Streptococcus oralis previously identified as uncommon ‘Streptococcus sanguis’ in Behçet's disease

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Pergamon

Archs oral Biol. Vol. 40, No. 8, pp. 685-690, 1995 0003-9969(95)00042-9

Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0003-9969/95 $9.50 + 0.00

S T R E P T O C O C C U S O R A L I S PREVIOUSLY IDENTIFIED AS UNCOMMON ' S T R E P T O C O C C U S S A N G U I S ' IN BEH(~ET'S DISEASE S. N A R I K A W A , 1 Y. S U Z U K I , l M. T A K A H A S H I , 2 A. F U R U K A W A , 1 T. S A K A N E l and Y. M I Z U S H I M A I ~Institute of Medical Science and Department of Internal and Laboratory Medicine, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216 and 2Department of Hygiene, Tsukuba College of Technology, 4-12 Kasuga, Tsukuba, Ibaraki 305, Japan

(Accepted 29 March 1995) Summary--The relation between the biochemical and serological properties of 'Streptococcus sanguis' was studied to characterize the strains isolated from dental plaque of patients with Beh~;et's disease and controls. Seven reference and I00 clinical strains preserved by the Beh~t's Disease Research Committee of Japan were identified using established criteria and differentiated with antisera against Strep. oralis ATCC 10557, Strep. sanguis ATCC 10556 and "Strep. sanguis' ST7, compatible with the criteria. Uncommon serovars (serotypes) KTH-1 (=ATCC 49298), KTH-2 (=ATCC 49296), KTH-3 (=ATCC 49295), KTH-4 (= ATCC 49297) and B220 (serovar KTH-1) with both IgA1 protease and neuraminidase (sialidase) were. identified as Strep. oralis, whereas common serovars ST3 with IgA1 protease alone and ST7 without both enzymes were identified as Strep. sanguis and Strep. gordonii, respectively. Isolates previously ranked as uncommon serovars were identified as Strep. oralis, whereas the rest ranked as common serovars were identified as the same species as those of the grouping strains. A soft-agar technique was available for species identification except for Strep. oralis serovar KTH-1 reacting with the antiserum against Strep. gordonii ST7. The frequency of isolation of Strep. oralis was higher in Beh~et's disease (52%) than in controls (38%), but no difference was observed between the properties of the two groups of isolates. Strep. oralis virulence factors may be involved in breach the mucosal barrier in patients with specific reactiviity to these antigens and inducing Behget's disease. Key words: Streptococcus oralis, Streptococcus sanguis, virulence, Behqet's syndrome.

INTRODUCTION Behget's disease is characterized by recurrent oral and genital ulcers, skin lesions and uveitis (Suzuki et al., 1992). 'Streptococcus sanguis' has been proposed as a potential agent in Behqet's disease based on higher isolation rates, skin reactivities and antibody titres in patients than control,,; (Mizushima et al., 1989; Isogai et al., 1990; Lehner et al., 1991). F r o m 1987 to 1991, hundreds of strains ol! streptococci producing dextran were isolated from tile teeth of patients and healthy controls, and collected in laboratories supported by the Beh~et's Disease Research Committee of Japan (Mizushima et al., 1989). The isolated strains were identified to the species level by the former taxonomic criteria ( H a m a d a et al., 1980). These 'Strep. sanguis' isolates were also examined serologically and found to belong mostly to undefined serovars in patients with B e h ~ t ' s disease (Okonogi et al., 1988; Isogai et al., 1990). Such isolates were named as K T H strains (Okonogi et al., 1988; Lehner et al., 1991).

