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

Black-pigmented Bacteroides 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-pigmented Bacteroides species have identified Bacteroides gingivalis as the most virulent species. B. gingivalis and Bacteroides intermedius have been associated with periodontal diseases; Bacteroides endodontalis is isolated specifically from infections in the oral cavity, and other black-pigmented Bacteroides species are recovered from oral mucous sites. DNA restriction endonuclease analysis was adapted for typing of B. gingivalis and B. intermedius.

Bacteroides↗

Surface antigens as virulence factors in infection with Bacteroides fragilis.

Organisms of the genus Bacteroides represent the major group of obligate anaerobes involved in human infections. Bacteroides usually cause either bacteremia or localized abscesses. Of the numerous species of Bacteroides, Bacteroides fragilis is the single most frequent clinical isolate. B. fragilis and Bacteroides melaninogenicus have chemically incomplete lipopolysaccharides as compared with the lipopolysaccharides (endotoxins) of aerobic bacteria, and the lipopolysaccharides of Bacteroides lack the biologic potency characteristic of endotoxin. This inactivity may account for the very infrequent occurrence of disseminated intravascular coagulation or purpura that can accompany sepsis due to these organisms. Furthermore, strains of B. fragilis have an immunologically common capsular polysaccharide. In an animal model of intraabdominal sepsis, the encapsulated strains caused abscesses when given without other organisms, but abscess formation from unencapsulated strains of Bacteroides generally required the administration of a synergistic aerobe. The abscesses caused by encapsulated strains were shown to be directly attributable to the capsular polysaccharide, which is an important virulence factor of this organism. Patients or experimental animals infected with B. fragilis develop antibodies to the capsular polysaccharide, and these antibodies can be detected in a radioactive antigen-binding assay.

Animals↗

Bacteroides dorei sp. nov., isolated from human faeces.

Two Gram-negative, anaerobic, non-spore-forming rod-shaped organisms were isolated from human faeces. These isolates were tentatively identified as Bacteroides based on morphological and biochemical criteria and appeared closely related to Bacteroides vulgatus ATCC 8482(T). The 16S rRNA gene sequence analysis showed that the isolates were highly related to each other (99.5 %) and confirmed their placement in the genus Bacteroides. 16S rRNA gene sequence similarity values with close phylogenetic neighbours Bacteroides vulgatus ATCC 8482(T) (96 %) and Bacteroides massiliensis CCUG 48901(T) (93 %) preliminarily demonstrated that the organisms represented a novel species. The results of phenotypic, chemotaxonomic and 16S rRNA gene sequence analyses, and DNA-DNA homology values provided evidence that these two unknown isolates represent a single species and should be assigned to a novel species of the genus Bacteroides, as Bacteroides dorei sp. nov. The type strain is JCM 13471(T) (=DSM 17855(T)).

Anaerobiosis↗

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.

Abdomen↗

The role of black-pigmented Bacteroides in human oral infections.

Today, 10 black-pigmented Bacteroides (BPB) species are recognized. The majority of these species can be isolated from the oral cavity. BPB species are involved in anaerobic infections of oral and non-oral sites. In the oral cavity, BPB species are associated with gingivitis, periodontitis, endodontal infections and odontogenic abscesses. Cultural studies suggest a specific role of the various BPB species in the different types of infection. Bacteroides gingivalis is closely correlated with destructive periodontitis in adults as well as in juveniles. Bacteroides intermedius seems to be less specific since it is found in gingivitis, periodontitis, endodontal infections and odontogenic abscesses. The recently described Bacteroides endodontalis is closely associated with endodontal infections and odontogenic abscesses of endodontal origin. There are indications that these periodontopathic BPB species are only present in the oral cavity of subjects suffering from periodontal breakdown, being absent on the mucosal surfaces of subjects without periodontal breakdown. BPB species associated with healthy oral conditions are Bacteroides melaninogenicus, Bacteroides denticola and Bacteroides loescheii. There are indications that these BPB species are part of the normal indigenous oral microflora. Many studies in the past have documented the pathogenic potential and virulence of BPB species. This virulence can be explained by the large numbers of virulence factors demonstrated in this group of micro-organisms. Among others, the proteolytic activity seems to be one of the most important features. Several artificial substrates as well as numerous biological proteins are degraded. These include anti-inflammatory proteins such as alpha-2-macroglobulin, alpha-1-antitrypsin, C3 and C5 complement factors and immunoglobulins. B. gingivalis is by far the most proteolytic species, followed by B. endodontalis. Like other bacteria, the lipopolysaccharide of B. gingivalis has shown to be active in bone resorption in vitro and is capable in stimulating interleukin-1 production in human peripheral monocytes. Based on the well documented association with periodontal disease and the possession of relevant virulence factors, BPB species must be considered as important micro-organisms in the etiology of oral infections. B. gingivalis seems to be the most pathogenic and virulent species.

