Demonstration of Serologically Different Capsular

Transcription

1 INFECTION AND IMMUNITY, Apr. 1971, p Copyright 1971 American Society for Microbiology Vol. 3, No. 4 Printed in U.S.A. Demonstration of Serologically Different Capsular Types Among Strains of Staphylococcus aureus by the Serum-Soft Agar Technique KOSAKU YOSHIDA Departmenit of Microbiology anid Immunology, Nippon Medical School, 113, Received for publication 9 November 1970 Tokyo, Japan Colonies of Staphylococcus aureus exhibiting diffuse-type growth in regular serumsoft agar containing 7.5% sodium chloride were isolated. After isolation, further identification of the encapsulated strains of S. aureus was performed. With this procedure, 19 encapsulated strains were obtained from 103 clinical specimens (18.4%). With these strains, three serologically distinct diffuse types of organisms were observed by the conversion of diffuse to compact type colonial morphology in serum-soft agar containing specific antidiffuse sera. Capsule-inhibiting activity of antisera was adsorbable with homologous encapsulated organisms and not adsorbed with either heterologous encapsulated organisms nor the derived compact variant, suggesting a specific activity for the antispecific capsular antibody. Fourteen strains were similar to the Smith diffuse-type strain, four strains were the same as NS58D, and one was identical to NS41D. These were provisionally designated as capsule types A, B, and C, respectively. Recently, Maverakis and Wiley (3) described four serologically different capsules, namely, the Smith and three other capsular types of animal strains, by using a "specific capsular reaction." However, the problems of specificity, occurrence, and conversion forms remained to be solved. Serological heterogeneity of the different capsule types of Staphylococcus aureus isolates from human sources other than the classical Smith or Smith-like strains has not been reported thus far. In an attempt to demonstrate the multiplicity of capsule types of strains of S. aureus, a serum-soft agar technique was applied, and the serologically different capsule types of the strains isolated from clinical specimens were demonstrated. This paper is concerned with these investigations. cedures described below. The classic Smith diffuse strain described elsewhere (8) was used for control and comparison purposes. Identification of encapsulated strains of S. aureus. Diffuse colonies were tested by Gram stain and for catalase and glucose oxidation/fermentation by using OF media (Difco). Strains showing both positive glucose oxidation and fermentation, which were also gram-positive cocci and were catalasepositive, were then tested for coagulase, clumping factor, deoxyribonuclease, mannitol fermentation, and acid phosphatase activities. To test for coagulase activity, 0.5 ml of an overnight culture in BHI broth was added to an equal volume of a 1:5 saline dilution of standard rabbit plasma (Difco) and incubated at 37 C for 1 to 24 hr. After 2, 4, and 8 hr, tubes showing visible coagulum were read as positive. To test for clumping factor activity, one colony of organisms cultured on BHI agar plates overnight at 37 C aerobically was emulsified in a drop of sterile MATERIALS AND METHODS water to which was added an equal drop of a 1:10 Isolation of the diffuse strains. Clinical specimens dilution of the same rabbit plasma used for the coagulase test. Any clumping appearing within 15 sec was taken from the nose, throat, pus, exudate, etc., (obtained in the Clinical Laboratory, Nippon Medical read as positive. When negative, however, a second School Hospital) on cotton swabs were rinsed off determination was made with undiluted fresh rabbit into 2.9 ml of physiological saline. The suspensions plasma. For deoxyribonuclease, mannitol fermentation, and acid phosphatase production, deoxyribo- were then diluted with physiological saline at a 2.0-ml volume to l:l0-4. A 0.1 -ml amount of each was then nuclease test media (Difco), salt-mannitol media inoculated into serum-soft agar by using Brain (Difco), and PP media (Nihon Eiken, Co., Ltd., Heart Infusion (BHI, Difco) containing 7.5% NaCI Tokyo) were used, respectively, and readings were by the method of Finkelstein and Sulkin (2). After 2 carried out according to instructions from the manufacturers. Based on these examinations, the coagulase- days of growth at 37 C, from one to five colonies were isolated and identified as S. aureus by the propositive strains were identified as S. aureus. Strains 535

