STAPHYLOCOCCUS AUREUS: SOME HOST-PARASITE INTERACTIONS

STAPHYLOCOCCUS A UREUS: SOME HOST-PARASITE INTERACTIONS

Frank A. Kapral

Department of Medical Microbiology The Ohio State University College of Medicine

Columbus, Ohio 43210

The accumulation of detailed knowledge on host-parasite interactions is obviously a complex and difficult task that requires multiple efforts and is often dependent on progress in other areas. It has become increasingly clear that staphylococcal infections actually represent a group of diseases, each with its own set of parameters, though the same host and bacterium is involved. There is no simple answer to the question of what is responsible for virulence or what constitutes an immune mechanism. The organism is capable of producing a large array of biologically active substances, and the host can manifest many responses; the importance of all of these can vary markedly with different types of staphylococcal disease.

THE ENTEROTOXIN AND ENTEROTOXINS

Early attempts to relate clinical disease to a particular staphylococcal product have for the most part proven fruitless, except in the case of staphylococcal food poisoning. One can question, perhaps, whether this entity quali- fies for inclusion in a discussion of host-parasite interactions, because infection of the host is not required. In any case, I do not wish to discuss the staphylococcal enterotoxins per se, since these are covered very aptly by Dr. Bergdoll later in this monograph. My purpose in broaching the subject concerns work by others who have demonstrated that organisms such as Vibrio cholerae, E. coli, Shigella dysenteriae, Clostridium perfringens, and possibly Vibrio para- hemolyticus elaborate substances capable of disturbing water and electrolyte transport in the small intestine. These substances have been named enterotoxins because of their direct action on the mucosa, which leads to fluid accumulation in the gut lumen. Despite the common label, these products exhibit an activity distinctly different from that of the classical staphylococcal enterotoxin.

The ability of S. aureus to proliferate in the gut and produce an enteritis or pseudomembranous enterocolitis is well recognized, particularly during the administration of broad-spectrum antibiotics, but factors involved in the pathogenesis of this disease remain obscure. It has been suggested that the enterotoxins are involved; however, most evidence supports the view that these substances bring about emesis by neural stimulation, either peripherally or centrally, but have no significant effect upon the intestinal mucosa.

Since &toxin is produced by most S. aureus strains and is capable of dis- rupting a wide variety of membrane-bound structures, we investigated the action of this toxin on the gut mucosa. Using the technique of McGonagle and colleagues,l in which intestinal segments (with blood supply intact) were perfused with saline solutions that contained 8-toxin, we were able to demonstrate that 8-toxin can interfere with water absorption in rabbit jejunum and

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268 Annals New York Academy of Sciences

ileum. In the jejunum this effect was more prominent in proximal segments than in distal segments, but in the ileum there was no difference between the responses of the proximal and distal segments.

It was also possible to demonstrate interference with water adsorption in guinea pig ileal segments maintained in vitro according to the method of Darlington and QuasteL2

Histological sections prepared from S-toxin-perfused segments after the termination of these experiments failed to reveal any visible effects on the intestinal mucosa.

When compared with cholera toxin or the E. coli enterotoxins, the action of S-toxin more nearly resembles that of the heat-stabile E. coli enterotoxin by virtue of the absence of a lag period, but it differs from it by exhibiting a sustained inhibition of water absorption for at least 6 hours.

Whether S-toxin stimulates adenyl cyclase activity in the mucosa, as does cholera toxin, remains to be seen. Furthermore, these findings do not necessarily implicate this toxin in the pathogenesis of staphylococcal enteritis, but merely suggest that this possibility should be explored. Nevertheless, one can pose the question, What is the staphylococcal enterotoxin: the classical enterotoxin, which has the prior claim to the name, or the S-toxin, which elicits the appropriate physiological response now associated with other enterotoxins?

EXFOLIATIN AND THE SCALDED SKIN SYNDROME

Let us return to the matter of associating a specific clinical response with a specific product.

