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The exotoxin A gene from Pseudomonas aeruginosa PAK was expressed in Escherichia coli from recombinant plasmids when transcription was initiated from a promoter in the cloning vector. The exotoxin A polypeptide synthesized was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of 66,000 daltons, identical in size to the mature exotoxin A made by P. aeruginosa. Analysis of the location of exotoxin A in various bacterial compartments by immunoblotting revealed that exotoxin A was exported by E. coli into its periplasmic space. Several functional assays, including analyses of disulfide bond formation, potentiation of ADP-ribosyltransferase activity, and HeLa cell cytotoxicity, were used to establish that the conformation of exotoxin A isolated from the E. coli periplasmic space is identical to that of exotoxin exported by P. aeruginosa to its extracellular space. Previous studies with recombinant plasmids expressing exotoxin A from P. aeruginosa PA103 (G. D. Gray, D. Smith, J. Baldridge, R. Markins, M. Vasil, E. Chen, and M. Heyneker, Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984) showed a complete lack of processing and export of pre-exotoxin A in E. coli, differing from results reported here. These discrepancies may be explained by observed differences in the sequence of signal peptides encoded by the exotoxin A genes of PAK and PA103 strains of P. aeruginosa.

Vibrio anguillarum was isolated as a pathogen in the commercial culture of oyster spat at Pigeon Point, Calif. A water-soluble, heat-stable exotoxin extracted from cultures of the vibrio inhibited larval swimming and contributed to larval mortality. Although the vibrio was insensitive to penicillin in standard plate testing, this antibiotic proved useful in preventing mass larval mortalities in the hatchery.

Sterile, concentrated culture supernatant from Pasteurella haemolytica (biotype A, serotype 1) strain 630 was subjected to physical, chemical, and immunologic treatments to determine their influence on leukotoxin (cytotoxin) activity contained in the supernatant. Each treated sample contained approximately 8 chemiluminescence inhibitory units of leukotoxin. Treatment effects were evaluated for their ability to inactivate leukotoxin activity. Leukotoxin activity in treated samples was determined by inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. Optimal leukotoxin synthesis by P haemolytica occurred when the bacteria were at the logarithmic growth phase, whereas stationary phase cultures contained minimal amounts of leukotoxin activity in their culture supernatant. Leukotoxin activity was heat labile; activity was substantially decreased when concentrated culture supernatant samples containing leukotoxin activity were incubated at 37 C for several hours. When concentrated culture supernatant was incubated at progressively decreasing temperatures, there was a progressive increase in the length of time that the leukotoxin retained its biologic activity. Samples stored at -70 C retained activity for at least 2 months. Leukotoxin activity was nondialyzable and was able to withstand considerable extremes in hydrogen ion concentration. Leukotoxin activity could not be pelleted when subjected to forces of 100,000 X g for 1 hour. Chemical and enzymatic studies suggested that P haemolytica leukotoxin contained carbohydrate and protein moieties. Chemical treatment with 0.2% sodium lauryl sulfate, 0.5% sodium deoxycholate, 7.5 mM EDTA and 8M urea with 8 mM 2-mercaptoethanol and enzymatic treatment with lipase, ribonuclease, and deoxyribonuclease had no discernible effect on leukotoxin activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Tampons were tested for effect on growth and production of toxic shock syndrome toxin 1 by Staphylococcus aureus. Under good growth conditions, regular absorbency tampons had little effect on bacterial growth and inhibited toxin production two- to fourfold. In contrast, higher absorbency tampons had three different effects: 1) some tampons had no effect on bacterial growth but inhibited toxin production; 2) many tampons inhibited both growth and toxin production; 3) one tampon inhibited growth but increased exotoxin per cell. These effects were independent of degree of saturation of the tampons and were observed at incubation times of six, 12, and 18 hours. In no instance was the production of toxic shock syndrome toxin 1 per milliliter increased in the presence of tampons when compared with control.

A two-step method is described for the production of exotoxin A by Pseudomonas aeruginosa in which a defined growth medium is modified for the toxin production phase. As a result, specific exotoxin A yields comparable to those obtained with complex media were achieved. In the development of this two-step process, several divalent metallic cations (Ca2+, Cu2+, and Mn2+), in addition to iron, were found to inhibit the yield of exotoxin A while Ca2+ and glycerol were found to increase yields. Northern blot analysis of total RNA isolates suggests that these effects on exotoxin A yields are based on events at the transcription level.

