biofilm formation of cocultures of S. pyogenes M49 (A) or M6 (B) and respiratory tract streptococci. After 72 h of incubation, the biofilms were quantifiedby safranin staining and absorption measurement at 492 nm. White bars represent the S. pyogenes monospecies biofilms, light gray bars represent the mono-species biofilms of the respiratory tract streptococci, and dark gray bars represent the mixed-species biofilms. Error bars resulted from three independentexperiments, asterisks indicate significant differences (Student t test: **, P � 0.01; ***, P � 0.001). (C) The panel below the graph shows SEM images ofmixed-species biofilms after 72 h of incubation in the combinations indicated.

Oral Streptococci and GAS Biofilm Formation

February 2013 Volume 79 Number 4 aem.asm.org 1269

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

combination. Consistently, artificial saliva did not change the twostreptococcal biofilm layers formed by the S. oralis and GASstrains (Fig. 5).

Effects of respiratory tract streptococci on GAS host cell ad-herence and internalization. After obtaining data on the mixed-species biofilm behavior of GAS and respiratory tract streptococcion inanimate supports, we were now interested in this interactionon host cell surfaces, which are typical targets for GAS adherenceand internalization. Different experimental setups were chosen toreflect all possible interaction scenarios: (i) GAS strains were firstallowed to contact the HEp-2 cells prior to adding the other respi-ratory tract streptococci; (ii) the other respiratory tract strepto-cocci were initially seeded to the HEp-2 cells, and only thereafterwere GAS introduced into this setting; and (iii) both species weresimultaneously added to the host cells. With each strategy, weexamined whether the respiratory tract streptococci could in-crease, delay, or even cure harmful effects inflicted by GAS on hostcells.

Compared to consequences after single-species exposure, onlystrategy ii had significant effects. The initial presence of the respi-ratory tract streptococci led to a marked reduction in HEp-2 celladherence and internalization for both GAS serotype strains (Fig.6). Consistently, cytotoxicity of GAS toward host cells was re-duced in this setting (see Fig. S5 in the supplemental material). Inconclusion, S. salivarius and both S. oralis strains protected HEp-2cells from the attack of both GAS strains to a comparable degree;however, this only happened when the respiratory tract strepto-cocci first interacted with the host cells.

Modifications of the experimental setup, such as a washingstep between the application of the different bacteria and a trans-well setup with respiratory tract streptococci in the upper com-partment and GAS plus host cells in the lower compartment, wereintroduced in order to uncover details of the protection mecha-nism. As a result of both modifications, an unchanged presence ofthe respiratory tract streptococci in the immediate vicinity of thehost cells is crucial for the observed protective effects (see, for

FIG 5 Impact of artificial saliva on biofilm formation of cocultures of S. pyogenes M49 (A) or M6 (B) and respiratory tract streptococci. After 72 h of incubation,the biofilms were quantified by safranin staining and absorption measurements at 492 nm. Light gray bars represent mixed-species biofilms in the presence ofartificial saliva, and dark gray bars represent mixed-species biofilms in the absence of artificial saliva. Error bars resulted from three independent experiments,asterisks indicate significant differences (Student t test: ***, P � 0.001). (C) The panel below the graph shows SEM images of the mixed-species biofilms after 72h of incubation in the presence of artificial saliva.

Fiedler et al.

1270 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

example, Fig. S6 in the supplemental material) for the adherenceand internalization of the M49 strain.

SEM pictures of the interaction of the respiratory tract strep-tococci with the HEp-2 cells (Fig. 7) demonstrated the presence of

high numbers of S. salivarius K-12 bacteria forming biofilm-likestructures on the host cells. Both S. oralis strains were present inonly low numbers on the host cell surfaces and did not attainbiofilm-like structures. This finding suggested that the similarprotective effects exerted by the respiratory tract streptococcicould be based on different functional or molecular backgrounds.The S. salivarius strain could potentially act as steric barrier,whereas the S. oralis strains could use molecular ways to influenceGAS-host cell interaction.

Transcriptional HEp-2 cell responses upon exposure tostreptococcal strains. In order to clarify some of the moleculardetails of the different mechanisms that the respiratory tract strep-tococci used for their similar protection of HEp-2 cells, we exam-ined the host cell transcriptomes after exposure to the protectivestreptococci by utilizing Affymetrix whole-genome transcriptomemicroarrays. HEp-2 cells were incubated with the M49 GAS strain

FIG 6 Impact of respiratory tract streptococci on adherence and internalization of S. pyogenes to/into HEp-2 cells. The numbers of adherent (dark gray bars) andinternalized (light gray bars) bacteria of S. pyogenes M49 (A) and M6 (B) alone on HEp-2 cells were set to 100%. The adherence and internalization values for bothS. pyogenes strains after pretreatment of the HEp-2 cells with the respiratory tract streptococci were related to the values in the absence of the respiratory tractstreptococci. Error bars reflect data from three independent experiments. (C) The adherence of both GAS strains (green) to HEp-2 cells (gray) is shown in afluorescence/light microscopy overlay picture.

FIG 7 Adherence of respiratory tract streptococci to HEp-2 cells. The SEMimages show the adherence of S. salivarius K-12 (A), S. oralis 4087 (B), and S.oralis DSMZ (C) strains to HEp-2 cells.