The occurrence of a scarlatiniform rash in patients who were suffering from staphylococcal infections had been sporadically r e p ~ r t e d . ~ - ~ Individuals had attempted to demonstrate the presence of an erythrogenic toxin in culture supernatants of S. aureus isolated from such cases, but it was difficult to inter- pret the reported findings, since both the ct- and S-toxins were capable of producing erythema in human skin, and no attempts were made to distinguish between reactions to these products and to the presumed erythrogenic toxin.

A generalized exfoliative dermatitis of infants and children (Ritter’s disease, staphylococcal scalded skin syndrome) was first described in 1888 and was recently reviewed by Lowney and colleagues.’ This disease had been associated with phage group 2 strains of S. aureus.

Melish and Glasgow considered the development of a scarlatiniform rash, generalized exfoliative dermatitis, and bullous impetigo as representing a spectrum of response to infection with this organism.’ They were able to produce a generalized exfoliation in newborn mice that they had infected with phage group 2 strains of S. aureus isolated from patients who exhibited these diseases. This reponse occurred only in mice less than 5 days old, and only with phage group 2 strains. Mice infected with these strains revealed a Nikolsky sign 12-16 hours after infection. Bullae appeared about 4 hours later, and extensive exfoliation occurred shortly thereafter. The animals generally died after complete exfoliation. This response was initiated by subcutaneous or intraperitoneal inoculation of the organisms, but the organisms were not necessarily localized in the skin. This suggested that the substance responsible might be a soluble product.

Following the demonstration by Melish and Glasgow that these S. aureus

Kapral : Some Host-Parasite Interactions 269

strains could cause a generalized exfoliation in neonatal mice, we were able to demonstrate that this response was mediated by a soluble protein. This protein was purified, characterized, and called exfoliatin.8 Subsequent reports by Melish and colleagues and Arbuthnott and colleagues lo have confirmed our findings.

The toxin has an estimated molecular weight of 23,500 and is relatively heat-stabile, but acid-labile. Exfoliatin is antigenic, and exfoliatin antitoxin is protective." Normal Swiss mice become refractory to exfoliatin as they de- velop a hair coat, but adult hairless mutant mice are susceptible after they lose their coat.'? Small doses of purified exfoliatin (0.3-6 pg) were shown to produce bullae in an adult human who lacked antibody to this toxin,13 thus indicating that the human is indeed responsive to this material.

Although soluble exfoliatin is antigenic in rabbits, alum-precipitated exfoliatin is a much better immunogen. This preparation is not suitable for human immunization, however, since it retains about 50% of its skin-loosening activity. Exfoliatin antitoxin prepared in rabbits will protect neonatal mice against lethal doses of exfoliatin, even if antitoxin administration is delayed until 15 min before exfoliation commences in control animals. This suggests that if a suitable antitoxin of human origin were available, it might be of value in treating patients early in the disease process. Although antibiotics are frequently used in the treatment of the scalded skin syndrome, they do not promptly halt the rapid and progressive exfoliation usually seen during the first 24-36 hours.

Previous studies suggested that only phage group 2 strains of S. aureus produce exfoliatin.' More recent studies by us reveal that of more than 200 phage group 2 strains examined, only 40% produce exfoliatin. Exfoliatin production was not associated with any particular phage type. Moreover, the amount of exfoliatin elaborated by various strains, under comparable conditions, varied markedly. Another study in which we utilized approximately 1,000 randomly selected S. aureus strains demonstrated that exfoliatin is not limited to phage group 2 strains. In all, 19 non-group 2 strains that produced exfoliatin were uncovered; three belonged to phage group 1, eight were of phage group 3, and eight isolates were untypable.

Preliminary studies in collaboration with Dr. Merlin Bergdoll revealed that many exfoliatin-producing strains elaborate a heretofore unknown enterotoxin. Purified exfoliatin, however, had no enterotoxin activity when fed to monkeys. Production of enterotoxin is usually associated with phage group 3 strains, and production of enterotoxin by a group 2 strain was previously considered a rarity. The significance of these relationships is not known at present, but it is of interest that enterotoxins B and C have some direct skin toxicity.1s

It must be noted that it is not possible at present to determine whether all exfoliatin-producing strains can cause the scalded skin syndrome. The conditions that determine the amount of toxin produced in vivo and its subsequent distribu- tion are not known. The importance of maternal antibody and active immunization as determinants is not established either. Most patients with the disease do not exhibit primary localized staphylococcal lesions: it is therefore presumed that the source of toxin is organisms that are carried in the upper respiratory tract, or possibly in the eyes. Such concepts must be confirmed, and if true, require study of the mode of toxin absorption. Variations in the above parameters may determine whether a patient responds with a rash, bullous impetigo, or frank exfoliation.


