Streptococcus Canis Sp. Nov.: a Species of Group G Streptococci from Animals

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1986, p. 422425 Vol. 36, No. 3 0020-7713/86/03O422-04$02.OO/O Copyright 0 1986, International Union of Microbiological Societies

Streptococcus canis sp. nov.: a Species of Group G Streptococci from Animals

LUC A. DEVRIESE ,l* JOZEF HOMMEZ,’ RENATE KILPPER-BALZ,3 AND KARL-HEINZ SCHLEIFER3 Faculty of Veterinary Medicine, University of Gent, B-9000 Gent, Belgium’; Regional Veterinary Investigation Centre, Torhout, Belgium2; and Lehrstuhl fur Mikrobiologie, Technische Universitat, Munich, Federal Republic of Germany3

f3-Hemolytic group G streptococci from cows with mastitis and from dogs with different pathological conditions were characterized and named Streptococcus canis sp. nov. These animal group G strains were differentiated from Streptococcus dysgalactiae (group G strains of human origin, as well as group L strains and certain animal and human group C strains) by simple physiological and biochemical tests. Deoxyribonucleic acid-deoxyribonucleic acid hybridization showed that the S. canis strains differed from S. dysgalactiae, Streptococcus pyogenes, Streptococcus equi, and Streptococcus salivarius. These organisms contain a rare cell wall peptidoglycan type, namely, Lys-Thr-Gly. The specific epithet of S. canis, which has been in use for many years in veterinary textbooks to designate group G strains isolated from dogs, was chosen for this proposed new species because it comprises animal but not human group G streptococci. The type strain is strain STR-T1 (= DSM 20715).

Streptococci belonging to Lancefield group G (15) form a Clark et al. (6), led us to examine the taxonomic status of heterogeneous collection of organisms. At least the follow- animal group G streptococci. ing three distinct groups can be found: (i) the large-colony form of P-hemolytic group G streptococci isolated from MATERIALS AND METHODS humans; (ii) large-colony P-hemolytic strains from animals which were first shown to differ from human group G strains Strains. A total of 28 group G strains from cases of bovine in their fibrinolytic activity (3) and later also in several other mastitis on 28 different farms were isolated in 1982, 1983, characteristics (6); and (iii) the “minute P-hemolytic col- and 1984; these strains were designated strains STR-T1, ony” group G strains from humans. STR-T2, STR-T3, STR-T4, STR-T5, STR-T7, STR-T32, Animal group G strains have been associated mainly with STR-T35, STR-T37, STR-T38, STR-T40, STR-T41, STR- genital, skin, and wound infections (2, 10) in dogs and with T42, STR-T43, STR-T44, STR-T45, STR-T47, STR-T50, bovine mastitis (6, 16). The naming of these strains has STR-T51, STR-T52, STR-T53, STR-T54, STR-T55, STR- always been confusing and contradictory. Stafseth et al. (20) T56, STR-T57, STR-T70, STR-T71, and STR-T72. The fol- first used the name “Streptococcus canis” to designate dog lowing three group G strains were isolated from dogs: strain strains belonging to group C. However, these strains had a STR-290 from wound exudate and strains STR-312 and characteristic carbohydrate fermentation pattern which was STR-313 from neonatal septicemia cases. A human group G found later only in animal group G strains (2). Widely used strain (strain G/D 166B) was obtained from D. Groothuis, veterinary textbooks (4,5) refer to group G strains from dogs Public Health Institute, Bilthoven, The Netherlands. Ten as “Streptococcus canis,” but this name has never been group L strains and group G strains from cases of bovine accepted officially. mastitis were used in comparative antibiotic susceptibility Recent nucleic acid hybridization and cell wall composi- tests. S. dysgalactiae DSM 20662T (T = type strain), Strep- tion studies (13) have shown that human large-colony group tococcus equi DSM 20561T, Streptococcus salivarius ATCC G strains are closely related to group L strains, the P- 13419 and ATCC 20560T, and Streptococcus pyogenes hemolytic group C organism “Streptococcus equisimilis,” NCTC 819gT, which were obtained from the Deutsche and a-hemolytic group C strains isolated from cases of Sammlung von Mikroorganismen (Gottingen, Federal Re- bovine mastitis and polyarthritis in lambs. Similar observa- public of Germany), the National Collection of Type Cul- tions led to an emended description of Streptococcus tures (Colindale, United Kingdom), and the American Type dysgalactiae (9) which fits these animal group C P-hemolytic Culture Collection (Rockville, Md.) were used in deoxyribo- “S. equisimilis,” group C a-hemolytic S. dysgalactiae, and nucleic acid (DNA)-DNA hybridization tests. group L strains, as well as human group C “S. equisimilis,” Physiological tests. Growth characteristics were investi- group L, and group G strains. The older description of S. gated in brain heart infusion medium (Oxoid Ltd., dysgalactiae given in Bergey ’s Manual of Determinative Basingstoke, United Kingdom), in Todd-Hewitt broth (Ox- Bacteriology, 8th ed. (7), applied only to the animal a- oid), and on 5% sheep blood agar containing tryptose blood hemolytic group C strains. agar base (Oxoid). Resistance to 6.5% NaCl was tested in However, it has been found that several characteristics of brain heart infusion medium, and resistance to 40% bile was animal group G strains in our collection did not correspond tested in bile esculin agar (Difco Laboratories, Detroit, to the characteristics given in the new description of S. Mich.) which was incubated for 48 h. Formation of acid, dysgalactiae. These observations, as well as the description reduction, and coagulation of litmus milk was studied in of the biotypes of animal and human group G strains by litmus milk medium (Difco). Decarboxylation of L-tyrosine was tested as described by Sharpe (19). Hyaluronidase activity was determined on blood agar that was swab inoculated with a Pasteurella multocida capsule type A strain * Corresponding author. and fibrinolysin on nutrient agar (Oxoid) containing 28 mg of

