Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-␤-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.It has long been recognized that bacteria can regulate gene expression in response to cell density, and quorum-sensing systems based on acylhomoserine lactone or peptide autoinducer (AI) molecules are widespread among gram-negative and gram-positive bacterial species, respectively. Cellular functions regulated by quorum sensing include the expression of virulence factors, competence for genetic transformation, conjugal DNA transfer, the production of antibiotics and secondary metabolites, and biofilm formation (14,16,48,69). More recently, a novel communication system was described in the marine bacterium Vibrio harveyi (61). Bioluminescence in V. harveyi is regulated by two distinct AI signaling molecules that are detected by independent signal transduction systems that subsequently converge in a common pathway to regulate gene expression (reviewed in reference 54). AI-1 is a well-characterized derivative of a homoserine lactone that is highly species specific for V. harveyi. In contrast, AI-2 is a furanosyl borate diester. AI-2 is formed chemically from 4,5-dihydroxy-2,3-pentanedione that is generated by the action of LuxS AI synthase on S-ribosylhomocysteine (8, 55). The luxS gene is highly conserved across a diverse range of gram-negative and gram-positive bacterial spe...

Porphyromonas gingivalis is an aggressive periodontal pathogen that persists in the mixed-species plaque biofilm on tooth surfaces. P. gingivalis cells attach to the plaque commensal Streptococcus gordonii and this coadhesion event leads to the development of P. gingivalis biofilms. Binding of these organisms is multimodal, involving both the P. gingivalis major fimbrial FimA protein and the species-specific interaction of the minor fimbrial Mfa1 protein with the streptococcal SspB protein. This study examined the contribution of the Mfa1-SspB interaction to P. gingivalis biofilm formation. P. gingivalis biofilms readily formed on substrata of S. gordonii DL1 but not on Streptococcus mutans cells which lack a coadhesion-mediating homologue of SspB. An insertional inactivation of the mfa1 gene in P. gingivalis resulted in a phenotype deficient in S. gordonii binding and unable to form biofilms. Furthermore, analysis using recombinant streptococci and enterococci showed that P. gingivalis biofilms formed on Enterococcus faecalis strains expressing SspB or translational fusions of SspB with SpaP (the non-adherent SspB homologue in S. mutans) containing the P. gingivalis adherence domain (SspB adherence region, BAR) of SspB. In contrast, an isogenic Ssp null mutant of S. gordonii DL1 was unable to support biofilm growth, even though this strain bound to P. gingivalis FimA at levels similar to wild-type S. gordonii DL1. Finally, site-specific mutation of two functional amino acid residues in BAR resulted in SspB polypeptides that did not promote the development of P. gingivalis biofilms. These results suggest that the induction of P. gingivalis biofilms on a streptococcal substrate requires functional SspB-minor fimbriae interactions.

For pathogens to survive in the human oral cavity, they must identify a suitable niche in the complex multispecies biofilm that exists on oral tissues. The periodontal pathogen Porphyromonas gingivalis adheres to Streptococcus gordonii by interacting with a specific region of the streptococcal SspB polypeptide, designated BAR. However, it does not adhere to Streptococcus mutans, which expresses SpaP, a highly conserved homolog of SspB. in SspB by site-specific mutation generated proteins that were predicted to assume an SpaP-like secondary structure, and the purified proteins did not promote P. gingivalis adherence. Furthermore, Enterococcus faecalis strains expressing the site-specific mutants did not support adherence of P. gingivalis cells. In contrast, P. gingivalis adhered efficiently to E. faecalis strains expressing intact SspB or SspB-SpaP chimeric proteins containing BAR. These results suggest that a region of SspB consisting of 26 amino acids is sufficient to mediate the adherence of P. gingivalis to S. gordonii and that the species specificity of adherence arises from its interaction with a discrete structural determinant of SspB that is not conserved in SpaP.Porphyromonas gingivalis is regarded as one of the primary pathogens contributing to adult periodontitis, one of the most common infectious diseases of adults (16,34). In the human oral cavity, this organism resides in a complex mixed-species biofilm that forms on the tooth surface and in the periodontal pocket (5,14,28,31). However, the specific mechanisms utilized by P. gingivalis to establish and maintain itself in the oral biofilm are not fully understood. Early events leading to biofilm development on oral tissues involve the interaction of gram-positive commensal organisms, e.g., streptococci and Actinomyces spp., with the salivary pellicle coating the tissue surface (10, 14). These primary colonizing organisms then provide an attachment substrate for the ordered accumulation of other gram-positive and gram-negative bacterial species, including Fusobacterium nucleatum and periodontal pathogens such as P. gingivalis (15,30,32). Thus, colonization of the developing oral biofilm by P. gingivalis likely involves its adherence to various antecedent bacteria such as the oral streptococci and/or F. nucleatum. The interaction of P. gingivalis with primary colonizing organisms such as streptococci may also be important in the invasion of dentinal tubules by P. gingivalis (21).The adherence of P. gingivalis to Streptococcus gordonii appears to occur through a protein-protein interaction requiring the SspB polypeptide of S. gordonii (17,18) and the minor fimbrial component of P. gingivalis (3). Indeed, expression of the sspB gene in Enterococcus faecalis resulted in a transformed cell that was capable of promoting P. gingivalis adherence (17). The SspB polypeptide is a multifunctional surface protein of S. gordonii and is a member of the highly conserved antigen I/II (27) family of cell surface proteins that are expressed by virtually all streptococci...

Minocycline-EDTA (M-EDTA) flush solution has been shown to prevent catheter-related infection and colonization in a rabbit model and in hemodialysis patients. We undertook this study in order to determine the activities of M-EDTA against organisms embedded in fresh biofilm (in vitro) and mature biofilm (ex vivo). For the experiment with the in vitro model, a modified Robbin's device (MRD) was used whereby 25 catheter segments were flushed for 18 h with 10 6 CFU of biofilm-producing Staphylococcus epidermidis, Staphyloccocus aureus, and Candida albicans per ml. Subsequently, each of the catheter segments was incubated in one of the following solutions: (i) streptokinase, (ii) heparin, (iii) broth alone, (iv) vancomycin, (v) vancomycin-heparin, (vi) EDTA, (vii) minocycline (high-dose alternating with low-dose), or (viii) M-EDTA (low-dose minocycline alternating with high-dose minocycline were used to study the additive and synergistic activities of M-EDTA). All segments were cultured quantitatively by scrape sonication. For the experiment with the ex vivo model, 54 catheter tip segments removed from patients and colonized with bacterial organisms by roll plate were longitudinally cut into two equal segments and exposed to either saline, heparin, EDTA, or M-EDTA (with high-dose minocycline). Subsequently, all segments were examined by confocal laser electron microscopy. In the in vitro MRD model, M-EDTA (with a low concentration of minocycline) was significantly more effective than any other agent in reducing colonization of S. epidermidis, S. aureus, and C. albicans (P < 0.01). M-EDTA (with a high concentration of minocycline) eradicated all staphylococcal and C. albicans organisms embedded in the biofilm. In the ex vivo model, M-EDTA (with a high concentration of minocycline) reduced bacterial colonization more frequently than EDTA or heparin (P < 0.01). We concluded that M-EDTA is highly active in eradicating microorganisms embedded in fresh and mature biofilm adhering to catheter surfaces. , abstr. 514, 1999). Staphylococcus epidermidis, Staphylococcus aureus, and Candida species are the leading organisms causing CRBSI (14,22,27).Because intraluminal colonization is the major source for the migration of organisms leading to bloodstream infections in long-term silicone catheters (19), recent guidelines have proposed the use of intraluminal antimicrobial lock solutions for the prevention and treatment of CRBSI (15, 17). Most long-term CVCs are flushed with heparin. An antimicrobialanticoagulant combination consisting of vancomycin and heparin with and without ciprofloxacin was used in several studies and was demonstrated to reduce the risk of catheter-related bacteremia caused by gram-positive organisms (2, 10, 26). However, with vancomycin-resistant gram-positive bacteria increasing, concerns have been raised over the use of vancomycin flush solutions and their potential for increasing the risk of vancomycin resistance (30).EDTA is a metal chelator with established anticoagulant activity and inhibitory activity...

Dental plaque is a complex biofilm that accretes in a series of discrete steps proceeding from a gram-positive streptococcus-rich biofilm to a structure rich in gram-negative anaerobes. This study investigated information flow between two unrelated plaque bacteria, Streptococcus cristatus and Porphyromonas gingivalis. A surface protein of S. cristatus caused repression of the P. gingivalis fimbrial gene (fimA), as determined by a chromosomal fimA promoter-lacZ reporter construct and by reverse transcription-PCR. Signaling activity was associated with a 59-kDa surface protein of S. cristatus and showed specificity for the fimA gene. Furthermore, P. gingivalis was unable to form biofilm microcolonies with S. cristatus. Thus, S. cristatus is capable of modulating virulence gene expression in P. gingivalis, consequently influencing the development of pathogenic plaque.The study of the ability of bacterial cells to communicate with one another and coordinate behavior is a burgeoning field with relevance to a number of microbial ecosystems (5,6,11,12,17). The plaque biofilm that accumulates on tooth surfaces comprises over 30 genera representing more than 500 species (9, 16). Despite this complexity, plaque formation is highly choreographed. Initial colonization by a group of gram-positive organisms, mainly streptococci, is followed by a succession of species that culminates in the arrival of gram-negative anaerobic bacteria such as Porphyromonas gingivalis, a predominant pathogen in severe adult periodontitis (13). Colonization of the dental biofilm by P. gingivalis is thus a pivotal event in the transition from a commensal plaque to a pathogenic entity. P. gingivalis colonization is contingent upon fimbria-mediated adhesion to oral surfaces (1, 7). The fimA gene that encodes the major subunit protein of fimbriae (FimA) can be regulated by environmental cues (2, 19). However, the extent to which plaque bacteria can modulate fimbrial gene expression in P. gingivalis through intercellular signaling mechanisms is largely unknown.Expression of fimA is regulated by S. cristatus. To identify signaling mechanisms of oral biofilm organisms that could affect expression of the fimA gene, we utilized P. gingivalis strain UPF, which contains a chromosomal fusion between the fimA promoter and a lacZ reporter gene (18). P. gingivalis UPF was grown in Trypticase soy broth (TSB) or on 1.5% TSB blood agar plates supplemented with yeast extract (1 mg/ml), hemin (5 g/ml), and menadione (1 g/ml) at 37°C in an anaerobic (85% N 2 , 10% H 2 , 5% CO 2 ) chamber. When appropriate, the culture medium contained the antibiotics erythromycin (20 g/ ml) and gentamicin (100 g/ml). The organisms tested for signaling activity were Streptococcus gordonii G9B and M5, Streptococcus sanguis 10556, Streptococcus mutans KPSK2, Streptococcus cristatus CC5A, and Actinomyces naeslundii NC-3, all of which were grown in Trypticase Peptone broth supplemented with yeast extract (5 mg/ml) and 0.5% glucose at 37°C aerobically; Treponema denticola GM-1, which was c...