It seems clear, however, that in the case of the scalded skin syndrome we can identify a particular staphylococcal toxin as being responsible for the major manifestations of the disease. This is not to say that exfoliatin is the only determinant involved, since further studies are required to give a better per- spective on the question. Neither may we infer that similar relationships necessarily exist with other types of staphylococcal infection.

COCCI IN CAVITIES

When a sufficient number of S. aureus (2 x los or more) are inoculated into the peritoneal cavity of mice or rabbits, the inoculum is promptly clumped, due to the interaction of the bound coagulase on the surface of the cocci with fibrinogen in the peritoneal cavity.l'-lz This phenomenon occurs only if the organisms possess the bound coagulase, and provided it is not covered by a capsule. If less than 2 x los are inoculated, clumping is ineffective and the organisms are readily phagocytized by the preexisting macrophages in the peritoneal cavity. Clumped organisms are effectively protected from phagocytosis. There is no evidence that these clumped organisms multiply in the peritoneal cavity for at least 12 hours.

Due to an infiux of neutrophils, the clumps of cocci become surrounded- by leukocytes 2-3 hours after inoculation. These clumps and associated leukocytes then aggregate into one or two large clusters by thC fifth hour. If the inoculum is capable of producing a lethal dose of either a-toxin or 8-toxin (15-20 HD,, or 200 HD,, respectively) within two hours after inoculation (before the clumps are surrounded by leukocytes), the animal dies by the sixth to eighth hour. Because a-toxin is much more potent than %toxin, the former is the major lethal factor in this type of infection; the latter becomes important only if a-toxin is not produced or is neutralized by antitoxin.

It was found that the synthesis of a-toxin is induced by CO, and repressed by glucose. Although it resembles typical enzyme induction in many respects, the mechanism for this effect is not understood. By growing the organism in chemostats under appropriate CO, tensions (0-15%) for at least four genera- tions, we were able to prepare inocula of the same S. aureus strain at different levels of a-toxin induction. When inoculated intraperitoneally, these prepara- tions exhibited a 15-fold difference between the LD,, of fully induced and noninduced inocula (5 x lo5 and 8 X lo9 cocci respectively). In vivo measure- ments revealed that noninduced or slightly induced inocula (5 X lo9 cocci) were unable to elaborate a lethal dose of a-toxin during the first 2 hours after inoculation, prior to being surrounded by leukocytes. The thick layer of leukocytes, once deposited, prevented further egress of a-toxin into the peritoneal cavity, and the hosts survived.

Certain strains of mice were unable to muster a significant neutrophil influx after intraperitoneal infection. In these animals clumping occurred as usual, but the clumps did not become surrounded by leukocytes. These mice proved susceptible to .smaller doses of staphylococci and exhibited a shorter survival time than mice with normal responses. Presumably the lack of leukocyte deposition about the clumped organisms permitted a more sustained absorption of a-toxin.

If the infecting strain possesses a capsule, then the bound coagulase- fibrinogen interaction does not occur, but in this case the capsule itself acts as


a protection by reducing phagocytosis of the dispersed inoculum. The Smith mucoid strain (a representative encapsulated strain) does multiply in the peritoneal cavity and continues to elaborate a-toxin in the process.

Initial comparisons of determinants that are important in the intrapleural infection and those known to be operative in the intraperitoneal infection reveal similarities, but also quantitative differences. Fewer organisms suffice to kill the host infected intrapleurally than the host infected intraperitoneally (LD,, = los versus 8 X los). Clumping of the inoculum occurs promptly in the pleural cavity, and a neutrophilic influx during the second to fourth hour causes the clumps to become surrounded by a leukocyte layer. a-Toxin elaborated during the initial hours (prior to neutrophil influx) is the prime but not the sole mechanism for subsequent death of the host. The absorption of a-toxin from the pleural cavity is more rapid than from the peritoneal cavity, and this is reflected by a shorter survival time in intrapleurally infected mice (with the same doses). Bacterial invasion of the lungs does not occur, but a prominent inflammatory response and some consolidation is evident in lung tissue removed at the time of death.

