Production of toxic shock syndrome toxin 1 by Staphylococcus aureus as determined by tampon disk-membrane-agar method.

The influence of 17 commercially available tampons on production of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus was investigated by using a tampon disk method. Filter membranes overlaying agar medium (with or without blood) in small petri dishes were spread inoculated with a TSST-1-producing strain of S. aureus. Disks cut from unrolled tampons were pressed and laid on the inoculated membranes; incubation was for 19 h at 37 degrees C with 5% CO2 in air. CFU on the membranes and in the disks were enumerated, and the presence of TSST-1 in the disks and in the agar layers was determined. Tampons made of different materials supported characteristic levels of cell growth and toxin production in the tampon. Colonization of the interface surface of the tampon disks was heavy. The number of CFU extracted from the tampon disks ranged from 5 X 10(10) to 82 X 10(10). There was little variation in the CFU recovered from the membranes ([1.9 +/- 0.4] X 10(11)). Sixty to 170 micrograms of TSST-1 was recoverable from the agar, with an additional 10 to 90 micrograms recoverable from tampon disks, depending on the type of tampon disk. The amount of toxin in the agar layer from the various tampon disks was relatively constant and indicated an important contribution of toxin from vaginal S. aureus cells not growing in the tampon. The main role of tampons in toxic shock syndrome may be that of providing a fibrous surface for heavy colonization and sufficient air for TSST-1 production.     

Vaginal isolates of Staphylococcus aureus associated with toxic shock syndrome.

Staphylococcus aureus has been isolated from vaginal fluids of women with toxic shock syndrome (TSS), a multisystem disease with onset usually during menses. A total of 15 vaginal isolates of S. aureus from TSS patients were compared with 18 vaginal isolates from women without TSS. Phenotypic traits which were significantly more frequent in the TSS group of strains than in the non-TSS group were arsenate resistance, proteolysis of hemoglobin, reduced hemolysis of sheep blood in agar medium, and lack of lethality of culture filtrates for chicken embryos and rabbits. In addition, isoelectric focusing of ethanol extracts of culture filtrates showed differences between the two groups in the occurrence of two proteins. All hemolytic and chicken embryo-lethal strains (3 TSS strains and 14 non-TSS strains) produced an extracellular protein with an isoelectric point of 8.6. In contrast, all TSS strains, but only one-half of non-TSS strains, released a protein with an isoelectric point of 7.0 and an apparent subunit molecular weight of 22,000.     

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.     

Production of toxic shock syndrome toxin 1 by Staphylococcus aureus restricted to endogenous air in tampons.

All types of four brands of tampons were tested in triplicate by a tampon sac method for their effect on production of toxic shock syndrome toxin 1 (TSST-1). In this method the available air is limited to that which is in the tampon sac. Tampons were weighed and inserted into dialysis sacs inoculated with a TSST-1-producing Staphylococcus aureus strain; the sacs were submerged into brain heart infusion agar, which was allowed to harden around the sacs, and were incubated for 18 h at 37 degrees C. The tampons were removed, weighed, and extracted; the CFU of staphylococci and the amount of toxin present in the extracts were determined. Glass wool was used in place of the tampons as one control, and inoculated empty sacs were used as a second control. The total CFU were consistently greater than 2 X 10(11) for the tampons and glass wool and less than or equal to 10(11) for the empty sac control. Total toxin production for all tampons tested and the glass wool was 2 to 10 times higher than the toxin produced with the empty sac control. These results indicate that tampons provide increased surface area for the staphylococci to grow and adequate oxygen for toxin production. No significant inhibition of growth of the staphylococci or TSST-1 production by any of the tampons tested was noted.     

Production of toxic shock syndrome toxin 1 by Staphylococcus aureus requires both oxygen and carbon dioxide

The effect of O(2) and CO(2) on expression of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus was investigated under controlled growth conditions with continuous-culture techniques. To stimulate TSST-1 production, air and anaerobic gas were premixed before delivery to the culture vessel. At a growth rate-or mass doubling time (t(d))-of 3 h, production of specific TSST-1 (expressed as micrograms per milligram of cell dry weight) was 5. 9-fold greater at an O(2) concentration of 4% than under anaerobic conditions. Increasing the O(2) concentration to 11% did not result in a significant increase (P> 0.05) in the rate of toxin production over that during growth in 4% O(2) but did result in a significant increase (4.9-fold; P<0.001) in the rate of toxin production over that during anaerobic growth. At a t(d) of 9 h, addition of 3.5% O(2) resulted in a 7.6-fold increase in specific TSST-1 production. When room air was sparged through a culture growing at a t(d) of 9 h, TSST-1 production increased significantly (by 3.4-fold) over that during anaerobic growth. When a growth environment of 4% O(2)-remainder N(2) was studied, no increase in TSST-1 production was observed; this was also the case with 8% O(2) at gas-flow rates of 0.1, 0.2, and 0.4 liters/min. In all experiments, production of biomass (expressed as milligrams of cell dry weight per milliliter) increased, indicating that O(2) was metabolized by S. aureus. Addition of CO(2) to the gas mix (4% O(2), 10% CO(2), 86% N(2)) resulted in a 5.1- to 6.8-fold increase in TSST-1 production over that during anaerobic growth and a 3.6-fold increase over that during growth in an environment of 4% O(2)-remainder N(2). The agr mutant strain tested produced 6.1-fold more specific TSST-1 in a growth environment of 4% O(2)-10% CO(2)-86% N(2) than during anaerobic growth. These data suggest that in this system, O(2) alone does not trigger production of TSST-1; rather, both CO(2) and O(2) are required.     

