Microbiol. Immunol., 44(12), 981-985, 2000

Serological Properties of and (Nutritionally Variant Streptococci)

Katsuhiro Kitada, Yasuko Okada, Taisei Kanamoto*, and Masakazu Inoue*

Department of Preventive Dentistry, Kagoshima University Dental School, Kagoshima, Kagoshima 890-8544, Japan

Received May 11, 2000; in revised form, August 15, 2000. Accepted September 9, 2000

Abstract: Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6-dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three-quarters of the , and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I-1 and/or I-5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype 1-5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Gran- ulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme-producing but vitamin B6-independent strains of , Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.

Key words: Abiotrophia sp., Granulicatella spp., Nutritionally variant streptococci, Serotyping

NVS comprise a part of the normal flora in the human gans (15) and Abiotrophia balaenopterae (11) have been mouth and are causative agents of reported, and we have proposed the presence of and other systemic infections (4, 16). They require vita- Abiotrophia paraadiacens sp. nov., which is genetical- min B6 analogs for growth and produce bacteriolyitc ly closely related to but distinct from A. adiacens (9). A enzyme (8, 13), and were originally assigned to aux- very recent study (3) has formally proposed that the otrophic variants of various (16). Abiotrophia is restricted to the type species A. NVS were once divided into 3 biotypes (2) but geneti- defectiva, and that A. adiacens, A. elegans and A. bal- cally into 2 species, Streptococcus defectivus and Strep- aenopterae are reclassified in a new genus, Granuli- tococcus adjacens (1). An earlier 16S rRNA gene catella gen. nov., as G. adiacens, G. elegans and G. sequence study determined the phylogenetic position balaenopterae. of NVS in the genus Streptococcus and proposed that Serological classification of microorganisms provides NVS be placed in a new genus, Abiotrophia, as important information in microbial , and the Abiotrophia defectiva and Abiotrophia adiacens (10). simple and rapid serotyping method is a little classical but In recent genetic studies, the new species Abiotrophia ele- a useful tool in ecological and epidemiological studies to determine sources or routes of transmission in systemic *Address correspondence to Dr. Masakazu Inoue, Department infections. NVS (i.e., Abiotrophia and Granulicatella of Preventive Dentistry, Kagoshima University Dental School, 8- spp.) have been reported to carry no Lancefield group 35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan. Fax: + 81-99-275-6188. E-mail: [email protected] Abbreviations: ATCC, American Type Culture Collections; u.ac.jp CCUG, Culture Collection University of Goteborg; DSM, *Present address: Department of Microbiology and Immunol- Deutsche Sammlung von Mikroorganismen; NCFB, National ogy, Kagoshima University Dental School, Kagoshima, Kago- Collections of Food ; NCTC, National Collection of shima 890-8544, Japan. Type Cultures; NVS, nutritionally variant streptococci.

981 982 K. KITADA ET AL antigens (16) and are divided into 3 serotypes (20). A. defectiva, G. adiacens, G. paraadiacens, G. elegans However, many streptococcus species are known to and G. balaenopterae, and 91 clinical isolates of the show large serological variety, whereas serotyping of Abiotrophia and Granulicatella spp. in our laboratory (8). the Abiotrophia and Granulicatella spp. has not been A. paraadiacens, a close relative to A. adiacens as pre- attempted extensively. We have recently isolated a large viously reported (9), is listed in the table tentatively as G. number of strains of the clinically important, unique paraadiacens although it has not been included in the cocci from the human mouth (8). The present study study for the proposal of the new genus Granulicatella demonstrates wide serological variations among (3). Thirty-three strains of 11 species in 5 related genera Abiotrophia and Granulicatella spp. were also used: the vitamin B6-independent but bacte- riolytic enzyme-producing strains of Streptococcus, Materials and Methods Enterococcus, Dolosigranulum and Aerococcus, the ref- erence strains of 18 serotypes in the Streptococcus mil- Bacterial strains. The bacterial strains employed are leri (S. anginosus) group (7), and the strains of Gemella. summarized in Table 1. A total of 103 NVS strains Most of the Abiotrophia and Granulicatella strains were used. They included 12 type or reference strains of used had been biotyped (8, 17) and genotyped (9), and