Mice and rabbits given a lethal dose of S. aweus intraperitoneally or intrapleurally exhibit marked respiratory distress (labored breathing and cyanosis) 3-4 hours prior to death. Death usually occurs during a tonic convulsion that lasts 30-60 sec, in which gasping is a prominent feature. These observations prompted an investigation to determine whether this infection could reduce the pulmonary surfactant and thus account for the respiratory symptoms.

Mice and rabbits were given a lethal dose of S. aureux via intraperitoneal injection. Immediately upon death the lungs, together with the trachea, were removed and compliance curves ( pressure-volume changes) were determined. Lungs obtained from normal animals sacrificed at the same time served as controls. There was no significant difference between the compliance curves obtained from infected animals and from controls; this indicated that the attendant respiratory symptoms are not mediated through a significant reduc- tion in the effectiveness of the pulmonary surfactant.

In summary, then, some determinants known to play a major role in the acute phase of either the intraperitoneal or the intrapleural infections are: (1 ) the production of a- and 8-toxins, (2) the bound coagulase, ( 3 ) capsules, and (4) the influx of neutrophils into the regions. None can be singled out as being more important than the others, and their roles must be considered in unison. Deficiencies in one factor may in some cases be compensated by other effects, thus leading to the same eventual outcome.

My main point, however, is not the aforementioned infections, since they are for the most part laboratory infections and are rather unlikeIy to occur naturally in humans. The most common human staphylococcal infection is the furuncle, yet attempts to produce abscesses suitable for study have by and large been stymied. One of the benefits to arise from the previous studies has been the realization that the intraperitoneal infection may be used to produce abscesses in a controlled fashion, under conditions amenable to continued study.

By employing noninduced inocula, it was possible to inoculate a sufficient quantity of organisms to achieve clumping, but since these could not elaborate a lethal dose of a-toxin, continued obskrvations on the fate of the inoculum could be made. Within 24 hours the clusters of organisms and leukocytes were covered with a layer of fibrin that contained scattered neutrophils. By 48 hours


deposition of connective tissue began, and this continued until the fourth day, when the lesions were characteristic of typical staphylococcal abscesses.

Once deposition of leukocytes about the clumped organisms has occurred, the foci can be removed readily from the peritoneal cavity and the organisms can be quantitatively recovered after homogenation in a tissue grinder. Such techniques have permitted studies on population changes within abscesses, as well as measurement of a-toxin production.

Three strains of S. aureus that differed in the maximum amount of a-toxin produced in vitro under optimal conditions were studied in this manner. To ensure host (mouse) survival, however, inocula were prepared so as to be minimally induced. More than 99% of the inoculum was recovered from the leukocyte-surrounded clumps (3 hours after inoculation). Subsequent survival of the staphylococcal population within abscesses was correlated with its ability to produce a-toxin. In vitro, upon induction, these strains produced maximal a-toxin concentrations of 10, 70, and 300 HD,,] per lo9 cocci. The correspond- ing half-lives of the population within abscesses were 1.5, 5, and 16 days.

Studies were also performed to measure the accumulation of a-toxin within such abscesses. Peak levels respectively averaging 80 and 900 HD,, per mouse were obtained with the two strains that had the lowest and highest capacities to produce a-toxin in vitro. These levels within abscesses were attained within 4-6 days after inoculation, and persisted until the 30th-35th day. During this interval the a-toxin concentrations within the abscesses remained constant, while at the same time the viable staphylococcal population was decreasing, although at different rates for the different strains.

These findings indicate that impressive quantities of a-toxin can accumulate within abscesses without destroying the host. Whether any toxin escapes from the abscess and affects the host more subtly has not been ascertained. The suggestion that an innate capacity to produce a-toxin may be related to the organism’s ability to survive within abscesses deserves further consideration.