Hemoglobin, but not albumin, has long been recognized as an infection potentiating factor in experimental Escherichia coli peritonitis, but the mechanism has defied definition. We have shown previously that stroma-free hemoglobin is not toxic to polymorphonuclear neutrophils. To test the hypothesis that hemoglobin provides a nutritional boost to the growth of E. coli in vivo, we inoculated E. coli into dialysis bags containing equivalent amounts of stroma-free hemoglobin or albumin. These bags were implanted into the peritoneal cavity of rats and at intervals the fluid was removed and the bacteria enumerated. This technique allows for intraperitoneal bacterial growth but eliminates the variables of lymphatic clearance and phagocytic ingestion. The growth rate of E. coli was the same irrespective of the nutritional supplement in the bag. Thus there is no experimental support for the notion that hemoglobin directly accelerates E. coli proliferation under in vivo conditions. To test the hypothesis that a leukocyte toxin may result from E. coli growth in hemoglobin, we exposed normal human neutrophils to the sterilized contents of the peritoneal dialysis bags. In vitro function of the neutrophils (viability, random migration, chemotaxis, phagocytosis, bacterial killing, and chemiluminescence) was significantly depressed by prior exposure to hemoglobin supernatants that had supported E. coli proliferation in vivo. Stroma-free hemoglobin had minimal adverse effects. Albumin supernatants that had supported E. coli proliferation in vivo had significantly less effect on neutrophil function even though the endotoxin levels were identical to the hemoglobin E. coli solutions. We must conclude that leukotoxins result from E. coli growth in solutions of pure hemoglobin. The data support the idea that the infection potentiating effect of hemoglobin in vivo is due to such leukotoxins.

The phaseolotoxin-producing Pseudomonas syringae pv. phaseolicola strain 1321 contains two ornithine carbamoyltransferases which differ in resistance to phaseolotoxin. Whereas ornithine carbamoyltransferase 1 (OCT 1) is inhibited at low concentrations of phaseolotoxin, ornithine carbamoyltransferase 2 (OCT 2) is insensitive to phaseolotoxin. The activity of the insensitive enzyme is correlated with the amount of toxin formed.

A medium for the accumulation of extracellular hemolysin (300 to 1,600 hemolytic units per ml) and protease (2 to 3 proteolytic units per ml) was developed for an anaerogenic strain of Aeromonas hydrophila. In this medium, growth yields were less but levels of accumulated toxin were greater or equivalent when compared with the same responses in brain heart infusion and nutrient broths. The medium was considered to be partially defined since the conditions for maximum observed hemolysin accumulation (1,600 hemolysin units per ml) were not identified. The results showed that iron and zinc contributed to the control of the extracellular accumulation of both toxins. Whereas iron exerted an inhibitory effect, zinc stimulated the accumulation of both toxins.

Isolates of Bacillus cereus from 118 cases, and two maternity unit outbreaks, of non-gastrointestinal infection were grouped on the basis of their estimated probable involvement in the infections from which they were isolated: (i) high probability--48 strains; (ii) intermediate--16 strains; (iii) low--7 strains; (iv) very low ("irrelevant")--49 strains. Rabbit skin test, haemolysin and phospholipase assays were used to determine exotoxin activities of strains within each group. The results suggest a significant relation between the virulence of an isolate as reflected in the degree to which it appeared responsible for the signs and symptoms of an infection, and its toxigenicity in the skin test. This is attributed to the ability of B cereus strains to synthesise, in varying degrees, a necrotic enterotoxin, possibly in conjunction with the primary haemolysin (cereolysin). The cases analysed in this study support the contention that B cereus, when isolated from an infection, may not be an inconsequential contaminant and should not be too readily dismissed as such.

Independently of their capacity to produce phytotoxins, strains of Pseudomonas syringae pv. phaseolicola contain two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) which differ in resistance to phaseolotoxin and Orn-P(O) (NH2)-NH-SO3 H (PNSOrn). At 18 degrees C, the optimal temperature for product formation, the balance of the two types of OCT was shifted in favour of the insensitive type in phaseolotoxin producing strains, and in favour of the sensitive one in strains with little or no toxin production. The results suggest a causal relationship between the existence of an insensitive enzyme and the synthesis of toxins.

