Eikenella corrodens in Conventional Hamsters

INFECTION AND IMMUNITY, Apr. 1982, p. 304-309 0019-9567/82/040304-06$02.00/0 Vol. 36, No. 1 Oral Implantation of Bacteroides asaccharolyticus and Eikenella corrodens in Conventional Hamsters TETSUJI NAGAHATA,1 TATSUO KIYOSHIGE,1 SHIRO TOMONO,1 RYUJI ABE,1 SHUJI SASAKI,'* AND ICHIRO TAKAZOE2 Lion Applied Research Laboratories, Lion Corporation, Odawara Kanagawa 256,1 and Department of Microbiology, Tokyo Dental College, Tokyo 101,2 Japan Received 5 August 1981/Accepted 27 November 1981 Oral implantation of Bacteroides asaccharolyticus 381-R' and Eikenella corrodens 1073S-R, which are highly resistant to streptomycin, was examined in conventional hamsters. The hamsters' first molars were ligatured with cotton threads preimmersed in bacterial suspensions. Bacterial inoculation was performed daily for 1 week, followed by a single weekly inoculation for 7 more weeks. Hamsters were fed Keyes' diet no. 2000 or ordinary powdered diet. Bacterial recovery, gingival histological changes, and immunological response were checked 2, 5, and 8 weeks after the daily serial inoculation. B. asaccharolyticus 381-R' was recovered from all hamsters during the experiment (6.7 x 106 to 6.3 x 108 colony-forming units). E. corrodens 1073S-R was recovered from the hamsters fed Keyes' diet no. 2000 throughout the experimental period (2.4 x 105 to 1.2 x 106 colony-forming units), but not in the group fed the ordinary powdered diet. The histological changes in gingival tissues at the second week showed no differences among the control group, the B. asaccharolyticus-inoculated group, and the E. corrodens-inoculated group. However, by the eighth week, the inflammation persisted only in the bacteria-inoculated groups, especially in the B. asaccharolyticus-inoculated group. A relatively increased serum antibody titer was also observed in the B. asaccharolyticus-inoculated group, but not in the E. corrodens-inoculated group. These findings indicate that B. asaccharolyticus 381- R' and E. corrodens 1073S-R can be implanted in hamsters' oral cavity with the aid of cotton thread ligature. It was also suggested that B. asaccharolyticus may have some pathogenic role in the destruction of periodontal tissue in hamsters. The role of gram-negative bacteria in the mechanisms of periodontal tissue destruction has been emphasized. Bacteroides asaccharolyticus has been shown to be a predominant isolate from the deep pockets of advanced periodontitis (19; A. Crawford, S. S. Socransky, and G. Bratthall, J. Dent. Res., 54A:97, abstr. no. 209, 1975) and from acute periodontal abscesses (11). Corroding bacteria, including Eikenella corrodens, have also been detected in pathological sites from all types of advanced periodontitis (Crawford et al., J. Dent. Res. 54A:97, abstr. no. 209, 1975). E. corrodens was also demonstrated in five cases of oral infection (3) other than advanced periodontitis. More recently, these bacteria have been shown to cause significant tissue destruction and alveolar bone loss when monoimplanted in gnotobiotic rats (4, 6, 10; J. Wennstrom, S. S. Socransky, L. Heijl, and J. Lindhe, J. Dent. Res. 58A:123, abstr. no. 122, 1979). Neither of these gram-negative bacteria, however, has been studied with respect to their implantation in the oral cavity of conventional animals. It has been reported that oral introduction of Actinomyces species into conventional hamsters with feeding of a high-sucrose diet resulted in massive plaque accumulation, severe periodontal tissue breakdown, and alveolar bone loss (5, 7). The purpose of the present study was to verify whether B. asaccharolyticus and E. corrodens can be implanted in the oral cavity of conventional hamsters when their mandibular teeth are ligatured and whether these bacteria show any pathogenicity in the periodontal tissues of hamsters. MATERIALS AND METHODS Experimental animals. Eight-week-old male golden hamsters were used. Experimental groups were fed Keyes' diet no. 2000 (8) or ordinary powdered diet (M powder diet; Oriental Yeast Co., Ltd., Tokyo, Japan). The control group was fed Keyes' diet no. 2000. All groups were composed of 10 hamsters, except for a group of 12 hamsters which were inoculated with B. asaccharolyticus 381-R' and fed Keyes' diet no. 2000. At 2 and 5 weeks after final serial daily inoculation, three hamsters from each group were sacrificed, and at 8 weeks the rest of the hamsters were sacrificed. Bacterial strains and culture conditions. Streptomy- 304