THE RENAL INFECTION

Upon intravenous inoculation of staphylococci more than 99% of the inoculum are cleared by the liver and spleen, but these organisms do not multiply, and are slowly eliminated.16, 1 7 Only in the kidneys is the multiplication of staphylococci regularly demonstrable.

In mice or rabbits, a consistent proportion of intravenously inoculated staphylococci (1 : 3,000) lodges in the kidneys. Some of these organisms in the kidneys proliferate and give rise to renal abscesses. Depending on the dose, either one or both kidneys may demonstrate this proliferation, but death results only with extensive involvement of both kidneys.

Using the criterion of “ability of the inoculum to multiply in the kidney,” a dose-response curve was established in mice with a strain of S. aureus capable of producing both the a- and &toxins. This relationship extended over a dosage range of several logs; 50% of kidneys exhibited some proliferation after an initial deposition of 7 x lo3 coccilg kidney. On the other hand, a dose- response curve that measured mortality as the effect ranged from a dose of 4 X lo3 to 4 X lo* (0-100% mortality) staphylococci originally deposited in the kidneys. Therefore it appears that although many organisms lodging in the kidneys can initiate multiplication, most of the resulting lesions heal spon- taneously.


In order to assess the importance of a-toxin in this type of infection, the behavior of three a-toxin-negative mutants was compared with that of their parent strains. It was found that initial deposition in the kidney was equal for mutant and parent (for the same i.v. dose), but the mutants failed to proliferate and were eliminated over a 1-2 week interval.

A similar study was done with a parent strain of S. aureus capable of producing large amounts of 8-toxin, but no u-toxin. This strain produces approximately 10-20 times the amount of &toxin usually produced by human isolates. Two mutants that lacked the ability to produce demonstrable 8-toxin were derived from this parent strain. The ability of the parent and mutant strains to grow within mouse kidneys was compared. Whereas the parent strain multiplied extensively, one mutant did not. The other mutant was capable of some multiplication, but never to the extent shown by the parent. Because of their different behavior, these mutants were examined for possible intracellular pools of 8-toxin and for the ability to elaborate small amounts of I-toxin that were undetectable by usual means. No intracellular pool was found, and culture supernatants had no demonstrable 8-toxin after a 200-fold concentration. It is possible that the one mutant capable of renal proliferation is producing a non- hemolytic yet biologically active molecular variant of 8-toxin, but until other means than hemolytic activity are available to characterize the &toxin this question cannot be resolved.

From the above studies it appears that the ability to produce a-toxin is important for initial multiplication in mouse and rabbit kidney, but the role of I-toxin is still undetermined.

A comparison of the survivaI in the kidney of u-toxin-induced and noninduced inocula revealed some differences in behavior. These differences were dose-dependent. Whereas induced inocula exhibited a 500- to 1,000-fold increase regardless of the initial renal dose, noninduced inocula manifested a 1-2 day lag before proliferating, and attained a renal concentration of about lo7 coccilg with both high and low infecting doses. The significance of these findings, however, cannot be appreciated fully without further studies on the pathogenesis of the renal infection.

Because most infected animals exhibit numerous renal abscesses upon death (at least in one kidney), the question was raised whether death resulted from renal failure. This initiated studies to measure the urea levels in blood during the course of the disease.

Groups of rabbits were bled three times a week until death ensued or the experiment was terminated. Irrespective of the dose, there were no significant changes in blood urea nitrogen values during this period. Therefore, in spite of extensive proliferation of the organisms, there is sufficient functional renal tissue to prevent uremia. It is of course possible that death results from dis- turbances in other renal functions, or that lesions in the kidneys simply serve as a source of staphylococcal products which act elsewhere.

CONCLUSIONS

Present evidence indicates that the prime clinical manifestations of the scalded skin syndrome can be attributed to the production of exfoliatin in vivo by certain strains of S . aureus. Such a simple and direct relationship between product and disease does not appear to exist in other types of staphylococcal


infection. I n a few systems that have been studied, the interplay of several determinants seems decisive.

The previous tendency has been to envision the role of certain products of the organism, notably the hemolysins, in terms of their direct toxic effects upon the host. Our data suggest that although these concepts may be pertinent under some conditions, it may be more important t o consider the role of these substances in the economy of the parasite under in vivo conditions, particularly as they influence multiplication and survival.

