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INT E RNAT IO NAL JOURNALOF S YS T E MAT IC B ACT E RIO L O G Y,Apr. 1987, p. 160-16200~~0-7713/87/020160-0302OO/OCopyright 0 1987, International Union of Microbiological SocietiesVol. 37, No. 2

Streptococcus suis sp . nov., nom. rev.RENATE KILPPER-BALZ AND KARL HEINZ SCHLEIFER

Lehrstuhl fu r Mikrobiologie, Technische Universitat Miinchen , Arcisstrasse 21, 0-8000Munich 2 , FederalRepublic of GermanyChem otaxonomic and d eoxyribonucleic acid homology studies were carried out on various Streptococcussuis strains to clarify their taxonom ic position. The results of the present study indicate that these strainsbelong to one species, namely, S. suis (ex Elliott) nom. rev. S train NCTC 10234 is proposed as the type strain.

Streptococcus suis is an important pathogen in pigs. Itwas originally isolated from cases of bacteremia and menin-gitis in piglets (2, 4, 15). More recently, however, S . suishas been isolated from pigs with respiratory diseases (5 , 11).The name S. suis has never been formally proposed ordescribed and is, therefore, not on the Approved Lists ofBacterial Names (14).Originally described strains were found to be serologicallygroupable into Lancefield groups R, S , RS, and T, and somewere nongroupable (3). Other authors (11, 15) describedeight different serovars of S . suis. All these strains aremlorphologically and biochemically very similar. In the pres-ent study, strains of the different Lancefield groups andserovars were investigated by deoxyribonucleic acid (DNA)hybridization and cell wall analyses to determine geneticrelationships of these serologically heterogeneous organismsanid to clarify their taxonomic status.

MATERIALS AND METHODSThe strains used in this study are shown in Table 1.Theywere cultivated in a medium consisting of (per liter ofdistilled water) 10 g of casein hydrolysate, 10 g of yeastextract, 5 g of D-glucose, 5 g of sodium chloride, 3 g ofsodium acetate, 0.5 g of L-cysteine hydrochloride, and 1mlof Tween 80 at pH 7.2 under microaerophilic conditions at35C for 18h. For the isolation of DNA, cells were harvestedat the end of the exponential growth phase after 2 h ofincubation with penicillin according to the method of Kilp-per-Balz and Schleifer (7). Methods for the preparation offilter-bound and radioactive DNA as well as optimal hybrid-

ization conditions and determination of guanine-plus-cytosine (G+C) contents by thermal denaturation have beendescribed previously (6, 9). Isolation of cell walls anddetermination of peptidoglycan types and cell wall sugarcompositions were done with cells in the stationary phase, aspreviously described (12, 13).

RESULTS AND DISCUSSIONThe results of DNA-DNA hybridization studies showedthcat all S. suis strains investigated are highly related,regardless of their Lancefield group or serovar. DNA relat-

* Corresponding author.

edness among different strains averaged more than 80%(Table 2), confirming their relationship at the species level.Cell wall composition and values of moles percent G+Chave been summarized in Table 1.Most strains of S . suisstudied contained the lysine-direct peptidoglycan type (di-rectly cross-linked stem peptides containing lysine as thediamino acid [13]). This peptidoglycan type was foundamong streptococci only in strains of S . suis and S . oralis(8). However, in contrast to S . oralis, strains of S . suiscontained rhamnose and lacked ribitol as major cell wallpolysaccharide constituents. In two strains of S . suis,namely, strain Henrichsen 11538 (serovar S) and strainHenrichsen 2651 (Lancefield group RS), at least part of thestem peptides of their peptidoglycan was not directly cross-linked but linked by an interpeptide bridge consisting ofalanine (ly~ine-[alanine]~-~;able 1).These two strains weregenetically closely related to typical strains of S. suis(Table 2) .Because of the reaction with group D antiserum, it hadbeen suggested that S. suis belongs to group D strepto-cocci together with S . bovis and even enterococci (2, 4,11). However, our hybridization studies demonstrated thatS. suis is not closely related to group D streptococci(DNA relatedness values between strains of S. uis andEnterococcus faecalis were less than 10%). This is consis-tent with the numerical taxonomic studies of Bridge andSneath (1) and the opinion of other authors who do notconsider the reaction with group D antiserum a specificcharacteristic for the identification of S. suis (3, 5 ) .As a result of this study it is confirmed that S . suis is agenetically homogeneous species. It has not yet been validlydescribed and is not on the Approved Lists of BacterialNames (14). On the basis of our results, the descriptionsgiven for group R and group S streptococci (2), and thecharacterizations given by other authors for R, S, R S, and Tstreptococci or S. uis (1,3 ,5,1 0, l l ), we propose S. suisas a valid species of the genus Streptococcus.Descriptionof Streptococcus suis (ex Elliott 1966)nom. rev.Streptococcus suis (suis. L. n. sus, a pig; L. gen. n. suis , ofa pig). Small ovoid cocci, less than 2 pm in diameter, occursingly, in pairs, or rarely in short chains. Some tendencytoward rod formation. Nonmotile. Gram positive. Chemo-organotrophs with fermentative metabolism. Facultativelyanaerobic. Catalase negative. Strains are groupable intoLancefield groups R, S, RS, and T or are nongroupable.They can also be subdivided into serovars 1 o 8 according tothe method of Windsor and Elliott (15) and Perch et al. (11).Acid from fermentation of D-glucose, sucrose, lactose,

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VOL.37, 1987 STREPTOCOCCUS SUZS sp. nov., NOM. REV. 161TABLE 1. Organisms studied, serological and cell wall data , and G + C conten ts

Lance- Lysine Cell wall polysaccharides containingfield Serovar peptido- mol%group glycan typeb Rhamnose Glucose Galactose GlcNH2 GalNH2 G + COrganism StrainS . suisS . suisS. suisS . suisS. suisS . suisS . suisS . suis S . suisS . suisS. suisS . suisS. suisS . suisS . viriduns 111S . pyogenesS . oralis

NCDO 2644 ( = NCTC 10237)Henrichsen S428 ( = NCTCHenrichsen S735 (= NCTCHenrichsen 4961Henrichsen 6407Henrichsen 11538Henrichsen 2524Henrichsen 8074Henrichsen 14636Henrichsen 2651CCUG 7984 (= NCTC 10234)CCUG 7985 (= NCTC 10237)CCUG 7986 (= NCTC 10446)CCUG 11673Kiel45527NCTC 8198TNCTC 11427T

10237)10234)

SSR

RSRST

A

DirectDirectDirectDirectDirect(AMl-2DirectDirectDirect(Awl-2DirectDirectDirectDirectDirect(Aldl-3Direct

NDN D+

N D+++++++N D++++( + I

N DN D+

N D( + I++++++N D+++-( + I

NDND+

N D++++++N D++++

-

-

N DN D+N D( + I++++++N D+++

( + I( + I

40.7ND40.639.939.140.841.040.340.242.038.4ND40.039.940.038.037.0

a NCDO, National Collection of Dairy Organisms, Shinfield, Reading, United Kingdom; Henrichsen, Streptococcus Department, Statens Serum Institut,Copenhagen, Denmark; CCUG, Culture Collection University of Goteborg, Sweden; Kiel, Streptokokkenzentrale, Kiel, Federal Republic of Germany; NCTC,National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom.Abbreviations of peptidoglycan types described by Schleifer and Kandler (13).GlcNH2, Glucosamine; GalNH2, galactosamine; -, absent; + , present; (+), present in minor amounts; ND, not determined.T A B L E 2. DNA-DN A relatedness values obtained at optimal hybridization conditions (25C below the melting temperature of DNA)

Source offilter-bound DNA% Relatedness with labeled DNA from:

S. suis S. suis type 4 S. suis S. suis S. oralisNCDO 2644 Henrichsen 6407 CCUG 7986 CCUG 11673 NCTC 11427TS . suis NCDO 2644S. suis type 2 Henrichsen S735S . suis type 3 Henrichsen 4961S. suis type 4 Henrichsen 6407S . suis type 5 Henrichsen 11538S. suis type 6 Henrichsen 2524 S . suis type 7 Henrichsen 8074S . suis type 8 Henrichsen 14636S. suis Henrichsen 2651S . suis CCUG 7984S. suis CCUG 7985S. suis CCUG 7986S. suis CCUG 11673S . viriduns 111 Kiel 45527S. oralis NCTC 11427TS . pyogenes NCTC 8198=

100858181919089931009710081100401514

89100

77758884

16

9377757694739810089100831003915

8280

808692100

14

19

162221

2019

202010017

maltose, salicin, trehalose, and inulin. No fermentation ofL-arabinose, D-mannitol, D-sorbitol, glycerol, melezitose, orD-ribose. Variable results in fermentation of raffinose andmelibiose and in production of hyaluronidase. Hydrolysis ofL-arginine, esculin, salicin, starch, and glycogen; no hydro-lysis of hippurate; no production of acetoin. Acid phospha-tase and alkaline phosphatase negative; L-ornithinedecarboxylase, N-acetylglucosaminidase, a-galactosidase,P-glucuronidase , and leucine arylamidase positive; p-galactosidase variable. Resistant to optochin. No growth at10or 45C, in 6.5% NaCl or 0.04% tellurite. Some strains areresistant to 40% bile (5) . Many strains produce P-hemolysison horse blood agar, and all are a-hemolytic on sheep bloodagar. Type of peptidoglycan is usually lysine direct, in a fewcases also lysine-(alanine)l-2. Cell wall polysaccharides con-tain rhamnose, glucose, galactose, and glucosamine. G+C

content of DNA is 38 to 42 mol% (T,,J. Important pigpathogen. The type strain is NCTC 10234 (= CCUG 7984 =Henrichsen S735).ACKNOWLEDGMENTS

We are grateful to J. Henrichsen, Statens Serum Insti tute,Cope nhag en, Den mar k, for providing us with strains of serovars 1 o8. This work was supported by a grant from the DeutscheForschungsgemeinschaft .LITERATURE CITED

1. Bridge, P. D., and P. H. A. Sneath. 1983. Numerical taxonomy2. Deibel, R. H., andH. W. Seeiey, Jr. 1974. Genus I. Streptococ-of Streptococcus. J. Gen. Microbiol. 129565-597.

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1.62 KILPPER-BA LZ AND SCHL EIFER INT. J . SYST. ACTERIOL.cu s Rosenbach 1884, 22, p. 490-509. In R. E. Buchanan andN. E. Gibbons (ed.), Bergey's manual of determinative bacte-riology, 8th ed. The Williams & Wilkins Co., Baltimore.3. De Moor, C. E. 1963. Septicaemic infections in pigs, caused byhaemolytic strep toco cci of new Lancefield groups designated R,S an d T. Antonie van Leeuwenhoek J . Microbiol. Serol.

4. Elliott, S. D. 1966. Streptococcal infection in young pigs. I. Animmunochemical study of the causative agent (PM streptococ-cus). J. Hyg. 64205-212.5. Hommez, J., L. A. Devriese, J. Henrichsen, and F. Castryck.1986. Identification and characterization of Streptococcus suis.Vet. Microbiol. 11:349-355.6. Kilpper, R., U. Buhl, and K. H. Schleifer. 1980. Nucleic acidhomology studies between Peptococcus saccharolyticus an dvarious anaerobic and facultatively anaerobic Gram-positivecocci. FE MS M icrobiol. L ett. 8:205-210.7. Kilpper-Balz, R ., and K. H. Schleifer. 1981. DNA-rRNA hybrid-ization studies among staphylococci and some other Gram-positive bacteria. FEM S Microbiol. L ett. 10:357-362.8. Kilpper-Balz, R ., P. Wenzig, and K. H. Schleifer. 1985. Molec-ular relationships and classification of some viridans strepto-cocci as Streptococcus oralis and emended description of Strep-tococcus oralis (Bridge and Snea th 198 2). Int. J. Syst. Bacteriol.

29:272-280.

3 5 4 8 2 4 8 8 .9. Kilpper-Balz, R. , B. L. Williams, R. Lutticken, and K . H.Schleifer. 1984. Relatedness of "Streptococcus milieri'' withStreptococcus anginosus an d Streptococcus constellatus. Syst.Appl. Microbiol. 5494-500.10 . Perch, B., E. Kjems, P. Slot, and K. B. Pedersen. 1981. Bio-chemical and serological properties of R , S , and RS s trepto-cocci. Acta Pathol. Microbiol. Scand. Sect. B 89:167-171.11. Perch, B., K . B. Pedersen, and J. Henrichsen. 1983. Serology ofcapsulated streptococci pathogenic for pigs: six new se rotypesof Streptococcus suis. J. Clin. Microbiol. 17:993-996.12 . Schleifer, K. H. , and 0.Kandler. 1967. Zur chemischen Zusam-mensetzung der Zellwand der Streptokokken. I. Die Amino-sauresequenz d es Mureins von Streptococcus thermophilus an dStreptococcus faecal i s . Arch. Mikrobiol. 57:335-364.13 . Schleifer, K. H ., an d 0. Kandler. 1972. Peptidoglycan types ofbacterial cell walls and their taxonomic implications. Bacteriol.Rev. 36:407477.14 . Skerman, V. B. D. , V. McGowan, and P. H. A. Sneath. 1980.Approved lists of bacterial names. Int. J. Syst. Bacteriol.30:225420.15 . Windsor, R. S., an d S. D. Elliott. 1975. Streptococcal infectionin young pigs. IV. An outbreak of streptoco ccal meningitis inweaned pigs. J. Hyg. 7569-78.