Rapid and Specific Detection of Toxigenic Staphylococcus aureus: Use of Two Multiplex PCR Enzyme Immunoassays for Amplification and Hybridization of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies.     

Involvement of Enterotoxins G and I in Staphylococcal Toxic Shock Syndrome and Staphylococcal Scarlet Fever

We investigated the involvement of the recently described staphylococcal enterotoxins G and I in toxic shock syndrome. We reexamined Staphylococcus aureus strains isolated from patients with menstrual and nonmenstrual toxic shock syndrome (nine cases) or staphylococcal scarlet fever (three cases). These strains were selected because they produced none of the toxins known to be involved in these syndromes (toxic shock syndrome toxin 1 and enterotoxins A, B, C, and D), enterotoxin E or H, or exfoliative toxin A or B, despite the fact that superantigenic toxins were detected in a CD69-specific flow cytometry assay measuring T-cell activation. Sets of primers specific to the enterotoxin G and I genes (seg and sei, respectively) were designed and used for PCR amplification. All of the strains were positive for seg and sei. Sequence analysis confirmed that the PCR products, corresponded to the target genes. We suggest that staphylococcal enterotoxins G and I may be capable of causing human staphylococcal toxic shock syndrome and staphylococcal scarlet fever.     

Staphylococcal and Streptococcal Pyrogenic Toxins Involved in Toxic Shock Syndrome and Related Illnesses

Abstract

Toxic-shock syndrome (TSS) is an acute onset, multiorgan illness which resembles severe scarlet fever. The illness is caused by Staphylococcus aureus strains that express TSS toxin-1 (TSST-1), enterotoxin B, or enterotoxin C. TSST-1 is associated with menstrual TSS and approximately one-half of nonmenstrual cases; the other two toxins cause nonmenstrual cases, 47% and 3%, respectively. The three toxins are expressed in culture media under similar environmental conditions. These conditions may explain the association of certain tampons with menstrual TSS. Biochemically, the toxins are all relatively low molecular weight and fairly heat and protease stable. Enterotoxins B and C, share nearly 50% sequence ho-mology with streptococcal scarlet fever toxin A; they share no homology with TSST-1 despite sharing numerous biological properties. Numerous animal models for development of TSS have suggested mechanisms of toxin action, though the exact molecular action is not known. The toxins are all potent pyr-ogens, induce T lymphocyte proliferation, requiring interleukin 1 release from macrophages, suppress immunoglobulin production, enhance endotoxin shock, and enhance hypersensitiv-iry skin reactions. The genetic control of the toxins has been studied and suggests the exotoxins are variable traits. Some additional properties of TSS S. aureus which facilitate disease causation have been clarified.     

Localization of a T-Cell Epitope of Superantigen Toxic Shock Syndrome Toxin 1 to Residues 125 to 158

Toxic shock syndrome toxin 1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. So far, little is known about T-cell epitopes on superantigens. In this study, we developed an improved method for localizing T-cell epitopes on superantigens that involved synthetic peptides plus costimulation by CD28 or phorbol myristate acetate. Using this method, we localized a T-cell epitope to a 34-residue region, TSST-1 (residues 125 to 158), which possessed only two of four TSST-1-targeted β-chain variable element (Vβ) specificities of T-cell receptors in humans and mice, human Vβ2 and murine Vβ15.     

Molecular and Serological Differentiation of Staphylococcal Exfoliative Toxin Synthesized Under Chromosomal and Plasmid Control

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by phage group II Staphylococcus aureus under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxin are distinct from each other is given. The plasmid-controlled toxin was synthesized along with the chromosomally controlled toxin by the group II UT0002 strain, whereas another group II strain, UT0007, synthesized only the plasmid-controlled toxin. The molecular weight of the plasmid-controlled toxin was slightly less than that of the chromosomally controlled type and could be separated from the latter on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide slab gels. On 7.5% SDS-polyacrylamide cylindrical gels there was no hint of heterogeneity, and both ETs migrated together as a single homogeneous band. The existence of two serotypes of ET among phage group II strains complicates interpretation of previous work in this field and makes necessary the preparation of two different antigens for radioimmunobinding assays. Discovery of these ET serotypes provided an explanation for previously reported low binding by rabbit hyperimmune serum (B. Wiley, L. Glasgow, and M. Rogolsky, Infect. Immun. 13:513-520, 1976) in the radioimmunobinding test. A molecular species of ET differing from each of the other two serotypes was isolated from cultures of a phage group III S. aureus. This ET produced scalding in suckling mice and was lower in molecular weight than the ET produced under plasmid control by group II strains. Preliminary serological studies indicated that the ET in the group III strain is closely related to or possibly identical to the group II toxin produced under plasmid control.     

