Early Studies Of Toxic Shock Syndrome Tss Biology Essay


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The early studies of toxic shock syndrome insightful the way to prompt public health interventions and provided the outcome for the prevention to the morbidity [1] Basically the toxic shock syndrome is the combination of sign and symptoms which occur due to the release of toxic substances and it cause life threatening condition. Todd and co-workers worked on TSS and gave the description about this disease. [2, 3, 4, 5] First case of toxic shocked syndrome was reported in Britain in November 1980. [6] In 1980 to 1985 the large outbreak had been observed in United States from [94]. There are many symptoms which has been observed in TSS such as high fever, hypotension, vomiting and diarrhea, conjunctival hyperernia, an erythematous rash followed by desquamation of hands and feet, multi-organ failure kidney-insufficiency and shock. Some evidence of impaired renal and hepatic functions, and occasionally death. [7, 8, 9, 10, 3, 12, 13, 14] Usually it said that TSS is a female disease but this disease has been observed in male and children as TSS has been reported to occur in males with localized S. aureus infections [15] and similarly It is possible that some cases of syndrome of hemorrhagic shock and encephalopathy represent a variant of TSS in small children. [119] Subsequently, the syndrome has been described in menstruating women and has been associated with tampon use and positive vaginal cultures for S. aureus. [15, 16, 17, 5, 20, 21, 22, 23, 24, 25]. TSS may occur in patients with mastitis, Bartholin's gland abscess, wound infection, or acute salpingitis. [154] Toxic shock syndrome (TSS) is a severe illness occur usually during menstruation but also in the postpartum period.[153] Subsequently, microbiological examinations showed that most of the neonates with this illness were colonized by methicillin-resistant S. aureus (MRSA) strains that produced TSST-1, suggesting that the pathogenesis of this illness is the same as that for TSS [94] An etiological role for Staphylococcus aureus was recognized in the earliest cases, but the pathogenesis of TSS remains to be elucidated. Since TSS is multisystemic and results from localized S. aureus infections, most attention has been directed toward the extracellular products of S. aureus as potential effectors of the disease.[13] As It has been observed in most of the cases that Staphylococcus aureus is responsible for many nosocomial and community-acquired infections. Its pathogenicity is attributed to its ability to produce many membrane- associated components and extracellular substances, several of which have been implicated as virulence factors. One of the most unique manifestations among the various staphylococcal infections is staphylococcal toxic shock syndrome (TSS). [28] Colonization or localized infection with Staphylococcus aureus has been demonstrated in 95 to 100% of those patients with toxic shock syndrome (TSS) who have had cultures taken before antibiotic therapy. S. aureus can be cultured from the vagina of most patients with menstrual TSS but it is also observed that staphylococcal bacteremia is uncommon in TSS patients.[29] but in sever condition bacteremia can be observe. The genetics of S. aureus enterotoxin production have been well studied [30, 31, 32, 33, 34, 35, 36, 37] Toxic shock syndrome can mimic many common diseases. Because it can be associated with a number of nonmenstrual-related conditions, patients with unexplained fever and rash and a toxic condition out of proportion to local findings should have the diagnosis of toxic shock syndrome in their differential diagnosis. Early recognition and aggressive management can decrease the overall morbidity and mortality. [27] Most of the people are confused in TSS and SSSS, Actually Staphylococcal toxic shock syndrome (TSS) and staphylococcal scalded skin syndrome (SSSS) are two distinct toxin-mediated syndromes with prominent cutaneous features. The exanthematous presentation of these syndromes places them in the broad category of childhood exanthems, and the ability to recognize these potentially devastating illnesses is essential for pediatricians and dermatologists who may encounter children with fever and rash. Recent advances in the understanding of the pathogenesis of these entities has helped to explain the distinctive clinical presentations of TSS and SSSS. [108] Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new entity of methicillin-resistant Staphylococcus aureus (MRSA) infection. Most of NTED cases reported previously in the literature were sporadic ones. Staphylococcus aureus produces a wide variety of exoproteins that contribute to its ability to colonize and cause disease in mammalian hosts.