2 536 YOSHIDA INFEC. JMMUN. exhibiting negative coagulase and deoxyribonuclease reactions and positive clumping factor reactions were excluded. Diffuse or counterpart compact variants, showing negative coagulase but positive deoxyribonuclease, mannitol fermentation, and acid phosphatase activities, were identified as S. aureus. However, for the purpose of this study, if capsules could not be observed under the light microscope with India ink techniques, the strains were excluded. Preparation of antisera. Encapsulated strains of S. aureus were cultured in BHI broth for 18 to 24 hr at 37 C aerobically. The organisms were harvested by centrifugation at 7,000 X g and were washed once with saline. A cell suspension containing approximately 108 to 109 organisms, determined rephelometrically, was then autoclaved at 121 C for 15 min. One to 1.5 ml of the preparation was then injected intravenously into rabbits on three successive days for 4 weeks. Ten days after the final injection, test bleedings were done, and then the animals were exsanguinated and the sera were separated. When serum activity was not sufficient, booster injections were given. Sera thus prepared were then tested for the ability to promote diffuse- to compact-type changes in colonial morphology in serum-soft agar which I designate as "converting activity." Homologous diffuse-strain cultures were employed according to the procedures described below. Determination of activity of antisera. Twofold dilutions of antisera were prepared with sterile physiological saline at dilutions up to 1:32. A 0.1-ml amount of each dilution, normal rabbit sera, and a dilution of organisms (10-6) cultured overnight at 37 C in BHI broth were combined with 10 ml of 0.15% BHI-soft agar. The mixture was cultured, and growth types were determined after 24 hr at 37 C. The minimum antiserum dilution producing complete compact-type colonial morphology after 24 hr of growth was expressed as 1 unit, and 2 units per 0.1 ml of the antisera was used in all further experiments. Determination of diffuse type. Two units of antisera, normal rabbit sera and a 10-6 dilution of organisms grown in BHI broth overnight at 37 C (contained in 0.1 ml) were combined with 10 ml of 0.15% BHI-soft agar and grown at 37 C. Normal rabbit serum was used as a control, and final determination was made after 24 hr of growth at 37 C. When diffuse colonies were converted to compact-type growth by the addition of antisera, the organisms were designated as the same type strain as that which had been used for rabbit immunization. Adsorption of converting activity of antisera. For the adsorption of transition activity of the antisera, the following adsorption tests were performed. To 1.0 ml of 2 units per 0.1 ml of antidiffuse sera, 1, 3, 10, and 30 mg of lyophilized encapsulated organisms, exhibiting different types in the above experiments, were added separately to the various antisera. After incubation at 4 C overnight, precipitates were removed by centrifugation at 7,000 X g, and the supernatants were filtered with a membrane filter (0.45,m, Millipore Filter Corp., Bedford, Mass.). A 10-ml amount of 0.15% BHI-soft agar was combined with 0.1 ml of treated immune sera, normal rabbit sera, and 0.1 ml of homologous organisms diluted (10-6) with saline after growth in BHI broth overnight at 37 C. Also, the same volume of encapsulated and derived counterpart unencapsulated organisms was used for the adsorption of the immune sera, and then the same techniques were repeated with homologous encapsulated organisms. Determinations were made after 24 hr of growth at 37 C aerobically. RESULTS Isolation of the encapsulated strains. With the isolation procedures used in these experiments, a high isolation rate of encapsulated strains of S. aureus was obtained. Encapsulated strains showing diffuse-type growth in BHI-serum-soft agar were isolated from 19 of 103 clinical specimens. As many as five colonies from each specimen could be isolated. These strains gave negative clumping factor reactions, and capsules could be demonstrated under the light microscope by using India ink preparations. Twelve strains had positive tube coagulase, deoxyribonuclease, and acid phosphatase reactions. Five strains were negative for mannitol fermentation. Five coagulase-negative strains were positive for deoxyribonuclease, mannitol fermentation, and acid phosphatase production. Two strains had negative tube coagulase and mannitol fermentation but gave positive deoxyribonuclease and acid phosphatase reactions and were identified as S. aureus because their counterpart-derived compact variants showed positive coagulase reactions. Concerning the isolation of compact variants from diffuse strains, all except three strains yielded compact variants. Effect of antisera on the colonial morphology FIG. 1. Colonial morphology of Smith diffuse strain in regular serum-soft agar containing (A) normal rabbit serum, (B) anti-smith diffuse serum, (C) anti- NS41D serum, and (D) anti-ns58d serum.

