T.J.M. van Steenbergen

Bacteroides endodontalis sp. nov. an Asaccharolytic Black- Pigmented Bacteroides Species from Infected Dental Root Canals

A new species, Bacteroides endodontalis, is proposed for black-pigmented asaccharolytic Bacteroides strains that have negligible deoxyribonucleic acid homology with either Bacteroides gingivalis or Bacteroides asaccharolyticus. Strain HG370 (= ATCC 35406) is the type strain. Unlike B. gingivalis, B. endodontalis does not agglutinate sheep erythrocytes or produce phenylacetic acid. B. endodontalis resembles B. asaccharolyticus physiologically but can be distinguished by its lower deoxyribonucleic acid guanine-plus-cytosine content (50 versus 53 to 54 mol%), by its serological reactions, by its electrophoretic patterns of proteins, and possibly by its menadione requirement.

Bacterial Colonization of Mineralized and Completely Demineralized Dentine in situ

The changing environment in a developing root lesion may result in a succession of the microbial flora in the dentine. As demineralization proceeds, the collagenous matrix is exposed, which could be c

An experimentally induced phlegmonous abscess by a strain of Bacteroides gingivalis in guinea pigs and mice.

The virulence of B. gingivalis strain W83 was studied in an experimental animal model. Cells grown overnight, washed and resuspended in broth, were injected intradermally or subcutaneously in the back of guinea pigs, rats and mice. This strain proved to be very virulent, causing a severe plegmonous abscess in guinea pigs. Also in mice, which are thought to be resistant to infections with black-pigmented Bacteroides strains, the same type of infection could be induced. Rats proved to be rather insensitive. The model presented can be used as a simple virulence test for these anaerobic bacteria.

Cytotoxic activity of Bacteroides gingivalis and Bacteroides asaccharolyticus.

Culture filtrates of all eight strains of Bacteroides gingivalis and all five strains of B. asaccharolyticus were toxic for Vero cells. Cytotoxicity was in general greater with material from cultures of B. gingivalis than from B. asaccharolyticus but none of the culture filtrates from eight strains of B. melaninogenicus showed activity in this test. The toxic material was released during prolonged incubation and more detailed study of preparations from one strain indicated that it had a molecular weight of less than 3500 and was heat stable.

Comparison of three molecular typing methods in studies of transmission of Porphyromonas gingivalis.

Porphyromonas gingivalis is associated strongly with severe periodontitis, but little information is available on possible transmission routes of this species. This study evaluated three DNA-based molecular typing methods for use in epidemiological surveys of P. gingivalis. In total, 32 isolates from eight married couples were investigated by: (i) restriction endonuclease analysis (REA) of whole chromosomal DNA; (ii) hybridisation of DNA fragments with ribosomal DNA (ribotyping); and (iii) amplification of DNA by the polymerase chain reaction with arbitrary primers (AP-PCR). The data obtained with the three methods were in broad agreement: in six of the eight couples, the isolates from husband and wife were indistinguishable, but isolates from unrelated individuals showed distinct types with all three methods. For some isolates, minor differences in REA pattern were obtained which could not be correlated with differences in ribotype or AP-PCR type. Ribotyping showed differences between isolates from one individual, which were indistinguishable with the other two methods. The patterns obtained with ribotyping or AP-PCR were simple in comparison to the relatively complex REA patterns. Although all three methods were concordant, AP-PCR was found to be the least time-consuming method. The data support the suggestion that P. gingivalis can be transmitted between spouses.

Confirmation of Bacteroides gingivalis as a Species Distinct from Bacteroides asaccharolyticus

We divided the asaccharolytic, black-pigmented Bacteroides strains into two groups on the basis of deoxyribonucleic acid (DNA) base ratios, DNA hybridization (S1 nuclease method) results, and direct hemagglutination. One homologous group of strains, which included the type strain of Bacteroides asaccharolyticus and had guanine-plus-cytosine contents of 52 to 54 mol%, contained only nonoral isolates. Another DNA homology group contained all of the strains from periodontal pockets and some nonoral isolates. These strains appeared to belong to the recently described new species Bacteroides gingivalis Coykendall et al. B. gingivalis strains had guanine-plus-cytosine contents of 48 to 50 mol%, showed high DNA homology values, and shared hemagglutinating activity. No DNA homology was observed between the two groups. Using a different method of analysis for DNA homology (S1 nuclease method), we confirmed the conclusion of Coykendall et al., who separated these two groups into different species, B. asaccharolyticus and B. gingivalis. Two strains from infected root canals could not be placed in either of these two species. On the basis of the DNA homology results, all asaccharolytic strains were distinguished clearly from the saccharolytic, black-pigmented Bacteroides strains, which at present are classified in the species Bacteroides melaninogenicus.

Bacterial colonization and degradation of demineralized dentin matrix in situ.

Lesion formation in dentin involves demineralization followed by degradation of the exposed organic matrix. Proteinases from microorganisms present in the dentin are believed to play an important role in the breakdown of the dentinal collagen. In this study, the microflora colonizing decalcified dentin matrix, placed in situ, was identified. The gelatinolytic activity of the isolated strains was assessed and related to the degradation of the dentin matrix. The predominant species found were Streptococcus mitis, Peptostreptococcus productus, Lactobacillus casei, Propionibacterium species and Veillonella parvula. Marked interindividual variation in the composition of the microflora was observed. The microflora possessed gelatinolytic activity, although no correlation was found with the severity of dentin matrix degradation. The chemically determined loss of collagen varied between 0 and 67 wt% per participant and corresponded with the extent of collagen degradation observed by transmission electron microscopy.