VOL. 36, 1982 BACTERIAL IMPLANTATION IN HAMSTERS 305 cin-resistant mutants of B. asaccharolyticus 381 and E. corrodens 1073S were used. Original strains were kindly provided by S. S. Socransky (Forsyth Dental Center, Boston, Mass.). Streptomycin-resistant mutants (2,000,ug/ml) were obtained by the transfer method (13), using blood agar containing various concentrations of streptomycin (50,ug/ml to 2 mg/ml). The medium for B. asaccharolyticus was Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with 1.5% agar, 5% sheep blood, sodium succinate (100,ug/ml), hemin (3,g/ml), and menadion (0.5,jig/ml). Tryptic soy agar (Difco) supplemented with 5% sheep blood was used for E. corrodens. Incubation was performed at 37 C in an anaerobic box containing 80% N2, 10% C02, 10% H2. Mutants designated B. asaccharolyticus 381-R' and E. corrodens 1073S-R were the same as the original strains with respect to their hemagglutination activity of human erythrocytes and biochemical characteristics. Cells cultured for 3 days on blood agar were suspended in Todd-Hewitt broth (optical density at 550 nm = 1.0) containing streptomycin (1 mg/ml), and 0.1 ml of these bacterial suspensions was inoculated into each cheek pouch and the oral cavity, a total inoculation of 0.3 ml. The second, fifth, and eighth week after the 7-day serial inoculation, the ligatured threads were carefully removed from the molar and suspended in 5 ml of Todd-Hewitt broth with small glass beads. The suspension was stirred for 30 s with a mixer. Immediately, 10-fold serial dilutions were prepared, and 0.1 ml of the diluent was spread on antibiotic medium plates and 5% blood agar plates. The plates were cultured anaerobically for 14 days. The black-pigmented colonies of B. asaccharolyticus developing on streptomycin (1 mg/ml) containing blood agar were enumerated. For the enumeration of E. corrodens, clindamycin (50,ug/ml)- and streptomycin (1 mg/ml)- supplemented blood agar plates were used. E. corrodens was confirmed by Gram stain, colony morphology, and cell morphology in the dark-field microscope and by the lysine-ornithine-decarboxylase test. Experimental schedule. The hamsters were anesthetized intraperitoneally with sodium pentobarbital (Somnopentyl; Pitman-Moore Inc., Washington Crossing, N.J.; 0.05 ml/100 g). The first right mandibular molar was ligatured with cotton threads (diameter, 0.19 mm) which had been immersed in a bacterial suspension (optical density at 550 nm = 1.0) containing streptomycin (1 mg/ml). Bacterial inoculation was performed daily for 1 week, followed by a single weekly inoculation for 7 more weeks. The animals were sacrificed at the second, fifth, and eighth week after the initial 7-day inoculation for the evaluation of bacterial implantation, serum antibody titer, and histological changes in periodontal tissues. In each experiment, the animals were sacrificed at 7 days after the final weekly inoculation. Serum antibody. Titers were determined by cell agglutination activity with a modified microtitration method (18). Three-day cultured cells on blood agar were suspended in 5 mm phosphate buffer (ph 7.2) containing 0.6% Formalin and washed twice with the same buffer. After centrifugation (10,000 rpm for 15 min), cells were suspended in the same buffer (optical density at 550 nm = 1.2). A mixture of serum diluent and the bacterial suspension was shaken for 1 min with a Tomy mixer (Tomy Seiko Co., Ltd., Tokyo, Japan), left at room temperature for 1 h, and then stored at 4 C for 18 h. The bacterial agglutination was examined under a dissection microscope. Histological examination. The right mandibles of hamsters were sampled at the second and eighth week after 7-day serial inoculation. The samples were fixed in 10% Formalin, decalcified with 