Oral Streptococci and GAS Biofilm Formation

February 2013 Volume 79 Number 4 aem.asm.org 1271

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

for 2 h as positive controls. In parallel, HEp-2 cells were preincu-bated for 2 h with respiratory tract streptococci, and then GASM49 was added, followed by incubation for another 2 h. In sub-sequent data analysis, at least 3-fold altered transcript amounts(signal log ratio � 1.58) were defined as a threshold for signifi-cance.

Pre-exposure of HEp-2 cells to S. oralis DSMZ revealed 10genes with annotated functions that were differentially expressedcompared to cells exposed to GAS M49 alone. Of these genes, onlyone (endothelin-2 gene EDN2) displayed an increased transcriptamount, while transcripts of nine genes were present at a lowerlevel (see Table S1 in the supplemental material). Among thegenes with decreased transcript amounts was PLG (plasminogen),which has been associated with bacterial adhesion and internal-ization (45).

Similarly, analysis of the HEp-2 cell transcriptomes after pre-incubation with S. salivarius and subsequent GAS M49 exposurecompared to GAS M49 exposure alone revealed 19 annotatedgenes with an at least 3-fold difference in transcript amounts. Sev-enteen of these genes exhibited lower transcript amounts, whereasonly two genes showed higher transcript amounts (see Table S2 inthe supplemental material).

DISCUSSION

S. pyogenes as an exclusive human pathogen uses various routes toenter the host. Uptake via airborne droplets is the predominantway of entering the respiratory tract and causing infections such aspharyngitis and tonsillitis. Many details of the first infection stepsat this anatomic site have been elucidated in the last several de-cades concerning the involved GAS virulence factors and mecha-nisms of pathogen-host cell interactions (3, 7, 9). However, beforeattaching to host tissues GAS will encounter the resident microbialbiome. The published information about that incident is stillscarce. Thus, the present work had several major objectives.

First, the consequences of mixing different GAS serotypestrains with diverse respiratory tract streptococci should be exam-ined and quantified in planktonic and biofilm states under aspectsof survival and virulence factor expression. Second, mixed-speciesbiofilm development with the different species should be scruti-nized since the specific GAS target epithelia on the pharynx andtonsils are especially covered by bacterial biofilms (46). Third, theeffect of respiratory tract streptococci on the interaction betweenGAS and host cells should be inspected with specific regard to anantecedent, simultaneous, or subsequent presence of the poten-tially protective streptococci. Since the findings could have thera-peutic implications, besides representatives of the leading localspecies, i.e., S. oralis (47, 48) and an established probiotic bacte-rium, the S. salivarius K-12 strain, were chosen as interaction part-ners.

Streptococci are the predominant bacteria in the oral cavityand are present in numbers ranging between 5 and 8 logs per ml offluid or mg of material (46). Much less is known about the num-bers of S. pyogenes bacteria when introduced into the pharynx.Most probably, S. pyogenes will not be taken up as single bacteriabut inhaled in droplets or ingested as dried material containingdensely packed bacteria that could equal several logs. To assessnumerical changes as a consequence of simultaneous presence ofpotential antagonists, S. pyogenes wild-type strains were mixedwith respiratory tract bacteria in various ratios to reflect the nu-merical variability of the natural environment. High numbers of

planktonic S. salivarius and S. oralis generally suppressed thegrowth of S. pyogenes—more pronounced for the M49 than forthe M6 serotype strain—in direct contact experiments. However,in transwell assays separating both growth partners, as well as inclassical bacteriocin assays on solid media, only S. salivarius dis-played this effect on the growth of both GAS serotype strains,implying the production of diffusible active substances specificallyby this species. The latter result was consistent with recent findingson this probiotic bacterium (49, 50), provided the S. pyogenesstrain was susceptible to the substance(s) and the cell numbers ofthe probiotic strain exceeded that of S. pyogenes by several logs.The present results extend the published data since thus fargrowth inhibition testing of S. pyogenes has only been conductedon solid media or in liquid assays using (semi)purified bacterio-cin. Several bacteriocin-producing S. salivarius strains wereshown to have probiotic effects and inhibit Gram-positive patho-gens of the upper respiratory tract such as S. pneumoniae or S.pyogenes (51), e.g., by the production of the lantibiotic salivaricinD (52). However, the S. salivarius-produced salivaricin could in-duce the production of defensive agents such as the S. pyogenesbacteriocin SalA1. In the present study, the growth of the respira-tory tract streptococci was not suppressed in the presence of dif-fusible substances from the GAS strains. In in situ studies, S. pyo-genes SalA1 expression was induced by a minimum of 8 � 105 S.salivarius cells per ml of saliva (49). This number is above the levelsof S. salivarius in saliva from healthy children, i.e., 105 CFU/ml(53) and 104 to 105 CFU/ml of saliva from adults treated with thecorresponding probiotic strains (54). Therefore, a GAS-associatedgrowth inhibition of respiratory tract streptococci could be irrel-evant for the natural encounter between both bacterial species intheir human hosts.

