JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1221-1224

Vol. 29, No. 6

0095-1137/91/061221-04$02.00/0 Copyright © 1991, American Society for Microbiology

Characterization of Staphylococcus hyicus with the ATB System and with Conventional Tests

32 Staph

CHRISTOPH LAMMLER

Institut fur Bakteriologie und Immunologie, Justus-Liebig-Universitat D-6300 Giessen, Germany

Giessen, Frankfurter Strasse 107,

Received 4 December 1990/Accepted 21 March 1991

The ATB 32 Staph system correctly identified 45 of 54 Staphylococcus hyicus cultures isolated from pigs and cattle. The biochemical profiles of the remaining nine cultures were not listed in the product data base. The 40 porcine and 14 bovine cultures resulted in three and seven different biochemical profiles, respectively. In parallel experiments, almost all S. hyicus cultures showed hemolytic reactions on chocolate agar, had CAMP-like reactivities in the zone of lysis of the staphylococcal beta-lysin, and had bacteriolytic properties on Micrococcus luteus cells. In addition, the S. hyicus cultures were unpigmented, were DNase positive, expressed an S. hyicus-specific teichoic acid, and were usually coagulase positive in porcine plasma. Most of the porcine strains were protein A positive, and two porcine cultures were clumping factor positive.

Staphylococcus hyicus is well known

as the causative usually acute and generalized dermatitis of pigs. This syndrome was originally described by Sompolinsky (29). Taxonomic problems followed, until the organism was eventually named S. hyicus (7). S. chromogenes, a closely related species, was formerly described as S. hyicus subsp. chromogenes (7) and has now been awarded specific status (11). S. hyicus has also been isolated from pigs with septic polyarthritis and from various other farm animals (5, 6, 8, 9, 25, 27, 30). Several commercial microbial identification systems have been used for the differentiation of coagulase-positive and coagulase-negative

agent of exudative epidermitis,

a

staphylococcal species associated with humans and animals (4, 13, 15, 20, 22, 34). These systems frequently demonstrate slightly lower accuracy levels for staphylococci isolated from animals, namely, S. hyicus and S. intermedius (31-33). The present study was designed to evaluate the efficacy of the ATB 32 Staph system in identifying cultures of S. hyicus. The biochemical profiles of the cultures were compared with other properties of this bacterial species. MATERIALS AND METHODS Bacterial cultures. A total of 40 S. hyicus isolates from pigs and 14 S. hyicus isolates from cattle were evaluated. The cultures included the S. hyicus reference strain NCTC 10350. Some of the cultures were kindly provided by R. P. Allacker, Hatfield Herts, United Kingdom; G. Amtsberg, Hanover, Germany; H. Wegener, Copenhagen, Denmark; and K. Yoshida, Kawasaki, Japan. Some of the S. hyicus cultures had been characterized previously (14-16). ATB 32 Staph system. The ATB 32 Staph system (API bio-Merieux, Niirtingen, Germany) contains 26 enzymatic tests in minitubes attached to each other. The enzymatic tests are for urease, arginine dihydrolase, ornithine decarboxylase, esculin, glucose, fructose, mannose, maltose, lactose, trehalose, mannitol, raffinose, ribose, cellobiose, nitrate (NIT), acetoin production (Voges-Proskauer [VP]), p-galactosidase, arginine arylamidase, alkaline phosphatase, pyrrolidonyl arylamidase, novobiocin resistance, saccharose, N-acetylglucosaminidase, turanose, arabinose, and

