JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1986, p. 940-9440095-1137/86/120940-05$02.00/0Copyright C 1986, American Society for Microbiology

Bacteroides buccae and Related Taxa inNecrotic Root Canal Infections

MARKUS HAAPASALO

Department of Cariology and Department ofBacteriology and Immunology, University of Helsinki,SF-00280 Helsinki 28, Finland

Received 16 April 1986/Accepted 26 August 1986

Fifty-seven adults with apical periodontitis were examined for the presence of nonpigmented Bacteroidesspecies in 62 infected root canals. Nonpigmented Bacteroides species were found in 35 canals. In four cases twononpigmented Bacteroides species and in one case three nonpigmented Bacteroides species were found. Speciesbelonging to the B. fragilis group were not isolated. The most frequently isolated species were B. buccae (15strains), B. oris (12 strains), and B. oralis (7 strains). oa-Fucosidase, D-N-acetylglucosaminidase, andP-xylosidase appeared to be useful in the identification of B. buccae and B. oris. Corroding Bacteroides specieswere not found; all corroding strains were identified as Wolinella recta. The occurrence of nonpigmentedBacteroides species was compared with the severity of the periapical infection. A total of 13 B. buccae strainswere found in acute infections and only 2 strains were found in asymptomatic infections, whereas othernonpigmented Bacteroides species were present in acutely infected and asymptomatic teeth with nearly equalfrequency. tJltrastructural study of 13 B. buccae strains showed that 8 strains had a crystalline proteinaceoussurface layer (S-layer) outside the outer membrane, but all 13 strains had areas of crystalline proteinthroughout in the outer membrane. The results suggest that B. buccae may have a specific role in thedevelopment of an acute opportunistic infection.

The genus Bacteroides is a large group of gram-negativeanaerobic rods which belong principally to the normal floraof humans (12). Among the best-known species are the B.fragilis group and black-pigmented Bacteroides species,which are the most commonly reported anaerobes in humanclinical infections (6). Bile-sensitive, nonpigmented Bacte-roides species are often found with black-pigmented Bacte-roides species (6).

Several new species of saccharolytic nonpigmenting Bac-teroides isolated from the human oral cavity have beendescribed during the last few years. Those that ferment thepentoses xylose and arabinose are B. buccae (13, 15, 16, 24),B. oris (13), and B. heparinolyticus (21). B. zoogleoformans,which also may ferment xylose and arabinose, occasionallyis isolated from the oral cavity (2). Newly identified sac-

charolytic species from the oral cavity that do not fermentthe pentoses are B. buccalis, B. veroralis, and B. oulorum(24, 25, 30). The occurrence, site specificity, and role ofmany of the newly identified species in human infections isnot yet established.A crystalline proteinaceous surface layer, the S-layer, is

knowti to occur in several gram-positive and gram-negativebacteria (27). Of the gram-negative anaerobic rods, theS-layer has been reported only in B. nodosus, B. buccae,Wolinella recta, and some spirochetes (7, 16, 17, 27). Inrecent studies of B. heparinolyticus and B. forsythus, struc-tures resembling the S-layer of B. buccae have been de-scribed, but these layers have not yet been studied in detail(21, 28). Ultrastructural studies of cells of B. buccae haverevealed that this species has an S-layer composed appar-ently of two separate lattices (7, 26). In this study theoccurrence of nonpigmented Bacteroides species in acuteand nonacute periapical infections is reported. Special atten-tion has been paid to strains with an S-layer.

MATERIALS AND METHODS

Subjects. Fifty-seven adult patients referred to the Depart-ment of Cariology and Endodontics, University of Helsinki,participated in the study. Sixty-two teeth with only one tootcanal with necrotic root canal infection and radiographicallydiagnosed apical periodontitis were included. Reasons forexclusion were (i) antibiotic therapy within the last 3months, (ii) a periodontal pocket over 4 mm deep, (iii)previous endodontic therapy of the affected tooth, (iv) vitalpulp tissue in the canal, and (v) perforated canals. The meanages of men and women were 42 and 36 years, respectively.Pain, swelling, or both; open sinus tract; and tenderness topercussion were regarded as signs of an acute infection.Sample collection and bacterial diagnosis. The pulp cavum

was perforated with sterile instruments, and samples were

obtained from root canals with sterile paper points andinoculated as described previously (8). Special precautionswere taken to avoid contamination. The following threemedia were used for the initial cultivation: (i) prereducedkanamycin (75 ,g/ml), vancomycin (7.5 ,ug/ml), laked blood(sheep blood) agar; (ii) a prereduced nonselective mediumcontaining horse blood (5%), bacteriological agar no. 1(Oxoid Ltd., Hampshire, England), yeast extract (10 g/liter;Oxoid), menadione (0.5 mg/liter), cystein (500 mg/liter),glucose (2 g/liter), laboratory lemco powder (10 g/liter;Oxoid), peptone (15 g/liter; Oxoid), and sodium chloride (5g/liter) (MCGP); (iii) a medium containing hemolysed horseblood (8%), Trypticase soy agar (BBL Microbiology Sys-tems, Cockeysville, Md.), Mueller-Hinton Agar (BBL), andIsoVitaleX (10 mg/liter; BBL). After inoculation the plateswere immediately placed in an anaerobic jar (BBL) andincubated in an anaerobic atmosphere (gas generating kit;Oxoid) at 37°C. For inocula for biochemical tests and forelectron microscopy studies, strains were grown on MCG