422 VOL. 36, 1986 STREPTOCOCCUS CANIS SP. NOV. 423

TABLE 1. Characteristics of 31 S. canis strains" human fibrinogen per liter or 6 mg of bovine fibrinogen per No. of strains liter. Pyrrolidonylarylamidase and P-glucuronidase activities Characteristic positivelno. of were tested by using media and reagents formulated by API strains tested System (La Balme les Grottes, France) and Rosco Catalase activity ...... 013 1 (Taastrup, Denmark), and L-alanine aminopeptidase activity Growth in 6.5% NaCl ...... 013 1 was tested with Bactident Aminopeptidase (E. Merck AG, Growth in 40% bile ...... 013 1 Darmstadt , Federal Republic of Germany). Esculin degrada- CAMP reaction ...... 013 1 tion was tested by using a API-20 Strep kit (API) and on agar Hippurate reduction...... 013 1 slants containing (per liter) 0.1 g of esculin, 0.1 g of ammo- Esculin degradation in agar medium ...... 31/31 nium ferric citrate, 10 g of tryptone, 5 g of peptone, 5 g of Esculin degradation in API ...... 31 (27 weak)/31 yeast extract, and 15 g of agar (pH 7.3). Starch hydrolysis Fibrinol y sin ...... 013 1 was evaluated by inoculating Mueller-Hinton agar (Oxoid) Tyrosine decarboxylation ...... 013 1 and flooding the plates with iodine after 24 h. Hippurate Hyaluronidase activity ...... 1 (weak)/31b Starch hydrolysis ...... 013 1 hydrolysis, the Voges-Proskauer reaction, a- and P-galac- L-Alanine aminopeptidase activity ...... 15/15 tosidases, alkaline phosphatase, leucine arylamidase, argi- Voges-Proskauer test ...... 013 1 nine dehydrolase, and acid production from carbohydrates Alkaline phosphatase activity ...... 3113 1 were tested by using API-20 Strep and API-50 CH systems. Leucine arylamidase activity...... 3 113 1 Antibiotic susceptibility testing. Overnight brain heart infu- Arginine dehydrolase activity ...... 3113 1 sion cultures were diluted 20-fold in buffered saline and Pyrrolidonylarylamidase activity ...... 013 1 inoculated onto Mueller-Hinton agar (Oxoid) and onto Iso- a-Galactosidase activity...... 2313 1' Sensitest agar (Oxoid) supplemented with 5% bovine blood P-galactosidase activity ...... 28/31' or laked horse blood and doubling dilutions of penicillin G, P-glucuronidase activity...... 4/31' lincomycin hydrochloride, or trimethoprim. The results Acid produced from: N-Acet ylglucosamine ...... 919 were read after 24 h at 37°C. These antibiotics were chosen Adonitol ...... 019 for quantitative testing because agar diffusion tests carried Am ygdaline ...... 019 out on Iso-Sensitest agar enriched with 7% lysed horse blood L- Arabinose ...... 013 1 by using Rosco susceptibility test tablets showed that the D- Arabinose ...... 019 inhibition zone diameters of these antibiotics were larger D- Arabitol ...... 019 with the animal group G strains than with group L S. Arbutin ...... 919 dysgalactiae strains. Cellobiose ...... 819' Serological tests. For serological tests we used the capil- Dulcitol ...... 019 Ery thritol ...... 019 lary tube precipitation method with Lancefield hot acid- D-Fructose ...... 919 extracted antigens (8), type B and C grouping antisera from D-Fucose ...... 019 Difco, and type A, B, C, E, F, G, and L grouping antisera L-Fucose ...... 019 from Wellcome Research Laboratories (Beckenham, United Galactose ...... 919 Kingdom), as well as coaggulation tests in which the P-Gentiobiose ...... 019 Streptex (Wellcome) and Streptococcal Grouping Kit (Ox- Gluconate ...... 019 oid) reagents and methods were used. 2-keto-gluconate ...... 019 Cell wall, DNA, and hybridization studies. Cell walls were 5-keto-gluconate ...... 2 (~eak)/9~ prepared and peptidoglycan types were determined as pre- D-Glucose ...... 919 viously described (17, 18). Isolation of DNA and DNA-DNA a-Methyl-D-glucoside ...... 6 (weak)/9' Glycerol (under paraffin)...... 9 (weak)/9 hybridization experiments were carried out by the filter Glycogen ...... 6 (weak)/31B method, as previously described (12, 13). DNA base com- Inositol ...... 019 position was determined by thermal denaturation with a Inulin ...... 013 1 model 2600 spectrophotometer (Gilford Instrument Labora- Lactose...... 30131 tories, Inc., Oberlin, Ohio) (11). D-Lyxose ...... 019 Maltose...... 919 Mannitol ...... 013 1 RESULTS AND DISCUSSION D-Mannose ...... 919 a-Methyl-D-mannoside ...... 019 All group G strains appeared as uniformly large p- Melezitose ...... '019 hemolytic colonies on blood agar, similar to typical S. Melibiose ...... 019 pyogenes colonies, and they produced deposits with clear D-Raffi no se ...... 013 1 supernatants in broth media. All strains produced acid and Rhamnose ...... 019 Ribose ...... 31/31 strong or weak reduction reactions in litmus milk, and 25 Salicin ...... 3113 1 Sorbitol ...... 019 a Only strains STR-290, STR-312, STR-313, STR-TIT, STR-T4, STR-T7, L-Sorbose ...... 019 STR-T32, STR-T35, and STR-T37 were included in most carbohydrate tests. Starch ...... 31/31 The following strains reacted differently: STR-T3 was weakly positive for Sucrose ...... 919 hyaluronidase; STR-TS, STR-T41, STR-T45, STR-T56, STR-TS7, STR-T70, D-Tagatose ...... 019 STR-T71, and STR-T72 were a-galactosidase negative; STR-TS, STR-T45, Trehalose...... 4 (2 weak)/31b and STR-290 were p-galactosidase negative; STR-T44, STR-T45, STR-T70, D-Turanose ...... 019 and STR-312 were p-glucuronidase positive; STR-312 was cellobiose nega- Xylitol ...... 019 tive; STR-290 and STR-313 were weakly positive for 5-keto-gluconate; D-Xylose ...... 019 STR-290, STR-312, and STR-313 were a-methyl-D-glucoside negative; STR- T37, STR-T38, STR-T41, STR-T51, STR-T55, and STR-T71 were weakly L-X ylose ...... 019 positive for glycogen; STR-312 was lactose negative; and STR-T3, STR-T4, P-Methyl-xyloside ...... 019 STR-T72, and STR-313 were trehalose positive. 424 DEVRIESE ET AL. INT. J. SYST.BACTERIOL.

strains coagulated the same medium. Other results of phys- TABLE 3. Characteristics useful for differentiation of S. canis iological and biochemical tests are shown in Table 1. from S. dysgalactiae" The minimal inhibitory concentrations of the three antibi- Characteristic S. canis S. dysgalactiae otics tested with the agar dilution method were slightly lower Hyaluronidase activity -b + with the animal group G strains than with group L S. Fibrinolysin - D" dysgalactiae strains; they ranged from 0.007 to 0.015 pg/ml a-Galactosidase activity Dt - with penicillin G, from 0.12 to 0.5 pg/ml with lincomycin, p-Galactosidase activity + and from 0.5 to 1 pg/ml with trimethoprim. All strains were p-Glucuronidase activity D- + interpreted as susceptible to penicillin G, methicillin, Acid produced from trehalose D- + ampicillin, erythromycin, lincomycin, and chloramphenicol ~~ ~ in agar diffusion tests. All strains were resistent to Based on results obtained with the S. canis strains described in this study and in references 2 and 6 and on the characteristics of group C, G, and L (S. sulfamides. It was not possible to interpret the zone diameter dysgalactiae) strains described in references 6 and 7. of diffusion tests with trimethoprim, tetracyclines, neo- -, More than 90% negative; + , more than 90% positive; D-, 75 to 90% mycin, streptomycin, gentamicin, colistin, and novobiocin negative; D + , 75 to 90% positive. because the interpretations which were made according to Group G S. dysgalactiae strains of human origin lyse human fibrin (6); a-hemolytic group C strains may lyse bovine fibrin but not human fibrin (7). the instructions of the manufacturer ranged from susceptible to intermediate to resistant without being reproducible. None of the acid extracts precipitated or coagglutinated that S. canis belongs to the pyogenic streptococci sensu with the group A, C, E, F, and L antisera used, but all strict0 (13). showed strong or weak precipitation reactions and uniformly The test results indicate that the animal group G strepto- strong coagglutination reactions with group G antisera. A cocci should be described as a new species different from S. total of 15 strains precipitated with Wellcome group B dysgalactiae, in which the human group G strains have antiserum, but the same strains were negative with group B found a place. Our results confirm and extend the findings of antiserum from Difco. Similar cross-reactions with group B other authors (1-3, 6, 21), who studied a considerable antiserum were seen in the coagglutination tests with number of strains. Special mention should be made of the Wellcome Streptex antiserum but not with the Streptococcal work of Biberstein et al. (2), who identified a large homoge- Grouping Kit from Oxoid. neous group with characteristics similar to those of the Cell walls were prepared from seven strains of S. canis strains studied here in their collection of dog strains. Clark et (strains STR-TIT, STR-T35, STR-T38, STR-T41, STR-T47, al. (6), who also studied a large number of strains, found STR-T72, and STR-290); all of them contained the rather several consistent differences between human and bovine rare peptidoglycan type Lys-Thr-Gly. This peptidoglycan group G strains. Host species other than dogs and cows may type has been found so far only in some strains designated harbor S. canis, and it remains to be seen whether the dog Streptococcus equinus or S. salivarius but not in human and cattle strains are epidemiologically related. group G strains (13, 18). Useful characteristics which differentiate S. canis from S. DNA-DNA hybridization studies were carried out by dysgalactiae are summarized in Table 3. using labeled DNA from S. canis and other streptococci. On the basis of the results obtained in this study we Table 2 shows that we found very high DNA homology propose that the group G streptococci which we examined values between the two strains of S. canis, confirming a be named Streptococcus canis. The specific epithet was relationship at the species level. S. canis STR-TIT shared chosen because it has been used unofficially in veterinary higher DNA homology (>25%) with some pyogenic strepto- texts for a long time. cocci (S. dysgalactiae, S. pyogenes, S. equi) than with Streptococcus canis sp. nov. Streptococcus canis (ca'nis. strains of S. salivarius, although S. sulivarius ATCC 13419 L.n. canis dog) cells are gram-positive cocci and occur in contains the same peptidoglycan type as S. canis strains. pairs or in chains. Wide zones of j3-hemolysis are produced. The results of the DNA-DNA hybridization studies indicate CAMP factor negative. Colonies are circular with entire edges. A precipitate is formed in broth cultures. Catalase negative. Facultatively anaerobic. Good growth occurs at 37°C. Not resistant to 6.5% NaCl or 40% bile. Negative for TABLE 2. DNA homology values for S. canis and some other hippurate, fibrinolysin, tyrosine, starch hydrolysis, Voges- streptococci" Proskauer, and pyrrolidonylarylamidase reactions. Positive Source of filter-bound DNA % Homology with 35S-labeled in L-aminopeptidase, alkaline phosphatase, leucine arylami- DNA from S. canis DSM dase, and arginine dehydrolase tests. Most strains produce a Organism Strainb 207LjT p-galactosidase, but few strains are hyaluronidase and p- S. canis DSM 2071jT 100 glucuronidase positive. Variable reactions in a-galactosidase (= STR-TIT) tests. S. canis DSM 20716 92 No acid is produced from adonitol, amygdalin, L-arab- ( = STR-290) hose, D-arabinose, D-arabitol, dulcitol, erythritol, D-fucose, S. dysgalactiae DSM 20662T 39 L-fucose, j3-gentiobiose, gluconate, 2-keto-gluconate, S. pyogenes NCTC 8198T 36 inositol, indin, D-lyxose, mannitol, a-methyl-D-mannoside, S. equi DSM 20561T 25 melezitose, melibiose, D-raffinose, rhamnose, sorbitol, L- S. salivarius ATCC 13419 13 sorbose, D-tagatose, D-turanose, xylitol, D-xylose, L-xylose, S. salivarius DSM 20560T 15 or P-methyl-xyloside. a Hybridization was carried out under optimal conditions (25°C below the Acid is produced from N-acetylglucosamine, D-fructose, melting point of DNA). galactose, D-glucose, maltose, D-mannose, ribose, salicine, DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany; NCTC, National Collection of Type Cultures, Central starch, and sucrose. Public Health Laboratory, London, England; ATCC, American Type Culture A few strains are trehalose positive, and some weak acid Collection, Rockville, Md. reactions may occur with glycogen. VOL. 36, 1986 STREPTOCOCCUS CANIS SP. NOV. 425

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