金黄葡萄球菌中毒休克综合征毒素1的研究

目的 应用PCR技术,检测金黄葡萄球菌的mecA基因和染色体上编码中毒休克综合征毒素1(TSST-1)的tst基因,了解tst基因的携带情况.方法 用PCR法对我院2006年8月-2007年5月临床分离的84株金黄葡萄球菌mecA基因和tst基因进行体外扩增,建立快速、特异、灵敏的检测产TSST-1耐甲氧西林金黄葡萄球菌(MRSA)的方法.结果 成功的对金黄葡萄球菌mecA基因和tst基因进行了检测,并进行基因测序.在我院84株受检金黄葡萄球菌中,41株金黄葡萄球菌的mecA基因扩增呈阳性,阳性株占48.81%.16株tst基因阳性,阳性株占19.05%.10株金黄葡萄球菌同时扩增出mecA基因和tst基因,阳性株占24.39%(10/41).结论 tst基因阳性株在临床分离的耐甲氧西林金黄葡萄球菌中占有较高的比例,应予以足够重视.     

Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.

Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.     

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.     

Serum antibodies to enterotoxins produced by Staphylococcus aureus with special reference to enterotoxin F and toxic shock syndrome.

The presence of antibodies to staphylococcal enterotoxins (enterotoxins A through F) in sera of healthy subjects (n = 567) and in sera of patients with toxic shock syndrome (n = 20) was determined. Furthermore, production of enterotoxins by Staphylococcus aureus isolated from humans was investigated. In 46, 86, 78, 41, 20, and 91% of the sera of healthy subjects, antibodies were found against enterotoxins A, B, C, D, E, and F, respectively. The high percentage of sera with antibodies against enterotoxin F correlated with the relatively high frequency of enterotoxin F-producing S. aureus isolated from humans (one-third of the isolates produced enterotoxin F). In patients with toxic shock syndrome, antibodies against enterotoxin F were not present or were present only at very low levels. An increase of antibodies after onset of the disease was observed in two of eight patients investigated. From the results, it can be concluded that only those humans who show low levels of antibodies are susceptible to toxic shock syndrome.     

Ovine-associated Staphylococcus aureus protein with immunochemical similarity to toxic shock syndrome toxin 1.

A toxic shock syndrome toxin 1 (TSST-1) antibody-binding protein produced by an ovine-associated strain of Staphylococcus aureus was examined. The protein showed total identity to TSST-1 by immunodiffusion analysis. Western blots (immunoblots) of proteins separated by isoelectric focusing revealed that the TSST-1 antibody-binding protein had a pI of 8.6 rather than 7.0, the pI of standard TSST-1.     

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.     

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.     

egc-Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin

PCR was employed to determine the presence of all known superantigen genes (sea, seq, and tst) and of the exotoxin-like gene cluster (set) in 40 Staphylococcus aureus isolates from blood cultures and throat swabs; 28 isolates harbored superantigen genes, five on average, and this strictly correlated with their ability to stimulate T-cell proliferation. In contrast, the set gene cluster was detected in every S. aureus strain, suggesting a nonredundant function for these genes which is different from T-cell activation. No more than 10% of normal human serum samples inhibited the T-cell stimulation elicited by egc-encoded enterotoxins (staphylococcal enterotoxins G, I, M, N, and O), whereas between 32 and 86% neutralized the classical superantigens. Similarly, intravenous human immunoglobulin G preparations inhibited egc-encoded superantigens with 10- to 100-fold-reduced potency compared with the classical enterotoxins. Thus, there are surprisingly large gaps in the capacity of human serum samples to neutralize S. aureus superantigens.     

Presence of toxic shock toxin in toxic shock and other clinical strains of Staphylococcus aureus

Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of the two. Three molecular weight variants of the protein were found in the two toxic shock syndrome strains that were studied, regardless of the purification method that was used. An isoelectric point of 7.15 and molecular weights of 21,400, 22,100, and 23,200 were determined for the different forms of the protein from electrophoresis data. A sedimentation coefficient of 2.3S was determined by sucrose gradient centrifugation, and a Stokes radius of 2 X 10(-7) cm was determined by gel filtration. An average molecular weight of 18,900 for all of the TST forms was calculated from these data by the Stokes-Einstein equation. A survey for TST in 32 control and 46 toxic shock strains of S. aureus by isoelectric focusing and by agarose gel double immunodiffusion with specific rabbit antiserum revealed that the isoelectric focusing method tends to overestimate the number of TST-positive strains because of the detection of non-TST, neutral staphylococcal proteins. Based on immunodiffusion data, the association of TST with toxic shock strains was found to be 100% in vaginal isolates and 62% in non-vaginal isolates. In the control strains, TST was found in 16% of the vaginal strains and 23% of the non-vaginal strains. The value of this toxin as a marker for toxic shock and its relationship to the pathogenesis of this disease are discussed.     

Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB, entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection of femA was used as an internal positive control. The multiplex PCR assay combined the primers for sea to see and femA in one set and those for eta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.     

Production of staphylococcal enterotoxin F and pyrogenic exotoxin C by Staphylococcus aureus isolates from toxic shock syndrome-associated sources.

A total of 136 isolates of Staphylococcus aureus were tested for production of staphylococcal enterotoxin F (SEF) and pyrogenic exotoxin C (PEC), both of which have been identified as reliable indicators of toxic shock syndrome (TSS)-associated strains. SEF and PEC production by isolates from TSS-associated and other sources was tested independently in two laboratories, after which the two sets of data were compared. A 100% concordance between SEF and PEC production was obtained. The TSS toxin candidates were produced by 30 of 136 isolates, and in all instances SEF and PEC were made concurrently by the same strains; in no case was one toxin made and not the other. In the five groups of S. aureus tested, toxins were detected as follows: 23 of 25 (92%) acute TSS isolates, 2 of 48 (4.2%) genital non-TSS isolates, 2 of 16 (12.5%) recovered TSS isolates, 1 of 23 (4.3%) clinical nongenital isolates, and 2 of 24 (8.3%) enterotoxigenic food outbreak isolates. Comparison of purified SEF and purified PEC by immunological and biochemical criteria by immunodiffusion, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis show that the toxins are immunologically identical and strongly suggest that the two nominal TSS toxins are in fact a single protein.     

金黄色葡萄球菌中毒休克综合征毒素1在大肠杆菌中的分泌型表达

中毒休克综合征毒素１（Ｔｏｘｉｃｓｈｏｃｋｓｙｎｄｒｏｍｅｔｏｘｉｎ－１，ＴＳＳＴ－１）在中毒休克综合征（Ｔｏｘｉｃｓｈｏｃｋｓｙｎｄｒｏｍｅ，ＴＳＳ）的发病过程中起重要作用。本研究应用ＰＣＲ和ＤＮＡ重组技术，构建了ＴＳＳＴ－１分泌型表达载体ｐＲＴＳ２０并在大肠杆菌细胞周质表达出分子量为２２×１０３的重组ＴＳＳＴ－１，表达量约占周质总蛋白的６％～８％。Ｗｅｓｔｅｒｎ印迹杂交表明，表达产物具有特异的抗原活性。对ＴＳＳＴ－１生物学特性、结构功能关系有待进一步研究。本文对ＴＳＳＴ－１抗毒素的制备具有一定的参考意义。     

Immunological protection of rabbits infected with Staphylococcus aureus isolates from patients with toxic shock syndrome.

Toxic shock syndrome toxin-1 (TSST-1) isolated from the growth medium of Staphylococcus aureus 1169 and 555 was used to immunize male rabbits before infection with either a TSST-1+ or a TSST-1- strain of S. aureus isolated from cases of TSS. None of the immunized rabbits died as a result of the infections, whereas 50% of the nonimmunized rabbits infected with the TSST-1- strain, D4508, and 75% of those infected with the TSST-1+ strain, 555, died. Western blots of crude extracellular protein preparations probed with sera from immunized rabbits indicated that the TSST-1- strain produces a 30,000-molecular-weight protein that cross-reacts with antiserum to TSST-1. Because both organisms caused similar diseases in rabbits, we propose to designate the cross-reacting protein as TSST-2.     

Oxygen and carbon dioxide regulation of toxic shock syndrome toxin 1 production by Staphylococcus aureus MN8

The production of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus MN8 exposed to a range of oxygen concentrations (0 to 21% [vol/vol]) was examined in batch and thin-film cultures. The response of S. aureus to this range of oxygen concentrations was studied in the absence and in the presence of 7% (vol/vol) carbon dioxide. In the absence of carbon dioxide, TSST-1 production in batch cultures increased from negligible levels in the presence of oxygen concentrations of 1% or less to 500 ng/ml in the presence of 2% oxygen and then decreased to 70 ng/ml or less in the presence of oxygen concentrations of 6% and higher. In the presence of carbon dioxide, however, toxin production increased from negligible levels in the presence of 1% oxygen to 1,900 ng/ml in the presence of 21% oxygen. In thin-film cultures, TSST-1 production increased from nearly undetectable levels under anaerobic conditions to 1 and 10 microg/ml under 21% oxygen in the absence and presence of carbon dioxide, respectively. This study demonstrates the controlling effects of both oxygen and carbon dioxide on TSST-1 production.