3 VOL. 3, 1971 S. AUREUS ISOLATION BY SERUM-SOFT AGAR METHOD 537 in serum-soft agar. With the addition of antidiffuse sera to serum-soft agar, homologous strains were converted from diffuse- to compacttype colonial morphology. This transition activity of the immune sera was titrated by the serumsoft agar technique by using homologous strains. The transition activity of the immune sera was 1:4 to 1:8 by the immunization schedule used in these experiments. However, the diffuse strains were not converted to compact type by heterologous strain antisera but only by the addition of homologous antisera. Thus, it was found that three diffuse types of the strains could be distinguished (Fig. 1 to 3). In these experiments, the Smith diffuse strain, strain NS58D (isolated from vaginal exudate), and strain NS41D (isolated from the nose) appeared to be FIG. 2. Colonial morphology of strain NS58D in regular serum-soft agar containing (A) normal rabbit serum, (B) anti-ns58d serum, (C) anti-smith diffuse serum, and (D) anti-ns41d serum. FIG. 3. Colonial morphology of strain NS41D in regular serum-soft agar containing (A) normal rabbit serum, (B) anti-ns41d serum, (C) anti-smith diffuse serum, and (D) anti-ns41d serum. TABLE 1. Designzationz of three different capsules of the strains of S. aureus and the number of strains isolated Capsule No. of typsule type Representative strain strains Per cent ~~~~~~~~isolated A Smith diffuse B NS58D C NS41D FIG. 4. Colonial morphology of Smith diffuse strain in regular serum-soft agar containing (A) normal rabbit serum, (B) anti-smith diffuse sera untreated, (C) anti-smith diffuse sera adsorbed with NS41D organisms, (D) anti-smith diffuse sera adsorbed with NS58D organisms, and (E) anti-smith diffuse sera adsorbed with Smith diffuse organisms. representative strains of each type which I provisionally designated as A, B, and C. With this classification, 73.7% of the strains were type A (Smith-like organisms), 21.0% of the strains were type B (NS58D), and 5.3% of the strains were type C (NS41D), respectively (Table 1). To elucidate further the heterogeneity of the diffuse strains, adsorption experiments with homologous and heterologous encapsulated organisms were carried out. With the addition of 3.0 mg (dry weight) of the Smith diffuse organisms to 20 units of rabbit anti-smith diffuse sera, the converting activity was completely adsorbed. However, the activity was not completely removed if less than 3.0 mg of Smith diffuse organisms was used. Further, the converting activity of the anti-smith diffuse sera was not adsorbed by either NS58D or the NS41D organisms regardless of the amount used (Fig. 4). In the case of the anti-ns58d, neither the Smith diffuse nor NS41D organisms adsorbed out the converting activities; finally, employing the anti-ns41d serum, the Smith diffuse and w4

4 538 YOSHIDA INFEC. IMMUN. TABLE 2. Growth types of the Smith diffuse strain, strain NS58D, and straini NS41D in serum-soft agar containing homologouis antisera absorbed with homologous anld heterologous organismsa Strain Homologous immune rabbit sera Normal absorbed with rabbit sera U- Smt NS58D NS4lD treated diffuse Smith diffuse D C D C C NS58D D C C D C NS41D D C C C D a D and growths. C mean diffuse- and compact-type FIG. 5. Colonzial morphology of Smith diffuse strain int regidar serum-soft agar conztaining (A) ntormal rabbit serum, (B) anti-smith diffuse sera untreated, (C) anti- Smith diffuse sera adsorbed with Smith compact organisms, and (D) anti-smith diffuse sera adsorbed with Smith diffuse organisms. NS58D organisms were unable to adsorb out the activity (Table 2). This indicated that there were three definite serological activities, namely, the Smith diffuse, NS58D, and NS41D converting activities. Determination of capsular antibody. These experiments were designed to elucidate the serological effects of the antisera on the conversion to colonial morphology in serum-soft agar. Counterpart compact, unencapsulated, and variant forms were isolated from the Smith diffuse strain, strain NS58D, and strain NS41D. In the treatment of 3 mg of Smith diffuse organisms with 20 units of anti-smith diffuse sera, converting activity was completely adsorbed out; however, the serum activity was not removed even though 30 mg of Smith compact organisms was used (Fig. 5). Similar experimental TABLE 3. Growth types of the Smith diffuse strain, NS58D straini, anid NS41D straini in serum-soft agar containinig homologous antisera absorbed with homologous diffuse and compact organismsa Strain Normal rabbit sera Immune rabbit sera absorbed with Untreated Diffuse Compact Smith diffuse D C D C NS58D D C D C NS41D D Ci D C a D and C mean diffuse- and compact-type growth. results were demonstrated with strains NS58D and NS41D (Table 3), suggesting that converting activity was a result of an anti-specific capsular antibody. The capsule types of the Smith diffuse strain, strain NS58D, and strain NS41D were then designated A, B, and C, respectively, as stated above. DISCUSSION Previously, it was reported that the generation times of some diffuse strains of S. aureus are longer than their counterpart