Virulent and avirulent staphylococcus aureus
Creators: name: Maverakis, Nick Harry
Publisher: name: University of Utah
Year: 2012

description: The term specific capsular reaction has been used in this thesis instead of capsular swelling reaction or "quelling" because no data were available to indicate that an increase in capsular size occurred when specific antiserum and encapsulated staphylococci were combined.  When specific antiserum and organisms were mixed, the antigen-antibody reaction occurred only at the surface of the capsule.  However, the cell wall, cytoplasmic matrix, and nuclear material were not affected.  The present study showed that erythrocytes sensitized with capsular material prepared from the wound strain of S. aureus were agglutinated by homologous staphylococcal antiserum. Gel double-diffusion tests showed three distinct precipitation lines when the partially purified capsular Material was diluted 1:1,000 in buffered saline pH 7.2 and only one precipitation line when the partially purified capsular material was diluted 1:10,000.  However. since the partially purified capsular material used for the sensitization of rabbit erythrocytes was diluted 1:10,000 in buffered saline and since it showed only one precipitation line at this concentration in gel double-diffusion tests. it was justifiable to ascribe the sensitization of rabbit erythrocytes as probably due to the capsular antigen. The specificity of the hemagglutination reaction was clearly shown by inhibition of hemagglutination by microgram quantities of the partially purified capsular material prepared from the wound strain of S. aureus.  Capsular material from the encapsulated wound strain and a nonencapsulated variant of it was detected in culture supernatant fluids by inhibition of indirect hemagglutination.   The encapsulated wound strain retained capsular material on its surface as shown by the specific capsular reaction while the nonencapsulated variant did not. The increased virulence of the encapsulated wound strain for embrjonated hens' eggs as compared to the nonencapsulated variant derived from it was believed to be due to a better retention of capsular material.  In the present work the specific capsular reaction was used exclusively to demonstrate capsules on the Smith diffuse variant and to demonstrate their absence on the compact variant of the Smith strain of S. aureus.  Morse (1962a),  Koenig (1962) and Modd and DeCourcy (1965) were unable to elicit a specific capsular reaction using the Smith strain. but workers in this laboratory succeeded in this endeavor by employing both rooster and rabbit antistaphylococeal serum.  Morse used the Smith diffuse strain of S, aureus in all of his experiments (personal communication).  India ink preparations of this strain showed that the organism was surrounded by an envelope structure (Morse, 1962a).  Mudd and DeCourcy (1965) reported that the encapsulated Smith strain of S.aureus grew as a compact colony in normal rabbit serum.  He reported that this staphylococcus was also surrounded by an envelope structure as demonstrated by the India ink method.  Although this thesis does not dispute the validity of these observations, his data tend to obscure the basic question, namely, whether or not the virulent form of the Smith strain is encapsulated and grows as a diffuse colony in serum or plasma soft agar.  Growth of the Smith strain of S. aureus in normal rabbit serum soft agar as a compact colony has usually been interpreted (Koenig, 1962; Koenig et al., 1962a, 1962b; Koenig and Melly, 1965) as an indication of nonencapsulation and characteristic of the avirulent form of the Smith strain of S. aureus, but Mudd and DeCourcy (1965) reported that their culture of the encapsulated Smith strain of S. aureus grew as a compact colony in normal rabbit serum soft agar.  Electron photomicrographs of the Smith diffuse variant of S. aureus showed the presence of a capsule (Koenig and Melly, 1965). However, Koenig (1962) was unable to find any difference in the diffuse or compact Smith strains of S. aureus when these strains were examined in India ink preparations. To use wet India ink preparations as the only criterion for the demonstration of capsules of S. aureus could lead to confusion.  It should be re-emphasized that the most definitiive method for the demonstration of capsules of S. aureus is the specific capsular reaction.  In his research Mudd (1965) maintained that the encapsulated Smith strain of S. aureus should be considered as the prototype of encapsulated S, aureus strains and that the wound strain isolated in this laboratory should be considered as a representative of a phenomenon which he called the extracellular peripheral precipitation reaction (EPPR).  The latter term, EPPR, seems to be only a circumlocution used to describe the specific capsular reaction,, Mudd and DeCourcy (1965) would exclude the wound strain of S. aureus as being truly encapsulated because they would restrict true encapsulation to virulent naturally occurring strains.  The wound strain in its encapsulated state was obtained by the method employed by Bigger„ Boland and 0'Mera (1927).  Its virulence in embryonated hens' eggs is indisputable (Wiley. 1961)„ Naturally occurring encapsulated S. aureus of the wound mucoid type-have been isolated from rabbits, roosters,  mice, and humans.  Each isolate exhibited a positive specific capsular reaction upon initial isolation (Wiley, 1961, 1963).  Wiley (1961) devised and employed a virulence test in embryonated hens' eggs which is well suited to the testing of the virulence of S. aureus isolates. In this test, the wound strain behaved as a virulent strain with an LD50 of less than 500 organisms. The finding of anticapsular antibodies that react with the wound strain in normal rabbits, roosters,  mice, and humans (Wiley, 1961, 1963) further supports the validity of this position.  Apparently, the wound strain is a major capsular type of S. aureus, but not the only one.  Evidence presented in this thesis indicates, for the first time, that the Smith diffuse variant is also encapsulated, as shown by the specific capsular reaction0 The wound mucoid strain and the RLM strain of Price and Kneeland represent members of a major capsular type against which antibodies are widely distributed (Wiley, 1961, 1963).  No detectable differences were found between the wound mucoid strain and the Smith diffuse variant, regarding the amount of hemolysin present in cultures.  Differences in the amount of coagulase formed by each strain were not great enough to explain the difference in virulence observed. Apparently, the best explanation is that the mice were resistant to the wound strain because they carried it„ and they had elaborated antibodies against it. Our tests did not reveal that mice carried the Smith capsular type of S, aureus, nor could we detect anticapsular antibodies against the latter.