pigmented Bacteroides (BPB) bacteria has been much im- proved, resulting in at least 10 recognized species. It has been demonstrated that most of these ...
Vol. 51, No. 3
INFECTION AND IMMUNITY, Mar. 1986, p. 972-974
0019-9567/86/030972-03$02.00/0
Copyright X 1986, American Society for Microbiology
Serological Characterization of Black-Pigmented Bacteroides endodontalis ARIE J. VAN WINKELHOFF,* NANCY KIPPUW, AND JOHANNES DE GRAAFF Research group for Commensal Infections, Departments of Oral and Medical Microbiology, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands Received 25 June 1985/Accepted 9 December 1985
Serological studies on the black-pigmented Bacteroides species B. endodontalis revealed three serotypes based on capsular determinants. A common antigen (0-antigen) could be demonstrated after decapsulation. Weak cross-reactivity was found with B. asaccharolyticus, but not with B. gingivalis. Similarity between the serology of Enterobacteriaceae and black-pigmented Bacteroides spp. is discussed.
overnight at room temperature. The titer of the serum was described as the reciprocal value of the highest dilution showing complete agglutination of the cells. All titer values were the results of at least two tests. For absorption of bacterial immunoglobulins in the test sera, bacterial cells were grown, killed, and washed as described previously for the vaccine preparation. A bacterial sediment containing about 5 x 1010 cells was added to 1 ml of serum, mixed on a vortex, ahd incubated at 0°C for 18 h. Bacterial cells were then separated from the serum by centrifugation (15 min at 13,000 x g). The same number of bacterial cells was added to the serum, and the suspension was incubated for 2 h at 37°C. Cell-free serum was used for the agglutination test. All titers were the result of at least two tests. None of the preimmune sera showed detectable antibody titers against the strains tested. Serum agglutination tests of the three test sera with six B. endodontalis strains resulted in a heterogeneous titer pattern (Table 1). Strains HG 370, HG 410, and HG 421 showed only high titers with antiserum HG 370, whereas strains HG 182 and HG 413 showed only high titers with antiserum HG 182. Strain HG 181 had high titers with all three antisera. These results suggested the presence of at least two serogroups within the species. Further tests were carried out with strains HG 370, HG 182, and HG 181. India ink preparation of cells of HG 370 and HG 182 clearly showed the presence of capsular layers, whereas no such structure was observed around cells of HG 181. From these observations, it was concluded that the low titer of antiserum HG 370 against HG 182, and vice versa, may be explained by the presence of serologically different capsules. The capsules of the strains tested keep the bacterial cells from agglutinating with antiserum HG 181, resulting in low titers, with the exception of the homologous strain (Table 1). Noncapsular, somatic antibodies (0-antibodies) in antisera HG 370 and HG 182 reacted with the exposed 0-antigens of HG 181 cells, resulting in high titers. The low titers of the strains with antiserum HG 181 may be the result of the presence of uncapsulated cells. The results of agglutination tests with whole bacterial cells also suggested the presence of common antigenic determinants (0-antigens). These common antigens were more clearly shown when the capsular structures were moved. Treatment of cells of HG 182 and HG 370 at 100°C for 60 min did not remove the capsular structures, as India ink preparations of heated cells showed. Decapsulation could only be achieved by heating cells for 135 min in an
In the past decade, the taxomony of the group of blackpigmented Bacteroides (BPB) bacteria has been much improved, resulting in at least 10 recognized species. It has been demonstrated that most of these species have specific antigenic compositions (5, 6, 8, 10), although serological cross-reactivity between BPB species has also been reported (1, 4, 8, 10). Most cross-reactivity was found within the group of saccharolytic BPB species, i.e., B. intermedius and B. melaninogenicus. It has been demonstrated that antigenic differences between BPB species may be based on immunochemical differences in the capsular layers (7). Antigenic heterogeneity within a BPB species has been reported for B. intermedius as determined by monoclonal antibodies (3) and has been suggested for B. melaninogenicus (1). B. endodontalis is a recently described asaccharolytic, black-pigmented Bacteroides species from infected dental root canals (14) and odontogenic abscesses (15). Previously we reported on the biochemical properties of this species (16). The purpose of the present study was to characterize the antigenic properties of B. endodontalis and to investigate the presence of common antigenic determinants with the two other asaccharolytic BPB species B. gingivalis and B. asaccharolyticus. For this purpose, methods were used which are applied for the serological typing of Enterobacteriaceae as described by Edwards and Ewing (2). The bacterial strains used in this study have been described previously (16). The B. endodontalis strains in this study were homogeneous as far as biochemical properties, guanine plus cytosine content, and homology of the DNAs are concerned (16). For antiserum preparations, whole cells of B. endodontalis HG 370 (ATCC 35406), HG 181 (H lla-c), and HG 182 (BN 11a-f) were used. Rabbit antisera were prepared as described previously (16). An agglutination test was used for the serological characterization of the strains (9). Before use, sera were incubated for 30 min at 56°C. Twofold serum dilutions were made in 0.1 ml phosphate-buffered saline (PBS) in round-bottomed microtiter wells. Microorganisms used for agglutination tests were cultured and killed as previously described for vaccine preparations (16). The bacterial cell suspension was washed three timnes in sterile PBS and suspended in PBS to a final optical density (X = 690 nm) of 0.2 to 0.3 (2 x 108 to 3 x 108 cells per ml). From this bacterial suspension, 0.1 ml was added to each of the wells, followed by incubation at 37°C for 2 h. For sedimentation, microtiter plates were stored *
