Microbiol. Immunol., 44(12), 981-985, 2000 Serological Properties of Abiotrophia and Granulicatella Species (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 Granulicatella adiacens, 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 Streptococcus, 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 infective endocarditis 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 viridans streptococci (16). genus 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 taxonomy, 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 Bacteria; 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