REFERENCES

1. MCGONAGLE, T. J., H. A. SEREBRO, F. L. IBER, T. M. BAYLESS & T. R. HENDRIX. 1969. Time of onset of action of cholera toxin in dog and rabbit. Gastroen- terology 57: 5.

2. DARLINGTON, W. A. & J. H. QUASTEL. 1953. Absorption of sugars from isolated surviving intestine. Arch. Biochem. Biophys. 43: 194.

3. STEVENS, F. A. 1927. The occurrence of Staphylococcus azlreus infection with a scarlatiniform rash. J. Amer. Med. Assoc. 88: 1957.

4. ARANOW, H. & W. B. WOOD. 1942. Staphylococcic infection simulating scarlet fever. J. Amer. Med. Assoc. 119: 1491.

5. DUNNET, W. N. & E. M. SCHALLIBAUM. 1960. Scarlet-fever-like illness due to staphylococcal infection. Lancet 2: 1227.

6. LOWNEY, E. D., J. V. BAUBLE, G. M. KREYE, E. R. HERRELL & A. R. MCKENZIE. 1967. The scalded-skin syndrome in small children. Arch. Dermatol. 95: 359.

7. MELISH, M. E. & L. A. GLASGOW. 1970. The staphylococcal scalded-skin syndrome. Development of an experimental model. New Engl. J . Med. 282: 1114.

8. KAPRAL, F. A. & M. M. MILLER. 1971. A product of Staphylococcirs aureus responsible for the scalded-skin syndrome. Infec. Immunity 4: 541.

9. MELISH, M. E., L. A. GLASG~W & M. D. TURNER. 1972. The staphylococcal scalded-skin syndrome: Isolation and partial characterization of the exfoliative toxin. J. Infec. Diseases 125: 129.

10. ARBUTHNOTT, J. P., J. KENT, A. LYELL & G. G. GEMMELL. 1971. Toxic epidermal necrolysis produced by an extracellular product of Sraphylococcus aureus. Brit. J . Dermatol. 85: 145.

1 1 . MILLER, M. M. & F. A. KAPRAL. 1972. Neutralization of Staphylococcus aureus exfoliatin by antibody. Infec. Immunity 6: 561.

12. KAPRAL, F. A. & M. M. MILLER. 1972. Skin lesions produced by Staphylococ- cus aureus exfoliatin in hairless mice. Infec. Immunity 6: 877.

13 . KAPRAL, F. A. 1973. Lesions produced in an adult human by Sraphylococcus aureus exfoliatin. J. Infec. Diseases. In press.

14. KAPRAL, F. A. 1966. Clumping of Sraphylococcus aureus in the peritoneal cavity of mice. J . Bacteriol. 92: 1166.

15. KAPRAL, F. A. 1966. An analysis of factors involved in experimental staphylococcal peritonitis and their possible role in other types of staphylococcal infections. Postepy Mikrobiologii 5: 297.

16. LI, I. W. & F. A. KAPRAL. 1962. Virulence and coagulases of Staphylococcus aureus. 11. Survival of certain coagulase negative mutants in the organs of intravenously infected rabbits. J. Infec. Diseases 111: 204.

17. KARAS, E. M. & F. A. KAPRAL. 1962. Virulence and coagulases of Staphylo- coccus aureus. 111. Survival of certain coagulase negative mutants in the organs of intravenously infected mice. J. Infec. Diseases 111: 209.

18. BERGDOLL, M. Personal communication.


DISCUSSION OF THE PAPER

DR. CHESBRO (University of New Hampshire, Durham, New Hampshire) : I was interested in something that you pointed out, Dr. Kapral, or perhaps it was implicit, because it seemed to bear on the dialogue that Dr. Park and Dr. Ekstedt had a while ago about physiological changes in the host versus the selection of preexistent mutants in the infective dose. In your longer-term experiments, in which you studied the strains that produce higher and lower amounts of a-toxin, one wonders whether you didn’t set up a selective condition.

