Phage group II Staphylococcus aureus has been identified as the etiological agent of the staphylococcal scaleded skin syndrome. The development of an animal model system permitted fulfillment of Koch's postulates and recognition of exfoliative toxin (ET) as being responsible for some of the clinical manifestations of this syndrome. Initial studies directed toward associating a lysogenic phage with the genetic control of ET synthesis failed to support this hypothesis. Growth of two Tox+ strains at 44 C was more effective than growth in ethidium bromide or sodium dodecyl sulfate in eliminating the ability to produce ET. The early and rapid accumulation of ET-negative (Tox-) variants during growth of strain UT 0007 at 44 C, the lack of any selective advantage of the Tox-variants over Tox+ cells during growth at 44 C, and an enhanced elimination frequency at 44 C of 97.9% over the spontaneous frequency of loss strongly suggest that the gene for ET synthesis is extrachromosomal. Additional evidence suggests that this gene is located on a plasmid which is not associated with genes for penicillinase synthesis and cadmium resistance. Two Tox+ strains harbored lysogenic phage capable of transducing cadmium resistance, but not penicillin resistance, to specific Tox-recipients.
Staphylococcal phage group 2 strain UT0007 was previously shown to contain a high-molecular-weight plasmid containing genes for exfoliative toxin (ET) and bacteriocin production. Phage group 2 strains UT0002 and UT0003 (Tox+Bac-) underwent a twofold and ninefold loss of ET activity, respectively, after growth at 44 C for 18 h. Strain UT0002 also lost total bacteriocin activity. Both strains contained (i) a 56S plasmid that was lost from those substrains showing reduced ET activity and (ii) a 21S plasmid with a gene for cadmium resistance that could be transduced into two recipient strains. Since the ET plasmid-negative substrains still made ET, it was postulated that this residual toxin was made from chromosomal genes. In characterizing the plasmid species from strains UT0002 and UT0003, the 21S but little or no 56S plasmid deoxyribonucleic acid could be isolated after centrifugation of cleared lysates from these strains on dye-buoyant density gradients. Treatment of cleared lysates from strain UT0002 with ethidium bromide, Pronase, or sodium dodecyl sulfate, but not heat at 60 C, induced conversion of the 56S closed circular ET plasmid to a 38S open circular form as determined after centrifugation on 5 to 20% neutral sucrose gradients.
Determinations of the ED50 of cultures of Mycoplasma arthritidis disclosed the existence of great diversity in the arthritogenic properties of the various strains. In most cases, the ED50 values were lowered after passaging in rats, and a more severe arthritis with a shorter period of onset was observed. Heavy suspensions of arthritogenic M. arthritidis did not appear to induce any toxic symptoms. Prior injections of endotoxin did not enhance the toxicity of M. arthritidis suspensions. The more arthritogenic strains grew more slowly in the basal medium and remained viable for longer periods of time than those with lower arthritogenic properties. All strains were identical on the basis of complement-fixation tests. Minor differences observed between strains during gel-diffusion studies could not be correlated with arthritogenic properties. Arthritogenic strains appeared to be less susceptible to the inhibiting action of rabbit antisera than were the nonarthritogenic strains. 538 on July 31, 2020 by guest http://iai.asm.org/ Downloaded from
Arthritis was produced in rats by the intravenous injection of Mycoplasma arthritidis. Metabolic inhibiting antibody and indirect hemagglutinating antibody could not be detected in the sera of arthritic or convalescent animals. Nonmurine species of mycoplasma were capable of inducing metabolic inhibiting antibody in the rat. A hypothesis based upon the possible occurrence of heterogenetic antigens common to M. arthritidis and rat tissue was brought forward to explain these findings. Complement-fixing antibody to M. arthritidis was detected 3 to 4 days after injection and subsequently rose to high levels, depending upon the severity of arthritis and number of organisms injected. Animals that had recovered from intravenous or subcutaneous inoculation with M. arthritidis were resistant to subsequent infections by the organism. Immunity could be passively transferred by the intravenous injection of convalescent serum. Adsorption of the convalescent serum with antigen greatly reduced the complement fixation titer but did not significantly alter the protective properties of the serum. The presence of complement-fixing antibody could not be related to the development of immunity. An avirulent strain of M. arthritidis and a strain previously classified as M. hominis type 2 were capable of inducing resistance to subsequent injection by virulent M. arthritidis. Histological studies of rat arthritis induced by Mycoplasma arthritidis have been well described in the literature (8, 15, 16, 21), but little detailed work has been reported on antibody response or the mechanisms involved in the development of immunity. Early studies by Collier (5) and Woglom and Warren (22) indicated that the subcutaneous (sc) or intravenous (iv) injection of rats with M. arthritidis resulted in the development of resistance to reinfection. Both of these studies failed MATERIALS AND METHODS Strains used. The sources of the mycoplasmas used in this study are listed in Table 1. The pathogenicity for rats of strains previously called M. hominis type 2, Campo, was established previously (4). To increase virulence, M. arthritidis strains 158 and 14124 were passaged in rats 10 times by sc injection. Mycoplasmas cultured from the resulting abscesses were injected sc into normal rats. The passaged strains were designated 158 PlO and 14124 PIO, respectively. The virulence of these organisms was similar to that of freshly isolated strains (B. C. Dole and L. Rowland, unpublished data). Unless otherwise stated, all experiments reported in this study were conducted with M. arthritidis strain 158 P10. Media. The medium used for maintenance of mycoplasma strains consisted of mycoplasma agar 930
The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.
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