,B-glucuronidase. Following the manufacturer's recommendations,

we

pre-

pared the inoculum for the ATB 32 Staph system with freshly subcultured bacteria adjusted in a suspension medium (API) to at least a 0.5 McFarland standard. The kit was inoculated with approximately 55 ,ll of the bacterial suspension, the urease, arginine dihydrolase, and ornithine decarboxylase wells were overlaid with 2 drops of mineral oil (API), and the kit was incubated for 20 to 24 h at 37°C under aerobic conditions. After incubation, NIT 1 and NIT 2 reagents were added to the NIT well, VP A and VP B reagents were added to the VP well, and FB reagent (API) was added to the P-galactosidase, arginine arylamidase, alkaline phosphatase, and pyrrolidonyl arylamidase wells. Reading of the kit resulted in an eight-digit numerical code which was used for species identification by comparison with code profiles listed in the ATB 32 Staph index (API). Conventional tests. The hemolytic reactions of the cultures were determined on normal and heated (chocolate) sheep blood agar plates. For preparation of the chocolate blood agar, defibrinated sheep blood (8%) was added to an agar base (Gibco Europe, Karlsruhe, Germany), and the mixture was heated at 85°C for approximately 10 min. For detection of synergistic hemolytic activities, the cultures were streaked on sheep blood agar at right angles to a betahemolytic S. aureus culture, incubated for 18 to 24 h at 37°C, and examined for CAMP-like reactivities (17). Pigment production was assayed on tryptone soya agar (Oxoid, Wesel, Germany), DNase activity was assayed on DNase test agar (Merck, Darmstadt, Germany), and bacteriolytic activities were assayed on tryptone soya agar containing a Micrococcus luteus reference strain (14). The S. hyicus-specific teichoic acid was extracted from the strains by autoclaving and subsequently detected by immunodiffusion reactions with a specific antiserum produced against S. hyicus reference strain NCTC 10350 (16). The coagulase test was performed as a tube test with porcine plasma diluted 1:5 in sterile NaCl (0.14 mol/liter) (18). The binding of plasma proteins to the staphylococcal surface was determined in slide agglutination reactions. The bacteria were incubated in 10 ml of brain heart infusion broth (Gibco), centrifuged, and resuspended at 1/10 the original volume in 0.14 M NaCl. The agglutination test was performed with 20 ,ul of the staphylococcal suspension, 20 ,ul of 1221

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J. CLIN. MICROBIOL. LAMMLERJ.CI.MRoo.

TABLE 1. Biochemical properties of S. hyicus isolates from pigs and cattle, determined with the ATB 32 Staph system Test

Urease

Arginine dihydrolase Ornithine decarboxylase Esculin Glucose Fructose Mannose Maltose Lactose Trehalose Mannitol Raffinose NIT VP

P-Galactosidase Arginine arylamidase Alkaline phosphatase Pyrrolidonyl arylamidase Novobiocin resistance Saccharose

N-Acetylglucosaminidase Turanose Arabinose

13-Glucuronidase

Ribose Cellobiose

% of positive S. hyicus isolates from: Cattle (n =14) Pigs (n = 40)

3 100 0 0 100 98 100 0 100 100 0 0 100 0 0 0 100 0 0 100 100 0 0 86 93 8

7 100 0 0 100 100 100 0 71 50 0 0 93 0 0 0 79 0 0 93 100 0 0 100 100 29

undiluted rabbit plasma, 20 p.I of a 0.2% human fibrinogen solution, 20 [LI of a 0.2% human fibronectin solution, and 20 RI of human erythrocytes sensitized with antibodies (Sangocell C; Behring Werke, Marburg, Germany). The fibrinogen preparation was purified from small amounts of fibronectin by successive passage through a Sepharose column containing gelatin (21). Fibronectin was isolated by affinity chromatography first on gelatin and then on heparin-Sepharose as described by Miekka et al. (23).

FIG. 1. Typical small zone of complete lysis after cultivation of S. hyicus on chocolate agar.

cultures reacted with fibronectin. Thirty-six of the porcine cultures and one of the bovine cultures showed protein A-like activities, visualized by a positive agglutination reaction with sensitized erythrocytes (Table 2). DISCUSSION

