Gram-positive and gram-negative bacteria use quorum sensingto coordinate population behavior . In several streptococci,quorum sensing mediated by competence-stimulating peptides [CSP]is associated with development of competence for transformation.We show here that a synthetic CSP favored the biofilm mode ofgrowth of Streptococcus intermedius without affecting the rateof culture growth.

 
In natural ecosystems, microorganisms grow preferentially attachedto surfaces, forming matrix-enclosed biofilms [3] . In this mode of growth, microorganisms exhibit increased resistance to antimicrobials and to host immune defense mechanisms [15] . Both gram-positiveand gram-negative bacteria communicate through quorum-sensing[QS] signals to coordinate population behavior . Biofilm maturationand expression of virulence factors are some of several identifiedQS-controlled behaviors [4, 7, 13] . Not surprisingly, QS has emerged as an attractive target for fighting biofilm infections.

The streptococci comprise a diverse group of gram-positive bacteria, ranging from pathogens to commensal organisms that may be involved in opportunistic infections . In the oral cavity, streptococci constitute approximately 60% of the initial biofilm microfloraon the tooth surfaces [16] . Members of the anginosus group, including Streptococcus intermedius, Streptococcus anginosus, and Streptococcus constellatus, are also found in the gastrointestinaland genitourinary tracts . The anginosus group may be associatedwith serious purulent infections, with S . intermedius exhibitingtropism for abscesses of the liver and brain [24].

While the QS signals employed by a large number of gram-negative bacteria are homoserine lactones, gram-positive bacteria typically use secreted peptides . The accumulated secreted peptides are generally detected via a sensor kinase receptor, inducing autophosphorylation of the kinase protein . The phosphoryl group is subsequently transferred to a response regulator that triggers, directlyor indirectly, the required changes in gene expression.

Bacterial natural transformation is thought to provide a selective advantage by allowing competent cells to acquire new traits,for example, antibiotic resistance, by incorporation of DNAreleased from other cells . Streptococci have a QS system thatregulates natural competence for genetic transformation . Thepeptide QS signal termed competence-stimulating peptide [CSP]has been identified in various strains of the mitis, anginosus[9, 10], and mutans groups of streptococci [9, 10] . In the anginosusgroup, distinct species commonly encode and respond to identicalCSPs . In other groups of streptococci, the CSPs are most oftenspecies specific and in several instances are specific to certaingroups of strains [9].

The disruption of QS genes in gram-negative bacteria, including Pseudomonas aeruginosa [4], Burkholderia cepacia [7], and Aeromonashydrophila [13], has been associated with altered biofilm formation.Similar findings have recently been reported for gram-positivebacteria, including the competence QS circuit in Streptococcusmutans [11] and Streptococcus gordonii [12], in which disruption of QS regulatory genes resulted in reduced biofilm formationor altered biofilm architecture . Initiation of a QS responseduring competence development is influenced by environmentalconditions . For S . intermedius, conditions that support endogenousinduction of competence development have been described [9, 19] . It appears, however, that the growth-dependent requirementsfor spontaneous competence can in several instances be overcomeby addition of synthetic CSP [8] . To determine whether the CSPby itself may mediate a response affecting S . intermedius biofilmformation, we examined the role of synthetic CSP under conditionsthat prevented spontaneous competence . We found that CSP-treatedcells formed more biofilm than untreated cells, whereas competencedevelopment occurred exclusively in the presence of syntheticCSP.

Environmental influence on S . intermedius biofilm formation and competence development. We carried out various assays to establish conditions in whichS . intermedius formed biofilm in the absence of spontaneouscompetence, while still responding to addition of syntheticCSP.

The biofilm assay was adapted from the method of O'Toole and Kolter [17], which is based on the ability of bacteria to formbiofilms on solid polystyrene surfaces . Overnight cells of S. intermedius NCTC 11324 grown in Todd-Hewitt broth [THB] [Difco Laboratories, Detroit, Mich.] supplemented with yeast extract were diluted 1:200 or 1:20 in trypticase soy broth [TSB] [Becton Dickinson and Company, Sparks, Md.]; 500 µl was inoculatedinto wells of polystyrene flat-bottom 24-well microtiter plates[Nunc, Copenhagen, Denmark] . The plates were incubated at 37°Caerobically or in a 5%-CO₂ aerobic atmosphere for 24 h . Theamount of biofilm formed in the wells was measured by stainingthe biofilm cells with 0.1% safranin . After wells were rinsedwith distilled H₂O, bound safranin was released from the stainedcells with 30% acetic acid, and the absorbance of the solutionwas measured at 530 nm . Alternatively, the biofilms were washedtwice and suspended in 500 µl of fresh TSB by scrapingthe bottom and lateral walls of the wells . To disperse clumpsand chains, the biofilm suspensions were gently sonicated for10 s at 4°C, and the optical density was measured at 600nm.