Antigens from serovars K T H and other control strains were prepared for skin testing, and the symptoms of Behqet's disease followed the skin test (Mizushima et al., 1989). Later we reported that lymphoproliferative responses to whole-cell antigens (Martin et al., 1979; Mizushima et al., 1989) from serovars K T H - I and KTH-3 were specific for Behqet's disease and stronger than responses to those from c o m m o n serovars (Suzuki et al., 1991), leading us to study the differences in properties between u n c o m m o n and c o m m o n serovars of 'Strep.

sanguis '. 'Strep. sanguis' was formerly classified into 'biotype A ' = ' t y p e I' and 'biotype B ' = ' t y p e II' (Hamada et al., 1980). Kilian, Mikkelsen and Henrichsen (1989) divided the biotype A group into Strep. sanguis and a new species, Strep. gordonff, and referred to the biotype B group as Strep. oralis, a later synonym of 'Strep. mitior' (French et al., 1989). Strep. oralis, proposed by Bridge and Sneath (1982) as a new species, resembles 'Strep. sanguis' type II. As the characteristics of Strep. oralis were originally variable, it was not easy to distinguish Strep. oralis from other viridans streptococci (Hardie, 1986). The description of Strep. oralis was later revised by Kilpper-Bfilz, Wenzig and Schleifer (1985) and by Kilian et al. (1989). The latter paper included

Abbreviations: ATCC, American Type Culture Collection; KTH, Kitasato-Ts~arumizu-Hashimoto; NCTC, National Collection of Type Cultures; TTY, trypticase tryptose yeast extract; TYC, trypticase yeast-extract cystine. 685

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well-defined differential charcteristics, which we have now used to identify 'Strep. sanguis' isolates. MATERIALS

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'Strep. sanguis' strains Reference strains used were KTH-1 (ATCC 49298) and KTH-2 (ATCC 49296) from patients with Kawasaki disease, KTH-3 (ATCC 49295), KTH-4 (ATCC 49297) and B220 (serovar KTH-1) from patients with Behqet's disease (Mizushima et al., 1989; Lehner et al., 1991). These were provided by Dr T. Tsurumizu, Kitasato Institute, before depositing in the ATCC. ST3 (serovar I) and ST7 (serovar IV) from healthy controls (Hamada et al., 1980) were provided by Dr S. Hamada, Osaka University Dental School. One hundred representative strains were isolated clinically from 50 patients with Behget's disease and 50 controls with TYC containing 5% sucrose medium in agar (Stoppelaar, Houte and de Moor, 1967). All strains were sampled from dental plaque of the labial smooth surface of upper incisors, except strain B220 which was taken from an aphthous lesion (Mizushima et al., 1989). Isolates had been previously grouped in serovars by the method of Isogai et al. (1990), and identified as 'Strep. sanguis' producing dextran, ct-haemolysis on blood agar and no acid from mannitol (Hardie, 1986). The type strains of Strep. sanguis ATCC 10556 (serovar I), Strep. oralis NCTC 11427 and Strep. gordonii ATCC 10558 (serovar I/II, = III) and the reference strain of Strep. oralis ATCC 10557 (serovar II) were provided by the Japanese Society for Bacteriology (NCTC 11427) and the other strains by Dr T. Tsurumizu, and used as controls for identification and serological grouping. Biochemical tests Biochemical properties (Table 1) including IgAi protease and neuraminidase (sialidase) were determined by standard procedures (Kilian et al., 1989).

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Preparation of rabbit antisera Formalin-treated vaccine (0.1 ml) prepared from ATCC 10557, ATCC 10556 and ST7 strains representing the three species of 'Strep. sanguis' was injected intravenously daily ( × 3) into each group of two rabbits weighing approx. 2 kg (Japanese white rabbit; Nihon Clea Co. Ltd, Tokyo, Japan) (Ichiman et al., 1981). In the second week, 0.5 ml of the vaccine was used followed by 1.0 ml weekly ( × 2). Two weeks after the final immunization, rabbits were exsanguinated, serum was separated and adsorbed with two heterogeneous organisms at 20mg/ml (dry weight) for 1 h at 36°C, and kept at 4°C overnight. Serological differentiation by the soft-agar technique The serum-soft-agar and soft-agar techniques as described originally by Finkelstein and Sulkin (1958) have been applied to the investigation of cell surface properties of non-motile bacteria including certain species of Staphylococcus, Streptococcus and Klebsiella (Chomarat, Ichiman and Yoshida, 1985). This procedure detects serologically distinct organisms by the conversion of diffuse to compact colonial morphology in serum-soft agar containing specific antisera (Yoshida, 1971).