Bacteroides↗

Black-pigmented Bacteroides spp. in human apical periodontitis.

The incidence of black-pigmented (BP) Bacteroides spp. in 62 human dental root canal infections (35 acute and 27 clinically asymptomatic cases of apical periodontitis) in 57 adults was studied. Altogether 37 strains of BP Bacteroides were found in 31 infections, always in mixed anaerobic infections. Two different BP Bacteroides species were present in six infections. B. intermedius was most frequently isolated (15 of 62 canals; 24%) followed by B. denticola which was present in 12 cases. Asaccharolytic BP Bacteroides species, B. gingivalis and B. endodontalis, were found in eight cases. BP Bacteroides species were found both from symptomatic and asymptomatic infections, but there were also several symptomatic cases from which BP Bacteroides species were not isolated. B. gingivalis and B. endodontalis were present only in acute infections, B. intermedius was found both in symptomatic and asymptomatic infections, and B. denticola occurred mostly in asymptomatic infections. BP Bacteroides species were isolated initially from 9 of the 11 teeth with symptoms at 1 week, but only from 22 of the 51 teeth that were symptomless at 1 week. Two strains of B. denticola were resistant to penicillin G at a concentration of 2.4 micrograms/ml, but the MIC of penicillin G for all other strains was 0.6 micrograms/ml or lower. Forty-two randomly selected patients received penicillin V (oral administration, 650 mg, three times daily) during the first week of endodontic therapy. Penicillin had no effect on the occurrence of symptoms after 1 week compared with the control group (20 patients).

Adult↗

Bacteroides buccae and related taxa in necrotic root canal infections.

Fifty-seven adults with apical periodontitis were examined for the presence of nonpigmented Bacteroides species in 62 infected root canals. Nonpigmented Bacteroides species were found in 35 canals. In four cases two nonpigmented Bacteroides species and in one case three nonpigmented Bacteroides species were found. Species belonging to the B. fragilis group were not isolated. The most frequently isolated species were B. buccae (15 strains), B. oris (12 strains), and B. oralis (7 strains). alpha-Fucosidase, beta-N-acetylglucosaminidase, and beta-xylosidase appeared to be useful in the identification of B. buccae and B. oris. Corroding Bacteroides species were not found; all corroding strains were identified as Wolinella recta. The occurrence of nonpigmented Bacteroides species was compared with the severity of the periapical infection. A total of 13 B. buccae strains were found in acute infections and only 2 strains were found in asymptomatic infections, whereas other nonpigmented Bacteroides species were present in acutely infected and asymptomatic teeth with nearly equal frequency. Ultrastructural study of 13 B. buccae strains showed that 8 strains had a crystalline proteinaceous surface layer (S-layer) outside the outer membrane, but all 13 strains had areas of crystalline protein throughout in the outer membrane. The results suggest that B. buccae may have a specific role in the development of an acute opportunistic infection.

Adult↗

Factors affecting production of catalase by Bacteroides.

Several variables affected the production of catalase by members of the "Bacteroides fragilis group" of anaerobic bacteria. Both media yielded higher catalase levels than the respective agar media. Addition of hemin to media after autoclave sterilization, rather than before, significantly increased production of catalase. Both of these variables could be related to the available hemin concentration present in the medium being tested. Significantly higher amounts of hemin were required for catalase production than were required for growth. For catalase production by B. fragilis ATCC 25285, 1 microgram of hemin per ml was required. Of the various media tested, the use of chopped meat broth resulted in the highest levels of catalase production (up to 50 to 60 U of catalase per mg of protein). Of the various species and DNA homology groups tested, strains of B. fragilis and Bacteroides distasonis were catalase positive. Strains of Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides eggerthi possessed variable catalase activity. Bacteroides vulgatus, Bacteroides uniformis, and DNA homology groups "3452A" and "subsp. a" were catalase negative. A catalase well test, in which equal volumes of 3% H2O2 and chopped meat culture are mixed, is described and recommended for routine catalase tests.

Anaerobiosis↗

Recovery of Bacteroides fragilis group from clinical specimens following antimicrobial therapy.

Over a period of 14 years (1973-1987), 3165 specimens submitted to the microbiology laboratory demonstrated the recovery of anaerobic bacteria. A total of 988 Bacteroides fragilis group isolates were recovered (0.3 isolates per specimen). Bacteroides fragilis accounted for 62% of the total of all B. fragilis group isolates, Bacteroides thetaiotaomicron for 15%, Bacteroides vulgatus for 8%, Bacteroides ovatus for 7%, Bacteroides distasonis for 6%, and Bacteroides uniformis for 2%. Of the 988 B. fragilis group isolates, 310 (31%) were recovered after the administration of antimicrobial therapy, and 129 (13%) were the single isolate recovered from the infected site at that time. The recovery rate of all members of B. fragilis group after the administration of antimicrobial therapy, when isolated alone or when mixed with other bacteria, was similar. The data illustrate the equal ability of all members of the B. fragilis group to persist in and to contribute to the inflammatory process; and provide further support for their pathogenic role.

Bacteroides Infections↗

Metronidazole and spiramycin therapy of mixed Bacteroides spp. and Neisseria gonorrhoeae infection in mice.

The in vitro and in vivo activity of metronidazole and spiramycin, used singly or in combination, was tested in the eradication of infection caused by Bacteroides spp. and Neisseria gonorrhoeae alone or in combination. The in vitro tests consisted of determinations of the minimal inhibitory concentrations (MIC), carried out with or without the addition of a constant amount of the other antimicrobials. The MIC of both Bacteroides bivius and Bacteroides fragilis for metronidazole were significantly reduced by the addition of spiramycin (from 0.5 to 0.125 micrograms/ml). The in vivo tests were carried out in mice and consisted of measurements of the effects of the antimicrobial agents on the bacterial contents of abscesses induced by subcutaneous injection of bacterial suspension. Synergism between metronidazole and spiramycin was noted against Bacteroides spp. in abscesses caused by either Bacteroides spp. alone, or in combination with N. gonorrhoeae. Furthermore, an additional reduction in the number of N gonorrhoeae was noted in mixed infection with Bacteroides that was treated with metronidazole alone. This study demonstrates the in vitro and in vivo efficacy of the combination of metronidazole and spiramycin in the treatment of infections caused by either Bacteroides spp. alone or in combination with N. gonorrhoeae.

Abscess↗

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water.

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.

Apoptosis↗

Antimicrobial susceptibilities of Eubacterium, Peptostreptococcus, and Bacteroides isolated from root canals of teeth with periapical pathosis.

Eubacterium, Peptostreptococcus, and Bacteroides were isolated in high frequency from root canals with acute periapical inflammation. The antimicrobial susceptibilities of these strains were studied by determining minimum inhibitory concentrations of different agents. Although all three kinds of isolates were susceptible to penicillins, the isolates other than black-pigmented Bacteroides were less susceptible to cephems, tetracyclines, and macrorides with several resistant strains. All strains were uniformly resistant to aminoglycosides. Some differences in susceptibilities were observed among species of Eubacterium and Peptostreptococcus, while penicillins were effective for both species. Black-pigmented Bacteroides showed good susceptibilities to all agents except for aminoglycosides. The susceptibility of Bacteroides gingivalis was superior to that of Bacteroides intermedius. There were many resistant strains in non-black-pigmented but not in black-pigmented Bacteroides isolates. Penicillins were the most effective for Eubacterium, Peptostreptococcus, and Bacteroides, indicating that penicillins are suitable for treatment of root canals with acute apical periodontitis.

Aminoglycosides↗

Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes.

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.

Adult↗

5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general.

The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

Algorithms↗

Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov.

The characteristics of the fusiform species Bacteroides forsythus, isolated from human periodontal pockets, were examined. 165 rDNA sequence analysis confirmed that B. forsythus was not a species within the genus Bacteroides sensu stricto. Although B. forsythus was phylogenetically related to Bacteroides distasonis and Bacteroides merdae in the phylogenetic tree, the ratio of anteiso-15:0 to iso-15:0 in whole-cell methanolysates of B. forsythus was different from those of B. distasonis, B. merdae and other Bacteroides species. B. forsythus did not grow on medium containing 20% bile, but members of the Bacteroides fragilis group did. B. forsythus was the only species tested that was trypsin-positive in API ZYM tests. The dehydrogenase enzyme pattern was of no use for the differentiation of B. forsythus and the B. fragilis group. On the basis of these data, a new genus, Tannerella, is proposed for Bacteroides forsythus, with one species, Tannerella forsythensis corrig., gen. nov., comb. nov. The type strain of Tannerella forsythensis is JCM 10827T (= ATCC 43037T).

