Characterization of staphylococci from patients with toxic shock syndrome.

Fifty staphylococcal strains that produced toxic shock syndrome (TSS) toxin 1 and that were isolated from patients with TSS were characterized. One strain had more properties that were characteristic of Staphylococcus hyicus than of Staphylococcus aureus. Forty-four strains had the same properties or differed in only one property. Thirty-five of the 50 strains produced either enterotoxin A or C or both in addition to TSS toxin 1.     

Involvement of coagulase-negative staphylococci in toxic shock syndrome.

Coagulase-negative staphylococci that produce toxic shock syndrome toxin 1 (TSST-1) or a staphylococcal enterotoxin or both were isolated from various sources. Coagulase-negative strains that produce TSST-1 alone or with enterotoxin A were the only staphylococci isolated from seven patients with toxic shock syndrome. Two other toxic shock syndrome patients had coagulase-positive staphylococci also, but only the coagulase-negative strains produced TSST-1. Coagulase-positive and coagulase-negative strains that produced TSST-1 were isolated from two other toxic shock syndrome patients. In addition, coagulase-negative staphylococci that produced toxins were isolated from patients with other staphylococcal infections and from food implicated in a case of food poisoning.     

Nonproduction of toxic shock syndrome toxin 1 by coagulase-negative staphylococci.

We tested 187 strains of coagulase-negative staphylococci (CNS) for the production of toxic shock syndrome toxin 1 (TSST-1). A total of 111 CNS strains were isolated from the tampons of menstruating women and 74 were isolated from unused tampons. Two strains were isolated from the genital tract of a patient with toxic shock syndrome. Strains were cultivated by the membrane-over-agar method to enhance production of TSST-1, and culture supernatants were tested by two exquisitely sensitive enzyme-linked immunosorbent assays. None of the 187 CNS strains produced TSST-1. We conclude that CNS colonizing the genital tracts of menstruating women and unused tampons produce TSST-1 infrequently, if ever, and are unlikely to play a role in toxic shock syndrome.     

Evaluation of coagulase-negative staphylococci for ability to produce toxic shock syndrome toxin 1.

A large and diverse group of coagulase-negative staphylococci were assayed for the ability to produce toxic shock syndrome toxin 1 (TSST-1) by immunological reactivity, and whole-cell DNAs from 33 of these strains were hybridized with a TSST-1-specific gene probe. None of the strains tested, including isolates that have been reported as TSST-1+, produced the exotoxin, and no DNA homology was found with the gene probe.     

Enterotoxin production by Staphylococcus aureus strains isolated from cases of chronic osteomyelitis.

Of 264 Staphylococcus aureus strains selected at random from 3,978 strains isolated from chronic osteomyelitis patients, 79 were found to produce enterotoxin. A majority of the strains (65%) were nontypable by phages belonging to the international phage set for human strain typing. The significance of these strains and their circulation among the population is discussed, especially from the standpoint of their possible role in the development of osteomyelitis.     

Rapid screening assay for toxic shock syndrome toxin production by Staphylococcus aureus.

A rapid immunoblot assay (TST-blot) was developed and used to screen Staphylococcus aureus isolates for toxic shock syndrome toxin (TST) production. Growth from an 18-h stab inoculum of S. aureus on brain heart infusion agar was transferred directly to a nitrocellulose sheet. Nonspecific protein binding sites were blocked with bovine serum albumin, and the nitrocellulose sheet was incubated with affinity-purified antibody to TST, followed by incubation with horseradish peroxidase-conjugated protein A. Toxin was visualized by detection of the peroxidase-conjugated protein A-anti TST-TST complex with 4-chloro-1-napthol. The sensitivities and specificities of the TST-blot and Ouchterlony microslide immunodiffusion assay were compared by screening 141 S. aureus isolates for TST production. In both assays, 53 of 141 isolates produced detectable levels of TST, whereas 88 isolates produced no toxin. A 100% concordance was found between the two assays. The TST-blot yielded the same results in less than 24 h as those yielded by the 3-day immunodiffusion assay. Thus, this rapid method for detection of TST in multiple samples appears to be well suited for diagnostic and epidemiological studies. Furthermore, it would appear to be ideal for use in TST genetics research.     

Enhancement of endotoxin-induced isolated renal tubular cell injury by toxic shock syndrome toxin 1.

The pathogenesis of toxic shock syndrome (TSS) remains unknown. On the basis of experimental data, it has been hypothesized that staphylococcal TSS Toxin 1 (TSST-1) may interact synergistically with low levels of endotoxin and give rise to many of the clinical findings. We have demonstrated previously that lipid A, the biologically active component of lipopolysaccharide (LPS), or endotoxin, induces dose-dependent necrosis of isolated rat renal tubular cells (RTCs). In the present studies, the authors investigated whether this injury could be augmented by TSST-1. The viability of RTCs was assessed by vital dye exclusion. Incubation of freshly isolated rat RTCs with either 1 ng/ml of TSST-1 or 0.1 ng/ml LPS or lipid A had minimal cytotoxicity (less than 6%). Exposure of RTCs to 1 ng/ml TSST-1 for 20 minutes, followed by washing, resulted in a significant enhancement of cytotoxicity when RTCs were exposed to 0.1 ng/ml LPS or lipid A. The sensitization of RTCs by TSST-1 to LPS- or lipid-A-induced injury was prevented by methylamine and chloroquine, two inhibitors of receptor-mediated endocytosis (RME). Chelation of extracellular calcium by 2 mM EGTA also blocked the TSST-1-induced sensitization of RTCs to LPS or lipid A. Inhibition of RTC arachidonic acid metabolism by methylprednisolone, indomethacin, ibuprofen, and piriprost significantly inhibited RTC necrosis induced by TSST-1 and LPS or lipid A by 33-62%. Thiourea and deferoxamine, agents which ameliorate oxidant injury, also inhibited this synergistic injury by 34-67%. Thus, TSST-1 enhanced the cytotoxic effects of LPS/lipid A, and the sensitization of RTCs appeared to involve RME or TSST-1. Oxidative metabolism of arachidonic acid and generation of reactive oxygen species appeared to participate in LPS/lipid-A-mediated RTC death.     

Enterotoxin production by coagulase-negative staphylococci in restaurant workers from Kuwait City may be a potential cause of food poisoning.

Staphylococcus aureus and coagulase-negative staphylococci (CNS) were isolated from the hands of food handlers in 50 restaurants in Kuwait City and studied for the production of staphylococcal enterotoxins, toxic shock syndrome toxin-1, slime and resistance to antimicrobial agents. One or a combination of staphylococcal enterotoxins A, B or C were produced by 6% of the isolates, with the majority producing enterotoxin B. Toxic shock syndrome toxin-1 was detected in c. 7% of the isolates; 47% produced slime. In all, 21% of the isolates were resistant to tetracycline and 11.2% were resistant to propamidine isethionate and mercuric chloride. There was no correlation between slime and toxin production or between slime production and antibiotic resistance. The detection of enterotoxigenic CNS on food handlers suggests that such strains may contribute to food poisoning if food is contaminated by them and held in conditions that allow their growth and elaboration of the enterotoxins. It is recommended that enterotoxigenic CNS should not be ignored when investigating suspected cases of staphylococcal food poisoning.     

Regulation of staphylococcal toxic shock syndrome toxin-1 and total exoprotein production by magnesium ion.

The effect of Mg2+ on in vitro production of extracellular proteins and, specifically, of toxic shock syndrome toxin-1 (TSST-1), by Staphylococcus aureus in a chemically defined medium was examined. As previously observed, the organisms did not proliferate in the absence of divalent cations. Low levels of Mg2+ (0.02 to 0.04 mM) permitted submaximal proliferation and elevated production of exoproteins. When the Mg2+ concentration was raised to 0.4 mM, multiplication was optimal and exoprotein levels were depressed. Ca2+ and Mn2+ diminished the effect of limiting Mg2+. The increased levels of exoproteins were not due to cell lysis or leakage since intracellular TSST-1 levels were not high enough to account for the increase in extracellular TSST-1 and since the intracellular enzyme, lactate dehydrogenase, was not found in culture supernatants. Cells cultured in low levels of Mg2+ remained in logarithmic growth longer than did those cultured in high concentrations of Mg2+ and, unlike the latter, produced exoproteins throughout the logarithmic growth phase. Low Mg2+ had no effect on cultures in the stationary phase, and organisms cultured in low Mg2+ recovered fully when transferred to high Mg2+. We conclude that, when cultured in medium deficient in Mg2+, S. aureus responds early in the growth cycle by increasing production of many extracellular proteins, including TSST-1.     