The identification of S. hyicus to the species level usually requires a large number of biochemical tests. These timeand material-consuming assays could be simplified by the use of the commercial ATB 32 Staph micromethod. As shown in the present study, this system allowed the correct identification of all mostly internationally collected porcine S. hyicus cultures, with almost identical biochemical profiles. The bovine cultures showed some variability in biochemical reactions resulting in seven different biochemical profiles. Some of the profiles were not listed in the index. In a recent study, the ATB 32 Staph system proved to be a reliable test for the differentiation of 24 different staphylococcal species; corresponding to the results of the present investigation, almost all S. hyicus cultures were correctly identified (2). Additional criteria for the differentiation of S. hyicus were the typical small zone of hemolysis on chocolate agar and, as tested with the beta-lysin of S. aureus, a CAMP-like synergistic hemolytic reaction. The hemolytic reaction on chocolate agar proved to be a common characteristic of S. hyicus (1). Comparable hemolysis could be observed on agar plates with blood from rabbits and humans, less pronounced hemolysis could be observed on agar plates with blood from horses, but no hemolysis could be observed on agar plates with blood from sheep and chickens (28). A CAMP-like zone

RESULTS

The biochemical properties of the S. hyicus cultures are summarized in Table 1. Most of the porcine S. hyicus cultures had the profile 26511264 (n = 35), and a few cultures had the profiles 26511260 (n = 4) and 36511260 (n = 1). The last is not included in the analytical profile index. Most of the bovine S. hyicus cultures had the profiles 26501064, 26511264, 26111264 (n = 3 each), and 26501264 (n = 2), and a few cultures had the profiles 26510264, 26501244, and 36501264 (n = 1 each). Some of these profiles (26501064, 26501264, 26510264, 26501244, and 36501264) are also not included in the index. S. hyicus was nonhemolytic on sheep blood agar. In contrast, almost all cultures produced a small zone of lysis when cultivated on chocolate agar and formed a CAMP-like zone of complete lysis in the zone of incomplete lysis of the staphylococcal beta-lysin (Fig. 1 and 2). S. hyicus was nonpigmented, was DNase positive, showed bacteriolytic activities on test agar plates containing M. luteus cells, and reacted specifically with antibodies against the S. hyicusspecific teichoic acid. Most of the cultures were coagulase positive, and two cultures reacted with fibrinogen, as evidenced by the positive clumping reaction. None of the

FIG. 2. CAMP-like synergistic hemolytic activities of S. hyicus (horizontal) in the zone of incomplete lysis of the staphylococcal beta-lysin (vertical) on sheep blood agar.

CHARACTERIZATION OF S. HYICUS WITH ATB 32 Staph SYSTEM

VOL. 29, 1991

TABLE 2. Properties of porcine and bovine isolates of S. hyicus, determined with conventional tests Test

Hemolysis on sheep blood agar Hemolysis on chocolate agar CAMP-like reactivity Pigment production DNase activity Bacteriolytic activity S. hyicus-specific teichoic acid Coagulase reaction 4h 24 h Clumping reaction Rabbit plasma

Fibrinogen Fibronectin Protein A

% of positive S. hyicus isolates from: Pigs (n = 40) Cattle (n = 14)