Isolation of bacteria from a field of bean plants (Phaseolus vulgaris L.) with conspicuous symptoms of halo blight disease resulted in 123 bacterial strains from which 57 were identified as Pseudomonas syringae pv. phaseolicola . At 18 degrees C the phaseolotoxin production of the isolated strains differs widely in submerse culture. Only few strains produce high amounts of phaseolotoxin being comparable with those of the reference strain 1321, while most of the strains show a low capability of phaseolotoxin production. Furthermore, we proved the stability of phaseolotoxin production of 29 strains. After one year about 50% of the strains were extremely reduced in their capability of phaseolotoxin production. We found that the reason for this reduction of toxin production is the appearance of Tox- segregants within a Tox+ clone. At 24 degrees C all strains (with one exception) show considerable lower amounts of toxin production or none at all: maximally 30% of the toxin amounts synthesized at 18 degrees C were produced. At 28 degrees C none of the isolated strains produce phaseolotoxin . The present data allow the conclusion to be drawn that in natural environments there exists a wide spread regarding the amount and stability of the phaseolotoxin production.

Strains of Staphylococcus aureus are known to differ in their ability to produce a number of toxins and digestive enzymes that may contribute to the organisms's virulence and invasive potential. Independent from these, however, certain strains produce a variety of specific toxins that cause specific clinical diseases, including staphylococcal food poisoning, staphylococcal enterocolitis, exfoliative skin disorders, and most recently, the toxic shock syndrome. This article reviews those diseases known to be mediated by specific staphylococcal toxins.

We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease activity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD.

The development of monoclonal antibody and enzyme-linked immunosorbent assay (ELISA) techniques has made possible the detection of specific antigens at extremely low concentrations. Diagnosis of recalcitrant diseases such as melioidosis depends upon either early isolation and identification of the causative organism or the identification of a specific marker antigen, Pseudomonas pseudomallei exotoxin, in serum; the latter is better because it allows more rapid and simple diagnosis. A method of detecting exotoxin concentrations of greater than 16 ng/ml by an ELISA based on a monoclonal antitoxin is here described; it is significantly more sensitive than the mouse lethality test (lower threshold 30 micrograms/ml) currently in use and an in-vitro cytotoxicity test (lower threshold 10 micrograms/ml) that we have developed and describe here.

The virulence of a protease-producing strain of Psuedomonas aeruginosa was compared with that of mutants that had lost the ability to produce proteases and other extracellular enzymes. Lethal infections were produced by inoculating mice intraperitoneally with bacteria in mucin, or by inoculating mice intraperitoneally or intravenously with bacteria 4 days after treatment with cyclophosphamide, 200 mg per kg body weight. No significant difference in virulence between the wild-type parent strain and some of its protease-deficient mutants was found. Histopatholoical examination of different organs in the cyclophosphamide-treated and infected mice showed striking fatty infiltration and focal necrosis of liver, multiple necrotic foci in the spleen and haemorrhagic cystitis with necorsis. The cystitis was produced by cyclophosphamide alone but was aggravated by the infection. In conclusion, no correlation between the production of protease in broth culture and the ability to produce lethal septicaemia in mice was found, and extracellular proteases probably didnot contribute to the virulence of P. aeruginosa. However, the histopathological changes in the liver suggested a role for exotoxin A insystemic infections.

We have recently isolated several groups of bacteriophages infecting Actinobacillus actinomycetemcomitans from periodontal lesions in patients with rapidly destructive periodontitis. Bacteriophage infection of these bacteria in these patients was restricted to periodontal pockets showing radiographic evidence of recent bone loss and suggests an association between phage-infected A. actinomycetemcomitans and active periodontal disease. On the basis of the biological activity of bacteriophages we propose a working hypothesis to explain the mechanism by which a phage may increase bacterial virulence in periodontal disease.

The effect of ten mineral salts on the productivity and toxin synthesis was studied in Bacillus thuringiensis IPM-1140. Exotoxin synthesis was stimulated by Zn2+, Mn2+ and NH4+ ions as well as by potassium phosphates. The direct correlation between the number of viable spores and the exotoxin accumulation was disordered at extreme salt concentrations. Optimal salt concentrations in the yeast-polysaccharide medium were found using the method of a fractional factor experiment, which made it possible to increase the productivity of the culture to 5 X 10(9) spores/ml and the yield of the exotoxin to 730 micrograms/ml. The thermoresistance and the entomopathogenic activity of crystals increased when B. thuringiensis IPM-1140 was grown in this medium.

