In vitro and ex vivo activities of minocycline and EDTA against microorganisms embedded in biofilm on catheter surfaces.

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, Staphylococcus 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.

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis.

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-beta-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.

Role of the Streptococcus gordonii SspB protein in the development of Porphyromonas gingivalis biofilms on streptococcal substrates.

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.

Biofilm on ventriculo-peritoneal shunt tubing as a cause of treatment failure in coccidioidal meningitis.

We describe a case of recurrent coccidioidal meningitis in which a fungal biofilm on the tip of ventriculo-peritoneal shunt tubing was likely responsible for a 4-year persistence of Coccidioides immitis, despite the patient's taking an adequate dosage of fluconazole. Fungal biofilms should be considered as a cause for treatment failure and fungal persistence, especially when artificial prostheses or indwelling catheters are present.