Nutrient competition-derived reduced growth of one speciesin the presence of large inocula of another species could be theexplanation for the observed effects of planktonic S. oralis on GASand vice versa. However, the strain specificity of this effect for S.pyogenes, as well as the exclusive occurrence in direct contact butnot in transwell system experiments, insinuated the presence ofadditional mechanisms. Such cell-associated but still undefinedfactors have also been observed in cocultures between S. oralis/Haemophilus influenzae or Moraxella catarrhalis (55) or cocul-tures between E. faecalis/S. pneumoniae, S. aureus, or Listeriaivanovii (56, 57).

S. pyogenes has been demonstrated to form single-species bio-films in vitro and in animal model infections (29, 32, 58). Theprotective function of a biofilm leading to an increased antibioticresistance level stimulated the idea that biofilms could be associ-ated with long-term persistence in asymptomatic carrier persons(59). However, in the pharynx, S. pyogenes encounters preexistingmixed-species biofilms on its target cells. Thus, it has to establishitself within and eventually to penetrate these biofilms. Based ondata from other pathogens, it is conceivable that both establish-ment and penetration will be influenced by many factors pro-duced by the resident microbial biome (60–63).

Consequently, respiratory tract streptococci were used to studydefined mixed-species biofilms with the serotype M49 and M6 S.pyogenes strains. Here, we could show that in general S. pyogenescan form mixed-species biofilms with the streptococcal speciesunder investigation. In most cases, increased biofilm masses weredetected. Of note, GAS M49, which forms scanty biofilms as singlespecies, was stimulated to a higher production rate in the presence

Fiedler et al.

1272 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

of S. oralis. Moreover, in all combinations with S. oralis strains, S.oralis formed the bottom layer and S. pyogenes formed the toplayer (Fig. 4). This is consistent with the pioneer character of S.oralis also observed in ex vivo samples from human volunteers(64) and in models of oral biofilms (65). The avidity of S. oralis forhuman cell surfaces seems to be associated with the activity ofspecific galactose- or sialic acid/N-acetylgalactosamine- bindinglectins (66, 67). Also, the interaction between S. oralis and S. pyo-genes could rely on the activity of lectins and appropriate sugarmoieties, since the latter species was shown to bind the lectin con-canavalin A (33), as well as several human glycoproteins (68).

Since pharyngeal biofilms would be bathed in saliva, the exper-iments were repeated in the presence of mucin as the major com-ponent of saliva (44). Generally, S. pyogenes should be able to dealwell with saliva. It was shown to bind to mucin by its M protein(69). Simultaneously, the two-component regulator SptRS is in-duced by the presence of saliva (70). This control circuit inducesvirulence factors such as Sic and SpeB, which inactivate antimi-crobial peptides normally contained in saliva (70–73). Other sali-va-driven regulatory circuits (MalR and CcpA) couple the usageof maltodextrin, the predominant sugar in saliva, as an exclusiveenergy source with the expression of several virulence factors andthereby support its respiratory tract persistence (74, 75). How-ever, in earlier publications it was shown that saliva decreased theability of S. pyogenes to attach to eukaryotic epithelial cells (76).

In testing single-species biofilms, S. pyogenes and S. salivariusbiofilm masses were strongly diminished by artificial saliva. Arti-ficial saliva has been described to facilitate S. salivarius growth butcounteracts its viability during stationary phase and under starva-tion conditions (77).

In mixed-species biofilms, biofilm masses were minimized forblends of both GAS isolates with S. salivarius, whereas in combi-nations with the S. oralis DSMZ strain, artificial saliva had com-paratively small or rather augmenting effects. This augmentingeffect of artificial saliva on biofilm formation of both S. oralisDSMZ single- and mixed-species biofilms is consistent with thespecies’ capacity to use mucin or the mucin component sialic acidas a carbon source by expressing secreted enzymes such as �-D-galactosidase, �-D-glucosidase, and �-N-acetyl-D-glucosamini-dase (78–80). However, we observed the contrary in the S. oralis4087 strain, on which saliva apparently exerts a negative influenceon its biofilm-forming capacity. Such strain-specific effects of sa-liva on S. oralis have recently been described and putatively beenassociated with the expression of specific adhesins (81).

Once integrated and established in the biofilms, GAS is locatedcloser to the underlying epithelial cell layers. Binding of S. pyo-genes to eukaryotic cells is indispensable for causing disease andpersisting in its human host (3). The influence of simultaneouslypresent bacteria on this interaction is not well studied. Thus far,only for Moraxella catarrhalis, another nasopharyngeal pathogen,was coaggregation with S. pyogenes reported to lead to increasedeukaryotic cell adherence but decreased internalization (82, 83).

To collect more data on the interaction of two bacterial andone eukaryotic partner, HEp-2 respiratory epithelial cells wereexposed to combinations of the two S. pyogenes strains and the S.oralis and S. salivarius strains. When testing the growth of singlespecies on the eukaryotic cells by SEM inspection, S. pyogenesstrains formed structures resembling microcolonies, S. salivariusformed large masses on the cell surface, while the S. oralis strainsonly occasionally adhered to the eukaryotic cells as single cells or

pairs (Fig. 7). Although the molecular basis for S. pyogenes eukary-otic cell attachment and microcolony formation is well established(28, 31, 33), for S. salivarius thus far only surface antigen C hasbeen determined as a responsible adhesin (84, 85).