Several groups of streptococci were tested for production of pyrogenic exotoxins (SPE) with Ouchterlony immunodiffusion, a newly developed passive hemagglutination inhibition assay, and an assay for pyrogenicity and capacity to enhance lethal endotoxin shock. With use of these assays, 68 (91%) of 75 group A streptococcal strains were positive for one or more of SPE types A, B, and C; seven were negative for both the known SPE types and antigenically unrelated pyrogenic exotoxins. Group A strains producing both SPE B and C were the most common, and strains producing A alone or AB and AC together were the least common. All of 11 rheumatogenic group A streptococci elaborated SPE C either alone or together with one or both of SPE types A and B. The 10 nephritogenic strains tested were positive for SPE B; five were positive for B alone. In contrast to group A streptococci, non-group A strains (41 tested) did not produce the known SPE types, and 19 of 19 tested were negative for antigenically unrelated pyrogenic exotoxins. Group A strains from Holland, India, and Japan also elaborated SPE. Several group A streptococci used widely in laboratory experiments were tested for SPE types produced.

Pseudomonas aeruginosa has emerged into the limelight mainly as a result of compromised host problems and the development of resistance leading to serious treatment difficulties. The organism possesses virulence factor that produce an effect in certain clinical situations. Changes in local anatomy, often with the presence of foreign bodies, are important (bladder and intravenous catheters, tracheostomy, burns, wounds, and injuries). Deficiencies in immune defense, particularly granulocytopenia, are almost a prerequisite for development of Pseudomonas septicemia and meningitis. The toxic factors of Pseudomonas organisms in combination with a disturbed defense mechanism produce characteristic necrotic skin lesions. A mucoid form of P aeruginosa is a characteristic feature of cystic fibrosis. Resistance of P aeruginosa to antibiotics is very definitely associated with overuse of broad-spectrum antibiotics in hospitals. New and more effective antibiotics may be needed.

Reference strains of Streptococcus pyogenes and strains from recent epidemics and sporadic cases of scarlet fever were examined for their ability to produce erythrogenic toxin type A (ET A) by ELISA and double immunodiffusion (Ouchterlony) using an anti-ET A antibody purified by affinity chromatography. Of the reference strains (most of them isolated before 1945) 16/51 produced more or less ET A (Table 1). ET A synthesis is strain-specific, but not type-specific. Well-known toxin producers like the strains NY-5; 594 or "Smith" produce up to 16.000 micrograms/l under optimal culture conditions. Type 3 strains isolated from scarlet fever patients during the outbreak 1972/73 seem to belong to one clone as evidenced by the uniform SDS-PAGE pattern: They were found to produce 5-200 micrograms/l (mean 68 micrograms/l) ET A only. Type 3 strains from sporadic cases, isolated 10 years later, produced 0-138 micrograms/l (mean 30 micrograms/l). Strains of the type 1 clone, causing the epidemic in 1982/83 produced only 0.75-10 micrograms/l (mean 8 micrograms/l) ET A (Table 3). Only a few strains of S. pyogenes isolated 1984 or later synthesized ET A but they were found more often to produce ET B (proteinase precursor) in batch cultures. S. pyogenes strains seem to have lost their ability to produce large amounts of ET A during the last decades. Because this toxin must be considered as a pathogenicity factor the decrease in toxin production may be one reason for the present mild form of scarlet fever.

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover.

Various procedures were studied for obtaining Bacillus thuringiensis strains of serotype I which synthesized exotoxins. Mutant clones with elevated exotoxin synthesis could be selected by treating the cells with N-methyl-N-nitro-N-nitrosoguanidine. The frequency of (+) variant selection was from 17 to 12 X 10(-2). The clones of S and R types differed in the insecticide activity of the exotoxin. Its yield could be increased by optimizing the composition of growth media. The strain specificity of B. thuringiensis producing the exotoxin was assayed in terms of carbon and nitrogen requirements.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Pseudomonas aeruginosa exotoxin A (toxin A) was developed. The level of toxin A produced by fresh P. aeruginosa isolates was compared to the level of toxin A produced by twelve-months-old subcultures derived from the same strains. Results obtained with the ELISA showed that toxin A was produced in similar amounts both by the fresh isolates and the subcultures. The production of toxin A seems to represent a stable property of the assayed P. aeruginosa strains. This is in contrast to other exoproducts from the same strains, such as extracellular proteinases. Some proteinases were produced at very different levels when freshly isolated P. aeruginosa cultures were compared with their twelve-months-old counterparts, confirming earlier reports in this respect.

