A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

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Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal a-toxin indicated the presence of a lytic factor other than a-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified a-toxin (HP a-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure a 12S-toxin. The interaction of HP a-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The a 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An a 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the a 12S molecule. Lipid monolayer experiments showed that HP a-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP a-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to a-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to a 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.

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PLATE VTHE haemolytic activity of Escherichia coli has been investigated by only a few authors. Although there are many reports in the literature about haemolytic E. coli, most authors have merely noted that haemolytic strains were isolated. Lovell and Rees (1960) and Smith (1963) first demonstrated the production of an extracellular haemolysin (designated a-haemolysin by Smith) in an alkaline meat-extract broth. Smith (1963) showed also that a cell-associated haemolysin (designated ,6-haemolysin) was produced by some strains. Other investigators (Snyder and Koch, 1966; Snyder and Zwadyk, 1969; Short and Kurtz, 1971) observed extracellular haemolytic activity when a strain of serogroup 0 6 was grown in chemically defined medium.There is little information about the purification of E. coli a-haemolysin. Zwadyk and Snyder (1971), like Lovell and Rees (1960), were unable to recover haemolytic activity after dialysis of ammonium-sulphate precipitates. Short and Kurtz (1971) found that two peaks of haemolytic activity appeared after chromatography of culture filtrates on Sephadex G-200 and Sepharose 6B. However, they gave no indication of the extent of purification and the activity of their starting material was low.Only one author, Smith (1963), has investigated the biological properties of a-haemolysin. He observed that the toxin was active against a range of erythrocyte species, that large amounts of haemolysin were required to kill mice and rabbits after intravenous injection, and that haemolytic culture filtrates did not cause dermonecrosis in the skin of rabbits or guinea-pigs. The haemolysin was unreactive after intragastric administration to rabbits.In the present study we have attempted to clarify some of the deficiencies of previous work. Our main objectives were as follows: (a) to produce large amounts of high-titre a-haemolysin, (b) to purify and characterise this agent, and (c) to investigate the biological properties of a-haemolysin in preparations with high specific activities. In an accompanying paper, the kinetics of erythrocyte lysis by a-haemolysin are reported (Rennie, Freer and Arbuthnott, 1974).

At least three different forms of staphylococcal a-toxin have been shown to exist: soluble active a-toxin (a 3S), soluble inactive a-toxin (al2s), and insoluble inactive aggregate. Aggregation to the insoluble, biologically inactive form could be induced by brief heating to 60 C. The aggregate was dissociated by treatment with 8 M urea with reappearance of biological activity. Subsequent removal of urea by dialysis resulted in some spontaneous reaggregation to the insoluble state. The supernatant

PLATE VITECHNIQUES for studying the mode of action of bacterial toxins at the molecular level are fast becoming valuable and necessary tools for the determination of the role of these agents as virulence factors in diseases of man and animals. The mechanism of action of Escherichia coli haemolysin is poorly understood. Although enough evidence now exists to conclude that calcium ions are required for haemolytic activity, only two groups of investigators have attempted to study the kinetics of erythrocyte lysis by E. coli a-haemolysin.The findings of Zwadyk and Snyder (1971) and Short and Kurtz (1971) indicated that lysis of sheep erythrocytes was dependent on haemolysin concentration, pH and temperature. Less haemolysis was observed as the concentration of red cells (RBC) was increased. It has been suggested that a complex consisting of haemolysin, calcium ions and RBC must be maintained until the lytic event (Short and Kurtz, 1971). These authors found that the addition of ethylenediaminetetraacetic acid (EDTA) at any time during the haemolytic reaction prevented subsequent haemolysis. They also noted that neither reducing agents nor lecithin or cholesterol affected a-haemolysin.Both groups of workers used methods that required centrifugation of reaction mixtures at different times, followed by spectrophotometric estimation of haemoglobin. In these processes there are unavoidable delays during which haemolysin-affected cells may be lysed.In this study, the turbidity of reaction mixtures was monitored continuously at 650 nm in a controlled-temperature spectrophotometer. By this technique it was possible to make rate measurements on haemolysis curves. MATERIALS AND METHODSHaemolysin production and purification. The preparations of E. coli u-haemolysin used in this study were produced in glucose-nutrient-broth medium and were purified by the three-stage method of Rennie and Arbuthnott (1974). Because it was more readily available, stage-I1 u-haemolysin was used for most assays. However, in each experiment, purified stage-III a-haemolysin was tested to confirm the results obtained with less pure preparations. (Rennie and Arbuthnott, 1974). Kinetic studies were carried out with silica cells of 1-cm light path in a controlled-temperature Unicam SP800 spectrophotometer, to which was attached a Unicam SP22 chart recorder (Pye-Unicam, Cambridge, England) set at a five-times multiplication factor. The spectrophotometer was adjusted so that extinction (E) was monitored at a constant wavelength of 650 nm.A 0.7% suspension of sheep erythrocytes (SRBC) was made in Veronal buffer, pH 7.3 (Cruickshank, 1969), containing lOmM CaCl2 (VC buffer). The spectrophotometer and chart recorder were then calibrated on a linear scale with the SRBC suspension. The contents of a 1-cm cell, containing 1-4 ml of VC buffer and 0.1 ml of 0.7% SRBC, were mixed by inversion and read at E 6 5 0 . The zero controls of the spectrophotometer and chart recorder were adjusted if necessary such that the E650 was 0.40. This represented 100% erythrocyte ...

PLATES XIII-XVIT is now generally accepted that strains of Staphylococcus aureus isolated from patients with staphylococcal toxic epidermal necrolysis of Ritter's type (TEN) or staphylococcal bullous impetigo produce an exotoxin, termed epidermolytic toxin, that is responsible for the intra-epidermal splitting in the plane of the stratum granulosum that characterises these lesions. In TEN, large areas of skin are affected and the loosened epidermis peels extensively. The discovery of epidermolytic toxin followed the important observation of Melish and Glasgow (1970) that staphylococci isolated from patients with TEN or bullous impetigo caused extensive epidermal splitting when injected into newborn mice. Subsequent work has been concerned mainly with the isolation and partial characterisation of the epidermolytic toxin (Arbuthnott et al. The toxin is a relatively thermostable antigenic protein with a molecular weight of approximately 25 000. The preparative isoelectric focusing studies of Arbuthnott et al. (1974) showed that the main form of epidermolytic toxin has PI = 7.0; a minor component having a PI = 6-0 was detected. In thinlayer-gel isoelectric focusing the main form exhibited complex microheterogeneity, and up to seven protein bands were detected between pH 6-2 and 7.0. Recently, Japanese workers have described the isolation of two serologically distinct forms termed A and B toxins (Kondo et al., 1974 and 1975), and the studies of Arbuthnott et al. (1974) also suggested the existence of two antigenically distinct forms of the toxin.The present study was undertaken with the aim of assessing the specificity and sensitivity of in-vitro methods for detecting and measuring epidermolytic toxin production by S. aureus. The strains were provided by Dr Elizabeth H.