0 93 93 0 100 100 100

0 100 100 0 100 100 100

45 55

21 79

5 5 0 90

0 0 0 7

of synergistic hemolysis had already been described for S. hyicus and for various coagulase-negative staphylococci (12). It was of interest that the lack of a synergistic hemolytic reaction for some of the strains used in this study was not related to the lack of hemolysis on chocolate agar. This result may indicate that two different hemolytically active substances are involved in these reactions. The hemolysin of S. hyicus and the coagulase-negative staphylococci responsible for the synergistic hemolytic activity showed some relationship to staphylococcal delta-hemolysin (12). All S. hyicus cultures were nonpigmented, in contrast to the usually clearly pigmented S. chromogenes (11). Additional criteria which allowed a preliminary differentiation of S. hyicus were the production of a bacteriolytic enzyme, the detection of an S. hyicus-specific teichoic acid, and the enhanced coagulase reaction of S. hyicus in porcine plasma (10, 14, 16, 18). The bacteriolytic enzyme of S. hyicus has been characterized and has already been used for the solubilization of group- and type-specific streptococcal antigens and for the isolation of chromosomal DNA from Streptococcus uberis (3, 19). The use of a specific antiserum against the teichoic acid from S. hyicus allowed the serological identification of this species. However, cross-reactivities to teichoic acids from S. chromogenes, S. xylosus, and S. saprophyticus have been described (14). Most of the S. hyicus cultures from pigs used in this study expressed surface receptors for immunoglobulin G, two cultures expressed surface receptors for fibrinogen, but none of the cultures expressed surface receptors for fibronectin. The protein A-like surface receptor for immunoglobulin G was usually not expressed by the bovine S. hyicus cultures, corresponding to the findings of Phillips and Kloos (25). DNA-DNA hybridization studies have indicated that S. hyicus isolates from pigs and cattle form different DNA homology groups, possibly representing not-yet-named subspecies (26). The protein A-like material from S. hyicus appeared to have a lower molecular weight and a more acidic isoelectric point than does protein A of S. aureus (24). The reaction of some S. hyicus cultures with fibrinogen was in contrast to the original description of this organism (7). The properties and the nature of the S. hyicus clumping factor remain to be elucidated. However, protein A- and clumping factor-positive staphylococcal species other than S. aureus must be considered in routine identification schemes.

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The results of the present study clearly indicate that the commercial ATB 32 Staph system can be used to identify porcine as well as bovine S. hyicus cultures. Hemolytic reactions, coagulase activity, and the binding of plasma proteins can be used to characterize further individual cultures of S. hyicus. Such characterization may be useful for epidemiological studies. The relationship of these characteristics to the pathogenicity of S. hyicus remains unclear. REFERENCES 1. Bisping, W., and G. Amtsberg. 1988. Farbatlas zur Diagnose bakterieller Infektionserreger der Tiere. Paul Parey, Berlin. 2. Brun, Y., M. Bes, J. M. Boeufgras, D. Monget, J. Fleurette, R. Auckenthaler, L. A. Devriese, M. Kocur, R. R. Marples, Y. Piemont, B. Poutrel, and F. Schumacher-Perdreau. 1990. International collaborative evaluation of the ATB 32 Staph gallery for identification of the staphylococcus species. Zentralbl. Bakteriol. 273:319-326. 3. Christ, D., S. Schwarz, and C. Lammler. 1988. DNA-fingerprinting of Streptococcus uberis. Med. Sci. Res. 16:1297-1298. 4. Crouch, S. F., T. A. Pearson, and D. M. Parham. 1987. Comparison of modified Minitek system with Staph Ident system for species identification of coagulase-negative staphylococci. J. Clin. Microbiol. 25:1626-1628. 5. Devriese, L. A. 1979. Identification of clumping-factor-negative staphylococci isolated from cows udder. Res. Vet. Sci. 27:313320. 6. Devriese, L. A., and J. Derycke. 1979. Staphylococcus hyicus in cattle. Res. Vet. Sci. 26:356-358. 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. chromogenes subsp. nov. Int. J. Syst. Bacteriol. 28:482-490. 8. Devriese, L. A., K. H. Schleifer, and G. 0. Adegoke. 1985. Identification of coagulase-negative staphylococci from farm animals. J. Appl. Bacteriol. 58:45-55. 9. Devriese, L. A., K. Vlaminck, J. Nuytten, and P. deKeersmaekker. 1983. Staphylococcus hyicus in skin lesions of horses. Equine Vet. J. 15:263-265. 10. Frede, C., D. Christ, and C. Lammler. 1989. Streptolytic activities of a lytic enzyme from Staphylococcus hyicus. Zentralbl. Bakteriol. 271:54-60. 11. Hajek, V., L. A. Devriese, M. Mordarski, M. Goodfellow, G. Pulverer, and P. E. Varaldo. 1986. Elevation of Staphylococcus

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Characterization of Staphylococcus hyicus with the ATB 32 Staph system and with conventional tests.

The ATB 32 Staph system correctly identified 45 of 54 Staphylococcus hyicus cultures isolated from pigs and cattle. The biochemical profiles of the re...
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