[44] Actually, the neonates exhibited a marked polyclonal expansion of v_2-positive T cells in the acute phase of this illness. As this neonatal disease did not match the clinical criteria for TSS, the disease was named neonatal toxic shock syndrome- like exanthematous disease (NTED)[94] Nearly all strains of MRSA causing NTED secrete a group of enzymes and cytotoxins which includes four hemolysins (alpha, beta, gamma, and delta), nucleases, proteases, lipases, hyaluronidase, and collagenase. [44] The main function of these proteins may be to convert local host tissues into nutrients required for bacterial growth. Some strains produce one or more additional exoproteins, which include toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEA, SEB, SECn, SED, SEE, SEG, SEH, and SEI), the exfoliative toxins (ETA and ETB), and leukocidin. Each of these toxins is known to have potent effects on cells of the immune system, but many of them have other biological effects as well. Their primary function in vivo may be to inhibit host immune responses to S. aureus. TSST-1 and the staphylococcal enterotoxins are also known as pyrogenic toxin superantigens (PTSAgs). Two former names for TSST-1 were staphylococcal pyrogenic exotoxin C and staphylococcal enterotoxin F. [44] SEB may be an important cause in TSS where TSST-1 is not elaborated, especially in nonmenstrual TSS isolates. [70]TSST-1 and SEA to SED have been linked to other staphylococcal syndromes such as staphylococcal scarlet fever (SSF) and recalcitrant erythematous desquamating disorder (REDD), both of which were suggested to be variants of TSS on the basis of toxin production and certain clinical similarities[150]S. aureus strains from patients with toxic shock syndrome produced enterotoxin F.[130] Jarraud et al. reported that most tst positive S. aureus strains are genetically related and have a type 3 accessory gene regulator (agr) allele. [17, 72] TSST-1 is encoded by tstH (where H refers to human isolate), which is present on the bacterial chromosome within a 15.2-kb mobile genetic element called staphylococcal pathogenicity island 1. [44, 38, 88, 89, 90] TSST-1, encoded by the tst gene, is a major virulence factor in toxic shock syndrome(TSS), staphylococcal scarlet fever, and neonatal toxic shock like exanthematous diseases (NTED) [17, 93, 94, 95] There are many biochemical and enzymatic reaction which occur during toxic shocked syndrome such as Cytokines produced by T cells and activated by TSST-1 are thought to cause the abnormal changes of TSS.[153] In obstetrics, the syndrome has been described following vaginal delivery, cesarean delivery, and spontaneous and therapeutic abortions. In the gynecologic patient, TSS has been documented as complicating surgical procedures such as tubal ligation, vaginal and abdominal hysterectomies, urethral and bladder suspensions, exploratory laparotomy, and therapy of extensive condyloma acuminatum. [154] Postoperative TSS can be particularly difficult to diagnose because patients often present with mild nonspecific, "flu like" symptoms and usually do not have evidence of local wound infection. It is therefore important to have a high suspicion index of occult wound infection complicated by TSS in postoperative patients with fever, rash, gastrointestinal symptoms or hypotension.[157] TSS is thought to be a superantigen-mediated disease. Toxins produced by Staphylococci and Streptococci act as superantigens that can activate the immune system by passing the usual antigen-mediated immune-response sequence.[26] TSS is caused by a toxin produced by certain types of Staphylococcus bacteria. A similar syndrome, called toxic shock-like syndrome (TSLS), can be caused by Streptococcal bacteria. [29, 42] The disease has been associated with the production of a toxin by the staphylococci isolated from TSS patients [21, 60, 61]. Typically, TSS strains of S. aureus are different from staphylococci isolated from asymptomatic carriers or from individuals with non-TSS staphylococcal infections. [122] TSS and menstrual TSS are now reported with almost equal frequency.[26] Nonmenstrual and menstrual TSS isolates also differ phenotypically in other characteristics, suggesting different origins or pathogenesis in these two variants of TSS.