A.J. van Winkelhoff

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.

Prevalence and Distribution of Six Capsular Serotypes of Porphyromonas gingivalis in Periodontitis Patients

Previous reports have described six serotypes based on K antigens in Porphyromonas gingivalis strains. The purpose of the present study was to investigate the prevalence and distribution of these serotypes in 185 patients with P. gingivalis-associated periodontitis. Polyclonal rabbit antisera, raised against each of the different type strains, were used in double-immunodiffusion and immunoelectrophoresis assays. In addition, a subset of 76 strains was investigated for the presence of capsular structures by means of the India ink and Bruce White staining techniques. These strains were also tested for auto-aggregation in phosphate-buffered saline (PBS). All six K serotypes were present in the study sample. In total, 84 (45.4%) patients were colonized with a K-typeable P. gingivalis strain with a predominance of types K5 (12%) and K6 (23.2%). A correlation was found between arbitrary age categories and the prevalence of currently known K serotypes, which were found in 60% of patients aged 12 to 30 years, in 49% of patients aged 31 to 50, and in 25% of patients aged 51 to 70 years. In the subset of 76 P. gingivalis strains, 32 (42.1%) were K-typeable. Fifty-three strains (69.7%) showed microscopic evidence of encapsulation, suggesting the existence of K serotypes other than K1 to K6. Twenty-one strains (27.6%) auto-aggregated in PBS and were not K-typeable, nor did they show any evidence of encapsulation. It was concluded that the majority of clinical P. gingivalis isolates is encapsulated and that encapsulation is associated with the presence of a K antigen. Auto-aggregation seems to be associated with the absence of a capsular structure and, consequently, the absence of a K antigen.

A Statistical Approach to the Ecology of Porphyromonas gingivalis

Previous studies have suggested that infections with Porphyromonas gingivalis, associated with periodontal disease, may consist of one clonal type. It has also been shown that each individual patient carries a unique clonal type of P. gingivalis, as assessed by DNA fingerprinting. This issue was further examined by random collection of multiple isolates ofP. gingivalis from multiple sites in several patients, and characterization of these isolates by DNA fingerprinting. Although most patients appeared to be infected exclusively by one clonal type of P. gingivalis, at least one patient was found to harbor two distinct clonal types. This indicates that the simultaneous presence of different clonal types of P. gingivalis can occur. A statistical method was developed for retrospective analysis of these data for estimation of whether the remainder of these patients were actually infected with single or multiple clonal types ofP. gingivalis. With this statistical method, a confidence interval was calculated for estimation of the true proportion of a single observed clonal type in the potential population of P. gingivalis that might be recovered from an infected patient. Statistically, the sampling of small numbers of sites per patient or isolates per site leads to a wide confidence interval for the estimated true proportion of the observed clonal type in the infecting P. gingivalis population. For example, when five sites in an oral cavity yield indistinguishable P. gingivalis isolates, then the true proportion of this clonal type in the total P. gingivalis population in the infected oral cavity is estimated to be in the interval between 55% and 100% (at a 95% confidence level). If the isolates from all sampled sites from a periodontal patient appear to be of the same clonal type, it can be calculated that at least 29 different sites must be sampled for this observed clonal type to represent at least 90% of the P. gingivalis population in this patient (95% confidence level); this was termed the LTP, or lowest true proportion. Such calculations can also be used for estimation of the LTP of a clonal type infecting a single oral site in a periodontal patient. These results confirm the commonly held opinion that studies investigating the ecology of P. gingivalis and other suspected pathogens must be designed on a large scale in order for statistically meaningful results to be obtained.

In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis.

Synergistic effects between amoxicillin, metronidazole, and the hydroxymetabolite of metronidazole against Actinobacillus actinomycetemcomitans.

Interactions between metronidazole and amoxicillin, metronidazole and its hydroxymetabolite, and amoxicillin and the hydroxymetabolite of metronidazole were investigated with checkerboard titrations in combination with accurately determined MICs and MBCs. Actinobacillus actinomycetemcomitans was used as the test organism. Synergism was found for all three combinations. Fractional inhibitory concentration indices and fractional bactericidal concentration indices varied from 0.3 to 0.7. These synergistic interactions between these antibiotics may explain the efficacy of the combination of metronidazole and amoxicillin in various bacterial infections, including periodontal disease.

Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species

The purpose of this study was to investigate the production of various enzymes by oral and non-oral black-pigmented Bacteroides species using chromogenic substrates. The 19 substrates present in the API ZYM system did not differentiate between B. melaninogenicus, B. denticola, B. loescheii and B. levii. The asaccharolytic black-pigmented Bacteroides species showed each species specific enzyme activity, however, differences were based on one enzyme only as far as B. asaccharolyticus and B. endodontalis are concerned. An extended number of 40 chromogenic substrates were tested in order to find more species specific enzyme. With a set of 20 substrates it appeared to be possible to discriminate between all species tested. The possibility to use enzymes for the identification of black-pigmented Bacteroides is discussed.

In vitro susceptibility of Helicobacter pylori to several antimicrobial combinations.

Synergy between metronidazole and its hydroxymetabolite and between each compound and amoxicillin or tetracycline-HCl was determined against Helicobacter pylori. Metronidazole plus its hydroxymetabolite and either compound combined with amoxicillin showed synergism against all 10 strains of H. pylori tested. Metronidazole plus tetracycline-HCl or the hydroxymetabolite plus tetracycline-HCl acted synergistically against seven and six strains, respectively, acted additively against three strains, and had no additional effect against one strain. These results may help to explain the in vivo efficacies of metronidazole combinations in the treatment of H. pylori-associated gastritis.

Pathogenic synergy: mixed infections in the oral cavity

In almost all infections in the oral cavity, mixed populations of bacteria are present. However, recent evidence points to a certain specificity in these infections:Streptococcus mutans is related to caries and black-pigmentedBacteroides species are suspected pathogens in periodontal disease. Periodontal diseases, endodontic infections and submucous abscesses in the oral cavity are probably mixed infections in which anaerobic bacteria together with facultatives or other anaerobes are present. In experimental mixed anaerobic infections black-pigmentedBacteroides strains have been shown to play a key role. Little is known about the pathogenic synergy between the bacteria involved in mixed infections. Important mechanisms could be nutritional interrelationships and interactions with the host defense. Within the group of black-pigmentedBacteroides B. gingivalis seems to be the most virulent species. These bacteria possess a great number of virulence factors, which might be important in the pathogenesis of oral infections.

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.

Cross-inhibition Between Black-pigmented Bacteroides Species

Cross-inhibition within the group of black-pigmented Bacteroides, including both oral and non-oral strains, was studied by means of a membrane filter technique. It was found that B. gingivalis possessed the most extended inhibitory capacity among all species tested. B. gingivalis showed inhibitory activity against B. intermedius, B. endodontalis, B. loescheii, and B. melaninogenicus. B. endodontalis was active against some B. intermedius strains. Among the saccharolytic species, some B . melaninogenicus strains were inhibitory for some B. endodontalis strains, some B. gingivalis strains, and some B. intermedius strains. These inhibitory activities observed in vitro may play a role in the colonization of the periodontal pocket.