S. salivarius and S. oralis strongly reduced S. pyogenes HEp-2cell adhesion and internalization if present for 2 h before S. pyo-genes was seeded (Fig. 6). Obviously, the established probiotic S.salivarius K-12 strain and the two S. oralis isolates could efficientlyprotect eukaryotic cells from S. pyogenes adherence, provided thestreptococcal strains have sufficient time to establish themselvesbefore S. pyogenes is inoculated. According to the results of thebacteriocin assays (Fig. 2), this protection was not associated todecreased GAS hemolysin production. Since S. salivarius wasdemonstrated by microscopy and quantitative measurements toform biofilm-like structures on eukaryotic cells, while S. oralishardly bound to this target, the protection is most probably basedon different mechanisms.

In addition to the killing of planktonic GAS (Fig. 1), the eu-karyotic cell protection exerted by S. salivarius could be due tosteric effects, i.e., the biofilm-like structure by which these bacteriacover the cells. Such protective effects of S. salivarius K-12 havealso been demonstrated against candidiasis in mouse models afteroral application (86). For the S. oralis strains, which, according tothe results from the cytotoxicity assays (see Fig. S5 in the supple-mental material), guard the HEp-2 cells even more efficiently,only small bacterial amounts bind to host cells, thereby precludingthe steric hindrance hypothesis. Thus, most probably anothermechanism applies. Potentially, regulatory and metabolic path-ways in the eukaryotic cells could be influenced by the presence ofthe S. oralis strains rendering the cells less susceptible to the S.pyogenes attack. Such beneficial effects on eukaryotic cells, as wellas the opposite, have been described for several viridans strepto-cocci (87–89). In contrast to the findings of Cosseau et al. (87)with the human bronchial epithelial cell line 16HBE14O, a nota-bly few number of transcripts showed significantly altered abun-dances in HEp-2 cells first exposed to respiratory tract strepto-cocci and then to M49 GAS compared to the positive control: 19 inthe case of S. salivarius and 10 in the case of S. oralis. Among thegenes repressed in the presence of S. oralis is PLG, encoding forplasminogen, which has been shown to be involved in the adher-ence and internalization of GAS M49 to or into host cells (45).Although the production of an active compound has been de-scribed in S. salivarius that inhibits the NF-�B pathway in humanepithelial cells and reduces secretion of the proinflammatory in-terleukin-8 (IL-8) (90), IL-8 transcript amounts were unaltered inour experimental setup. Both streptococcal species lead to adownregulation of genes involved in cGMP, GTPase, RAB, andRAS signaling (GFM1, HRSALS, GUCA1B, RAB6A, andRAPGEF2), as well as a changed expression of genes involved inapoptosis regulation (XRCC5, CCAR1, and MNDA). Further-more, upregulation of the expression of endothelin-2 in the case ofS. oralis could lead to an enhanced activation and acquisition ofneutrophils and macrophages in vivo.

Taken together, the study showed that S. oralis could induceprotection of the eukaryotic cells even without largely binding tothe cells or producing bacteriocins affecting S. pyogenes. Also,transcriptome analysis does not uncover apparent mechanisms bywhich S. oralis could protect the eukaryotic cells. The surprisinglyless effective protection of HEp-2 cells by S. salivarius also in-volved expression changes in only a restricted panel of genes and,

Oral Streptococci and GAS Biofilm Formation

February 2013 Volume 79 Number 4 aem.asm.org 1273

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

instead, could predominantly be exerted by building an almostimpermeable, potentially bacteriocin-producing wall of S. sali-varius biofilm in front of the host cell target.

We demonstrated here that S. pyogenes can establish itself inmixed-species biofilms with typical species of the resident pharyn-geal microbial biome. However, the species, i.e., S. oralis, whichmost efficiently supports S. pyogenes growth in mixed-species bio-films and simultaneously does not affect virulence factor produc-tion in the beta-hemolytic streptococci, also most efficiently pro-tects underlying epithelial host cells from S. pyogenes-inflicteddamages.

ACKNOWLEDGMENTS

This study was financially supported by the German Ministry of Educa-tion and Research (BMBF, SysMO-LAB2, ERA-NET1, and ERA-NET2initiatives) and the German Academic Exchange Service (DAAD).

We thank Gerhard Fulda, Wolfgang Labs and Michael Laue from theElectron Microscopy Centre of the University of Rostock for their helpwith generating SEM pictures and Jana Normann for excellent technicalassistance.

REFERENCES1. Cunningham MW. 2000. Pathogenesis of group A streptococcal infec-

tions. Clin. Microbiol. Rev. 13:470 –511.2. Carapetis JR, Steer AC, Mulholland EK, Weber M. 2005. The global

burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685– 694.3. Kreikemeyer B, Klenk M, Podbielski A. 2004. The intracellular status of

Streptococcus pyogenes: role of extracellular matrix-binding proteins andtheir regulation. Int. J. Med. Microbiol. 294:177–188.

4. Hynes W. 2004. Virulence factors of the group A streptococci and genesthat regulate their expression. Front. Biosci. 9:3399 –3433.