Comparison of pathogenic factors expressed by group A Streptococci isolated from patients with streptococcal toxic shock syndrome and scarlet fever.

Streptococcal toxic shock syndrome (STSS) is an illness with high mortality. To obtain clues to understanding the pathogenesis of STSS, we investigated the expression of several pathogenic factors in ten group A streptococcus (GAS) isolates from ten patients with STSS in Japan, in comparison with ten GAS isolates from children with scarlet fever. The ten scarlet fever-derived GAS isolates were equally low in lethality and anti-phagocytic activity in mice and in the production of streptolysin O (SLO), and equally high in production of superantigenic exotoxins (SAGTs) and cysteine proteinase. By comparison, the ten STSS-derived GAS isolates were heterogeneous in the expression of the above pathogenic factors, which ranged from low to high values. Most of the ten STSS-derived isolates were higher in lethality and anti-phagocytic activity and production of SLO, and lower in the production of SAGTs and cysteine proteinase than the ten scarlet fever-derived isolates. The results suggest that the lethality and anti-phagocytic activity examined in mice and SLO may be involved mainly in the development of most of the ten STSS cases.     

Effect of environmental conditions on production of toxic shock syndrome toxin 1 by Staphylococcus aureus.

The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in a fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger.     

The experimental production of mastitis in sheep by intramammary inoculation of Pasteurella haemolytica.

Acute mastitis was consistently produced in primiparous ewes by inoculation of the mammary gland, via the teat canal, with an isolate of Pasteurella haemolytica, serotype A9, originating from a field case of ovine mastitis. Mastitis developed following the inoculation of as few as 10 colony forming units of this isolate, suggesting that only a small number of organisms would be required to initiate the disease under natural conditions provided they were already beyond the teat canal. Clinical signs and macroscopic lesions were well developed within 24 h of inoculation and were similar to those found in the naturally-occurring disease. The ability to reproduce mastitis consistently will facilitate studies of the pathogenesis of the disease and the comparison of different isolates of P. haemolytica with respect to virulence determinants.     

Sequence of the toxic shock syndrome toxin gene (tstH) borne by strains of Staphylococcus aureus isolated from patients with Kawasaki syndrome.

To explore whether a novel staphylococcal clone or structural variant of toxic shock syndrome toxin 1 is associated with Kawasaki syndrome, six toxigenic strains of Staphylococcus aureus from Kawasaki syndrome patients were studied. The strains were divisible into two groups based on phenotypic and genotypic characteristics and are therefore unequivocally not clonal. Portions of the tstH genes of each strain were sequenced. Three were sequenced in their entirety, while the remainder were sequenced from codon 66 to codon 137 of the mature protein only. Two of the former group differed slightly in the sequences of their signal peptides relative to the sequence published for the tstH signal peptide. Those differences did not affect toxin processing or secretion. The sequenced portions of the regions encoding mature toxic shock syndrome toxin 1 were identical in all six strains and corresponded exactly to the published sequence of tstH. No evidence was found for the existence of a structural variant of tstH uniquely associated with Kawasaki syndrome.     

Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome.

Menstrually associated toxic shock syndrome (TSS) is attributed primarily to the effects of staphylococcal exotoxin toxic shock syndrome toxin 1 (TSST-1). A region of the 194-amino-acid toxin spanning residues 115 through 144 constitutes a biologically active site. Several point mutations in the TSST-1 gene in that region result in gene products with reduced mitogenic activity for murine T cells. In this study we evaluated the toxicity of recombinant TSST-1 and several mutants of TSST-1 made by transformed Staphylococcus aureus during in vivo growth in a rabbit infection model of TSS. The toxicities of the transformed strains of S. aureus for rabbits correlated with the mitogenic activities of the recombinant toxins. An isolate originally obtained from a patient with a confirmed case of TSS (S. aureus 587) implanted in a subcutaneous chamber served as a positive control. TSST-1 produced in vivo led to lethal shock within 48 h, and a TSST-1-neutralizing antibody (monoclonal antibody 8-5-7) administered to rabbits challenged with S. aureus 587 prevented fatal illness. Rabbits infected with transformed S. aureus RN4220 expressing wild-type toxin (p17) or mutant toxins retaining mitogenic activity for T cells succumbed within a similar time frame. Blood chemistries of samples obtained from infected animals before death indicated abnormalities in renal and hepatic functions similar to those induced by parenteral injection of purified staphylococcal TSST-1. Mutant toxin 135 (histidine modified to alanine at residue 135) possessed only 5 to 10% of the mitogenic activity of wild-type toxin. Rabbits challenged with transformed S. aureus RN4220 expressing mutant toxin 135 exhibited only mild transient illness. Mutant toxin 135 retained reactivity with monoclonal antibody 8-5-7 and by several criteria was conformationally intact. Toxin from a double mutant, 141.144, with alanine substitutions at residues 141 (histidine) and 144 (tyrosine), also was devoid of mitogenic activity. In this case, antibody recognition was lost. Mutant toxins 115 and 141 were found to possess approximately half-maximal mitogenic activity. Rabbits challenged with S. aureus RN4220 expressing either 115 or 141 toxin succumbed to lethal shock. We conclude that the ability of TSST-1 to activate murine T cells in vitro and its expression of toxicity leading to lethal shock in rabbits are related phenomena.     

