Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR.

An in vitro plaque model based on the use of human salivary bacteria and tooth-like surfaces was previously developed for studying the formation of oral biofilm and its use for pre-clinical testing of candidate antimicrobial or antiplaque agents. In this study, a quantitative Taqman PCR assay (QPCR) was developed to compare the bacterial compositions of in vitro biofilms to parent saliva samples, and to determine the relative contributions of different species in the formation of the oral biofilm. In addition, the growth inhibition of saliva-derived plaque was evaluated by chlorhexidine. With this assay, which consisted of primer/probe sets targeting either 16S rDNA sequences present in public databases or cloned ribosomal intergenic spacer region (ISR) sequences, 15 oral bacteria derived from saliva as well as those that were responsible for biofilm formation in an in vitro plaque model were rapidly identified and quantified. Among the target organisms were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Lactobacillus acidophilus, Micromonas micros, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythensis, and Veillonella parvula. Primer and probe sets developed were both sensitive and specific. The relative profiles of a number of bacteria in 45-h-old biofilms were determined and, when compared to saliva samples, it was found that most of the bacteria identified in saliva also populated the in vitro plaque, including some anaerobes. Brief exposure of biofilms to chlorhexidine resulted in significant losses in viability. This new broad spectrum QPCR assay in combination with the in vitro plaque model will be of significant value in the quantitative study of the microbial composition of human saliva, saliva-derived plaque, and pre-clinical evaluation of potential antimicrobial and antiplaque molecules.

Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10(7) CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.

FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis.

Adherence of microorganisms to damaged heart tissue is a crucial event in the pathogenesis of infective endocarditis. In the present study, we investigated the role of the FimA protein as a potential virulence factor associated with Streptococcus parasanguis endocarditis. FimA is a 36-kDa surface protein that is a recognized adhesin in the oral cavity where it mediates adherence to the salivary pellicle. An insertion mutant and a deletion mutant of S. parasanguis were employed in the rat model of endocarditis to determine the relevance of FimA in endocarditis pathogenesis. Catheterized rats were infected with either the fimA deletion mutant VT929, the fimA insertion mutant VT930, or the isogenic, wild-type S. parasanguis FW213. Rats inoculated with FW213 developed endocarditis more frequently (50.9%) than animals inoculated with either the deletion mutant (2.7%) or the insertion mutant (7.6%) (P < 0.001). A series of in vitro assays were performed to explore the mechanism(s) by which FimA enhanced the infectivity of S. parasanguis. FimA did not inhibit the uptake or the subsequent killing of S. parasanguis by phagocytic granulocytes. Similarly, FimA did not play a role in the adherence to or the aggregation of platelets. Significant differences were noted between FW213 and VT929 (P < 0.05) and FW213 and VT930 (P < 0.001) in their abilities to bind to fibrin monolayers. The mean percent adherence of FW213 to fibrin monolayers (2.1%) was greater than those of VT929 (0.5%) and VT930 (0.12%). Taken together, these results indicate that FimA is a major virulence determinant associated with S. parasanguis endocarditis and further suggest that its role is associated with initial colonization of damaged heart tissue.