Identification of Genes Encoding Two-Component Lantibiotic Production in Staphylococcus aureus C55 and Other Phage Group II S. aureus Strains and Demonstration of an Association with the Exfoliative Toxin B Gene

The production of exfoliative toxin B (ET-B), but not ET-A, was shown to be specifically associated with production of a highly conserved two-component lantibiotic peptide system in phage group II Staphylococcus aureus. Two previously studied but incompletely characterized S. aureus bacteriocins, staphylococcins C55 and BacR1, were found to be members of this lantibiotic system, and considerable homology was also found with the two-component Lactococcus lactis bacteriocin, lacticin 3147. sacalphaA and sacbetaA, the structural genes of the lantibiotics staphylococcins C55alpha and C55beta and two putative lantibiotic processing genes, sacM1 and sacT, were localized together with the ET-B structural gene to a single 32-kb plasmid in strain C55. Irreversible loss of both ET-B and two-component lantibiotic production occurs during laboratory passage of ET-B-positive S. aureus strains, particularly at elevated temperatures.     

Identification and Characterization of Staphylococcal Enterotoxin Types G and I from Staphylococcus aureus

Staphylococcal enterotoxins are exotoxins produced by Staphylococcus aureus that possess emetic and superantigenic properties. Prior to this research there were six characterized enterotoxins, staphylococcal enterotoxin types A to E and H (referred to as SEA to SEE and SEH). Two new staphylococcal enterotoxin genes have been identified and designated seg and sei (staphylococcal enterotoxin types G and I, respectively). seg and sei consist of 777 and 729 nucleotides, respectively, encoding precursor proteins of 258 (SEG) and 242 (SEI) deduced amino acids. SEG and SEI have typical bacterial signal sequences that are cleaved to form toxins with 233 (SEG) and 218 (SEI, predicted) amino acids, corresponding to mature proteins of 27,043 Da (SEG) and 24,928 Da (SEI). Biological activities for SEG and SEI were determined with recombinant S. aureus strains. SEG and SEI elicited emetic responses in rhesus monkeys upon nasogastric administration and stimulated murine T-cell proliferation with the concomitant production of interleukin 2 (IL-2) and gamma interferon (IFN-γ), as measured by cytokine enzyme-linked immunoassays. SEG and SEI are related to other enterotoxins of S. aureus and to streptococcal pyrogenic exotoxin A (SpeA) and streptococcal superantigen (SSA) of Streptococcus pyogenes. Phylogenetic analysis and comparisons of amino acid and nucleotide sequence identities were performed on related staphylococcal and streptococcal protein toxins to group SEG and SEI among the characterized toxins. SEG is most similar to SpeA, SEB, SEC, and SSA (38 to 42% amino acid identity), while SEI is most similar to SEA, SEE, and SED (26 to 28% amino acid identity). Polyclonal antiserum was generated against purified histidine-tagged SEG and SEI (HisSEG and HisSEI). Immunoblot analysis of the enterotoxins, toxic-shock syndrome toxin 1, and SpeA with antiserum prepared against HisSEG and HisSEI revealed that SEG shares some epitopes with SEC1 while SEI does not.     

金黄色葡萄球菌及其肠毒素B对慢性实验性哮喘小鼠的影响

目的探索金黄色葡萄球菌肠毒素B（SEB）对实验性支气管哮喘小鼠肺组织Th0细胞分化的在体调节和对气道炎症的作用。方法32只15～17dBALB／c小鼠，随机分为4组：对照组、模型组、金黄色葡萄球菌组、金黄色葡萄球菌肠毒素B（SEB）组。用卵蛋白（OVA）致敏和激发建立小鼠慢性哮喘模型，在致敏前14d对幼年鼠做金黄色葡萄球菌或SEB预防性腹腔注射。观察记录小鼠的耗氧量、肺组织切片、支气管肺泡灌洗液（BALF）中炎性细胞及相关细胞因子等的改变。结果金黄色葡萄球菌组与模型组相比，肺组织病理切片结构紊乱减轻、炎症细胞明显减少，小鼠耗氧量降低（5．24±0．12vs5．59±0．18），BALF中IL-4水平明显下降（44．94±4．51vs29．37±4．17），IFN-γ水平明显增加（19．61±3．83vs29．33±4．04），SEB组也有上述作用（耗氧量：5．09±0.20；细胞介素4：36．68±5．10；γ干扰素：24．27±3．46）。结论金黄色葡萄球菌能明显减轻实验性哮喘的气道炎症，纠正哮喘小鼠肺组织中Th1／Th2细胞因子的失衡，其作用可能是通过其分泌的SEB。     

A Toxic Shock Syndrome Toxin - 1 Peptide That Shows Homology to Amino Acids 180-193 of Mycobacterial Heat Shock Protein 65 IS Presented as Conventional Antigen

Peripheral blood mononuclear cells (PBMC) from the majority of adults (12 out of 18 subjects tested) showed an in vitro proliferative response to a 20 amino acid long peptide (peptide 1, a.a 18-37) derived from TSST-1. In contrast, thymocytes and PBMC from cord blood did not proliferate to this peptide. TSST-1 peptide 1 did not induce IL-1β mRNA in monocytes indicating that it does not behave as a superantigen. Proliferation of PBMC to peptide 1 could be blocked by anti-HLA-DR, but not by anti-HLA DP or DQ monoclonal antibodies suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide. Peptide 1 shows homology to a.a 180-193 of mycobacterial hsp 65 and was shown to stimulate the proliferation of T cell lines and T cell clone specific for the purified protein derivative (PPD) of Mycobacterium tuberculosis. This cross reactivity may confer on TSST-1 the potential to trigger self reactivity and may also contribute to the natural immunity against TSST-1.     

Complete Nucleotide Sequence of a Staphylococcus aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C

The complete nucleotide sequence of pETB, a 38.2-kb Staphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.     

Pyrogenic Specificity of Streptococcal Exotoxins, Staphylococcal Enterotoxin, and Gram-Negative Endotoxin

Streptococcal exotoxin and staphylococcal enterotoxin share several biological properties, including pyrogenicity, lymphocyte mitogenicity, and enhancement of gram-negative endotoxin lethality. These analogies of the toxins prompted comparative pyrogenic studies. When American Dutch rabbits were immunized by repeated intravenous injections of small amounts of staphylococcal enterotoxin, they exhibited a decreased febrile response upon challenge when compared with control animals not previously injected. The same animals responded similarly to control groups when challenged with streptococcal exotoxin A, B, or C. No cross-reactivity was observed in reciprocal cross-tests, using animals immune to the streptococcal toxins and challenging with staphylococcal enterotoxin. No cross-reactivity between either the streptococcal or staphylococcal toxins and bacterial endotoxin was observed.     

Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates

Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.     

抗金黄色葡萄球菌C_1型肠毒素McAb的制备及初步应用

金葡菌Ｃ１型肠毒素免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞与Ｓｐ２／０小鼠骨髓瘤细胞融合选出了能分泌高滴度ＭｃＡｂ的１８个克隆株，对其中１０株进行了鉴定，６株属ＩｇＧ１，４株属ＩｇＧ３。用双抗体夹心法和特异性中和抑制试验比较了ＭｃＡｂ和ＰｃＡｂ的敏感性和特异性，并用制备的ＭｃＡｂ诊断试剂对ＳＥＣＩ污染食品进行了检测应用，可特异检出ｌｕｇＳＥＣ１／０．５ｇ食物／ｍｌ。     

产肠毒素D金黄色葡萄球菌的筛选及其基因克隆和蛋白表达

目的克隆金黄色葡萄球菌肠毒素D基因（entD），表达和纯化其重组蛋白（rSED），以进一步制备抗rSED抗体，研制SED金标免疫快速检测试纸条。方法利用PCR技术．从野生型金黄色葡萄球菌中筛选产肠毒素D金黄色葡萄球菌标准株，扩增entD基因，克隆至pGEM—T Easy载体．亚克隆至原核表达载体pET28a，重组质粒转化到大肠杆菌BL21（DE3），IPTG诱导表达蛋白，Ni-NTA亲和层析纯化蛋白，免疫印迹检测抗原活性。结果分离获得1株产肠毒素D金黄色葡葡球菌标准株．成功克隆entD序列．测序表明该基因共684bp，与其他entD序列（GenBank登陆号：M28521）具有99％的同源性。rSED蛋白在大肠杆菌中得到高效的可溶性表达。免疫印迹结果表明．rSED蛋白能被抗天然SED抗体识别。结论通过基因重组技术制备的重组SED蛋白具有良好的免疫反应性。     

A 21-Mer Synthetic Peptide of Toxic Shock Syndrome Toxin 1, TSST-1[58-78], Activates T Cells by Binding to MHC Class II and by an MHC Unrestricted Xenostimulatory Pathway

Toxic shock syndrome toxin-1 (TSST-1) is a “superantigen” which binds to MHC class II molecules and induces a polyclonal stimulation of T cells. In this communication by using a FACS technique and a 21-mer synthetic peptide from the primary sequence of TSST-1 (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1 (58-78), we demonstrated binding of the peptide only to cells bearing MHC class II. The proliferative effect of TSST-1 (58-78) on human T cells was shown to be inhibited much more by anti-HLA-DR than by anti-HLA class I antibody. Furthermore, human monocytes were able to present TSST-1 (58-78) to a mouse VSV specific T cell clone by a xenostimulatory mechanism. These data indicate this peptide to contain an active site of the TSST-1 holotoxin.     