Table 1. List of the bacterial strains used

°' M. luteus cell-lytic enzyme production: positive (+) or negative (-). b'Strains provided by: #1 , American Type Culture Collections (Rockville, Md., U.S.A.); #2, National Collection of Type Cultures (London, U.K.); #3, National Collections of Industrial and Marine Bacteria (Aberdeen, U.K.); #4, Deutsche Sammlung von Mikro-organismen and Zellkulturen GmbH (Braunschweig, Germany); #5, Dr. A. Bouvet (Assistance Publique, Hopitaux de Paris, Paris, France); #6, Dr. I. van de Rijn (Wake Forest University Med- ical Center, Winston-Salem, N.C., U.S.A.); #7, Dr. E. Falsen (Culture Collection, University of Goteborg, Goteborg, Sweden); #8, Dr. S. Hamada (Osaka University Dental School, Osaka, Japan); #9, Dr. H. Takada (Tohoku University School of Dentistry, Sendai, Japan); and (7) and (8), stock cultures or clinical isolates in our laboratory. SEROTYPE OF ABIOTROPHIA AND GRANULICATELLA SPP. 983 then classified into 4 species, A. defectiva, A. adiacens, and then serum was separated by centrifugation (1,700 X A. paraadiacens or A. elegans. Strain ATCC 27527 is g, 20 min, 4 C). When required to prepare a serotype- listed as Gemella morbillorum in the current ATCC cat- specific antiserum, 1 ml of a crude antiserum was added alog but has been reclassified physiologically and genet- to 50 mg (dry weight) of whole cells of the cross-react- ically as A. (G.) paraadiacens (9). Some other strains ing strain(s) and incubated with occasional shaking at 37 were examined first for vitamin B6 dependency for C for 1 hr and at 4 C overnight. Finally, the cells were growth, Micrococcus luteus cell-lytic activity, and chro- removed by centrifugation (11,000 X g, 5 min, 4 C). mophore production, as well as being examined in a The absorption procedures were repeated until all non- Rapid ID32 STREP System (ver. 1.0; Bio Merieux SA, specific antibodies were removed. The antiserum was Marey-1'Etoile, France) as described previously (8). concentrated by ultrafiltration if necessary. They were then classified as mentioned above. G. bal- Preparations of Lancefield group A to F antisera were aenopterae CCUG 37380T was confirmed to be physio- purchased from Difco Laboratories (Detroit, Mich., logically different from the other Abiotrophia and Gran- U.S.A.). The 17 typing antisera specific to S. milleri ulicatella spp. and shown to be a bacteriolytic enzyme serotypes (a to k, Osano-I to -III and Ottens-I to -IV) non-producer (unpublished). were described previously (7). The Abiotrophia and Granulicatella strains were Preparation of antigen extracts. Cell surface antigens grown anaerobically (10% H2+ 10% CO, +80% N,) at were extracted from the lyophilized whole cells in saline 37 C for 18 hr in Todd-Hewitt broth (BBL Microbiology (20 mg/ml) by autoclaving as described by Rantz and Systems, Cockeysville, Md., U.S.A.) supplemented with Randall (14) (designated R-R extracts). R-R extracts 0.001% pyridoxal HC1 or occasionally with 5% horse from S. milleri group strains were prepared previously serum, and strains of 11 other species of the 5 genera in (7). The Lancefield group A to G antigen preparations Todd-Hewitt broth. Cells were harvested by centrifu- were purchased from Difco Laboratories. gation (6,000 X g, 20 min, 4 C), washed 3 times in dis- Immunodiffusion. Reactivity of antigen extracts with tilled water, and lyophilized. the typing antisera was examined in a capillary precipitin Preparation of typing antisera. A total of 12 test. The double-immunodiffusion analysis was per- Abiotrophia and Granulicatella strains were selected as formed in a 1.2% Noble Agar gel (Difco Laboratories) in immunogens to prepare serotyping antisera (Table 2). 0.01 M phosphate buffer, pH 7.2. Cells of the strains were suspended to a concentration of 1.0 mg (dry weight)/ml in saline containing 0.05% NaN,. Results and Discussion Rabbits (female, 2.5-3.0 kg; Japan SLC, Hamamatsu, Japan) were given intravenous injections of whole-cell Immune sera were prepared by immunizing rabbits suspensions for 4 weeks, during which 0.5 ml was inject- with whole cells of 6 types or reference strains and 6 oral ed on alternate days on the 1st week (3 times), then a 1- isolates of Abiotrophia and Granulicatella spp. The week rest, and 1.0 ml in the 3rd and 4th weeks (6 times). antisera against 2 G. adiacens strains and those against 2 Blood was collected usually 2 days after the last injection G. elegans strains were shown to be identical, respec-

Table 2. The type strains and typing antisera°' of the serotypes in Abiotrophia and Granulicatella species

