Factors Affecting the Lysis of Erythrocytes Treated With Staphylococcal Β Toxin

Abstract

The action of staphylococcal /8 toxin on the erythrocytes of a number of species of animal was described by Bryce and Rountree (1936). These workers found that when the j8 toxin was mixed with the erythrocytes of certain species and the mixture held at 37° C. for one hour there was no lysis, but if it was then transferred to the cold the cells lysed. Christie, Atkins and Munch-Petersen (1944) showed that this lysis could be completed in ruminant erythrocytes at 37° C. by the action of a factor produced during the growth of Streptococcus agalactiae. Wilson and Slavin (1950) and Murphy, Stuart and Reed (1952) showed that certain other organisms, notably some strains of Streptococcus uberis, gave the same reaction, whilst not all strains of Strep, agalactiae did so. Murphy et al. designated the factor responsible for this reaction CAMP. This factor has no demonstrable effect on cells not treated with /3 toxin.During the development in this laboratory of a "spot" plate test to detect Strep, agalactiae in contaminated cultures, and a tube test for the assay of CAMP, several interesting reactions were observed. These reactions form the basis of the work described in this paper. MATERIALS AND METHODS. staphylococcal p toxin.This was prepared from a growth of the pure /3 toxin producing staphylococcus S32A of Bryce and Rountree, kindly supplied by E. Munch-Petersen. A sloppy agar culture of S32A, incubated for 48 hours at 37° C. in an atmosphere of 20 p.c. CO2 was filtered through muslin, centrifuged at about 3,500 r.p.m. for 20 minutes, and filtered through a Seitz E.K. pad. The filtrate, referred to as p toxin, was stored in bottles at 4° C. Standardisation of p toxin.Serial double dilutions of p toxin were prepared in 0-5 ml. quantities in normal (physiological) saline. To each tube was added 0-5 ml. of a suspension of 3 p.c. washed sheep orythrocytes in a 1 in 4 dilution in saline of neutral CAMP factor. Lysis in this system was found to run parallel with titration of p toxin by the usual "hot-cold" method. The last tube showing complete lysis was taken as the end point. CAMP factor.This was produced by growing Strep, agalactiae in crystal violet broth. For stock CAMP factor our strain 519/13/1 was used, the culture being filtered through a Seitz E.K. pad and the filtrate stored in the cold.

Streptococcus pyogenes streptolysin O as a cause of false-positive CAMP reactions

“…However, SO produced in broth cultures is never entirely oxidized, and some activity is present even in the absence of reducing agents (11). Sphingomyelinase-depleted SRBC (f-SRBC) are highly susceptible to lysis by a wide variety of physical, chemical, and biological agents (14) and are also more liable to lysis by SO than SRBC not exposed to staphylococcal sphingomyelinase (Table 1). It would seem that the SO produced by group A streptococci in the CAMP test system has sufficient activity to lyse the fragile 3-SRBC used.…”

“…It has been shown that the staphylococcal beta-lysin is a sphingomyelinase (9) which depletes the erythrocyte membrane of lipids, making the erythrocytes susceptible to subsequent lysis by a variety of physical, chemical, or biological agents. These agents include mechanical damage, temperature or pH changes, and EDFA as well as the CAMP factor of GBS (14,16). The extracellular CAMP factor is now known to be a thermostable protein with a molecular weight of 23,500 and the ability to lyse beta-lysin-sensitized, but not intact, SRBC (1).…”

Rapid tube CAMP test for identification of Streptococcus agalactiae (Lancefield group B)

“…Further, the requirements for optimal CAMP factor production (12), in particular pH, peptone, and carbohydrate content, must be met by the same medium. In addition, even in the absence of CAMP factor, physical factors, including alterations in temperature, pH, and osmolarity, may induce lysis of the beta-lysin-affected sheep RBC (11,16). This multiplicity of variables helps to explain the vagaries of CAMP tests, without even considering the problems of lysis due to non-group B streptococci.…”

Nature and mechanism of action of the CAMP protein of group B streptococci

“…However, the cells do not lyse unless a further condition is imposed upon the system. The additional requirement can be met in a variety of ways, as by chilling (5), addition of ethylenediaminetetraacetate (22), alteration of pH or salt concentration (20,25), or addition of a specific extracellular product of streptococci belonging to Lancefield group B (Streptococcus agalactiae) (8). The last agent provides the basis for a laboratory test for the presumptive identification of group B streptococci, used in veterinary microbiology since the pioneering work of Christie et al in 1944 (8).…”