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. beta-galactosidase synthesis in the strains (cointegrates) carrying the toxA-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa. Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of beta-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of beta-galactosidase and ETA synthesis were abolished.

The characteristics of exotoxin inhibition of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase isolated from Escherichia coli and Bacillus thuringiensis were investigated. RNA polymerase isolated from a variety of growth stages was partially purified and assayed using several different native and synthetic DNA templates, and exotoxin inhibition patterns were recorded for each. Although 8 to 20-h RNA polymerase extracts of E. coli retained normal sensitivity to exotoxin (50% inhibition at a concentration of 7.5 X 10(-6) M exotoxin), RNA polymerase isolated from late exponential and ensuing stationary-phase cultures of B. thuringiensis were nearly 50% less sensitive than exponential RNA polymerase activity. Inhibition patterns relating culture age at the time of RNA polymerase extraction to exotoxin inhibition suggested a direct correlation between diminishing exotoxin sensitivity and sporulation. Escherichia coli RNA polymerase could be made to mimic the B. thuringiensis exotoxin inhibition pattern by removal of sigma from the holoenzyme. After passage through phosphocellulose, exotoxin inhibition of the core polymerase was 30% less than the corresponding inhibition of E. coli holoenzyme. Heterologous enzyme reconstruction and assay were not possible due to loss of activity from the B. thuringiensis preparation during phosphocellulose chromatography, apparently from the removal of magnesium. In enzyme velocity studies, inhibition with exotoxin was noncompetitive with respect to the DNA template in the RNA polymerase reaction.

Toxic shock-like syndrome isolates of group A streptococci were evaluated for production of pyrogenic exotoxins (also called SPEs, scarlet fever toxins, and erythrogenic toxins). The isolates were consecutively obtained during 1987 and 1988. Of these isolates, 23 of 26 made SPE type A, 10 of 26 made SPE B, and 8 of 26 made SPE C. SPE A was produced in significantly greater amounts than SPEs B and C (3.2 micrograms/ml of culture fluid compared with 0.7 and 0.6 microgram/ml, respectively). SPE A, administered in miniosmotic pumps implanted subcutaneously in rabbits, was significantly more toxic than SPE C; seven of eight rabbits succumbed after challenge with 150 or 300 micrograms of SPE A, compared with one of six after challenge with SPE C.

In studies of Pasteurella haemolytica type 1 cytotoxin, filter-sterilized culture supernatants from organisms grown in RPMI-1640 tissue culture medium generally have been used. Supplementation of the medium with 7% bovine fetal serum was shown to be necessary for maximal cytotoxin production, as measured by percentage of bovine peripheral blood leukocytes that were killed. The serum-induced increase in cytotoxicity could not be explained simply by a greater percentage of increase in the number of viable organisms produced in the enriched medium. There also was no correlation between encapsulation of the organisms and cytotoxin production. Several natural iron-containing proteins including transferrin, lactoferrin, conalbumin, and hemoglobin stimulated cytotoxin production in lieu of bovine fetal serum, leading to the conclusion that one function of serum supplementation may be to increase the medium's iron concentration. A number of additional iron-containing and iron-chelating compounds were tested, with the conclusion that the iron concentration of the growth medium, as well as the presence of a suitable carrier molecule, may be critical for efficient cytotoxin production by P haemolytica.

We describe the detection and quantitation of type A streptococcal exotoxin (erythrogenic toxin, streptococcal pyrogenic exotoxin) by an enzyme-linked immunosorbent assay. This sensitive and specific technique detected microgram amounts of type A exotoxin and was useful for studying the kinetics and regulation of type A exotoxin production during the growth of Streptococcus pyogenes NY5. Maximum production of type A exotoxin was observed during the mid-log phase of growth, similar to the production of other streptococcal extracellular products. When S. pyogenes NY5 was grown at 42 degrees C, decreases in both growth and type A exotoxin production were observed. The results obtained when we studied the influence of nutrient additives and metal ions on the production of type A exotoxin led to the conclusion that none of these factors significantly affected type A exotoxin synthesis and that regulation was constitutive.

The activity of exotoxin A in culture filtrates prepared from cultures obtained by growing P. aeruginosa strains PA-7 and PA-103 in Martin's broth containing iron at a concentration of 0.08 microgram/ml, 0,05 M sodium glutamate and 1% of glycerin has been shown to be 1.5 times higher than that in filtrates prepared from cultures obtained by growing the above strains in a medium containing soybean tryptic digestion (USA). The optimun conditions for the production of exotoxin A by these strains are achieved during their cultivation in a fermenter at a temperature of 32 degrees C for 18 hours with simultaneous stirring (800 r. p. m.) and oxygenation (450 m3/h). Under these conditions the biological activity of the filtrates is 200 LD50/ml, their ADP-ribosyltransferase activity is 9500 c. p. m. and a sharply defined precipitation line appears in the double diffusion test in gel with monospecific antiserum to purified toxin, used in a dilution of 1:8.

Two ornithine carbamoyltransferases (OCT 1 and OCT 2) were isolated from Pseudomonas syringae pv. phaseolicola and purified by precipitation with ammonium sulfate, heat denaturation, chromatography on DEAE-Sephadex A-50 and Sephadex G-200. Molecular weights of both enzymes: 110,000; optimal activity: pH 8.5 to 9.5 (OCT 1), pH 8.4 (OCT 2); apparent Km for ornithine: 7 X 10(-4) (both enzymes); apparent Km for carbamoyl-phosphate: 7 X 10(-4) (OCT 1), 2.8 X 10(-3) (OCT 2). Both enzymes possess only an anabolic function. OCT 1 is highly inhibited by low concentrations of phaseolotoxin and Orn-P(O)(NH2)-NH-SO3H, OCT 2 is insensitive to both compounds. The inhibition of OCT 1 is reversible.

Twenty-one cases of infection with Bacillus cereus are summarised. The histories supplied showed that at least 15 of these were associated with severe or potentially severe symptoms including two deaths. Analysis of the production of exotoxins, including haemolysin and phospholipase, by these strains is given, and the relevance of these metabolites to the severity of the condition is discussed. Three incidents of bovine mastitis resulting from B. cereus and involving three deaths are also included. The observations presented here together with those of previous reports which are reviewed indicate that B. cereus may be of clinical importance, not just an opportunist but also as an agent of potentially severe infections in its own right.

Pseudomonas aeruginosa elaborates a number of extracellular products which have been shown to play a role in the pathogenesis of disease caused by this organism. In this study, we showed that the host environment markedly affects the levels of exoproducts produced. We compared the phenotypes of a number of P. aeruginosa strains obtained from a variety of clinical sources, including burn wounds, skin wounds, urine, cystic fibrosis sputum, acute pneumonia sputum, and blood. The clinical isolates were examined quantitatively for levels of total protease, elastase, phospholipase C, exotoxin A, and exoenzyme S produced in vitro under defined conditions. The exoproduct levels varied significantly, depending on the site of isolation. Elevated levels of elastase were demonstrated in strains isolated from acute lung infections, phospholipase C levels were elevated in urinary tract and blood isolates, exotoxin A levels were elevated in blood isolates, and exoenzyme S levels were increased in acute pneumonia isolates. Isolates from cystic fibrosis sputum produced low amounts of virtually all of the tested exoproducts, particularly as compared with sputum isolates from acute P. aeruginosa lung infections.

Production of group A streptococcal pyrogenic exotoxins (SPE) type A and C was transferred from toxigenic streptococcal strains to nontoxigenic strains by lysogeny. Lysogens were tested for SPE with Ouchterlony immunodiffusion on Todd-Hewitt agar plates; toxin diffusing from isolated colonies reacted with specific hyperimmune antisera to SPE. Phage prepared from strains T25(3) (T12gl) and 3GL16, both yielding SPE type A, formed plaques on T25(3) (NONLYSOGENIC) lawns. Over 90% of the colonies picked from the plaque centers yielded A toxin, suggesting SPE type A was transferred by lysogenic conversion. SPE type C formation was transferred to nontoxigenic strains T25(3) and K56 with supernatant fluids from mitomycin C-induced cultures of CS112, producing SPE types B and C. All lysogens tested were positive for SPE type C, indicating that C toxin induction also was transferred by lysogenic conversion. SPE type B formation was not transferable by lysogeny with the strains tested.