°' The anti-G . adiacens G40 serum was serologically identical to the anti-C50 serum, and the anti-G. elegans YTM1 serum to the anti-HHC5 serum. 11Not absorbed . 984 K. KITADA ET AL tively. Thus, 10 specific antisera were obtained (Table 2), able. The G. balaenopterae type strain was not serotyp- each of which produced only a single precipitin line able. The A. defectiva strains were all serotype I, sug- with its homologous antigen in immunodiffusion (Fig. gesting that strains of the species or the genus 1). None of the serotyping antiserum preparations were Abiotrophia have a common antigen. Several A. defec- reactive with the Lancefield group A to G antigen prepa- tiva serotype I strains were divided further into sub- rations, nor the Lancefield group A to F specific antisera serotype I-1 and/or 1-5. The G. adiacens strains were with R-R extracts of the serotype reference strains, indi- generally serotype II or III and the G. paraadiacens cating that the 10 Abiotrophia and Granulicatella strains were serotype IV, V or VI. All G. adiacens and serotypes are independent from these Lancefield G. paraadiacens serotype II strains were also subserotype serogroups. I-5. The G. elegans strains were serotype VII or VIII. These typing antisera divided 72 of the 103 strains As far as we have tested, the serotypes of Abiotrophia tested into major serotypes I through VIII, and 24 of the and Granulicatella spp. were undetectable among strains 31 serotype I and II strains into subserotypes I-1 and/or of the other streptococcus species and related genera. 1-5 (Table 3). All A. defectiva strains, all except one G. The M. luteus cell-lytic enzyme-producing strains of elegans strain, three-quarters of the G. adiacens strains, Lancefield group D enterococci (E. faecalis, E. hirae, E. and half of the G. paraadiacens strains were serotyp- durans), D. pigrum, A. urinae, and S. intermedius (8) had no antigen reactive with the Abiotrophia and Granuli- catella serotyping and subtyping antisera. Neither did the vitamin B6-independent, bacteriolytic enzyme non-pro- ducing bona fide G. morbillorum strains, which are bio- chemically closely related to some G. elegans strains (Gemella-like NVS) (8), nor G. haemolysans strains. In addition, antigen extracts from the reference strains of 18 S. milleri group serotypes (normally 9, 2 and 7 serotypes in S. anginosus, S. constellatus and S. inter- medius, respectively) (7) did not react with any of the 10 typing antisera. Extracts of the 10 Abiotrophia and Granulicatella serotype or subserotype reference strains were not reactive with any of the 17 S. milleri group Fig. 1. Specificity of one typing antiserum for serologically dif- serotyping antisera (7) tested. Thus, the Abiotrophia ferentiating Abiotrophia and Granulicatella strains. In the immun- and Granulicatella serotypes and subserotypes are con- odiffusion in agar gel, the serotype II typing antiserum (wells A) sidered to be independent from the 18 S. milleri group reacted only with R-R extracts from the homologous vaccine serotypes. strain G. adiacens ATCC 491757 (serotype II). Wells 1 to 11: R- Van de Rijn and his colleagues have divided NVS R extracts of the 10 vaccine strains and 1 other. Well 1, NVS-47 strains into 3 serotypes, I to III (20). We demonstrated (serotype I); 2, ATCC 491757 (serotype II); 3, C50 (serotype here that Abiotrophia and Granulicatella spp. are sero- III); 4, HKTI-1 (serotype IV); 5, HHT2 (serotype V); 6, TKTI logically more variable. The van de Rijn serotype I (serotype VI); 7, HHC5 (serotype VII); 8, DSM 11693 (serotype VIII); 9, ATCC 491767 (subserotype 1-1); 10, YK3 (subserotype and II antigens are shown to be ribitol teichoic acid 1-5); 11, ATCC 27527 (serotype V). containing galactose, or galactose and galactosamine

Table 3. Distribution of various serotypes among the Abiotrophia and Granulicatella species

°' Untypable . ]: Double count of the numbers with asterisk. " Four of the A. defectiva strains were also reactive with the serotype 1-5 or I-1 subtyping antiserum or both. mThe 20 G. adiacens or G. paraadiacens strains were also reactive with the serotype I-5 subtyping antiserum. SEROTYPE OF ABIOTROPHIA AND GRANULICATELLA SPP. 985

