Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell‐surface anchorage signals within the primary sequence. Full‐length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid‐phase fibronectin, in a heparin‐sensitive interaction, and blocked binding of wild‐type pneumococcal cells to fibronectin. However, a C‐terminally truncated PavA′ polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2–2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2–2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C‐terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell‐surface physico‐chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104‐fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin‐binding protein plays a direct role in the pathogenesis of pneumococcal infections.

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A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely,Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping forL. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical toL. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiatingLactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.

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Yeasts are being increasingly identified as important organisms in human infections. Adhesive interactions between yeasts and bacteria may contribute to yeast retention at body sites. Methods for studying adhesive interactions between bacterial strains are well known, and range from simple macroscopic methods to flow chamber systems with complex image analysis capabilities. The adhesive interactions between bacteria and yeasts have been studied employing several of the methods originally developed for studying adhesive interactions between bacteria. However, in many of the methods employed the larger size of the yeasts as compared with bacteria results in strong sedimentation of the yeasts, often invalidating the method adapted. In addition, most methods are semi-quantitative and do not properly control mass transport. Consequently, adhesive interaction mechanisms between yeasts and bacteria identified hitherto, including lectin binding and protein-protein interactions, must be regarded with caution. Extensive physico-chemical characteristics of yeast cell surfaces are not available and a physico-chemical mechanism has not yet been put forth. A new method for quantifying adhesive interactions between yeasts and bacteria is proposed, based on the use of a parallel plate flow chamber, in which the influence of adhering bacteria upon the kinetics of yeast adhesion and aggregation of the adhering yeasts is quantitatively evaluated, under carefully controlled mass transport.

Lactobacilli can interfere with the adhesion of uropathogens to uroepithelial cells and catheter materials through a variety of mechanisms, such as adhesion. Lactobacillus adhesion to substratum surfaces has been theorized to result from the physicochemical properties of the interacting surfaces. In this paper physicochemical cell-surface properties, including hydrophobicity (determined by water contact angles), pH dependence of zeta potentials, elemental surface composition (determined by X-ray photoelectron spectroscopy), and adhesion to hexadecane, of four genotypically characterized Lactobacillus species (eight L. acidophilus, eight L. casei, four L. fermentum, and seven L. plantarum strains) were determined to see whether a grouping of the strains according to their phenotypes could be obtained that corresponded with the genotypic characterization of the strains. The strains showed major differences in physicochemical cell-surface properties: at the species level relationships could be observed between water contact angles, isoelectric points, and the N/C and O/C elemental surface concentration ratios, with nitrogen-containing groups (proteins) being responsible for increased hydrophobicities and isoelectric points, and oxygen-containing groups (phosphates and polysaccharides) yielding decreased hydrophobicities and isoelectric points. A hierarchical cluster analysis grouped all L. acidophilus strains in one well-separated cluster that also included two L. casei and two L. fermentum strains. Separation of L. acidophilus from the other species was done predominantly on the basis of increased cell surface hydrophobicity (average water contact angle of 63°) and isoelectric point (approximately pH 3.3) as compared with the other species, which had lower water contact angles and isoelectric points, and corresponded with the observation that only L. acidophilus strains adhered in measurable numbers to hexadecane. Also, the L. plantarum strains were grouped closely together in one cluster, but this cluster was heterogeneous due to the inclusion of L. casei and L. fermentum strains.Key words: Lactobacillus, surface properties, hydrophobicity, zeta potential, adhesion.

Urinary biomaterials are compromised by device-related urinary tract infections, bacterial biofilm formation, and biomineral encrustation. In the absence of urinary infection, calcium oxalate is the prevalent encrustation mineral formed. Considering this, a novel approach was taken in the study reported here, in that an oxalate-degrading enzyme, oxalate oxidase (OXO), was immobilized on the surface of silicone elastomer (PDMS), a common urological biomaterial. It was hypothesized that the enzymatic action of OXO could lower urinary oxalate levels near the device surface, thereby preventing calcium oxalate crystal formation. The PDMS surface was functionalized with the use of radio-frequency plasma discharge (RFPD) in the presence of water vapor, then coated with 3-aminopropyltriethoxysilane (AMEO). The resulting aminated surface was covalently coupled with OXO via glutaraldehyde bioconjugation. The ability of the OXO-coated PDMS to prevent calcium oxalate encrustation was evaluated with the use of a modified Robbins device (MRD) encrustation model. RFPD performed on PDMS resulted in an increase in the hydrophilicity of treated surfaces, as measured by contact angle. X-ray photoelectron spectroscopy studies showed increases in elemental oxygen, after water-vapor plasma, and in nitrogen after AMEO derivatization. The immobilized enzyme was shown to retain 47.5% of its specific enzymatic activity as compared to free enzyme. In vitro experiments for 6 days, with the use of a MRD, showed 53% less encrustation deposits on discs coated with OXO than control discs. The results from the current study suggest that PDMS-immobilized oxalate-degrading enzymes are active against calcium oxalate encrustation.

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