10% formic acid, and sectioned. Sectioned periodontal tissues were stained with hematoxylin and eosin. Histological changes at the site of the ligature were focused on the loss of gingival attachment, polymorphonuclear leukocyte infiltration into oral epithelium and connective tissue, disorder of collagen fiber line, and alveolar bone resorption. RESULTS Bacterial implantation in the oral cavity of hamsters. The numbers of bacteria recovered from the ligatured cotton thread are summarized in Table 1. The total bacterial cell counts in the samples varied from 2.6 x 108 to 2.3 x 1010 colony-forming units. The number of B. asaccharolyticus 381-R' varied from 6.7 x 106 to 6.3 x 108 colony-forming units. B. asaccharolyticus 381-R' was recovered from all hamsters fed either ordinary powdered diet or Keyes' diet no. 2000 during the experiment, although there was a little variation in the recovered bacterial cell numbers. E. corrodens 1073S-R was also recovered from all hamsters fed Keyes' diet no. 2000. The recovered bacterial cell numbers were 2.4 x 105 to 1.2 x 106 colony-forming units. In the ordinary powdered diet group, however, the bacteria (7.0 x 105) were recovered only at the second week, but not at the fifth or eighth week. In the control group, black-pigmented colonies and E. corrodens were not found. Serum antibody titers. Titers against the two strains of bacteria varied from animal to animal and with the period of time after inoculation. Agglutination titers against B. asaccharolyticus 381-R' rose. Titer were 1:16 at maximum (Table 2). No agglutination titer against E. corrodens 1073S-R was detected during the experiment. Furthermore, no clear differences in agglutination activity were found between the two diet groups (Tables 2 and 3). In the control group the titers against B. asaccharolyticus 381-R' and E. corrodens 1073S-R were less than 1:2 in all animals. Histological findings. The loss of gingival attachment, polymorphonuclear leukocyte infiltration into epithelium and connective tissue, and disorder of collagen fiber line associated with alveolar bone resorption in connective tissue were observed. Severe vertical and horizontal alveolar bone resorption was also observed in all groups. By the second week, there were no distinct histological differences between the control group and the bacteria-inoculated groups after either B. asaccharolyticus 381-R' (Fig. 1) or E. corrodens 1073S-R inoculation. However,

306 NAGAHATA ET AL. TABLE 1. Recovery of inoculated bacteria from ligatured threads in hamsters INFECT. IMMUN. No. of bacteria recovered' after serial inoculation at wk: Bacterium Diet 2 5 8 Control No. 2000 0 0 0 2.7 ± 1.2 x 188 1.0 ± 0.3 x 109 9.0 ± 2.1 x 108 B. asaccharolyticus 381-R' M powderb 2.0 ± 0.4 x 107 (3/3) 1.6 ± 0.3 X 107 (3/3) 3.4 ± 1.2 X 107 (4/4) 3.4 ± 0.4 x 108 6.9 ± 3.0 x 108 2.6 ± 0.3 x 108 No. 2000 2.3 ± 1.2 x 107 (3/3) 6.7 ± 0.3 x 106 (2/2) 6.3 ± 5.8 x 108 (5/5) 3.8 ± 0.3 x 108 3.2 ± 1.1 x 108 2.3 ± 0.5 x 1010 E. corrodens 1073S-R M powder 7.0 ± 4.5 x 105 (3/3) 0 (0/3) 0 (0/4) 1.9 ± 0.6 x 109 5.9 ± 0.8 x 108 3.1 ± 0.6 x 108 No. 2000 3.4 ± 1.9 x 105 (3/3) 2.4 ± 0.4 x 105 (3/3) 1.2 ± 0.8 x 106 (4/4) 5.3 ± 1.7 x 108 1.2 ± 0.7 x 109 4.5 ± 1.6 x 108 a The first value is mean cell numbers of the inoculated strains (B. asaccharolyticus 381-R' or E. corrodens 1073S-R) recovered on the antibiotics agar (streptomycin agar or streptomycin and clindamycin agar) ± standard error of the mean. The second value is mean total bacteria recovered on the blood plate ± standard error of the mean. The value in parentheses is the ratio of animals in which inoculated bacteria were recovered to the total in which bacteria were inoculated. b Ordinary powdered diet. the degree of polymorphonuclear leukocyte infiltration was lower in the E. corrodens-inoculated group than in the control or the B. asaccharolyticus-inoculated group. The vertical resorption was especially notable at the site of the first molar with the appearance of several osteoclasts. By the eighth week, the histological changes became less severe in the control (Fig. 2A). In contrast, inflammation still