S . intermedius growth and biofilm formation in TSB was negligible under aerobic conditions . When grown in 5% CO₂, S . intermediusformed homogeneous biofilms . The largest amount of biofilm wasformed with inoculums of cells diluted 1:200.

To examine whether competence could be initiated by endogenous CSP, the plasmid pVA838 expressing erythromycin resistance [14] was added at the time of inoculation [final concentration, 0.2 µg/ml] . After a 24-h incubation, transformants were selectedby growth on THB agar plates containing the selective antibiotic. Biofilm cells grown in THB supplemented with horse serum [9, 19] were included as positive controls . Cells grown in THB supplementedwith horse serum showed spontaneous competence development,with a transformation yield of 40 transformants/ml, whereasgrowth in TSB resulted in no transformants . Competence developmentin TSB could, however, be induced by addition of synthetic CSPas described below . Based on these preliminary observations,growth of cells diluted 1:200 in TSB incubated at 37°C ina 5% CO₂ incubator was chosen to further examine the effectof CSP on biofilm formation.

Synthetic CSP in the nanomolar concentration range enhances S . intermedius biofilm formation and induces competence. The peptide CSP 11325 [amino acid sequence: NH₂-DSRIRMGFDFSKLFGK-COOH], which induces competence in S . intermedius NCTC 11324, was synthesized[MedProbe] [9] . CSP concentrations varying between 0.2 and 100nM were tested for their effect on biofilm formation and competencedevelopment in TSB medium after 24 h . CSP was added immediatelyafter dilution of the cells [1:200] . Competence was assayedby exposure of the cells to pVA838 [final concentration, 1 µg/ml]during culture growth and selection of transformants on THBagar containing the selective antibiotic.

A relationship between the amount of CSP and the degree of CSP stimulating activity was observed [Fig . 1a and b] . The steep increase in biofilm formation [Fig . 1a] and competence induction[Fig . 1b] in the concentration range between 0.2 and 1.6 nMis consistent with the CSP autoinducing response mechanism.

 
We also investigated the proportion of competent cells in thebiofilm and planktonic fractions, respectively, upon additionof 100 nM CSP . At 24 h, 0.032% of the cells in the biofilm fractionwere transformants, in contrast to only 0.003% of the cellsin the planktonic fraction.

The enhancement of biofilm formation by CSP was verified by scanning electron microscopy . Biofilms were grown in TSB orin minimal medium [MM] [6] containing 10 mM glucose as with the biofilm assay described above, except that a polystyrene disk [Nunc] was immersed in each well before inoculation . After24 h the disks were removed, rinsed with distilled H₂O, and fixed with 2.5% glutaraldehyde in 0.1 M Sørensen buffer.Dehydrated samples were obtained through a series of ethanolrinses and dried at the critical point with liquid CO₂ . CSP-treatedcells formed more biofilm than untreated cells during growthin both TSB and MM [Fig . 2] . Increased chain formation has been associated with the deletion of the CSP-encoding gene in S.mutans [11] . It was not, however, apparent in the SEM images whether the CSP influenced chain formation in S . intermedius. Additionally, in liquid culture growth, long chains were observed in both the presence and the absence of CSP by phase-contrast microscopy, with no observable difference in chain size [datanot shown].

 
CSP promotes the biofilm mode of growth without affecting the growth rate. To examine whether enhancement of biofilm formation was related to an effect on bacterial growth rate, the growth rate in liquid culture was monitored in the absence or presence of 100 nM synthetic CSP . Optical density measurements at 600 nm were taken at different time intervals until stationary phase . No effect on the rateof culture growth was observed.

We also determined the relative distributions of planktonicand biofilm cells during growth in the wells after 4, 6, 8,and 24 h [Fig . 3] . The 500-µl planktonic fraction was transferred to a cuvette, and the optical density at 600 nmwas measured . The biofilm fraction was resuspended in 500 µlof fresh TSB and sonicated as described above, and the opticaldensity was measured at 600 nm . After 4 h, no difference inbiofilm formation was detected between growth in the presenceor absence of CSP . After 6 h, the biofilm formed in the presenceof CSP was 22% higher than without the CSP, and after 8 h, anincrease of 29% was observed . After 24 h, an increase of approximately100% was observed . Notably, the total growth, assessed as thesum of planktonic and biofilm values, was not affected by additionof CSP at any time.