5. Olsen RJ, Shelburne SA, Musser JM. 2009. Molecular mechanisms un-derlying group A streptococcal pathogenesis. Cell Microbiol. 11:1–12.

6. McIver KS. 2009. Stand-alone response regulators controlling global vir-ulence networks in Streptococcus pyogenes. Contrib. Microbiol. 16:103–119.

7. Fiedler T, Sugareva V, Patenge N, Kreikemeyer B. 2010. Insights intoStreptococcus pyogenes pathogenesis from transcriptome studies. FutureMicrobiol. 5:1675–1694.

8. Dmitriev AV, Chaussee MS. 2010. The Streptococcus pyogenes proteome:maps, virulence factors, and vaccine candidates. Future Microbiol.5:1539 –1551.

9. Bessen DE, Lizano S. 2010. Tissue tropisms in group A streptococcalinfections. Future Microbiol. 5:623– 638.

10. Podbielski A, Kreikemeyer B. 2001. Persistence of group A streptococciin eukaryotic cells: a safe place? Lancet 358:3– 4.

11. Osterlund A, Engstrand L. 1997. An intracellular sanctuary for Strepto-coccus pyogenes in human tonsillar epithelium: studies of asymptomaticcarriers and in vitro cultured biopsies. Acta Otolaryngol. 117:883– 888.

12. Facinelli B, Spinaci C, Magi G, Giovanetti E, Varaldo E. 2001. Associ-ation between erythromycin resistance and ability to enter human respi-ratory cells in group A streptococci. Lancet 358:30 –33.

13. Reid G. 2005. The importance of guidelines in the development andapplication of probiotics. Curr. Pharm. Des. 11:11–16.

14. Tagg JR, Dierksen KP. 2003. Bacterial replacement therapy: adapting‘germ warfare’ to infection prevention. Trends Biotechnol. 21:217–223.

15. Walls T, Power D, Tagg J. 2003. Bacteriocin-like inhibitory substance(BLIS) production by the normal flora of the nasopharynx: potential toprotect against otitis media? J. Med. Microbiol. 52:829 – 833.

16. Rosenkranz W, Grundmann E. 1994. Immunomodulator action ofliving, nonpathogenic Enterococcus faecalis bacteria from humans.Arzneimittelforschung 44:691– 695. (In German.)

17. Habermann W, Zimmermann K, Skarabis H, Kunze R, Rusch V. 2002.Reduction of acute recurrence in patients with chronic recurrent hyper-trophic sinusitis by treatment with a bacterial immunostimulant (Entero-coccus faecalis Bacteriae of human origin. Arzneimittelforschung 52:622–627. (In German.)

18. Fiorentino TR, Beall B, Mshar P, Bessen DE. 1997. A genetic-based

evaluation of the principal tissue reservoir for group A streptococci iso-lated from normally sterile sites. J. Infect. Dis. 176:177–182.

19. Moore WE, Moore LV. 1994. The bacteria of periodontal diseases. Peri-odontol. 2000 5:66 –77.

20. Kolenbrander PE. 2000. Oral microbial communities: biofilms, interac-tions, and genetic systems. Annu. Rev. Microbiol. 54:413– 437.

21. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. 2005. Defining thenormal bacterial flora of the oral cavity. J. Clin. Microbiol. 43:5721–5732.

22. Grice EA, Segre JA. 2011. The skin microbiome. Nat. Rev. Microbiol.9:244 –253.

23. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC,Bouffard GG, Blakesley RW, Murray PR, Green ED, Turner ML, SegreJA. 2009. Topographical and temporal diversity of the human skin micro-biome. Science 324:1190 –1192.

24. Wade W. 2002. Unculturable bacteria–the uncharacterized organismsthat cause oral infections. J. R. Soc. Med. 95:81– 83.

25. Parahitiyawa NB, Scully C, Leung WK, Yam WC, Jin LJ, SamaranayakeLP. 2010. Exploring the oral bacterial flora: current status and futuredirections. Oral Dis. 16:136 –145.

26. Guglielmetti S, Taverniti V, Minuzzo M, Arioli S, Stuknyte M, Karp M,Mora D. 2010. Oral bacteria as potential probiotics for the pharyngealmucosa. Appl. Environ. Microbiol. 76:3948 –3958.

27. Guglielmetti S, Taverniti V, Minuzzo M, Arioli S, Zanoni I, StuknyteM, Granucci F, Karp M, Mora D. 2010. A dairy bacterium displays invitro probiotic properties for the pharyngeal mucosa by antagonizinggroup A streptococci and modulating the immune response. Infect. Im-mun. 78:4734 – 4743.

28. Akiyama H, Morizane S, Yamasaki O, Oono T, Iwatsuki K. 2003.Assessment of Streptococcus pyogenes microcolony formation in infectedskin by confocal laser scanning microscopy. J. Dermatol. Sci. 32:193–199.

29. Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreike-meyer B. 2006. Characterization of biofilm formation by clinically rele-vant serotypes of group A streptococci. Appl. Environ. Microbiol. 72:2864 –2875.

30. Swidsinski A, Goktas O, Bessler C, Loening-Baucke V, Hale LP, AndreeH, Weizenegger M, Holzl M, Scherer H, Lochs H. 2007. Spatial organi-zation of microbiota in quiescent adenoiditis and tonsillitis. J. Clin.Pathol. 60:253–260.