Animals↗

Bacteroides intestinalis sp. nov., isolated from human faeces.

During studies of the microbiota of human faeces, five strains of Gram-negative anaerobic rods were isolated following growth in a polyamine-deficient medium. These strains belonged to the genus Bacteroides on the basis of 16S rRNA gene sequence data. 16S rRNA gene sequence similarity between one of the strains, 341T, and recognized species within the genus Bacteroides was <95 %. The DNA G+C content (44 mol%) and major fatty acid composition (anteiso-C(15 : 0), 32.0 %) supported the affiliation of strain 341(T) to the genus Bacteroides. Biochemical tests and DNA-DNA hybridization analysis demonstrated that strain 341T was distinct from Bacteroides uniformis and Bacteroides helcogenes, to which it was related most closely. On the basis of these data, a novel Bacteroides species, Bacteroides intestinalis sp. nov., is proposed with strain 341T (= JCM 13265T = DSM 17393T) as the type strain.

Bacteroides↗

Bacteriocin production by strains of Bacteroides isolated from human feces and the role of these strains in the bacterial ecology of the colon.

Several human fecal isolates of Bacteroides have been found to produce bacteriocins. The bacteriocin-producing strain T1-1 was studied in the most detail. Strain T1-1 belongs to the 0061-1 deoxyribonucleic acid (DNA) homology group of Bacteroides. This homology group phenotypically resembles Bacteroides thetaiotaomicron but has little DNA homology with it. The bacteriocin-producing strains T1-12 and T1-48 belong to the 3452-A DNA homology group. This group has DNA homology with B. thetaiotaomicron and Bacteroides ovatus. The bacteriocin-producing strain T1-42 remains unidentified in that it does not belong to any recognized DNA homology group of the saccharolytic intestinal bacteroides. The extracellular bacteriocin produced by strain T1-1 was specifically bactericidal for other bacteria within the genus Bacteroides. The highest bacteriocin titers (32 to 64) were produced in complex media, with only trace amounts being produced in a defined medium. The bacteriocin appeared to have a high molecular weight (>/=300,000) and was unusual because it was stable from pH 1 to 12 and only a 50% reduction in activity resulted after 15 min at 121 degrees C in an autoclave. It was inactivated by trypsin and Pronase. Strain T1-1 was isolated from all three fecal samples obtained over a 25-week period from an individual who was part of a National Aeronautics and Space Administration mock Skylab flight. Strains T1-12, T1-48, and T1-42 were isolated only from the first fecal sample. Each of these strains was immune to the bacteriocins produced by the others. These strains were found to coexist in the colon with a larger population of non-bacteriocin-producing, bacteriocin-susceptible strains of Bacteroides.

Bacteriocins↗

gyrA mutations associated with quinolone resistance in Bacteroides fragilis group strains.

Mutations in the gyrA gene contribute considerably to quinolone resistance in Escherichia coli. Mechanisms for quinolone resistance in anaerobic bacteria are less well studied. The Bacteroides fragilis group are the anaerobic organisms most frequently isolated from patients with bacteremia and intraabdominal infections. Forty-four clinafloxacin-resistant and-susceptible fecal and clinical isolates of the B. fragilis group (eight Bacteroides fragilis, three Bacteroides ovatus, five Bacteroides thetaiotaomicron, six Bacteroides uniformis, and 22 Bacteroides vulgatus) and six ATCC strains of the B. fragilis group were analyzed as follows: (i) determination of susceptibility to ciprofloxacin, levofloxacin, moxifloxacin, and clinafloxacin by the agar dilution method and (ii) sequencing of the gyrA quinolone resistance-determining region (QRDR) located between amino acid residues equivalent to Ala-67 through Gln-106 in E. coli. Amino acid substitutions were found at hotspots at positions 82 (n = 15) and 86 (n = 8). Strains with Ser82Leu substitutions (n = 13) were highly resistant to all quinolones tested. Mutations in other positions of gyrA were also frequently found in quinolone-resistant and -susceptible isolates. Eight clinical strains that lacked mutations in their QRDR were susceptible to at least two of the quinolones tested. Although newer quinolones have good antimicrobial activity against the B. fragilis group, quinolone resistance in B. fragilis strains can be readily selected in vivo. Mutational events in the QRDR of gyrA seem to contribute to quinolone resistance in Bacteroides species.

Amino Acid Substitution↗