Production of a toxic shock syndrome toxin variant by Staphylococcus aureus strains associated with sheep, goats, and cows.

A toxic shock syndrome toxin (TSST) variant with an isoelectric point (pI) of 8.6 produced by an ovine-associated Staphylococcus aureus strain was described previously. Analysis of additional strains associated with sheep, goats, cows, and humans by isoelectric focusing with immunoblotting using monoclonal antibodies revealed that all 18 strains associated with sheep and all 12 strains associated with goats produced the TSST variant. Only 1 of 10 bovine-associated strains and no human-associated strains produced the variant, whereas the others produced TSST-1 (pI between 7.0 and 7.2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting indicated that both TSST-1 and the TSST variant had a molecular size of 24 kilodaltons.     

Production of gamma-hemolysin and lack of production of alpha-hemolysin by Staphylococcus aureus strains associated with toxic shock syndrome.

The hemolytic activity of toxic shock syndrome isolates of Staphylococcus aureus is enhanced when agarose is substituted for agar in blood plates or when strains are grown in liquid culture in the presence of 20% (vol/vol) CO2 in air. Hemolytic activity of a representative panel of toxic shock syndrome isolates was rigorously assessed both on blood agar and in liquid culture to unequivocally identify the predominant hemolysins produced. As determined by isoelectric focusing and Western immunoblotting, 15 of 15 TSS isolates produced gamma-lysin and 10 of 15 produced delta-lysin. None produced beta-lysin, and only 2 of 15 produced alpha-lysin. The low rate of alpha-lysin production was a most striking characteristic, since all strains were found to have the alpha-lysin gene by Southern blot hybridization.     

Involvement of staphylococcal enterotoxins in nonmenstrual toxic shock syndrome.

Adequate evidence is available to show that the major toxin responsible for toxic shock syndrome (TSS) is TSS toxin 1 (TSST-1). More than 90% of the staphylococcal strains isolated from TSS patients produce this toxin. However, approximately 60% of these strains produce one or more of the staphylococcal enterotoxins, with a number of them producing only enterotoxin, primarily enterotoxin B. Of 55 staphylococcal strains isolated from nonmenstrual cases, 46 produced TSST-1; 42 produced one of the enterotoxins, including 8 that produced only enterotoxin B. The fact that the enterotoxins can produce in monkeys many signs and symptoms similar to those observed in TSS in humans implicates them as the cause of some cases of TSS.     

Slime production, hemolysis and proteolysis in coagulase-negative staphylococci isolated in hemoculture from humans

Of 162 strain of coagulase negative staphylococci isolated from haemocultures most frequently the following were identified: S. epidermidis (56.8%), S. haemolyticus (17.3%) and S. hominis (14.2%). By means of the STAPHYtest first 138 strains were assessed (85.2%). Mucus formation was proved in 26 strains (16.0%) of six different species. Haemolytic activity was present in 131 strains (80.9%) in seven species, significantly more frequently in S. haemolyticus. Proteolytic activity was displayed by 118 strains (72.8%) of all nine identified species, significantly more often in S. epidermidis. Mucus and protease formation, haemolysin and protease formation as well as simultaneous formation of all three factors was observed most frequently in S. epidermidis. Strains of S. epidermidis thus have more frequently signs considered virulence factors than strains of the other species of coagulase negative staphylococci.     

Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli.

It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.