31. Frick IM, Mörgelin M, Björck L. 2000. Virulent aggregates of Streptococ-cus pyogenes are generated by homophilic protein-protein interactions.Mol. Microbiol. 37:1232–1247.

32. Cho KH, Caparon MG. 2005. Patterns of virulence gene expression differbetween biofilm and tissue communities of Streptococcus pyogenes. Mol.Microbiol. 57:1545–1556.

33. Manetti AG, Zingaretti C, Falugi F, Capo S, Bombaci M, Bagnoli F,Gambellini G, Bensi G, Mora M, Edwards AM, Musser JM, Graviss EA,Telford JL, Grandi G, Margarit I. 2007. Streptococcus pyogenes pili pro-mote pharyngeal cell adhesion and biofilm formation. Mol. Microbiol.64:968 –983.

34. Manetti AG, Köller T, Becherelli M, Buccato S, Kreikemeyer B, Pod-bielski A, Grandi G, Margarit I. 2010. Environmental acidification drivesStreptococcus pyogenes pilus expression and microcolony formation onepithelial cells in a FCT-dependent manner. PLoS One 5:e13864. c18 doi:10.1371/journal.pone.0013864.

35. Kimura KR, Nakata M, Sumitomo T, Kreikemeyer B, Podbielski A,Terao Y, Kawabata S. 2012. Involvement of T6 pili in biofilm formationby serotype M6 Streptococcus pyogenes. J. Bacteriol. 194:804 – 812.

36. Maddocks SE, Wright CJ, Nobbs AH, Brittan JL, Franklin L, StrombergN, Kadioglu A, Jepson MA, Jenkinson HF. 2011. Streptococcus pyogenesantigen I/II-family polypeptide AspA shows differential ligand-bindingproperties and mediates biofilm formation. Mol. Microbiol. 81:1034 –1049.

37. Podbielski A, Woischnik M, Leonard BA, Schmidt KH. 1999. Charac-terization of Nra, a global negative regulator gene in group A streptococci.Mol. Microbiol. 31:1051–1064.

38. Cisar JO, Kolenbrander PE, McIntire FC. 1979. Specificity of coaggre-gation reactions between human oral streptococci and strains of Actino-myces viscosus or Actinomyces naeslundii. Infect. Immun. 24:742–752.

39. Köller T, Manetti AG, Kreikemeyer B, Lembke C, Margarit I, Grandi G,Podbielski A. 2010. Typing of the pilus-protein-encoding FCT region andbiofilm formation as novel parameters in epidemiological investigationsof Streptococcus pyogenes isolates from various infection sites. J. Med. Mi-crobiol. 59:442– 452.

Fiedler et al.

1274 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

40. Abbott JD, Shannon R. 1958. A method for typing Shigella sonnei, usingcolicine production as a marker. J. Clin. Pathol. 11:71–77.

41. Molinari G, Rohde M, Talay SR, Chhatwal GS, Beckert S, Podbielski A.2001. The role played by the group A streptococcal negative regulator Nraon bacterial interactions with epithelial cells. Mol. Microbiol. 40:99 –114.

42. Fiedler T, Kreikemeyer B, Sugareva V, Redanz S, Arlt R, Standar K,Podbielski A. 2010. Impact of the Streptococcus pyogenes Mga regulator onhuman matrix protein binding and interaction with eukaryotic cells. Int. J.Med. Microbiol. 300:248 –258.

43. Palmer RJ, Jr, Gordon SM, Cisar JO, Kolenbrander PE. 2003. Coaggre-gation-mediated interactions of streptococci and actinomyces detected ininitial human dental plaque. J. Bacteriol. 185:3400 –3409.

44. Amerongen AV, Veerman EC. 2002. Saliva: the defender of the oralcavity. Oral Dis. 8:12–22.

45. Siemens N, Patenge N, Otto J, Fiedler T, Kreikemeyer B. 2011. Strep-tococcus pyogenes M49 plasminogen/plasmin binding facilitates keratino-cyte invasion via integrin-integrin-linked kinase (ILK) pathways and pro-tects from macrophage killing. J. Biol. Chem. 286:21612–21622.

46. Wilson M. 2005. Microbial inhabitants of humans: their ecology and rolein health and disease. Cambridge University Press, Cambridge, UnitedKingdom.

47. Kreth J, Merritt J, Qi F. 2009. Bacterial and host interactions of oralstreptococci. DNA Cell Biol. 28:397– 403.

48. Rosan B, Lamont RJ. 2000. Dental plaque formation. Microbes. Infect.2:1599 –1607.

49. Wescombe PA, Upton M, Dierksen KP, Ragland NL, Sivabalan S,Wirawan RE, Inglis MA, Moore CJ, Walker GV, Chilcott CN, JenkinsonHF, Tagg JR. 2006. Production of the lantibiotic salivaricin A and itsvariants by oral streptococci and use of a specific induction assay to detecttheir presence in human saliva. Appl. Environ. Microbiol. 72:1459 –1466.

50. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR.2007. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded atadjacent loci on a 190-kilobase transmissible megaplasmid in the oralprobiotic strain Streptococcus salivarius K-12. Appl. Environ. Microbiol.73:1107–1113.

51. Santagati M, Scillato M, Patane F, Aiello C, Stefani S. 2012. Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens.FEMS Immunol. Med. Microbiol. 65:23–31.

52. Birri DJ, Brede DA, Nes IF. 2012. Salivaricin D, a novel intrinsicallytrypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolatedfrom a healthy infant. Appl. Environ. Microbiol. 78:402– 410.

53. Carlsson J, Grahnen H, Jonsson G, Wikner S. 1970. Early establishmentof Streptococcus salivarius in the mouth of infants. J. Dent. Res. 49:415–418.

54. Horz HP, Meinelt A, Houben B, Conrads G. 2007. Distribution andpersistence of probiotic Streptococcus salivarius K-12 in the human oralcavity as determined by real-time quantitative polymerase chain reaction.Oral Microbiol. Immunol. 22:126 –130.

55. Bernstein JM, Faden HS, Scannapieco F, Belmont M, Dryja D, Wolf J.2002. Interference of nontypeable Haemophilus influenzae and Moraxellacatarrhalis by Streptococcus oralis in adenoid organ culture: a possiblestrategy for the treatment of the otitis-prone child. Ann. Otol. Rhinol.Laryngol. 111:696 –700.

56. Bottone E, Allerhand J, Pisano MA. 1971. Characteristics of a bacteriocinderived from Streptococcus faecalis var. zymogenes antagonistic to Diplo-coccus pneumoniae. Appl. Microbiol. 22:200 –204.

57. Galvez A, Valdivia E, Abriouel H, Camafeita E, Mendez E, Martinez-Bueno M, Maqueda M. 1998. Isolation and characterization of enterocinEJ97, a bacteriocin produced by Enterococcus faecalis EJ97. Arch. Micro-biol. 171:59 – 65.

58. Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. 2004.Single species biofilm-forming ability of root canal isolates on gutta-percha points. Eur. J. Oral Sci. 112:523–529.

59. Baldassarri L, Creti R, Recchia S, Imperi M, Facinelli B, Giovanetti E,Pataracchia M, Alfarone G, Orefici G. 2006. Therapeutic failures ofantibiotics used to treat macrolide-susceptible Streptococcus pyogenes in-fections may be due to biofilm formation. J. Clin. Microbiol. 44:2721–2727.

60. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Green-berg EP. 1998. The involvement of cell-to-cell signals in the developmentof a bacterial biofilm. Science 280:295–298.

61. Federle MJ, Bassler BL. 2003. Interspecies communication in bacteria. J.Clin. Invest. 112:1291–1299.

62. Stoodley P, Sauer K, Davies DG, Costerton JW. 2002. Biofilms ascomplex differentiated communities. Annu. Rev. Microbiol. 56:187–209.

63. Suntharalingam P, Cvitkovitch DG. 2005. Quorum sensing in strepto-coccal biofilm formation. Trends Microbiol. 13:3– 6.

64. Li J, Helmerhorst EJ, Leone CW, Troxler RF, Yaskell T, Haffajee AD,Socransky SS, Oppenheim FG. 2004. Identification of early microbialcolonizers in human dental biofilm. J. Appl. Microbiol. 97:1311–1318.

65. Sanchez MC, Llama-Palacios A, Blanc V, Leon R, Herrera D, Sanz M.2011. Structure, viability and bacterial kinetics of an in vitro biofilm modelusing six bacteria from the subgingival microbiota. J. Periodontal Res.46:252–260.

66. Murray PA, Levine MJ, Tabak LA, Reddy MS. 1982. Specificity ofsalivary-bacterial interactions. II. Evidence for a lectin on Streptococcussanguis with specificity for a NeuAc�2, 3Ga1�1, 3Ga1NAc sequence.Biochem. Biophys. Res. Commun. 106:390 –396.

67. Weerkamp AH, McBride BC. 1980. Adherence of Streptococcus salivariusHB and HB-7 to oral surfaces and saliva-coated hydroxyapatite. Infect.Immun. 30:150 –158.

68. Hytonen J, Haataja S, Finne J. 2006. Use of flow cytometry for theadhesion analysis of Streptococcus pyogenes mutant strains to epithelialcells: investigation of the possible role of surface pullulanase and cysteineprotease, and the transcriptional regulator Rgg. BMC Microbiol. 6:18. doi:10.1186/1471-2180-6-18.

69. Ryan PA, Pancholi V, Fischetti VA. 2001. Group A streptococci bind tomucin and human pharyngeal cells through sialic acid-containing recep-tors. Infect. Immun. 69:7402–7412.

70. Shelburne SA, Sumby P, III, Sitkiewicz I, Granville C, Deleo FR, MusserJM. 2005. Central role of a bacterial two-component gene regulatory sys-tem of previously unknown function in pathogen persistence in humansaliva. Proc. Natl. Acad. Sci. U. S. A. 102:16037–16042.

71. Fernie-King BA, Seilly DJ, Davies A, Lachmann PJ. 2002. Streptococcalinhibitor of complement inhibits two additional components of the mu-cosal innate immune system: secretory leukocyte proteinase inhibitor andlysozyme. Infect. Immun. 70:4908 – 4916.