remained in the bacteria-inoculated groups, particularly in the B. asaccharolyticus-inoculated group (Fig. 2B through D). The degree of change in the B. asaccharolyticus-inoculated group was slightly greater than that in the E. corrodens-inoculated group fed Keyes' diet no. 2000. The change in the E. corrodens-inoculated group fed ordinary powdered diet was similar to that of the control group. No remarkable signs of regeneration of vertically resorbed alveolar bone were observed in the B. asaccharolyticus 381-R'-inoculated group, but such changes occurred in the E. corrodens 1073S-R-inoculated group and in the control group. Regeneration of horizontally resorbed alveolar bone was not observed in any group. Lymphocyte and plasma cells were not TABLE 2. Serum agglutination titers against inoculated B. asaccharolyticus 381-R' Time after No. of animals with Diet. serial reciprocal titer of: Total inoculation (wk) <2 2 4 8 16 M powdera 2 3 0 0 0 0 3 5 2 0 1 0 0 3 8 1 1 1 1 0 4 No. 2000 2 1 1 0 0 1 3 5 0 1 1 0 1 3 8 1 0 3 1 0 5 aordinary powdered diet. observed in any sample during the experiment. Hair or food impaction was not found in any animals. DISCUSSION It has been extremely difficult to establish human oral microorganisms in the oral cavity of conventional animals (22). In previous studies, oral implantation was unsuccessful even when the flora was suppressed by means of continuous antibiotic application, using the same inoculation schedule as this experiment. There were no histological changes in the periodontal tissues of these hamsters (T. Nagahata, unpublished data). The present study indicated that the oral implantation of some microorganisms can be successful when the ligature is applied. The effects of ligature on the development of periodontal tissue destruction have been reported by some authors (17, 21). Ligature placement contributes to a fast plaque accumulation which can induce periodontal disease in conventional animals. The adsorptive activity of bacteria is undoubt- TABLE 3. Serum agglutination titers against inoculated E. corrodens 1073S-R Time after No. of animals with Diet serial reciprocal titer of: Total inoculation (wk) <2 2 4 8 16 M powder' 2 3 0 0 0 0 3 5 2 1 0 0 0 3 8 3 1 0 0 0 4 No. 2000 2 1 2 0 0 0 3 5 2 1 0 0 0 3 8 4 0 0 0 0 4 a Ordinary powdered diet.

VOL. 36, 1982 BACTERIAL IMPLANTATION IN HAMSTERS 307 K. 'O1 70 ;%F v~~~~~~- 4 t s pg.. 48' - D >,'g~*'.t s: XA -77ml r4 >- 'z ^ ***7 Jf -< ul gr..-.. + fde _w -k.)- I.,. v.,-- 'I FIG. 1. Mesio-distal cross section of interproximal periodontal tissue between the mandibular first and second molars. (A) Three weeks after the ligature in control group. x67. (B) Enlargement of (A). x134. (C) Three weeks after the ligature in the B. asaccharolyticus 381-R'-inoculated group. x 67. (D) Enlargement of (C). x 267. Note alveolar bone resorption, polymorphonuclear leukocyte (P) infiltration in epithelium and underlying connective tissue and the area of collagen fiber line disorder associated with alveolar bone resorption (D) in both groups. edly a key to the colonization of the bacteria in the oral cavity. B. asaccharolyticus has been shown to adhere to epithelial cells and several strains of gram-positive bacteria in vitro (20). Similar findings have been obtained with E. corrodens (20, 23). Also, in in vivo experiments, high numbers of streptomycin-labeled cells of B. asaccharolyticus were recovered from preformed dental plaque 150 min after introduction (20). Numerous indigenous bacteria formed plaque with inoculated bacteria around the ligatured thread (Table 1, Fig. 2D). The implantation of B. asaccharolyticus 381-R' and E. corrodens 1073S-R in the oral cavity of conventional hamsters observed in the present study can be explained by the adsorptive activity of test microorganisms to the dental plaque preformed by the ligature. Direct or indirect adsorptive