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TABLE 1. General bacteriological findings in 62 necrotic rootcanal infections (apical periodontitis)

Percentage of allstrains isolated

AnaerobesNonpigmented Bacteroides spp................... 16Black-pigmented Bacteroides spp.a ....... ........ 14Fusobacterium spp . .............................. 12Gram-negative motile rods........................ 9Gram-positive cocci and rods..................... 33

Facultative anaerobesca-Hemolytic streptococci ........... .............. 10Eikenella corrodens............................... 1Capnocytophaga sp............................... 1Actinobacillus actinomycetemcomitans ........... <1a See Haapasalo et al. (8).

agar (MCG agar is MCGP agar without peptone, laboratorylemco powder, or sodium chloride). Colony morphology wasstudied also on laked rabbit blood agar.Anaerobic jars were opened after 7 days, and subcultures

were made. Several representative samples of each colonytype were characterized. The identification of nonpigmentedBacteroides species was based on the following tests:aerotolerance, Gram stain, susceptibility to kanamycin (100,ug/ml) and vancomycin (5 ,ug/ml), bile sensitivity (growth onBBE agar [18]), colony morphology, hydrolysis of starch andesculin (22), gelatin liquefaction (API 20A system; API,Montalieu-Vercieu, France), production of indole (22), fer-mentation of carbohydrates (API 20A system), and produc-tion of glycosidase enzymes with chromogenic substratetablets (Rosco, Taastrup, Denmark). B. buccae ATCC33574T, NP333 and WPH78 (formerly designated B.pentosaceus), ATCC 33690 and ATCC 33691 (formerlydesignated B. capillus), and B. oris ATCC 33573T and ATCC27518 were used as reference organisms in glycosidase tests.B. oralis strains and reference strains B. heparinolyticusATCC 35895T and B. buccalis DSM 20616T were also testedwith the RapID-ANA system (Innovative Diagnostic Sys-tems, Inc., Atlanta, Ga.). Corroding strains were also stud-ied for urease production, by using both chromogenic diag-nostic tablets (Rosco) and urease tubes (22), and for nitratereduction (22).

Electron microscopy. The cells were collected with aplatinum loop from the MCG plates (anaerobic incubationfor 3 days) and placed into 0.1 M phosphate buffer (pH 7.2).

Intact cells were negatively stained with 2% phosphotungsticacid (pH 6.5). The samples for thin sectioning were fixed andprepared as described previously (19) and stained withtannic acid (29). For freeze-fracturing, glycerol was added tothe suspension to a final concentration of 30% (vol/vol). Thepellet was collected by centrifugation (12,000 rpm; Ep-pendorf centrifuge 5414) and was frozen in liquid Freon 22(E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.)cooled by liquid nitrogen. The fracturing in a freeze-etchingunit (BAF 400T; Balzers, Liechtenstein) was performed at-120°C, and platinum shadowing was done at an angle of 40degrees. The electron micrographs were taken with JEM-100CX (negative staining) and JEM-1200EX (thin sectionsand replicas) transmission electron microscopes at 80 and 60kV, respectively.

RESULTSThe general bacteriological findings of this study are

summarized in Table 1. The number of anaerobes in onecanal was usually from three to seven. Forty-onenonpigmented Bacteroides species were isolated from 35 of62 root canals in mixed anaerobic infections; none of thenonpigmented Bacteroides strains was found in pure culture.Thirty-seven black-pigmented Bacteroides species werefound in 31 teeth (8). B. fragilis was not found. Fusobacteria,anaerobic gram-negative motile rods, and gram-positive rodsand cocci were also frequently isolated; but these groupswere not analyzed in detail. One or two facultativeanaerobes were present in less than half of the canals.Eikenella corrodens and Capnocytophaga sp. were isolatedin two teeth each, and Actinobacillus actinomycetemcomi-tans isolated from one tooth was the only pure culture in thesamples studied.The two most frequently found nonpigmented Bacteroides

species were B. buccae and B. oris. The frequencies and keycharacteristics of the nonpigmented Bacteroides speciesisolated are given in Table 2. The glycosidase reactions ofreference strains of B. buccae and B. oris were as describedabove for clinical strains. Five nonpigmented Bacteroidesstrains could not be identified to the species level becausethe reactions did not fit to any existing taxa. In addition, intwo cases a new anaerobic gram-negative rod with fimbriaewas found. The new strains had a few characteristics incommon with the genus Bacteroides, but also many differ-ences were found, and it has been suggested that the strainsshould be placed into a new genus (9).