72. Fernie-King BA, Seilly DJ, Lachmann PJ. 2004. The interaction of strep-tococcal inhibitor of complement (SIC) and its proteolytic fragments withthe human beta defensins. Immunology 111:444 – 452.

73. Frick IM, PÅkesson Rasmussen M, Schmidtchen A, Björck L. 2003. SIC,a secreted protein of Streptococcus pyogenes that inactivates antibacterialpeptides. J. Biol. Chem. 278:16561–16566.

74. Shelburne SA, Keith D, III, Horstmann N, Sumby P, Davenport MT,Graviss EA, Brennan RG, Musser JM. 2008. A direct link between car-bohydrate utilization and virulence in the major human pathogen group AStreptococcus. Proc. Natl. Acad. Sci. U. S. A. 105:1698 –1703.

75. Shelburne SA, Okorafor N, III, Sitkiewicz I, Sumby P, Keith D, Patel P,Austin C, Graviss EA, Musser JM. 2007. Regulation of polysaccharideutilization contributes to the persistence of group A streptococcus in theoropharynx. Infect. Immun. 75:2981–2990.

76. Courtney HS, Hasty DL. 1991. Aggregation of group A streptococci byhuman saliva and effect of saliva on streptococcal adherence to host cells.Infect. Immun. 59:1661–1666.

77. Roger P, Delettre J, Bouix M, Beal C. 2011. Characterization of Strep-tococcus salivarius growth and maintenance in artificial saliva. J. Appl.Microbiol. 111:631– 641.

78. Byers HL, Homer KA, Beighton D. 1996. Utilization of sialic acid byviridans streptococci. J. Dent. Res. 75:1564 –1571.

79. van der Hoeven JS, van den Kieboom CW, Camp PJ. 1990. Utilizationof mucin by oral Streptococcus species. Antonie Van Leeuwenhoek 57:165–172.

80. van der Hoeven JS, Camp PJ. 1991. Synergistic degradation of mucin byStreptococcus oralis and Streptococcus sanguis in mixed chemostat cultures.J. Dent. Res. 70:1041–1044.

81. Dorkhan M, Chavez de Paz LE, Skepo M, Svensater G, Davies JR. 2012.Effects of saliva or serum coating on adherence of Streptococcus oralisstrains to titanium. Microbiology 158:390 –397.

82. Brook I, Gober AE. 2006. Increased recovery of Moraxella catarrhalis andHaemophilus influenzae in association with group A beta-hemolytic strep-tococci in healthy children and those with pharyngo-tonsillitis. J. Med.Microbiol. 55:989 –992.

83. Lafontaine ER, Wall D, Vanlerberg SL, Donabedian H, Sledjeski DD.2004. Moraxella catarrhalis coaggregates with Streptococcus pyogenes andmodulates interactions of S. pyogenes with human epithelial cells. Infect.Immun. 72:6689 – 6693.

Oral Streptococci and GAS Biofilm Formation

February 2013 Volume 79 Number 4 aem.asm.org 1275

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from

84. Weerkamp AH, Jacobs T. 1982. Cell wall-associated protein antigens ofStreptococcus salivarius: purification, properties, and function in adher-ence. Infect. Immun. 38:233–242.

85. Weerkamp AH, Handley PS, Baars A, Slot JW. 1986. Negative stainingand immunoelectron microscopy of adhesion-deficient mutants of Strep-tococcus salivarius reveal that the adhesive protein antigens are separateclasses of cell surface fibril. J. Bacteriol. 165:746 –755.

86. Ishijima SA, Hayama K, Burton JP, Reid G, Okada M, Matsushita Y,Abe S. 2012. Effect of Streptococcus salivarius K-12 on the in vitro growthof Candida albicans and its protective effect in an oral candidiasis model.Appl. Environ. Microbiol. 78:2190 –2199.

87. Cosseau C, Devine DA, Dullaghan E, Gardy JL, Chikatamarla A, Gel-latly S, Yu LL, Pistolic J, Falsafi R, Tagg J, Hancock RE. 2008. The

commensal Streptococcus salivarius K-12 downregulates the innate im-mune responses of human epithelial cells and promotes host-microbehomeostasis. Infect. Immun. 76:4163– 4175.

88. Stinson MW, Alder S, Kumar S. 2003. Invasion and killing of humanendothelial cells by viridans group streptococci. Infect. Immun. 71:2365–2372.

89. Hasegawa T, Minami M, Okamoto A, Tatsuno I, Isaka M, Ohta M.2010. Characterization of a virulence-associated and cell-wall-locatedDNase of Streptococcus pyogenes. Microbiology 156:184 –190.

90. Kaci G, Lakhdari O, Dore J, Ehrlich SD, Renault P, Blottiere HM,Delorme C. 2011. Inhibition of the NF-�B pathway in human intestinalepithelial cells by commensal Streptococcus salivarius. Appl. Environ. Mi-crobiol. 77:4681– 4684.

Fiedler et al.

1276 aem.asm.org Applied and Environmental Microbiology

on May 8, 2021 by guest

http://aem.asm

.org/D

ownloaded from