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Streptococcus oral& in Beht;et's disease

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Table 2. Serovar, source and identity of 100 clinically isolated strains previously identified as "Strep. sanguis' No. of strains used

Serovar KTH- 1 KTH-2 b KTH-3 KTH-3 and -4c KTH-4

Strep. oralis Strep. sanguis Strep. gordonii

21 0 5 3 13

I

II III III and IVd IV

No. of strains identified as:

12/8a 0 3/2 2/l 7/6

0 0 0 0 0

1/0 0 0 0 0

4

0

1/3

0

4 0 34 16

2/2 0 0 0

0 0 0 0

0 0 14/20 8/8

aNo. of strains from 50 patients with Beh~;et's disease/no, of strains from 50 controls. bNo isolate of serovar KTH-2 was available, being rarely isolated from patients with Behq,et's disease or controls. cStrains reacting with both anti-KTH-3 and anti-KTH-4 sera. dStrains reacting with anti-serovar III serum also reacted with anti-serovar IV serum. Organisms grown overnight at 36°C in TTY medium ( H a m a d a and Torii, 1978) were diluted to 10 - 6 with sterile saline:. Aliquots o f 0.1 ml were mixed with 0.1 ml o f a d s o r b e d rabbit antiserum or normal serum a d s o r b e d with all strains, then mixed with 10ml o f T T Y dialysate medium containing 0.08% agar (Bacto agar, Difco, MI, U.S.A.), kept at 4°C for 30 min to solidify the agar, then cultured at 36°C for 2 4 h (Yoshida, 1971; Ichiman et al., 1981). The difference in colonial m o r p h o l o g y between the organisms cultured with antiserum and normal serum was determined visually. RESULTS

Biochemical properties Table 1 shows the biochemical properties o f all strains corresponding to 'Strep. sanguis'. Reference strains KTH-1 to K T H - 4 and B220 were identified as Strep. oralis, whereas strains ST3 and ST7 were identified as Strep. sanguis biovar 1 and Strep. gordonii biovar 2 or 3,, respectively.

Relation between serovar and identity in the clinical strains A total o f 45 isolates previously classified as serovars K T H and serovar II were identified as Strep. oralis, four isolates classified as serovar I as Strep. sanguis, and 50 isolates classified as serovars III a n d / o r IV and one isolate as serovar K T H - I as Strep. gordonii (Table 2). The m o s t frequent isolated species in patients with Behqet's disease was Strep. oralis, but no significant differences in serological or biochemical properties (including the activity o f two enzymes) were observed between the isolates from patients and controls. Strept. mitis (Kilian et al., 1989) and Strept. parasanguis (Whiley et al., 1990), neither o f which produces extracellular polysaccharide, were excluded from the test strains.

Serological differentiation by the soft-agar technique Strains exhibiting diffuse colonial m o r p h o l o g y in soft-agar medium containing normal serum were converted to the c o m p a c t state only by the addition

Table 3. Serological differentiation of Strep. oralis, Strep. sangu& and Strep. gordonii by the soft-agar technique with antisera against strains ATCC 10557, ATCC 10556, and ST7, respectively Conversion of colonial morphology from diffuse to compact growth with antisera against:

Species

'Strep. sanguis'

No. of strains used

ATCC 10556

ST7

Strep. oralis

KTH-1 KTH-2 KTH-3 KTH-3 and -4 KTH-4 II

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Strep. sangttis Strep. gor&Jnii

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-

34 16 1

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Serovar of

Ill and IV IV KTH- 1

alncludes tl:Le reference strains KTH-I and B220. bThe reference strain. Clncludes the reference strain KTH-3. dlncludes the reference strain KTH-4.