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.

A bacteriophage, designated phi Aa17, was isolated by mitomycin C induction from leukotoxic Actinobacillus actinomycetemcomitans strains 651. Electron microscopy of the virus revealed particles with regular, nonelongated, polyhedral heads, and tails consisting of a contractile sheath and core. Spikes emanated from the base of the tail. The head had a diameter of 70 nm. The fully extended tail sheath had a length of 127 nm and a diameter of 22 nm. In its contracted form, the tail sheath measured 47 nm in length and 25 nm in diameter. The phage had a buoyant density of 1.370 in CsCl, and its genome was found to be double-stranded DNA. A single-cycle growth curve revealed that the phage had a latent period of 30 min and a burst size of 435 PFU per cell. The host range of the phage was examined, and A. actinomycetemcomitans strains ATCC 29523 and ATCC 29524 were found to be phage sensitive, whereas strains Y4, ATCC 29522, 2043, 652, 651, 627, 2097, N27, 2112, and 511 were resistant. The host range of this virus does not suggest any association between the phage and leukotoxin production.

Single strains of 5 different P. haemolytica serotypes (1, 2, 5, 6 and 9) and an untypable strain were compared in an attempt to detect differences which might be related to virulence. All but the untypable strain caused extensive lesions when injected into the lungs of healthy cattle. Each strain was found to be encapsulated and to be toxic in vitro for bovine leukocytes. Each strain also produced leukotoxin in vitro. The toxins varied, however, in total toxic activity and in the kinetics of leukotoxin production. Vaccination of cattle with each of the serotype strains elicited antibodies to organism somatic antigens and, to various degrees, the production of leukotoxin-neutralizing antibodies which showed no strain specificity in cross-neutralization studies. Although each of the serotype strains appeared to be a potential bovine pathogen, subtle differences were observed which may explain the importance of Serotype 1 strains in bovine pneumonic pasteurellosis.

The article specifies the mechanisms of toxigenicity in clinical streptococci strains. The production of type A erythrogenic toxin is found to be related to the toxigenicity gene expression localized in moderate bacteriophage genome. The possibility of using DNA probes to assess the degree of toxigenicity is discussed together with the relationship between the toxigenicity and the gene dose.

There is concern that group A streptococci, which have caused less serious infections in developed countries in recent decades, may be acquiring greater virulence. We describe 20 patients from the Rocky Mountain region who had group A streptococcal infections from 1986 to 1988 that were remarkable for the severity of local tissue destruction and life-threatening systemic toxicity. Among the 20 patients (median age, 36), necrotizing fasciitis with or without myositis was the most common soft-tissue infection (55 percent). Nineteen patients (95 percent) had shock, 16 (80 percent) had renal impairment, and 11 (55 percent) had acute respiratory distress syndrome. The mortality rate was 30 percent. All patients but 1 had positive tissue cultures for Streptococcus pyogenes; 12 had positive blood cultures. Most of the patients had no underlying disease; 2 used intravenous drugs. Strains of group A beta-hemolytic streptococci isolated from 10 patients were not of a single M or T type; however, 8 of the 10 strains produced pyrogenic exotoxin A (scarlet fever toxin A, a classic erythrogenic toxin), which has rarely been observed in recent years. From our study of this cluster of severe streptococcal infections with a toxic shock-like syndrome, we conclude that in our region, more virulent group A streptococci have reappeared that produce the pyrogenic toxin A associated with scarlet fever.

A. actinomycetemcomitans is a facultative Gram-negative coccobacillus which has been implicated in the etiology and pathogenesis of localized juvenile periodontitis and has also been recognized for its potential to cause serious extraoral infections, particularly endocarditis. The polymorphonuclear neutrophil has been suggested to play a key role in host resistance to periodontopathic organisms, as indicated by the association between defective production or function of these phagocytic cells and severe periodontal disease. This association has engendered interest in the study of the interaction between neutrophils and A. actinomycetemcomitans, as well as the role of immunoglobulin and complement in facilitating this interaction. The objective of this review is to summarize current knowledge of the nature and consequences of the interaction between A. actinomycetemcomitans and the host defense triad consisting of neutrophils, complement and immunoglobulin.