New group D streptococcal species

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1 Indian J Med Res 119 (Suppl) May 2004, pp New group D streptococcal species Laurent Schlegel *, Francine Grimont, Patrick A. Grimont & Anne Bouvet* * Centre National de Référence des Streptocoques, Service de Microbiologie, Hôtel Dieu AP-HP, Université Paris V & Unité Biodiversité des Bactéries Pathogènes Emergentes, INSERM U389, Institut Pasteur, Paris, France Received August 6, 2003 Background & objectives: The heterogeneity of group D streptococci led to the identification of various biotypes of Streptococcus equinus and Streptococcus bovis and to the description of new species. The objective of the present study was to improve the phenotypic delineation between species and to clarify their respective phylogenetic position. Methods: Physiological and genomic analyses were carried out in 84 representative strains of the group D streptococci. Biotypes were determined with the API 20 strep and rapid ID 32 STREP systems of identification. Quantitative DNA-DNA hybridization under stringent conditions and values of the DT m allowed to delineate species and subspecies. The phylogenic position of the different genomic groups was determined by comparing the sequences of their 16S rdna. Results: Four DNA-clusters, including seven species or subspecies, were characterized. Differential associations of biochemical characters allowed their identification. S. equinus and the type strain of S. bovis belonged to a single species. S. gallolyticus, S. bovis biotype II.2, and S. macedonicus formed a single DNA-cluster including three different subspecies. These were designated as S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, and S. gallolyticus subsp. macedonicus. The two other DNA-clusters corresponded to the two subspecies of S. infantarius, and to S. alactolyticus. Interpretation & conclusion: This study presented a new classification associated with an identification scheme of group D streptococci. The changes in this classification demonstrate the interest of a polyphagic approach of the bacterial identification. Key words Group D streptococci - Streptococcus bovis - S. equinus - S. gallolyticus - S. infantarius - taxonomy Group D streptococci, the common inhabitants of the intestinal flora of vertebrates, were first identified by their antigenic and biochemical characteristics 1,2. Genomic studies confirmed the separation of enterococci from non enterococci 3,4. The group D streptococcal strains were then commonly designated as S. equinus or S. bovis depending on their origin and on their biochemical characteristics. Identification of S. bovis strains was based on their phenotype. Biotypes I and II designated mannitol-positive and mannitol-negative strains, respectively. Biotype II included two different phenotypes according to the presence (biotype II.2) or absence (biotype II.1) of b-glucuronidase In 1984, a genomic classification of the S. equinus / S. bovis complex delineated six different DNAgroups 6. The group 1 included both S. equinus and some biotype II.1 S. bovis strains. The groups 2, 3, and 4 corresponded to different unnamed genospecies. The group 6 was called S. alactolyticus, whereas the group 5 (S. saccharolyticus) was further included in the Enterococcus genus 7. Correlation studies between the genomic classification and the biotypes led to identify phenotypic variants, which were progressively described as individual species or subspecies. The DNA-group 2 (S. bovis biotype I) was