ATCC 10557

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I

B

a

b

a

b

Fig. 1. Colonial morphologies of Strep. gordonii ST7 (A) and Strep. oralis KTH-I (B) in soft agar containing normal rabbit serum [a] and rabbit anti-ST7 serum adsorbed with cells of Strep. oralis ATCC 10557 and Strep. sanguis ATCC 10556 [b]. Strain KTH-1 exhibiting diffuse colonial morphology in serum-soft agar was converted to compact growth by the addition of anti-ST7 serum, as was the case with strain ST7.

of homologous antisera (Table 3 and Fig. 1A). A m o n g the Strep. oralis strains, however, two reference and 20 isolated strains comprising serovar K T H 1 reacted with the antiserum against Strep. gordonii ST7 (Table 3 and Fig. 1B). DISCUSSION

Following the application of the criteria described by Kilian et aL (1989), we found that 41 of 42 s e r o v a r - K T H strains were identified as typical Strep. oralis. In the soft-agar technique, Strep. oralis strains (except serovar KTH-1) reacted with the anti-Strep. oralis A T C C 10557 serum, although most strains did not react with the same antiserum in Ouchterlony's

agar diffusion (Isogai et al., 1990). Some u n c o m m o n serotypes of 'Strep. sanguis' might be due to an ambiguous description of Strep. oralis. However, the serovar-KTH-1 strain identified as Strep. gordonii shows that the species sometimes has antigens reacting with anti-Strep, oralis KTH-1 serum. Reference strains ST3 and ST7 were originally used for serogrouping (Torii, 1978; H a m a d a et al., 1980), and later in the study of Beh~et's disease (Mizushima et al., 1989; Isogai et al., 1991; Lehner et al., 1991). We identified strain ST3 as Strep. sanguis biovar 1 as well as the type strain A T C C 10556. Strain ST7 was identified as Strep. gordonii biovar 2 or 3, both sharing the same differential characteristics. As serovar-III 'Strep. sanguis' ( A T C C 10558) is Strep.

Streptococcus oralis in Behqet's disease gordonii biovar 2, ti~e different serovar-IV Strep. gordonii ST7 is assumed to be biovar 3. A previous report suggested that anti-Strep, oralis A T C C 10557 serum did not react with 9 of 18 test strains of Strep. oralis (Kilian et al., 1989). We obtained similar results where anti-Strep, gordonii ST7 serum reacted with Strep. oralis serovar KTH-1, but anti-ATCC 10557 serum did not. We therefore introduced a soft-agar technique for dividing Strep. oralis strains broadly into two groups with antisera against A T C C 10557 and ST7. Kilian et al. (1989) differentiated certain oral streptococci by assaying IgA1 protease and neuraminidase, both of which were regarded as virulence factors. IgA1 proteases are extracellular enzymes that cleave serum IgA1 and, to a limited extent, secretory IgA (Plaut, Gilbert and Wister, 1977; Kilian and Holmgren, 1981). The cleavage of IgA1 by enzyme results in a marked decrease in antibody activity (Plaut et al., 1977). Neuraminidases, which cleave the ct-ketosidic linkage between sialic acids and the glycosyl residues of glycoproteins, glycolipids or colominic acids (Moncla and Braham, 1989), have also been considered to function as virulence factors in mucosal commensals and pathogens (Varki and Diaz, 1983; Moncla and Braham, 1989). As sialic acid plays a part in the host defence mechanisms such as immunoglobulins, leucocytes, erythrocytes and mucus, bacterial neuraminidases might affect the response to infection (Moncla, Braham and Hillier, 1990). Bacterial antigens inducing Behget's disease (Mizushima et al., 1989; Pervin et al., 1993) might penetrate the oral mucosa, where they would encounter the innate host defence mechanisms. We have confirmed th~.t the frequency of isolation of Strep. oralis in plaque flora is higher in patients than controls. However, we could not distinguish between the isolates from patients and controls. The bacterial antigen, of Strep. oralis might be effective in penetrating the mucosal barrier, eliciting a specific immune response and inducing Beh~et's disease. REFERENCES