Agar-corroding, obligately anaerobic gram-negative rodswere present in six infected root canals. The strains, identi-

TABLE 2. Frequencies and key characteristics of the nonpigmented Bacteroides species isolated in 62 cases of apical periodontitis

Acid froma: EnzymesSpecies No. of EsculinSpecies strains Arabinose Xylose Salicin Cellobiose hydrolysis P-N-Acetylglucos- ai-Fucosidase 3-Xylosidase 1-Glucosidase

aminidase

B. buccae 15 + + + + + _ _ +b +B. oris 12 + + + + + + +C + +B. oralis 7 - - + + + + + - +B. oulorum 1 - - - - + + + - +B. biviusd 1 - - - - - + + - -Bacteroides spp. 5e _ - - V + + V - + (w)

a All strains produced acid from glucose, lactose, maltose, and mannose. Abbreviations: +, positive reaction; -, negative reaction; V, variable reaction;w, weak reaction.

b The P-xylosidase reaction by B. buccae was more rapid than that by B. oris.c Two strains were negative.d B. bivius was the only strain which repeatedly gave a strong positive reaction for gelatin hydrolysis.e Strains hydrolyzed starch, and fermentation reactions were variable.

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fied as Wolinella recta, were straight and tiny rods andurease negative, and they reduced nitrate. All strains were

motile, and negatively stained cells in the electron micro-scope showed flagella and a crystalline surface structuresimilar to that reported for W. recta (17).

Thin-sectioned cells of 13 B. buccae strains studied underan electron microscope revealed an S-layer outside the outermembrane in only eight strains (Fig. 1). By using a freeze-fracture technique, a crystalline outer membrane proteinwas found in all 13 strains, regardless of the presence of theS-layer. The crystalline outer membrane protein was alwayssituated in the concave fracture face of the outer membrane(Fig. 2).Of the 62 infections, 35 were acute at the time of sampling.

The occurrence of nonpigmented Bacteroides species insymptomatic and asymptomatic cases is shown in Table 3.

DISCUSSION

When B. oris and B. buccae were described as separatespecies, they were determined to be distinct by means ofDNA homology studies and polyacrylamide gel electropho-resis patterns (13). It has been suggested that the lack off-glucosidase in B. oris would be of diagnostic value todistinguish between these two species (6, 10, 11), butDellinger and Moore (4) recently showed that both speciesproduce this enzyme. This is expected, because both B. orisand B. buccae hydrolyze esculin (6,7-dihydroxycoumarin-p-D-glucose), which can be hydrolyzed by 3-glucosidase (5).The two pentose-fermenting species in this study were

differentiated by ot-fucosidase and f-N-acetylglucosamin-idase (4). Moreover, B. oris and B. buccae were the onlyspecies isolated in this study that produced P-xylosidase(Table 2). The enzyme profile of B. heparinolyticus ATCC35895T, which was studied as a reference, was identical withthat of B. oris, with the exception of the glycine aminopep-tidase test, which was positive for B. heparinolyticus. More

FIG. 1. A thin-sectioned cell of B. buccae. The S-layer is clearlyseen outside the outer membrane. Bar, 0.2 ,um. S, S-layer; OM,outer membrane; CM, cytoplasmic membrane.

FIG. 2. A freeze-fractured cell of B. buccae that reveals theconcave fracture faces of the cytoplasmic membrane (CM) and theouter membrane (OM). Crystalline structures are clearly seen on theOM fracture face. Bar, 0.2 p.m.

B. heparinolyticus strains must be studied to confirm theusefulness of this enzyme in the differentiation of these twospecies. The glycosidase enzyme profiles of B. zoogleo-formans have not been reported, but the species is probablyidentified already by its unique growth characteristics (2).The differentiation between B. oralis and B. buccalis was

done on the basis of production of glycine aminopeptidase,which was produced by all seven strains isolated in thisstudy. B. buccalis DSM 20616T, which was studied as areference, gave a negative result for this test. This result is ingood agreement with that of Dellinger and Moore (4). In theoriginal description of B. buccalis the organism was negativefor starch hydrolysis (24). However, B. buccalis strainsstudied later by Holdeman et al. (10) hydrolyzed starch likeB. oralis. So, it is uncertain whether starch hydrolysis is ofdiagnostic value with these two taxa.The data of the occurrence of nonpigmented Bacteroides

species in endodontic infections is limited. Identification ofbile-sensitive nonpigmented Bacteroides species has often

TABLE 3. Occurrence of symptoms in teeth from whichnonpigmenting Bacteroides species were isolated

No. with or without symptoms:Species

+ (n = 35a) -(n = 275)

B. buccae 13 (37)c 2 (7)B. oris 7 (20) 5 (19)B. oralis 5 (14) 2 (7)B. bivius 1 (4)B. oulorum 1 (3)Bacteroides spp. 1 (3) 4 (15)

aTotal number of acute infections.bTotal number of asymptomatic infections.c Numbers in parentheses are percentage of samples from which species

were isolated.

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been done only to the genus level. On the other hand, thetaxonomy of bile-sensitive nonpigmented Bacteroides spe-cies has changed considerably during the last few years, andreference to the newly established species cannot be foundin older reports. Pentose-fermenting species were reportedusually as B. ruminicola subsp. brevis, but may often havebeen either B. buccae or B. oris. Williams et al. (31) studied10 dentogenous abscess aspirates and isolated B. ruminicolasubsp. brevis in two cases. Chow et al. (3) aspirated 31infections, including facial abscesses, osteomyelitis, anddentogenous abscesses, and found three B. ruminicolasubsp. ruminicola strains. Wittgow and Sabiston (32) tooksamples from 32 intact necrotic root canals and reported B.ruminicola subsp. brevis in 8 canals. Sabiston et al. (23)found B. ruminicola subsp. brevis in 8 of 58 dentogenousabscess aspirates. Aderholt et al. (1) reported B. ruminicolasubsp. ruminicola in only 1 of 50 dentogenous abscesses,and Oguntebi et al. (20) found no pentose-fermenting Bacte-roides species in 10 periapical abscesses. It is difficult toevaluate whether the great variation in isolation frequenciesis real or reflects differences in sampling procedures anddiagnostic methods. In some studies the main interest mayhave been directed toward some other groups of bacteria,e.g., black-pigmented Bacteroides species.

In this study the two most frequently isolated species ofnonpigmented Bacteroides fermented xylose and arabinose.B. buccae was almost always associated with the presence ofacute symptoms, whereas B. oris occurred with nearly equalfrequency both in acute and asymptomatic infections (Table3). It is possible that this difference is suggestive of thedifferent virulence capacities of these two species. In aprevious study of the ultrastructure of several of the Bacte-roides species it was found that B. buccae (mostly referencestrains) was the only species which had an S-layer (7). In thisstudy, in which more clinical strains of B. buccae werestudied, not all strains had the S-layer. B. buccae strains hadbeen preserved at -70°C in glycerol-milk before the ultra-structural studies, and it is possible that some strains lost theability to express the S-layer. On the other hand, the type ofthe medium used for growing the strains or the number oftransfers after removing the strains from -70°C had no effecton the S-layer. However, a crystalline outer membraneprotein was seen in all B. buccae strains in freeze-fracturedspecimens. Kerosuo et al. (E. Kerosuo, M. Haapasalo, H.Ranta, and K. Lounatmaa, submitted for publication) havestudied further the ultrastructure and biochemistry of cellwalls of several oral Bacteroides and Fusobacterium speciesand have not found crystalline structures in the outer mem-branes of other species, including strains of B. oris. Thefunction of the S-layer is poorly understood (27). The onlyreport of the S-layer as a possible virulence factor is aboutAeromonas salmonicida, which causes furunculosis in fish;only strains with an S-layer are virulent (14). Preliminaryresults with purified S-layer protein from one B. buccaestrain in this laboratory suggest that it is highly immunogenicin rabbits.

In a study of black-pigmented Bacteroides species inendodontic infections it was found that of all black-pigmented Bacteroides species the two asaccharolytic spe-cies B. gingivalis and B. endodontalis occurred only in acuteinfections (8). Only 2 of 15 B. buccae strains were foundtogether with B. gingivalis (one case) or B. endodontalis(one case); thus, the symptoms in most teeth in which B.buccae was isolated cannot be explained by the presence ofasaccharolytic oral black-pigmented Bacteroides species.

This study confirms the finding that B. fragilis is not

routinely isolated from uncomplicated endodontic infections(1, 20, 23, 31). Instead, bile-sensitive, nonpigmented Bacte-roides species are frequently present. It is also suggestedthat B. buccae may have a special role in the development ofacute infections. The crystalline structures both outside andin the outer membrane of B. buccae cells can be regarded aspossible virulence factors, and the function of these struc-tures needs to be studied further.

ACKNOWLEDGMENTS

I thank Helena Ranta and Eero Kerosuo for taking most of theelectron micrographs and Olli-Veikko Renkonen for helpful discus-sions. Katsuji Okuda kindly gave me the type strain of B.heparinolyticus.

This study was supported by the Finnish Dental Society.

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