 
Specificity of the CSP response. The specificity of the CSP effect on biofilm formation was testedwith both S . intermedius and S . mutans . S . intermedius biofilmformation was unaffected by exposure to up to 400 nM S . mutanssynthetic CSP 159 [synthesized by MedProbe; amino acid sequence,NH₂-SGSLSTFFRLFNRSFTQALGK-COOH] . We also tested whether S . mutansbiofilm formation was enhanced by CSP 159 and whether S . intermediusCSP 11325 affected S . mutans biofilm formation . S . mutans LT-11 biofilm formed in the presence of CSP 159 was enhanced by approximately 85% but was unaffected by S . intermedius CSP 11325 . Thus, the CSP response was species specific for both S . intermedius and S . mutans.

DNA structural role in S . intermedius biofilm. DNA has recently been reported as an important component ofthe P . aeruginosa biofilm structure [25] . It was suggested that the origin of the DNA could be a result of active DNA transport from the P . aeruginosa cells into extracellular vesicles . Such a mechanism has not been reported for gram-positive microorganisms. Induction of lysis of a subfraction of the cells has been observed, however, during Streptococcus pneumoniae competence development [21].

To test whether DNA could be an important component of the biofilm formed by S . intermedius, DNase I [20 U/µl; Roche] wasadded to the initial inoculums at 40, 200, or 400 U/ml [final concentration] . The inhibitory effect of DNase I on biofilmformation was observed after growth in both the presence andthe absence of CSP [Fig . 4] . With the maximum concentrationof DNase I [400 U/ml], biofilm formed in the presence of CSPwas reduced by 45% [standard error, 9.5] and without CSP by52% [standard error, 3.8], a difference that was not statisticallysignificant [t test; P = 0.419] . The results indicate that DNAmay play an important role in the structure of S . intermediusbiofilm . We are currently investigating whether the expressionof DNA-binding proteins up-regulated by CSP [2] may be involvedin the observed increased biofilm formation in response to theCSP.

 
The QS-biofilm connection is clear for several bacteria [4, 5, 7, 13] . Mutants deficient in QS, however, can still formbiofilms, although in smaller amounts or lacking the typicalarchitecture of mature biofilms . For S . gordonii [12] and S. mutans [11], for instance, inactivation of competence-regulatorygenes does not abolish the ability of the cells to form biofilms,although the biofilms differ from the wild types . Deletion ofregulatory genes involved in QS responses, however, may resultin secondary mutations to compensate for disruption of QS regulons,thus confusing interpretation of results [22] . In our studywe investigated directly the role of the CSP signal in biofilmformation and competence development of the wild-type strain. We found that the respective synthetic CSPs favored the biofilm mode of growth for both S . intermedius and S . mutans, and we confirmed the role of CSP in S . intermedius competence development [9, 19].

For the mitis and anginosus groups of streptococci, the genes encoding the CSP, the kinase receptor, and the cognate response regulator are organized in an operon . For S . mutans, however, the promoter for the CSP-encoding gene is distinct from thepromoter for the kinase receptor and the response regulator.Moreover, inactivation of the genes encoding the histidine kinaseor the response regulator results in complete abolishment ofcompetence for S . pneumoniae [18], while for S . mutans competenceis reduced or not affected [1, 10] . This indicates that themechanisms of regulation may vary among different streptococcalspecies . Despite such differences, both S . intermedius and S.mutans responded to their respective CSPs by exhibiting increasedbiofilm formation.

The S . pneumoniae CSP response during competence development is well characterized . Genome-scale studies have recently shown, however, that among the CSP-inducible genes, more than halfare dispensable for transformation, while several induced genesare stress related [20] . Stress responses are also related to biofilm formation . Environmental conditions, such as nutritional limitation, appear to trigger biofilm formation by several bacterial species [23] . We are currently investigating whether S . intermediusexposed to the synthetic CSP may sense stress conditions notactually present in the environment and whether this in turnmay favor the biofilm mode of growth . In the biofilm, the cellsare probably able to cope more efficiently with stress conditions.Elucidation of how cells communicate to regulate such groupbehavior may lead to novel strategies for control of bacterial infection.

 
This work was supported by a postdoctoral fellowship from The Norwegian Research Council.

 
* Corresponding author . Mailing address: Department of Oral Biology, Faculty of Dentistry, University of Oslo, P.O . Box 1052, Blindern, N0316 Oslo, Norway . Phone: 47 22840352 . Fax: 47 22840302 . E-mail: cpaiva@odont.uio.no .

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