Corresponding author. 972
VOL. 51, 1986
NOTES
TABLE 1. Serum agglutination titers of three antisera raised against three B. endodontalis strains Serum agglutination titer of antiserum against: HG 181 HG 182 HG 370
Strain
HG HG HG HG HG HG
4 64 64
16 64 64
2,048 2.048
1,024
2,048 2,048
370 410 421 181 182 413
2,048 512 4 2
8 64
2,048
973
TABLE 3. Serum agglutination titers of three antisera against B. endodontalis HG 370, HG 182. and HG 181 tested on B. gingivalis and B. asaccharolvticus strains Serum agglutination titer of antiseruIm HG 370
against: HG 182
HG 181
0 0 0
0 0 0
0-2 0-2 0-2
0 0 32 32 8
0 0 64 32 4
0 0 128 32 16
Strain
B. gingivalis HG66 HG76 HG184
B. asaccharolwticus
autoclave at 121°C. This procedure is often used in the serological classification of Enterobacteriaceae if decapsulation at 100°C is not effective (2). Gram-stained preparations of autoclaved cells subsequently washed twice in PBS showed intact gram-negative coccobacillar rods. Serum agglutination tests with autoclaved bacterial cells revealed comparable titers with all three immune sera. An exception was antiserum against HG 370, which showed a decreased titer against the homologous strain and strain HG 181 but a clearly elevated titer against strain HG 182 (Table 2). From these tests it was concluded that the strains tested possess a common, heat-stable, somatic antigen (0-antigen). For further confirmation of these common antigenic determinants, we incubated some antisera with heterologous B. endodontalis strains to remove specific immunoglobulins. Antisera against HG 182 and HG 181 were immunosorbed with decapsulated HG 370 cells. Both absorbed sera were tested on decapsulated cells of strains HG 370, HG 182, and HG 181. Results revealed titers of 0 and 16 for HG 182 and HG 181 antisera, respectively. Furthermore, both absorbed antisera showed decreased titers with decapsulated cells (Table 2). These results indicated that the heat-stable antigen is a common antigen of the three tested B. endodontalis strains. The results of these experiments showed the presence of three serotypes of B. endodontalis, which can be characterized as O1 K1 (HG 370, HG 410, and HG 421), 01 K2 (HG 182 and HG 413), and 01 K- (HG 181). This serological typing is based on the absence or presence of serologically different capsules which are highly heat stable. This type of capsule has been described previously for Escherichia coli and Salmonella spp. (2). To our knowledge, this is the first observation of different serotypes within a black-pigmented Bacteroides species based on agglutination of whole bacterial cells. Results of agglutination tests with cells of the two other human asaccharolytic BPB species B. gin gixalis and B. asaccharolyticius are summarized in Table 3. Four of the seven B. gingivalis strains tested appeared to be autoagglutinable in PBS. Strains HG 66, HG 76, and HG 184 did not agglutinate with antisera HG 370 and HG 182 and hardly TABLE 2. Serum agglutination titers of three B. endodonitilis antisera with decapsulated B. endodontali.s cells Serum agglutination titer of antiserum against: Decapsulated
cells of strain
HG 370
HG 182
HG 370 HG 182 HG 181
256 64 256
2.048
1.024
HG 182
(370)" 0 4 4
HG 181
2,048 1.024
1,024 2,048 cells. "Antiserum absorbed wvith decapsulated HG 370 ND, Not done.
HG 181
(37t0)" 16 8
NDD"
HG 9 HG107 HG 71 HG 111 HG129
agglutinated with antiserum HG 181. Autoclaving B.
gingii'alis cells did not result in detectable titers, indicating that there is no serological relationship between B. endodontalis and B. gin givalis. Some of the tested B. asaccharolvticus strains showed weak to moderate titers with the three test sera. These titers did not change if these B. asaccharolyticus cells were autoclaved, whereas autoclaved HG 107 cells weakly reacted with all three antisera, resulting in titers of 8 to 16. These results indicate that there are certain common antigenic determinants between B. endodontalis and B. asaccharolvticus. The presence of a capsular structure around HG 182 cells has been reported by Sundqvist et al. (12). Sundqvist et al. (13) also demonstrated that this strain is more virulent that the uncapsulated strain HG 181 by using experimental infections. Furthermore, strain HG 182 also showed low chemiluminescence and very limited phagocytosis by human polymorphonuclear leukocytes in comparison with strain HG 181. Our findings suggest that different serotypes of B. endodontalis may be correlated with differences in pathogenicity and virulence based on the presence of capsular structures. We thank P. A. M. Guinee and W. H. Jansen of the Rijksinstituut voor Volksgezondheid en Milieu for their valuable suggestions.
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