Toxin Levels in Serum Correlate with the Development of Staphylococcal Scalded Skin Syndrome in a Murine Model

Staphylococcal scalded skin syndrome (SSSS) is an exfoliative dermatitis that results from infection with exfoliative toxin-producing Staphylococcus aureus. SSSS is seen primarily in infants and children. Here we ask if there is a specific maturation process that protects healthy adults from this syndrome. For these studies, an active recombinant exfoliative toxin A (rETA) was used in a neonatal mouse model. A time course generated on the susceptibility to the toxin as a function of mouse age indicated that BALB/c mice developed the characteristic symptoms of SSSS until day 7 of life. Between day 7 and day 8 of life there was a dramatic decrease in susceptibility, such that mice at day 9 of life were resistant to the effects of the toxin. This time course corresponds approximately to the time needed for maturation of the adaptive immune response, and SSSS in adults is often identified with immunocompromised states. Therefore, mice deficient in this response were examined. Adult mice thymectomized at birth and adult SCID mice did not develop the symptoms of SSSS after injection with the toxin, indicating that the adaptive immune response is not responsible for the lack of susceptibility observed in the older mice. SSSS in adults is also associated with renal disorders, suggesting that levels of toxin in serum are important in the development of the disease. rETA was not cleared as efficiently from the serum of 1-day-old mice compared to clearance from 10-day-old mice. Ten-day-old mice were given repeated injections of toxin so that the maximal level of toxin was maintained for a sustained period of time, and exfoliation occurred in these mice. Thus, whereas the adaptive immune response is not needed for protection of adult mice from SSSS, efficient clearance of the toxin from the bloodstream is a critical factor.     

Structural and Functional Properties of Antibodies to the Superantigen TSST-1 and Their Relationship to Menstrual Toxic Shock Syndrome

Menstrual toxic shock syndrome (mTSS) is an acute febrile disease accompanied by hypotension and multiple organ involvement. Infection with Staphylococcus aureus producing the superantigen toxic shock syndrome toxin-1 (TSST-1) vaginally is necessary; however, only a small fraction of those infected with TSST-1 producing bacteria actually develop mTSS, suggesting that host factors modulate disease susceptibility. Serum antibodies to the toxin protect against development of the syndrome, but not all antibodies can neutralize the toxin. We set out to determine whether risk of developing the syndrome is related to the absence of neutralizing antibody and if antibody isotypes influence the neutralization capacity. In healthy subjects, TSST-1-binding serum antibodies were exclusively of the IgG and IgM classes; however, toxin-neutralizing capacity was correlated to the TSST-1-specific IgG1 and IgG4 antibodies (r (2)=0.88, p<0.0001 and 0.33, p<0.0086, respectively) but not with IgM antibodies. Specific IgA was not detectable. Compared to healthy matched controls who were colonized vaginally with S. aureus, IgG1 anti-TSST-1 antibodies and toxin neutralizing activity was lacking in all of the acute phases and in the majority of convalescent sera, suggesting that these patients may be incapable of generating TSST-1 neutralizing antibodies. These new findings support the hypothesis that host factors are important in the development of mTSS and that the anti-toxin isotype impacts antibody functionality.     

Toxin Genes and Other Characteristics of Staphylococcus aureus Isolates from Milk of Cows with Mastitis

In the present study, 103 Staphylococcus aureus strains isolated from milk samples from 60 cows with mastitis from eight different farms in seven different locations in one region of Germany were compared pheno- and genotypically and by identification of various toxins. On the basis of culture and hemolytic properties and by determination of the tube coagulase reaction, all of the isolates could be identified as S. aureus. This could be confirmed by PCR amplification of species-specific parts of the gene encoding the 23S rRNA. In addition, all of the S. aureus isolates harbored the genes encoding staphylococcal coagulase and clumping factor and the genes encoding the X region and the immunoglobulin G binding region of protein A. These four genes displayed size polymorphisms. By PCR amplification, the genes for the toxins staphylococcal enterotoxin A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be detected. To analyze the epidemiological relationships, the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNAs. According to the observed gene polymorphisms, the toxin patterns, and the information given by macrorestriction analysis of the isolates by pulsed-field gel electrophoresis, a limited number of clones seemed to be responsible for the cases of bovine mastitis on the various farms.     

Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness.