the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the ..... coccus Rosenbach 1884, HAL, (Nom.
INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY, Oct. 1990, p. 409-411 OO20-7713/90/O4O409-03$02.OO/O Copyright 0 1990, International Union of Microbiological Societies
Vol. 40, No. 4
Staphylococcus schleiferi subsp. coagulans subsp. nov. Isolated from the External Auditory Meatus of Dogs with External Ear Otitis SHIZUNOBU IGIMI,l* EIJI TAKAHASHI,2 A N D TOMOTARI MITSUOKA3 Department of Biomedical Research on Food, National Institute of Health, 2-10-35 Kamiosaki, Shinagawa-ku, Tokyo 141 , l and Department of Veterinary Microbiology2 and Department of Biomedical S ~ i e n c e , ~ Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan A new subspecies, Staphylococcus schleiferi subsp. couguluns, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi NS50274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, P-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S . schleiferi subsp. cougulans from the other coagulase-positivestaphylococci is presented. The type strain is GA211 (=JCM 7470).
of catalase, oxidase, coagulase, clumping factor, heat-stable nuclease ,hyaluronidase , arginine dihydrolase ,phosphatase , urease, and acetoin, anaerobic growth in thioglycolate medium, hemolysis, nitrate reduction, carbohydrate reactions, antibiotic susceptibilities, and enzymatic reactions were determined as described previously (11). Isolation of DNA was accomplished by the method of Saito and Miura (16). Lysostaphin (Sigma Chemical Co., St. Louis, Mo.) was used for cell disruption. The moles percent G+C in DNA was determined by the thermal denaturation method of Marmur and Doty (14). DNA-DNA hybridization experiments were performed as described by Crosa et al. (4).
The coagulase-positive species Staphylococcus intermedius is often isolated from veterinary clinical specimens from dogs (1-3,15) and is sometimes isolated from those from cats (2, 8). In the course of our taxonomic studies on staphylococci isolated from dogs and cats, a group of unidentified coagulase-positive strains was discovered. These strains were isolated from the external auditory meatus of dogs suffering from external ear otitis. Though strains from this group were phenotypically similar to strains of S . intermedius, we cannot identify these strains as S . intermedius, because acetoin is produced and acid is not generated from trehalose, unlike the reactions of S . intermedius. In the course of DNA-DNA hybridization studies, our unidentified group of strains revealed high levels of homology with S . schleiferi N850274T. S . schleiferi was reported by Freney et al. in 1988 as a new species isolated from human clinical specimens. It produced no coagulase in tube tests (9). Despite a relatively high level of DNA homology, phenotypic characteristics of S . schleiferi do not match those of our strains.
RESULTS AND DISCUSSION Except for a few carbohydrate reactions, the 21 strains are phenotypically homogeneous. All produce coagulase and heat-stable nuclease. The most distinctive characteristic is lack of acid production from trehalose. DNA-DNA hybridization studies among coagulase-positive species of the genus Staphylococcus are listed in Table 1. These results indicate that all of seven strains tested are closely related, whereas the levels of DNA homology of these strains with other coagulase-positive staphylococci were rather low (5 to 27%). DNA-DNA hybridization of S. schleiferi N850274T DNA with labeled DNA from these strains revealed high levels of homology and indicated that these strains are closely related to S. schleiferi at the species level. However, we propose to classify them as a subspecies of S . schleiferi because the phenotypic characteristics do not match the description of S. schleiferi (9). If we consider the test tube coagulase test, clumping factor, colony diameter, and acid production from sucrose, D-mannitol, D-trehalose, and D-ribose as variable characteristics of S. schleiferi, we could hardly differentiate S . schleiferi from the other staphylococci. The test tube coagulase test is the most important and significant test for identification of staphylococcal strains isolated from clinical specimens. Our 21 strains produce coagulase, but S . schleiferi does not. Our strains were isolated mainly from canine otitis, whereas S. schleiferi is isolated from human clinical specimens. For epidemiolog-
MATERIALS AND METHODS Bacterial strains. Twenty-one strains (GA11, GA64, GA89, GA116, GA124, GA199, GA211, GA222, GA227, GA238, GA247, GA288, GA337, GA347, GA390, GA400, GA412, GA416, GA456, GA483, and GA499) were isolated between 1982 and 1988 from discharges of the external ear of dogs diagnosed as suffering from external ear otitis. The isolation medium was Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% defibrinated horse blood. Cultures were maintained at -80°C in skim milk (Difco Laboratories, Detroit, Mich.). Unless noted otherwise, inocula for biochemical tests were prepared from overnight cultures on P agar plates (13), and incubation for tests was at 37°C. The type strains of various Staphylococcus species were obtained as previously described (11). Methods. Cell and colony characteristics, the production
* Corresponding author. 409
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IGIMI ET AL.
TABLE 1. Levels of DNA-DNA relatedness between S. schleiferi subsp. coagulans and other species in the genus Staphylococcus % Relatedness" with DNA of S. schleiferi subsp. coagulans strain:
Source of unlabeled DNA
S . schleiferi subsp. coagulans GA211 S. schleiferi subsp. coagulans GAll S . schleiferi subsp. coagulans GA64 S. schleiferi subsp. coagulans GA238 S. schleiferi subsp. coagulans GA288 S. schleiferi subsp. coagulans GA390 S. schleiferi subsp. coagulans GA400 S. schleiferi subsp. schleifer N850274= S . aureus subsp. aureus CCM 885T S . aureus subsp. anaerobius MVF-7= S. intermedius JCM 2422T S . hyicus JCM 2423=
GA211
GAll
GA64
GA288
GA390
100 92 76 96 98 96 76 62 10 7 25 15
94 100 72 96 67 81 90 65 12 9 22 15
88 88 100 111 108 105 95 70 5 6 27 10
85 90 85 92 100 95 89 73 11 8 21 11
75 95 69 98 99 100 92 55 8 10 22 12
a Level of relatedness after reassociation for 24 h at 58°C. Labeled DNA of strain GA211 shows 16% relatedness with DNA of S. felis ATCC 49168T and 13% relatedness with DNA of S. carnosus DSM 20501*.
ical purposes, it is desirable to distinguish between our strains and S. schleiferi. Therefore, we propose the following. Staphylococcus schleiferi subsp. coagulans subsp. nov. coagulans (co.a'gu.lans. L. adj. coagulans curdling, coagulating). The following description of S . schleiferi subsp. coagulans is based on a total of 21 strains, unless noted otherwise. Cells are gram-positive cocci, 0.8 to 1.2 pm in diameter, occurring singly, in pairs, and, predominantly, in irregular clusters. Nonmotile. Nonsporeforming. Colonies on horse blood agar after 24 h at 37°C are circular, entire, 1.5 to 2.0 mm in diameter, slightly convex, and opaque with a smooth glistening surface. Not pigmented. Facultatively anaerobic. Growth occurs in both the aerobic and anaerobic portions of semisolid thioglycolate medium. All strains produce free coagulase (test tube coagulase test with rabbit plasma) but fail to produce fixed coagulase (clumping factor with human plasma). All strains produce
p-hemolysin (hemolysis on sheep blood agar and hot-cold reaction), but a-hemolysin and 8-hemolysin are not detected. All strains reduce nitrate and produce heat-stable nuclease, acetoin, phosphatase, arginine dihydrolase, and urease. All strains are hyaluronidase and oxidase negative. All strains are susceptible to 1.6 pg of novobiocin per ml . All strains produce acid from glucose, mannose, fructose, galactose, and glycerol. Acid is usually produced from ribose (95%) (negative for strain GA288) and lactose (86%) (negative for strains GA390, GA400, and GA416). Some strains produce acid from mannitol (48%) (strains GA11, GA222, GA227, GA238, GA247, GA288, GA412, GA456, GA483, and GA499) and sucrose (24%) (GA64, GA89, GA337, GA347 and GA400). No acid is produced from arabinose, xylose, rhamnose, maltose, cellobiose, trehalose, raffinose, melibiose, melezitose, glycogen, sorbitol, erythritol, inositol, adonitol, dulcitol, or xylitol. The G,+C content of DNA, as determined on the basis of
TABLE 2. Characteristics on the basis of which S. schleiferi subsp. coagulans subsp. nov. can be distinguished from other coagulase-positive Staphylococcus species and S . schleiferi subsp. schleiferi" Reactionbfor: Characteristic
Aerobic growth Colony diameter >5 mmc Pigment Rabbit plasma coagulated Clumping factor Heat-stable nuclease Hemolysins Acetoin production Hyaluronidase production Acid produced from: Sucrose Maltose Galactose D-Mannitol D-Trehalose D-Ribose
~
~~
~
~~~~~~
~
S . schleiferi subsp. coagulans
S . schleiferi subsp. schleiferi
+ ++ + + +
+-
+ + + + ND
d
+ d -
+
S . aureus subsp. aureus
S . aureus subsp. anaerobius
-Iw
-
+ + ++ + +
~~~~~~~~
"
*'
+ + + d + +
hyicus
" delphini
+ + +
-
+ND
+ + d + +
-/w
+ + ND +
ND
Data are from references 5-7, 9, 10, 12, and 17. + , More than 90% of the strains are positive; - , more than 90% of the strains are negative; d, 11 to 89% of the strains are positive; w, weak reaction; -/w, negative or weak reaction; ND, result not determined. Colony diameter is determined after incubation on Pagar (13) at 34 to 35°C for 3 days and at room temperature for an additional 2 days. Under these conditions colonies of S. schleiferi subsp. coagulans are 5.0 to 8.0 mm in diameter.
VOL. 40, 1990
STAPHYLOCOCCUS SCHLEIFERI SUBSP. COAGULANS SUBSP. NOV.
the melting temperature in three strains (strains GA211, GA11, and GA64), ranges from 35 to 37 mol%. The characteristics of the type strain, GA211 (=JCM 7470), are the same as these described above. The guanineplus-cytosine content of its DNA is 35 mol%. The type strain was isolated from canine external ear otitis. Selected characteristics that are useful in the identification of S. schleiferi subsp. coagulans and in distinguishing this taxon from other coagulase-positive Staphylococcus species are listed in Table 2. S. schleiferi subsp. coagulans is easily dserentiated from coagulase-negative S. schleiferi subsp. schleiferi on the basis of its test tube coagulase test reaction and from the other coagulase-negative species on the basis of its production of coagulase, heat-stable nuclease, and phemolysin. S . schleiferi subsp. coagulans can be distinguished from other coagulase-positive species on the basis of its acetoin production, P-hemolysin production, negative hyaluronidase activity, and lack of acid production from trehalose or maltose. LITERATURE CITED 1. Berg, J. N., D. E. Wendell, C. Vogelweid, and W. H.Fales. 1984. Identification of the major coagulase-positive Staphylococcus sp. of dogs as Staphylococcus interrnedius. Am. J. Vet. Res. 45: 1307-1309. 2, Biberstein, E. L., S. S. Jang, and D. C. Hirsh. 1984. Species distribution of coagulase-positive staphylococci in animals. J. Clin. Microbiol. 19:61M15. 3. Cox, H. U., S. S. Newman, A. F. Roy, and J. D. Hoskins. 1984. Species of Staphylococcus isolated from animal infections. Cornell Vet. 74:124-135. 4. Crosa, J., H. Braener, and S. Falkow. 1973. Use of a singlestrand specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexes. J . Bacteriol. 115:904-911. 5 . de la Fuente, R., G. Suarez, and K. H. Schleifer. 1985. Staphylococcus aureus subsp. anaerobius subsp. nov., the causal agent of abscess disease of sheep. Int. J. Syst. Bacteriol. 35:99-102. 6. Devriese, L. A. 1977. Isolation and identification of Staphylo-
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coccus hyicus. Am. J. Vet. Res. 38:787-792. 7. Devriese, L. A., V. Hajek, P. Oeding, S. A. Meyer, and K. H. Schleifer. 1978. Staphylococcus hyicus (Sompolinsky 1953) comb. nov. and Staphylococcus hyicus subsp. chrornogenes subsp. nov. Int. J. Syst. Bacteriol. 2k482-490. 8. Devriese, L. A., D. Nzuambe, and C. Godard. 1984. Identification and characterization of staphylococci isolated from cats. Vet. Microbiol. 9:279-285. 9. Freney, J., Y. Brun, M. Bes, H. Meugnier, F. Grimont, P. A. D. Grimont, C. N e d , and J. Fleurette. 1988. Staphylococcus lugdunensis sp. nov. and Staphylococcus schleiferi sp. nov., two species from human clinical specimens. Int. J. Syst. Bacteriol. 38:168-172. 10. Hhjek, V. 1976. Staphylococcus interrnedius, a new species isolated from animals. Int. J. Syst. Bacteriol. 26:401-408. 11. Igimi, S., S. Kawamura, E. Takahashi, and T. Mitsuoka. 1989. Staphylococcus felis, a new species from clinical specimens from cats. Int. J. Syst. Bacteriol. 39373-377. 12. Kloos, W. E., and K. H. Schleifer. 1986. Genus JV. Staphylococcus Rosenbach 1884, HAL, (Nom. Cons. Opin. 17 Jud. Comm. 1958, 153), p. 1013-1035. I n P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 13. Kloos, W. E., T. G. Tornabene, and K. H. Schleifer. 1974. Isolation and characterization of micrococci from human skin, including two new species: Micrococcus lylae and Micrococcus kristinae. Int. J. Syst. Bacteriol. 24:79-101. 14. Marmur, J., and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5109-118. 15. Raus, J., and D. N. Love. 1983. Characterization of coagulasepositive Staphylococcus interrnedius and Staphylococcus aureus isolated from veterinary clinical specimens. J. Clin. Microbiol. 18:789-792. 16. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 17. Veraldo, P. E., R. Kilpper-Balz, F. Biavasco, G. Satta, andK. H. Schleifer. 1988. Staphylococcus delphini sp. nov., a coagulasepositive species isolated from dolphins. Int. J. Syst. Bacteriol. 38:436439.