2 called S. gallolyticus 8 ; it is associated with endocarditis and colonic carcinoma 9. The DNA-group 4 was called S. infantarius 10 ; it includes two subspecies of medical importance 11. Two additional species S. macedonicus 12 and S. waius 13 were further described. Using DNA-DNA hybridization and 16S rdna sequencing, we confirmed that S. macedonicus and S. waius were subjective synonyms, and demonstrated that S. bovis biotype II.2 and S. macedonicus were subspecies of a same DNA-cluster, which included the previously described S. gallolyticus 14. Here, we reappraise the classification of the S. bovis/s. equinus complex with the objective to improve the phenotypic delineation between species and to clarify their respective phylogenetic position. Material & Methods Representative strains (n=84) of the DNA-groups 1, 2, 4, and 6, and of the new species were obtained from various collections. They were identified as group D streptococci by conventional methods 15. Production of enzymes and fermentation of carbohydrates were determined using both the Api 20 Strep and rapid ID32 STREP identification systems (BioMérieux, France). Hydrolysis of methyl-gallate was assayed by the colorimetric test 8. The DNA was extracted and purified using the phenol/chloroform method 16. DNA from type or reference strains were labeled by using the Megaprim DNA labeling reaction kit (Amersham, UK). Quantitative DNA-DNA hybridization was carried out in a liquid medium under stringent conditions of 60 C for 16 h, according to a modification of the S1-nuclease/ trichloracetic acid precipitation method 17. The temperature (T m ), at which 50 per cent of the reassociated DNA is hydrolysed by the S1-nuclease, was determined. The difference (DT m ) between the melting temperatures of homo- and heteroduplexes allowed to quantify the DNA divergence between strains with high level of DNA homology 17. Polymerase chain amplification of 16S rrna encoding DNA, and sequencing of amplified fragments were carried out as previously described 18. Alignment SCHLEGEL et al: GROUP D STREPTOCOCCI 253 with a selection of the available sequences of 16S rdna from GeneBank and phylogenetic analysis of the 16S rdna data were performed by using the Clustal multiple sequence method with the Megalign program from the DNAstar package. A distance matrix was computed using a Kimura model for nucleotide substitution. A phylogenetic tree was generated from the distance matrices using the neighbour joining method. Results & Discussion Phenotypic characterization: The phenotypic tests confirmed that all strains were group D streptococci. None of them grew on bile-esculine agar and in 6.5 per cent NaCl broth. They produced leucine aminopeptidase, alanyl-phenylalanyl-proline arylamidase, and acetoin. They did not produce pyrrolidonyl-arylamidase and arginine dihydrolase. Our results confirmed that S. equinus and S. bovis differed by classical characters. The species and subspecies exhibited noticeable differences (Table). The major differential associations were: (i) acidification of mannitol and hydrolysis of gallate by S. gallolyticus ; (ii) presence of b-glucuronidase and b-mannosidase, and acidification of trehalose by S. gallolyticus subsp. pasteurianus (S. bovis biotype II.2); (iii) absence of b-glucosidase and b-glucuronidase, with presence of b-galactosidase by S. gallollyticus subsp. macedonicus; (iv) absence of b-glucosidase by most strains of S. infantarius subsp. infantarius, associated with the absence of b-glucuronidase and b-galactosidase, and with the acidification of glycogen and raffinose; (v) presence of b-glucosidase, and acidification of methyl-b-glucopyranoside, but not of glycogen and raffinose by S. infantarius subsp. coli; (vi) absence of acidification of most of the carbohydrates, including lactose by S. alactolyticus. DNA-DNA hybridization: The existence of four DNA homology groups within the S. bovis/s. equinus complex was demonstrated. DNA-cluster I is formed by the strains of S. equinus and the type-strain of S. bovis (91 to 100% DNA homology) Type strains of the former S. gallolyticus, S. macedonicus, and S. waius species, and the strains of S. bovis biotype II.2 belonged to the same DNA-cluster II. This cluster contained three different subgroups. The level of homology between strains was over 77 per cent (DT m 2 C) within each subgroup, and ranged from 48 to 93 per cent (DT m 6 C)

3 254 INDIAN J MED RES (SUPPL) MAY 2004 Table. Differential biochemical characteristics of group D streptococcal strains S. equinus S. gallolyticus subsp. S. infantarius subsp. S. alactolyticus / S. bovis gallolyticus pasteurianus macedonicus infantarius coli No. of strains n = n = 13 n = 21 n = 5 n =10 n = 14 n = 4 Hydrolysis of Aesculine V + + Gallate Production of ß-glucosidase V + + ß-glucuronidase a-galactosidase - / + + v v ß-galactosidase (ß-GAR) ß-galactosidase (ß-GAL) v ß-mannosidase - V Acidification of Starch - / v - Glycogen - / Inulin - / Lactose - / Mannitol Meth-ß-D-glucopyranoside V Raffinose - / + + v Trehalose v , ³ 80% of positive reactions; -, 20% of positive reaction; v, 21 to 79% of positive reactions

4 between the different subgroups. The first subgroup includes the reference strain of the DNA-group 2 of Farrow et al 6, and the type strains of S. gallolyticus and S. caprinus. The second subgroup included the type strains of S. macedonicus and S. waius. The last subgroup was formed by S. bovis II.2, including the type strain of S. pasteurianus described independently as a new species 19. The DT m 6 C indicating the strong affinity of heteroduplexes constructed with the strains of the different subgroups, we proposed to name the three subspecies: S. gallolyticus subsp. gallolyticus, macedonicus, and pasteurianus. The DNA-cluster III included S. infantarius subsp. infantarius and subsp. coli. The DNA-cluster IV corresponded to S. alactolyticus. 16S rdna sequencing and phylogenetic analysis: We added in the Genbank databank the sequences of 16S rdna that we have determined 14. The 99 per cent similarity of the sequence of 1455 base fragments of the type strains of S. equinus and S. bovis confirmed them to belong to the same species. Similarly, the 99.6 per cent homology between the two type strains of S. alactolyticus 6 and S. intestinalis 20 was in agreement with their closely related whole-cell proteins patterns 21. We constructed a phylogenetic tree from the sequences of 350 to 400 bp from our results and from those from previouly described type strains (Fig.). Three major divisions corresponding to the four DNA-clusters were observed. The first division included the type strains of S. equinus and S. bovis (DNA-cluster I), and the two subspecies of S. infantarius (DNA-cluster III). The second division includes the three subspecies of Fig. Simplified phylogenetic tree of group D streptococci based on comparative analysis of bp 16S rdna sequences. SCHLEGEL et al: GROUP D STREPTOCOCCI 255 S. gallolyticus (DNA-cluster II). Within this cluster II, the divergence between the 16S rdna sequences from the type or reference strains of S. gallolyticus, S. macedonicus, and S. bovis biotype II.2 were in agreement with the delineation of the three subspecies, whereas the 99.5 per cent similarity of sequences between S. waius and S. macedonicus confirmed that they belonged to a single genomic group, as recently reported by Manachini et al 22. The third division included S. alactolyticus and the subjective synonym S. intestinalis (DNA-cluster IV). This study presents a new classification associated with an identification scheme of group D streptococci. Seven species or subspecies could be identified according to differential biochemical reactions. The changes in this classification of the S. bovis/s. equinus complex demonstrate the interest of a polyphasic approach of the bacterial identification. References 1. Lancefield RC. A serological differentiation of human and other groups of haemolytic streptococci. J Exp Med 1933; 57 : Facklam RR. Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl Microbiol 1972; 23 : Kilpper-Bälz R, Fischer G, Schleifer KH. Nucleic acid hybridization of group N and group D streptococci. Curr Microbiol 1982; 7 : Bentley RW, Leigh JA, Collins MD. Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rrna sequences. Int J Syst Bacteriol 1991; 41 : Coykendall AL, Gustafson KB. Deoxyribonucleic acid hybridizations among strains of Streptococcus salivarius and Streptococcus bovis. Int J Syst Bacteriol 1985; 35 : Farrow JAE, Kruze J, Phillips BA, Bramley AJ, Collins MD. Taxonomic studies on Streptococcus bovis and Streptococcus equinus: description of Streptococcus alactolyticus sp. nov. and Streptococcus saccharolyticus sp. nov. System Appl Microbiol 1984; 5 : Rodriguez U, Collins MD. Phylogenic analysis of Streptococcus saccharolyticus based on 16S rrna sequencing. FEMS Microbiol Lett 1990; 71 : Osawa R, Fujisawa T, Sly LI. Streptococcus gallolyticus sp. nov.; gallate degrading organisms formerly assigned to Streptococcus bovis. System Appl Microbiol 1995; 18 : Klein RS, Recco RA, Catalano MT, Edberg SC, Casey JI, Steigbigel NH. Association of Streptococcus bovis with carcinoma of the colon. New Eng J Med 1977; 297 :

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