The protective effect of intravenously administred rabbit antitoxin serum was studied in lethal Pseudomonas aeruginosa bum infections in mice. Survival after infection with 2 median lethal doses of a toxigenic, low-protease-producing strain (PA103) was enhanced in antitoxin-treated mice, as compared with controls that had received anti-bovine serum albumin serum (P = 0.0004). Survival time was prolonged in other antitoxin-treated mice infected with toxigenic, highprotease-producing strains (PA86 and PA220, P = 0.0003 and P = 0.01, respectively). In contrast, antitoxin had no protective effect in mice challenged with a nontoxigenic strain (WR 5, P = 0.57). There were fewer viable bacteria in blood and liver of antitoxin-treated mice than in those of anti-bovine serum albumintreated controls after infection with toxigenic organisms, whereas there were no significant differences between the two groups after challenge with the nontoxigenic strain. These data suggest that P. aeruginosa exotoxin A contributes to lethality in this burn infection model, and this effect is diminished by passive immunization with antitoxin.

A trypsin-sensitive, heat-labile exotoxin of Pseudomonas aeruginosa strain P-A-103 has been purified by a procedure that includes membrane ultrafiltration, hydroxylapatite chromatography, ion-exchange cellulose chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the exotoxin with a 40-fold increase in specific activity (micrograms of protein per median lethal dose [LD5J). The mean lethal dose of the purified toxin administered intravenously into mice weighing 20 g was approximately 6 'sg of protein. The toxin contained virtually no nucleic acid, detectable pigment, or lipopolysaccharide. When subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the toxin separated into at least six protein components which appeared to have similar molecular weights. The estimated molecular weight of the toxin is 54,000, and its isoelectric point is 5.0

Antibody to Pseudomonas aeruginosa exotoxin was detected in human sera by using a cytotoxicity-neutralization assay. Serum antitoxin was present in high titer in all 14 patients who recovered from serious pseudomonas infections (log2 of 50% neutralization titer, mean + standard deviation = 6.0 + 1.2). In contrast, serum antitoxin was present in lower titer in four of seven patients with fatal pseudomonas infections (3.3 + 2.7, P < 0.005), in 3 of 7 patients with non-pseudomonas infections (1.4 ± 0.6, P < 0.001), and in 6 of 14 normal control subjects (2.0 ± 1.3, P < 0.001). Fourfold or greater serum antitoxin rises were demonstrated in two survivors of acute infections, and toxin-neutralizing activity was associated with the immunoglobulin G fraction of human sera. Immunization of rabbits with purified exotoxin also induced high antitoxin titers.

Seventy-five consecutive clinical Pseudomonas aeruginosa isolates were tested for in vitro exotoxin production. Exotoxin was demonstrated in culture filtrates biologically, by its ability to produce characteristic dermonecrotic lesions in guinea pigs, and serologically, by counterimmunoelectrophoresis (ClE) with rabbit antiserum elicited with purified exotoxin. By these two methods, exotoxin was detected in 87 and 89% ofP. aeruginosa strains, respectively (r = 0.48, P < 0.001). Although less sensitive than CIE in detecting exotoxin, immunodiffusion demonstrated a reaction of antigenic identity in most cases. Exotoxin was produced by all seven Fisher-Devlin immunotypes and by untypable strains. In contrast, exotoxin was not detected in the culture filtrates of 16 non-aeruginosa pseudomonas isolates and 48 non-pseudomonas organisms. The production of biologically similar and antigenically closely related exotoxins is thus a characteristic of the majority of P. aeruginosa strains derived from diverse clinical sources.

Pseudomonas aeruginosa exotoxin has been purified to a specific activity of 12,000 to 16,000 mouse median lethal doses/mg of protein. Total recovery was about 25%, and the degree of purification was approximately 3,000-fold. Preparative polyacrylamide gel electrophoresis greatly facilitated purification. As judged by analytical disc gel electrophoresis, the purified toxin contained one major band of protein and only a negligible amount of contamination. Antiserum prepared against the purified toxin neutralized the lethal activity of crude toxin preparations and reacted by double immunodiffusion with a single component of concentrated broth cultures of P. aeruginosa isolates obtained from a clinical source.