compact variants (10). To isolate as many encapsulated strains as possible, single diffuse colonies were isolated from clinical specimens. These colonies were then tested for speciation as S. aureus. With this procedure, encapsulated strains of S. aureus were isolated from 19 of 103 clinical specimens (18.4%.). This isolation ratio was considerably higher than that reported by Rogers (4), 0.4%, and that reported by Yoshida et al. (9), 4%. The differential identification procedure for encapsulated organisms of S. aureus used by Wiley (6), which has been known for more than 10 years, is called the "specific capsular reaction." With this procedure, Maverakis and Wiley (3) have described four serologically different types of encapsulated strains of S. aureus which originated primarily from mice. In addition, Wiley and Maverakis (7) observed 37 carriers of encapsulated S. aureus strains in 60 mice. However, Mudd and DeCourcy (4) postulated that the capsule swelling phenomenon is not a true swelling of the capsule and designated it as an "extracellular peripheral precipitin reaction." Therefore, identification of the capsule and the demonstration of heterogeneity in capsular types were accomplished in this investigation by the serum-soft agar technique. Conversion of colonial morphology, i.e., diffuse to compact type in

5 VOL. 3, 1971 S. AUREUS ISOLATION BY SERUM-SOFT AGAR METHOD 539 serum-soft agar, with the addition of homologous antisera has already been described by others (1, 2). Eda and Iwata (1) regarded this phenomenon as an antigen-antibody reaction. However, the Smith diffuse strain had been assumed as unique among strains of S. aureus, and the presence of human source encapsulated strains of S. aureus other than the Smith or Smith-like strains had not been described at that time. In a previous paper (9), the numbers of Smith-like organisms found among human source isolates of S. aureus were reported. Although the majority, 73.7%, of encapsulated strains of S. aureus were also Smith-like in this experiment, two additional serological types distinct from the Smith or Smith-like strains were observed. However, these types were found in much lower numbers. These serological specificities were determined by experiments on the adsorption of the converting activity of the immune sera with both homologous and heterologous organisms. A significant finding was that the converting activity of the antidiffuse sera was adsorbed with the homologous diffuse organisms but was not absorbed with the compact strains derived from the homologous diffuse strains. This suggests that converting activity of the immune sera would react with the capsular component only and was not associated or reactive with cell wall or cell membrane components. With regard to the relationship between capsule types 1 to 4 of Wiley and my scheme (A, B, C), type 2 corresponds to type A; however, the relationship of the other types is not known presently. In earlier investigations of the pseudocompact-type organisms (8), these variants, isolated from the diffuse strains used in this experiment, exhibited serological patterns similar to their parent diffuse organisms. The biochemical properties and immunological characteristics of the antigenic substances associated with presently known capsular types are currently under investigation, and these findings will be published in future reports. ACKNOWLEDGMENTS The author thanks Y. Kimura, Nippon Medical School, for advice and encouragement during the course of this work. Also, he is deeply indebted to M. R. Smith, U.S. Army, Sagamihara, Kanagawa-ken, for help in the preparation of the manuscript. LITERATURE CITED 1. Eda, T., and K. Iwata Studies on unique staphylococcal strains exhibiting high virulence for mice by intraperitoneal inoculation. 5. Serological properties. Jap. J. Bacteriol. 23: Finkelstein, R. A., and S. E. Sulkin Characteristics of coagulase positive and coagulase negative staphylococcus in serum-soft agar. J. Bacteriol. 75: Maverakis, N. H., and B. B. Wiley Evidence for a multiplicity of capsular types among Staphylococcus aureus strains. J. Bacteriol. 96: Mudd, S., and S. J. DeCourcy, Jr Interaction of viscid material of Staphylococcus aureus with specific immune sera. J. Bacteriol. 89: Rogers, D. E Experimental observations on staphylococcal diseases, p Symposium on staphylococci and staphylococcal infections. Pantowowe Wydawnictwo Naukowe Warszewa. 6. Wiley, B. B A new virulent test for Staphylococcus aureus and its application to encapsulated strains. Can. J. Microbiol. 7: Wiley, B. B., and N. H. Maverakis Virulent and avirulent encapsulated variants of Staphylococcus aureus. J. Bacteriol. 95: Yoshida, K., M. Takahashi, and Y. Takeuchi Pseudocompact-type growth and conversion of growth types of strains of Staphylococcus aureus in vitro and in vivo. J. Bacteriol. 100: Yoshida, K., M. R. Smith, and Y. Naito Biological and immunological properties of encapsulated strains of Staphylococcus aureus from human sources. Infec. Immun. 2: Yoshida, K., and Y. Takeuchi Comparison of compact and diffuse variants of the strains of Staphylococcus aureus. Infec. Immun. 2: