The characteristics of a group of slow-growing, fusiform, fastidious anaerobes isolated from advanced periodontal lesions in human oral cavities were examined. Our results indicated that 12 fusiform Bacteroides strains belong to a new species in the genus Bacteroides. The name Bacteroides forsythus is proposed for these isolates. The type strain is strain ATCC 43037.Anaerobic, gram-negative, fusiform organisms which did not resemble previously recognized speqies were isolated from deep periodontal pockets of humans that showed progressive tissue loss (3,15,17). The cells appeared to be long filaments or medium rods with tapered (fusiform) or rounded ends. Some cells had central swellings. Large spheroids were frequently associated with the cells, particularly the cells with filamentous morphology. These strictly anaerobic fusiform Bacteroides strains grew slowly on blood agar plates and grew poorly in all broth media tested and on most agar media tested. Biochemical tests gave inconsistent results and were usually negative. For this reason, the fusiform Bacteroides strains were characterized without using most of the conventional biochemical tests, Deoxyribonucleic acid (DNA) guanine-plus-cytosine (G +C) content, DNA-DNA homology, cell wall ultrastructure, serology, API enzyme substrate tests (Analytab Products, Plainview, N.Y.), and protein profiles of whole sonicated cells obtained by using polyacrylamide gel electrophoresis (PAGE) indicated that these organisms comprise a distinct species in the genus Bacteroides. The name Bacteroides forsythus is proposed for this new species.Strains and inocula. The strains which we used are listed in Table 1. These organisms were maintained on Trypticase soy agar plates supplemented with 5% sheep blood (TSBA; BBL Microbiology Systems, Cockeysville, Md.) at 35 to 37°C in an atmosphere containing 10% H2, 10% CO2, and 80% N2. The fusiform Bacteroides strains were maintained on TSBA plates; a fusiform Bacteroides strain was streaked on one-half of each plate, and a strain of Fusobacterium nucleaturn was streaked on the other half of the plate. The strains were stored in liquid nitrogen by using previously described methods (14). Inocula for tests were obtained by scraping cells from the surfaces of TSBA plates. At least 100 plates of fusiform Bacteroides strains were used to obtain sufficient cells for DNA extraction. Two to four plates were used to provide the inoculum for an enzyme-linked immunosorbent assay and for each of the tests in the API enzyme substrate test series.DNA-DNA hybridization. Cells of reference strains were grown in 2-to 3-liter broth cultures (mycoplasma broth [BBL] supplemented with 5 mg of hemin per liter) for extraction of DNA. The DNA was extracted by using a method modified (13) from the procedure of Marmur (9). G+C contents were determined by the thermal denaturation method (1). Levels of DNA-DNA homology were determined by using the initial renaturation rates (2).API enzyme substrate tests. API ZYM and API An-Ident enzymatic substrate tests were ...

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 (≥103 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.

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.

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.

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.