Bridge P. D. and Sneath P. H. A. (1982) Streptococcus gallinarum sp. nov. and Streptococcus oralis sp. nov. Int. J. syst. Bacteriol. 32, 410-415. Chomarat M., Ichiman Y. and Yoshida K. (1985) Pseudodiffuse-type growth of a Staphylococcus aureus strain in serum-soft agar. J. clin. Microbiol. 22, 132-133. Finkelstein R. A. and Sulkin S. E. (1958) Characteristics of coagulase positive and coagulase negative staphylococcus in serum-soft agar. ,L Bacteriol. 75, 339-344. French G. L., Talsania H., Charlton J. R. H. and Phillips I. (1989) A physiolog;Lcalclassification of viridans streptococci by use of the A]PI-20STREP system. J. reed. Microbiol. 28, 275-286. Hamada S. and Torii M. (1978) Effect of sucrose in culture media on the location of glucosyltransferase of Streptococcus mutans and cell adherence to glass surfaces. Infect. Immun. 20, 592-599. Hamada S., Torii M., Tsuchitani Y. and Kotani S. (1980) Isolation and immunological classification of Streptococcus sanguis from human tooth surface. J. clin. Microbiol. 12, 243-249. Hardie J. M. (1986) Oral streptococci. In Bergey's Manual of Systematic Bacteriology (Eds Sneath P. H. A., Mair

689

N. S., Sharpe M. E. and Holt J. G.), Vol. 2, pp. 1054-1063. The Williams & Wilkins Co., Baltimore. Ichiman Y., Kono E., Kushiro H., Ito S. and Yoshida K. (1981) Serological differentiation of strains of group B streptococci by the soft-agar technique. Zbl. Bakt. Hyg., L Abt. Orig. A. 249, 184-189. Isogai E., Ohno S., Kotake S., Isogai H., Tsurumizu T., Fujii N., Yokota K., Syuto B., Yamaguchi M., Matsuda H. and Oguma K. (1990) Chemiluminescence of neutrophils from patients with Behqet's disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora. Archs oral Biol. 35, 43-48. Isogai E., Isogai H., Yokota K., Hayashi S., Fujii N., Oguma K., Yoshikawa K., Sasamoto Y., Kotake S. and Ohno S. (1991) Platelet aggregation induced by uncommon serotypes of Streptococcus sanguis isolated from patients with Beh~et's disease. Archs oral Biol. 36, 425-429. Kilian M. and Holmgren K. (1981) Ecology and nature of immunoglobulin A1 protease-producing streptococci in the human oral cavity and pharynx. Infect. lmmun. 31, 868-873. Kilian M., Mikkelsen L. and Henrichsen J. (1989) Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended description of Streptococcus sanguis (White and Niven, 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes and Horder, 1906). Int. J. syst. Bacteriol. 39, 471-484. Kilpper-B/ilz R., Wenzig P. and Schleifer K. H. (1985) Molecular relationship and classification of some viridans streptococci as Streptococcus oralis and emended description of Streptococcus oralis (Bridge and Sneath, 1982). Int. J. syst. Bacteriol. 35, 482-488. Lehner T., Lavery E., Smith R., van der Zee R., Mizushima Y. and Shinnick T. (1991) Association between the 65kilodalton heat shock protein, Streptococcus sanguis, and the corresponding antibodies in Beh~et's syndrome. Infect. Immun. 59, 1434-1441. Martin D. K., Nelms D. C., Mackler B. F. and Peavy D. L. (1979) Lymphoproliferative responses induced by streptococcal antigens in recurrent aphthous stomatitis and Behqet's syndrome. Clin. Immun. Immunopathol. 13, 146-155. Mizushima Y., Hoshi K., Matsuda T., Takahashi M., Takada H., Kuribayashi S., Hamada S., Hashimoto T., Takeuchi A., Ichikawa Y., Arimori S., Ujihara H., Miyanaga Y., Kogure M., Wakatsuki F., Ohno S. and Sasamoto Y. (The Behqet's Disease Research Committee of Japan) (1989) Skin hypersensitivity of streptococcal antigen and the induction of systemic symptoms by the antigens in Behqet's disease---A multicenter study. J. Rheumatol. 16, 506-511. Moncla B. J. and Braham P. (1989) Detection of sialidase (neuraminidase) activity of Actinomyces species by using 2'-(4-methylumbelliferyl)ct-D-N-acetylneuraminic acid in a filter paper spot test. J. clin. Microbiol. 27, 182-184. Moncla B. J., Braham P. and Hillier S. L. (1990) Sialidase (neuraminidase) activity among Gram-negative anaerobic and capnophilic bacteria. J. clin. Microbiol. 28, 422-425. Okonogi H., Nakai M., Shimizu H., Futakami K., Tsurumizu T., Hashimoto T., Isogai E. and Sato M. (1988) Characteristics of Streptococcus sanguis isolated in Behqet's disease. J. den. Hlth 38, 562-563. Plaut A. G., Gilbert J. V. and Wister R. Jr (1977) Loss of antibody activity in human immunoglobulin A protease of Neisseria gonorrhoeae and Streptococcus sanguis. Infect. Immun. 17, 130-135. Pervin K., Childerstone A., Shinnick T., Mizushima Y., van der Zee R., Hasan A., Vaughan R. and Lehner T. (1993) T cell epitope expression of mycobacterial and homologous human 65-kilodalton heat shock protein peptides in

