Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples.

The purpose of this study was to compare DNA probe analyses to cultural methods for detecting three periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius, in human subgingival plaque. Subgingival sites from patients diagnosed as either healthy or showing evidence of gingivitis or juvenile or adult periodontitis were sampled using two paper points. The number of these pathogens from one paper point was determined using microbiologic media and speciated by biochemical tests. Results were then compared to bacterial numbers obtained from the other paper point using species-specific DNA probes. In 60 samples from the disease group, DNA probe analysis demonstrated 100% effectiveness in detecting A. actinomycetemcomitans and B. intermedius and 91% effectiveness in detecting B. gingivalis at culture positive levels (greater than or equal to 10(3) cells). In addition, probe assays frequently identified these pathogens in samples that were culture negative. Probe analysis revealed a better correlation between presence of a pathogen and clinical evidence of disease on an individual patient basis. In contrast, most samples taken from sites of healthy individuals showed undetectable levels of all three pathogens as determined by both techniques. These results suggest that DNA probe technology is at least equivalent and often superior to cultural methods for detecting A. actinomycetemcomitans, B. gingivalis, and B. intermedius in human subgingival plaque samples.

Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate.

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

A cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis.

In this study, we evaluated the sensitivity and specificity of whole genomic DNA probes for the periodontal pathogens Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis. By means of these probes, DNA hybridizations were performed against other organisms found in the oral cavity and organisms previously determined to be genetically similar. All three probes were sensitive to 10(3) cells for their respective organism. The H. actinomycetemcomitans probe cross-reacted with several haemophilus strains, Wolinella, and Campylobacter, indicating that H. actinomycetemcomitans-specific sequences would have to be identified and cloned for accurate detection of this organism in heterogeneous patient samples. Only very low levels of cross-reactivity were observed between the B. intermedius probe and representative black-pigmented Bacteroides. This low level of cross-reactivity did not interfere with the accurate identification of B. intermedius in sample evaluations. The B. gingivalis probe showed no cross-reactivity. Whole genomic probes will be used for the detection of B. intermedius and B. gingivalis in patient samples.

API ZYM and API An-Ident reactions of fastidious oral gram-negative species.

API ZYM and API An-Ident enzymatic substrate tests were done on six oral species which are difficult to characterize with conventional biochemical tests. "Bacteroides forsythus, the "fusiform" Bacteroides species (A. C. R. Tanner, M. A. Listgarten, M. N. Strzempko, and J. L. Ebersole, manuscript in preparation), is difficult to cultivate in broth media, yet it gave 15 positive tests in these series. The tests were able to separate this new species from species of Capnocytophaga and Fusobacterium. "B. forsythus" reactions were similar but not identical to those of reference Bacteroides species. Positive reactions for alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and alpha-glucuronidase suggest that "B. forsythus" may be saccharolytic. It was the only species tested which was trypsin positive. Wolinella species, Campylobacter concisus, B. gracilis, and Eikenella corrodens are asaccharolytic, and characterization relies heavily on sensitivities to inhibitory agents. These species reacted weakly in the API ZYM and API An-Ident enzymatic substrate tests, and the reactions were not useful for separating these species. The enzyme reactions differentiated Wolinella recta and C. concisus from Selenomonas sputigena, another oral motile but saccharolytic organism.