DR. KAPRAL: I think this is a real possibility. It may not be evident with strains that produce very large or very small amounts of a-toxin, but if YOU recall the data with the 182 strain, where the population within abscesses first decreased rapidly for four days, then increased until the seventh day, and later decreased slowly, this may represent the overall response of a mixed population, in which one organism can survive better than the other. We don’t have any data to substantiate this at present, however.

It is important to realize that in vivo conditions are radically different from those we usually employ in the laboratory. It was under in vivo conditions that we first noted induction of a-toxin. This was later shown to result from an increase in CO, tension and a decrease in glucose concentration, brought on by the inflammatory response.

DR. MELISH (Hawaii) : I want to congratulate you on the extension of the work with the scalded skin syndrome. I think your observation that the epi- dermolytic toxin, as I prefer to call it, is produced by strains other than those in phage group 2, is very important. As you know, this is not what I found, but I used a much smaller group of non-phage group 2 organisms. Recently, I have found that it was a mixed group 2 and group 3 phage pattern.

Were all of your nontypable strains tested with lOOX routine test phage dilution?

DR. KAPRAL: Yes, all strains were tested with lOOX routine test dilution. DR. M. E. MELISH: And you had 19 out of 200 or 6 out of 200? DR. KAPRAL: No, 19 out of about 1,000 strains. About half of these

randomly selected strains were from Ohio State University Hospital, and the other half were from the Cincinnati General Hospital.

DR. DERO (Downstate Medical School, Brooklyn, New Y o r k ) : I would also like to comment on the scalded skin syndrome. I think that here, too, the host is extremely important. The very same clinical and histologic picture is seen in adults, and there it is caused not by staphylococci, but by drugs.

DR. KAPRAL: I agree. When we’re talking about the scalded skin syndrome, however, I think most now tend to consider it as being of staphylococcal etiology. The other situation, Lyell’s syndrome, is perhaps mediated through drug-hypersensitivity,

DR. MELISH: We’ll be talking a little more about this later in this monograph, but I think that they are not clinically and histologically identical. In the scalded skin syndrome as it is seen in children, the histologic lesion is high in the epidermis. The lesion seen in adults is generally lower in the epidermis, at the dermal-epidermal junction. There has been a lot of confusion about that up to this time. There is also quite a difference in the mortality rates, and it’s possible that some of the things seen in adults may be due to staphylococci as


well. But I think they are probably different syndromes; they are clinically quite close, but histologically different.

DR. ADLAM ( Wellcome Research, England) : Dr. Kapral, when you induce the production of a-toxin in the 182 organism, are other components induced at the same time? Have you looked, for example, for coagulase or leucocidin production?

DR. KAPRAL: We haven’t looked at this critically, but I suspect that the leucocidins are probably induced.

The effect is that when the organism is exposed to appropriate concentrations of CO, in the presence of oxygen and in the absence of appreciable glucose, it begins to produce a-toxin at a very rapid rate. The extent of this response is dictated by the genetic capabilities of the strain. We refer to this as induction. But I suspect this is a more general phenomenon, and probably affects the synthesis of more than one component.

DR. ADLAM: May I say a few words about the differences between staphylococci grown in vivo and in vitro? I think a lot of evidence is now accumu- lating that there are considerable differences between organisms harvested directly from the animal and organisms that are grown in broth (Symp. Soc. Gen. Microbiol. 22: 1 ) .

First of all there is the work of Gellenbeck (J . Bacteriol 83: 450) and Beining and Kennedy (J . Bacteriol. 85: 732) , who showed that there are marked differences between the aerobic respiration, antigenic composition, and virulence of the organisms harvested from the animals and the same organisms grown in vitro. Then there is the work of Gladstone and Glencross, who showed that staphylococci grown in implanted dialysis sacs in vivo produced more a-toxin than organisms grown in vitro. Finally, in some of the work that we did three or four years ago, we showed that staphylococci grown in vivo were more resistant than organisms grown in vitro to intracellular killing by polymorpho- nuclear leucocytes and to killing by serum and phagocyte lysate bactericidins (J. Med. Microbiol. 3: 147 and 157).

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STAPHYLOCOCCUS AUREUS: SOME HOST-PARASITE INTERACTIONS