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S. Narikawa et al.

short term cell lines from patients with Behget's disease. J. Immunol. 151, 2273-2282. de Stoppelaar J. D., Houte J. V. and de Moor C. E. (1967) The presence of dextran-forming bacteria, resembling Streptococcus sanguis, in human dental plaque. Archs oral Biol. 12, 1199 1201. Suzuki Y., Ishimaru A., Nakagawa M., Aoyama E., Narikawa S., Hoshi K. and Mizushima Y. (1991) Response of peripheral blood lymphocytes to bacterial antigens in patients with Behqet's disease. Annual Report of Beh¢et's Disease Research Committee of Japan (Ed. Mizushima Y.), pp. 44-46 and 167. The Ministry of Health and Welfare, Tokyo. Suzuki Y., Hoshi K., Matsuda T. and Mizushima Y. (1992) Increased peripheral blood 76 + T cells and natural killer cells in Beh~et's disease. J. Rheumatol. 19, 588-592.

Torii M. (1978) Isolation and classification of Streptococcus sanguis from human tooth surfaces and their adherence ability to smooth glass surfaces: I. Serological and biological typing of the isolated strains and cariogenicity of the selected strains in SPF rat. Jap. J. oral Biol. 20, 341-349. Varki A. and Diaz S. (1983) A neuraminidase from Streptococcus sanguis that can release o-acetylated sialic acids. J. biol. Chem. 258, 12,465-12,471. Whiley R. A., Fraser H. Y., Doublas C. W. I., Hardie J. M., Williams A. M. and Collins M. D. (1990) Streptococcus parasanguis sp. nov., an atypical viridans Streptococcus from human clinical specimens. FEMS microbiol. Lett. 68, 115 122. Yoshida, K. (1971) Demonstration of serologically different capsular types among strains of Staphylococcus aureus by the serum-soft agar technique. Infect. lmmun. 3, 535-539.