Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites.

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species-specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.

Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses.

Restriction endonuclease analysis, rRNA gene restriction analysis (ribotyping), multilocus enzyme electrophoresis and lipase production were investigated for their potential to differentiate isolates belonging to the closely-related species Prevotella intermedia and Prevotella nigrescens. Of 122 strains identified originally as P. intermedia, 52 were assigned to P. intermedia and 68 to P. nigrescens using multilocus enzyme electrophoresis. All 39 P. intermedia and 52 out of 53 P. nigrescens tested produced lipase. Restriction endonuclease analysis identified clonal variants, but did not facilitate the differentiation of strains into species. Taq I ribotyping of 99 strains revealed that all P. intermedia demonstrated a species-specific fragment of 0.40 kbp, which was always associated with a second fragment of 0.57 kbp, and all P. nigrescens tested shared a species-specific fragment of 2.21 kbp. Two strains atypical by multilocus enzyme electrophoresis had none of the above species-specific fragments. Thus, lipase production and restriction endonuclease analysis did not distinguish between P. intermedia and P. nigrescens, but Taq I ribotyping did and also allowed the characterization of individual strains.

Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens.

Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.

Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections.

The occurrence and surface properties of prevotella intermedia and P. nigrescens in healthy sites and in periodontic and endodontic infections were studied among 73 strains, tentatively identified as P. intermedia. Fifteen strains were from necrotic root canal infections, 41 were from periodontal samples, and 17 isolates were obtained from healthy gingival sites. Identification of isolates as either P. intermedia or P. nigrescens was based on differences in malate and glutamate dehydrogenase electrophoretic mobilities which allowed unambiguous separation of P. intermedia and P. nigrescens. The majority of strains from periodontal samples were P. intermedia (29 of 41 strains). In endodontic samples only 4 out of 15 isolates were P. intermedia, while all except 1 of 17 strains from healthy gingival sites were identified as P. nigrescens. SDS-PAGE of whole cell proteins revealed 31 and 38 kDa proteins in P. nigrescens which were not detected in P. intermedia. Surface biotinylation of cells, followed by Western blotting and detection by alkaline phosphatase conjugated extravidin, showed strong staining of the 31 kDa protein in P. nigrescens indicating that this protein is located on the surface of the cell. Corresponding staining was not seen in P. intermedia. Fimbria-like projections were observed using electron microscopy of negatively-stained cells of P. nigrescens. The results show that P. intermedia and P. nigrescens may have different site specificities and surface properties and thus emphasize the need for accurate identification of these two species for the evaluation of their role in the pathogenesis of oral infections.

Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains.

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, BglII, and HinfI on a collection of Candida albicans strains comprising eight strains randomly selected from clinical microbiology laboratory specimens, three reported azole-resistant strains from treatment failures, and several subcultures of the azole-resistant strain NCPF 3310 (also known as the Darlington strain) received from different laboratories. The results demonstrated a diversity of the restriction fragment length polymorphism patterns that were obtained and revealed that two of the proposed Darlington subcultures had patterns distinct from each other and from those of the other Darlington isolates; both were also found to have lost their azole resistance.