(5, 6, 18), and the chemical structure of the type II anti- Isolation and properties of bacteriolytic enzyme-producing gen has been reported (19). It is possible that the van de cocci from the human mouth. FEMS Microbiol. Lett. 144: Rijn serotype I antigen (5) and our serotype I antigen are 135-140. identical, both being found with A. defectiva NVS-47. 9) Kanamoto, T., Sato, S., and Inoue, M. 2000. Genetic het- However, association between their serotypes I to III erogeneities and phenotypic characteristics of strains of the genus Abiotrophia and proposal of Abiotrophiaparaadiacens and the 10 serotypes or subserotypes we demonstrated sp. nov. J. Clin. Microbiol. 38: 492-498. here requires further study. 10) Kawamura, Y., Huo, X.G., Sulutana, F., Liu, S., Yamamoto, The serological typing of Abiotrophia and Granuli- H., and Ezaki, T. 1995. Transfer of Streptococcus defec- catella isolates from various clinical specimens would be tives and Streptococcus adjacens to Abiotrophia gen. nov. as useful, for example, as a clue to evaluate a potent risk of Abiotrophia adiacens comb. nov. and Abiotrophia defectiva bacterial endocarditis. Indeed, the Abiotrophia and comb. nov., respectively. Int. J. Syst. Bacteriol. 45: 798- Granulicatella strains of only serotypes I, II and 111, 803. 11) Lawson, P.A., Foster, G., Falsen, E., Sjoden, B., and Collins, and none of the other serotypes, are generally highly M.D. 1999. Abiotrophia balaenopterae sp. nov., isolated infective in the experimental bacterial endocarditis model from the minke whale (Balaenoptera acutorostrata). Int. J. in rats (12). Syst. Bacteriol. 49: 503-506. 12) Okada, Y., Kitada, K., Takagaki, M., Ito, H.-O, and Inoue, M. References 2000. Endocardiac infectivity and binding to extracellular matrix proteins of oral Abiotrophia species. FEMS Immunol. 1) Bouvet, A., Grimont, F., and Grimont, P.A.D. 1991. Med. Microbiol. 27: 257-261. Intraspeciesvariations in nutritionallyvariant streptococci: 13) Pompei, R., Caredda, E., Piras, V., Serra, C., and Pintus, L. rRNAgene restrictionpatterns of Streptococcus defectivus 1990.Production of bacteriolytic activity in the oral cavity by and Streptococcusadjacens. Int. J. Syst.Bacteriol. 41: 483- nutritionally variant streptococci. J. Clin. Microbiol. 28: 486. 1623-1627. 2) Bouvet,A., Villeroy, F., Cheng,F., Lamesch, C., Williamson, 14) Rantz, L.A., and Randall, E. 1955. Use of autoclaved extracts R., and Gutmann,L. 1985.Characterization of nutritionally of hemolytic streptococci for serological grouping. Stan- variant streptococciby biochemicaltests and - ford Med. Bull. 13: 290-291. bindingprotein. J. Clin. Microbiol.22: 1030-1034. 15) Roggenkamp, A., Abele-Horn, M., Trebesius, K.-H., Tretter, 3) Collins, M.D., and Lawson, P.A. 2000. The genus U., Autenrieth, LB., and Heesemann, J. 1998. Abiotrophia Abiotrophia(Kawamura et al.)is not monophyletic:proposal elegans sp. nov., a possible pathogen in patients with culture- of Granulicatellagen. nov.,Granulicatella adiacens comb. negative endocarditis. J. Clin. Microbiol. 36: 100-104. nov., Granulicatellaelegans comb. nov. and Granulicatella 16) Ruoff, K.L. 1991. Nutritionally variant streptococci. Clin. balaenopteraecomb. nov.Int. J. Syst. Evol.Microbiol. 50: Microbiol. Rev. 4: 184-190. 365-369. 17) Sato, S., Kanamoto, T., and Inoue, M. 1999.Abiotrophia ele- 4) George,R.H. 1974. The isolationof symbioticstreptococci. gans strains comprise 8% of the nutritionally variant strep- J. Med. Microbiol.7: 77-83. tococci isolated from the human mouth. J. Clin. Microbiol. 5) George,M., and vande Rijn,I. 1988.Purification of Serotype 37:2553-2556. I antigen from nutritionally variant streptococci. Infect. 18) Sieling, P.A., and van de Rijn, 1. 1991. Purification and Immun.56: 1222-1231. characterization of Streptococcus adjacens (nutritionally 6) George,M., and van de Rijn, I. 1988..Nutritionally variant variant streptococcus serotype II) group antigen. Infect. streptococcal serotype I antigen. Characterization as a Immun. 59: 592-599. lipid-substitutedpoly (ribitolphosphate). J. Immunol.140: 19) Sieling, P.A., Thomas, M.J., and van de Rijn, I. 1992. Char- 2008-2015. acterization of the Streptococcus adjacens group antigen 7) Inoue, M., Eifuku-Koreeda,H., Kitada, K., Takamatsu- structure. J. Bacteriol. 174: 349-354. Matsushita,N., Okada, Y., and Osano, E. 1998. Serotype 20) van de Rijn, I., and George, M. 1984. Immunochemical variationin Streptococcusanginosus, S, constellatus and S. study of nutritionally variant streptococci. J. Immunol. 133: intermedius.J. Med. Microbiol.47: 435-439. 2220-2225. 8) Kanamoto,T., Eifuku-Koreeda,H., and Inoue, M. 1996.