308 NAGAHATA ET AL. INFECT. IMMUN. I Wr C ~~~~~~~~~~~~~~~~r FIG. 2. Mesio-distal cross section of interproximal periodontal tissue between the mandibular first and second molars. (A) Nine weeks after the ligature in the control group. x 67. Note disappearance of polymorphonuclear leukocyte infiltration in epithelium and in underlying connective tissue. (B) Eight weeks after serial inoculation with B. asaccharolyticus 381-R'. x 67. Note obscure outline of alveolar bone and polymorphonuclear leukocyte (P) infiltration in epithelium and just underlying connective tissue. (C) Eight weeks after serial inoculation with B. asaccharolyticus 381-R'. x27. The ligatured cotton thread was not removed in this sample. This preparation was not included in any data. Note resorbed alveolar bone. (D) Enlargement of (C). x 134. Note accumulated plaque (PL) around the thread (T), pus (PS) around the plaque, and polymorphonuclear leukocyte (P) infiltration in underlying connective tissue. activity to the other microorganisms in preformed dental plaque must be critical. The large amount of pus observed around the ligatured threads presumably results in good nutritional resources for the growth of inoculated bacteria, as is observed at the site of the human periodontal disease (1). Plaque accumulated as a result of the ligatured threads around the subgingival site in hamsters might contribute to the anaerobiosis Ṫhe increase in antibody titer after the inoculation of B. asaccharolyticus 381-R' was quite

VOL. 36, 1982 low. However, the fact that there was no increase of titer in the control group and also the apparent difference in the histological findings between the control and B. asaccharolyticus 381-R'-inoculated group collectively suggest that the specific reaction occurred due to the implantation. On the other hand, no humoral responses to E. corrodens 1073S-R were found. This might be due to an insufficient number of implanted bacteria for raising antibody titer, weak antigenicity, or antigenic competition (16) between preexisting bacteria and inoculated microorganisms. The application of ligature around the neck of the tooth of an experimental animal has been shown to cause severe destruction of periodontal tissue and alveolar bone loss (2, 9, 12, 14, 21). In the present study, similar histological findings were observed by the third week after ligature in both the control and bacteria-inoculated groups. However, apparent effects of bacterial inoculation appeared at the later stage of the experiment. The histological findings in the control group showed a clear regeneration of the periodontal tissues by the ninth week after ligature in spite of the accumulated plaque, suggesting that hamster periodontal tissues are tolerant of the plaque formed by indigenous bacteria or that the bacterial plaque itself is not so severely pathogenic. In bacteria-inoculated groups, the inflammation continued in the epithelium and connective tissues, especially in the B. asaccharolyticus 381-R'-inoculated group. In this group, however, no regeneration of the alveolar bone was observed, unlike the control and E. corrodens 1073S-R-inoculated groups. Persistent inflammation observed in the E. corrodensinoculated group might reflect the strong biological activity of E. corrodens lipopolysaccharides which has been recently demonstrated (15). The present findings suggest that B. asaccharolyticus 381-R' and E. corrodens 1073S-R have some pathogenicity toward periodontal tissues of conventional hamsters. The establishment of inoculated bacteria and histological changes after removal of the ligature are under investigation. LITERATURE CITED 1. Akiyoshi, M. 1977. Clinical pathology of periodontal disease, p. 19-41. In T. Imada (ed.), Clinical aspects of periodontics. Dental Outlook Special Issue. 2. Deporter, D. A., and D. Y. Brown. 1980. Fine structural observations on the mechanism of loss of attachment during experimental periodontal disease in the rat. J. Periodontal Res. 15:304-313. 3. Goodman, A. D. 1977. Eikenella corrodens isolated in oral BACTERIAL IMPLANTATION IN HAMSTERS 309 infections of dental origin. Oral Surg. 44:128-134. 4. Irving, J. T., M. G. Newman, S. S. Socransky, and J. D. Heeley. 1975. Histological changes in experimental periodontal disease in rats mono-infected with a gram-negative organism. Arch. 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