Taxonomy, virulence and epidemiology of black-pigmented bacteroides species in relation to oral infections

SummaryBlack-pigmentedBacteroides species are recognized as suspected pathogens of oral infections. Developments in the taxonomy of this group include description of a new asaccharolytic species,Bacteroides salivosus, and proposal for the reclassification of the asaccharolytic species into a separate genus,Porphyromonas. Studies on the pathogenicity and virulence of black-pigmentedBacteroides species have identifiedBacteroides gingivalis as the most virulent species.B. gingivalis andBacteroides intermedius have been associated with periodontal diseases;Bacteroides endodontalis is isolated specifically from infections in the oral cavity, and other black-pigmentedBacteroides species are recovered from oral mucous sites. DNA restriction endonuclease analysis was adapted for typing ofB. gingivalis andB. intermedius.ZusammenfassungSchwarz-pigmentierteBacteroides-Spezies stehen im Verdacht, orale Infektionen zu verursachen. Die Weiterentwicklung der Taxonomie dieser Gruppe schließt die Beschreibung der neuen asaccharolytischen Spezies,Bacteroides salivosus, ein sowie den Antrag auf Umklassifizierung der asaccharolytischen Spezies in ein separates Genus,Porphyromonas. Studien der Pathogenität und Virulenz der schwarz-pigmentiertenBacteroides-Spezies wiesenBacteroides gingivalis als die virulenteste Spezies aus.B. gingivalis undBacteroides intermedius sind in Zusammenhang mit periodontalen Erkrankungen gebracht worden.Bacteroides endodontalis konnte bei oralen Infektionen und andere schwarz-pigmentierteBacteroides-Spezies aus oraler Mukosa isoliert werden. Die DNA-Restriktions-Endonuklease-Analyse ist für die Klassifizierung derB. gingivalis undB. intermedius angepaßt worden.

Description of two morphotypes of Peptostreptococcus micros.

Peptostreptococcus micros is often isolated from abscesses in several parts of the human body. The oral cavity is considered the natural habitat for the species, which has been implicated as a periodontal pathogen. In plaque samples from periodontitis patients we observed the presence of a rough morphotype of P. micros in addition to the previously recognized smooth morphotype. The rough morphotype has not been described previously. Both morphotypes are frequently isolated simultaneously from the same patient. In this paper strains of both morphotypes are described. The smooth morphotype, represented by the type strain, grew as small, dome-shaped, bright white, nonhemolytic colonies. The rough morphotype grew as equally white dry colonies which were hemolytic and had wrinkled edges. DNA-DNA reassociation studies revealed homology at the species level between the two morphotypes; in addition, no differences in physiological characteristics were observed when the organisms were tested with API-32A and API-ZYM kits. The rough cells had long, thin fibrillar structures outside the cell envelope when they were stained negatively for electron microscopy. In the smooth morphotype these structures were not present. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole-cell extracts were different for the two morphotypes. In xylene-water phase partition studies, the smooth morphotype was found to be hydrophobic, whereas the rough morphotype was found to be relatively hydrophilic. The distinct morphotypes were stable on blood agar; however, the rough morphotype changed to a nonfibrillar type with a smooth colony morphology after repeated subculturing in broth.

Pathogenicity of Peptostreptococcus micros morphotypes and Prevotella species in pure and mixed culture

Recently, an atypical rough colony morphotype of Peptostreptococcus micros, a species which is found in ulcerating infections, including periodontitis, was isolated. The virulence of morphotypes alone and in combination with Prevotella intermedia and P. nigrescens was investigated both in vivo and in vitro. All strains tested induced abscesses containing fluid pus in a mouse skin model, and lesions caused by monocultures of the rough morphotype strains of P. micros were statistically significantly larger than those induced by the smooth morphotype strains. Inocula containing both morphotypes produced similar sized abscesses compared to mono-inocula containing the same bacterial load. Both Prevotella species induced small abscesses when inoculated alone, and when Pr. nigrescens was inoculated with one of the other strains, the abscesses were not significantly different from the abscesses induced by the mono-infections of this strain. Synergy, in terms of higher numbers of colony forming units (cfu) in the mixed inocula, was found for all combinations of the rough morphotypes of P. micros and both Prevotella spp. Pus from abscesses caused by combinations of Peptostreptococcus and Prevotella spp. transmitted the infection to other mice, but no abscesses were formed in mice inoculated with pus induced by mono-inocula. These results demonstrated synergic activity between both rough and smooth P. micros strains and oral Prevotella strains. The in-vitro co-culture experiments produced no evidence of growth stimulation. The effect of P. micros strains on the immune system was investigated by testing their ability to initiate luminol-dependent chemiluminescence of polymorphonuclear leucocytes in the presence and absence of human serum. In the latter, the rough morphotype strains initiated higher counts than the smooth morphotype strains. Further work is needed to elucidate the difference in virulence between the smooth and the rough morphotype cells of P. micros and the nature of the interaction with the Prevotella spp.