[139] MRSA clones have been characterized on the basis of several other characteristics. For instance, MRSA detected in Japan was shown to produce toxic shock syndrome toxin 1, a superantigenic toxin family member [66, 67, 68] The first methicillin-resistant S. aureus (MRSA) isolates were detected in the hospital setting in the early 1960s. These epidemic MRSA strains of hospital origin have also been detected in the community, infecting patients with risk factors associated with hospital-acquired MRSA infection (H-MRSA), such as recent hospitalization. [17] Since the introduction of penicillin in the 1950s, the control and treatment of S. aureus infection has improved dramatically, but the emergence of methicillin-resistant S. aureus (MRSA) has become a serious threat. [63 , 64]. Methicillin resistance is encoded by the mecA gene carried by a mobile genetic element, namely, staphylococcal cassette chromosome mec (SCCmec). [63, 65] S. aureus consists of elements that encode resistance factors. Among those, the staphylococcal chromosome cassette methicillin-resistance islands (SCCmecs) have gained most attention, as they represent the key determinant conferring resistance to methicillin and other beta-lactam antibiotics and are thus responsible for the occurrence of the infamous MRSA (methicillin-resistant S. aureus). Remarkably, pathogenicity islands such as the SaPIs have so far been not known to contain resistance factors, while the SCCmec elements were assumed to contain only resistance but no toxin genes[11] SCCmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins (SEs), exfoliative toxins (ETs) including S. intermedius exfoliative toxin (SIET), and toxic shock syndrome toxin 1 (TSST 1). These toxins are associated with atopic dermatitis, mastitis, food poisoning, canine pyoderma, and chronic otits [96] The contribution of agr to S. aureus virulence has been clearly linked to its implication in gene regulation and bacterial interference. S. aureus agr is a 3-kb locus showing highly conserved and hyper variable regions among S. aureus strains. The sequence of this hypervariable segment is the target of PCR amplification for defining agr types .[103]Over the last few decades, there has been an enormous increase and emergence of S. aureus strains resistant to the antibiotic methicillin (MRSA strains), particularly in nosocomial settings. The intrinsic resistance to these antibiotics is attributed to the presence of mecA, whose product is a 78-kDa protein called penicillin binding protein 2a. Identification of mecA in such MRSA strains has led to some knowledge regarding the use of the antibiotic vancomycin. The femA gene encodes a factor which is essential for methicillin resistance and is universally present in all S. aureus isolates. The femA gene product, a 48-kDa protein, has been implicated in cell wall metabolism and is found in large amounts in actively growing cultures.[66] However, the presence of common geographically-independent subtypes was detected. Recent studies have reported that methicillin resistance (Mec[r]) may be present in clinically derived TSST-1-producing S. aureus isolates from Japan and Europe. [69] The genetic properties of extracellular proteins in S. aureus are reviewed as possible models for the acquisition or expression of this toxic shock antigen. [38]. Epidemiology studies found that women who developed mTSS were more likely to be white and were users of barrier contraceptives or tampons.[ 14, 78, 79] Although some high-absorbency tampons appeared to increase the relative risk of TSS, epidemiologic studies did not resolve the issue of which specific tampon characteristic was associated with this increased risk. [9] This protein is synthesized by almost all menstrual and many nonmenstrual strains of S. aureus isolated from patients with the clinical manifestations of TSS, although it is likely that staphylococcal enterotoxins other than TSST-1 play an important role in the disease [2] Toxic shock syndrome toxin 1 (TSST-1) is a superantigenic toxin secreted by some S. aureus isolates [17] Subsequently, other exoproteins have been described (including the previously described epidermal toxin) which may also play roles in the pathophysiology of TSS. [99] TSST-1 positive isolates showed interesting characteristics which all of the isolates produce both SEC and coagulase type VI [98] Staphylococcus aureus synthesizes a large number of extracellular proteins that are important during pathogenesis. These include several cytolytic toxins (a-, i-, oy- and 6- hemolysin), toxic shock syndrome toxin-I (TSST-1), enterotoxins, leucocidin, the immunoglobulin binding protein A, coagulase which activates prothrombin, several hydrolytic enzymes, and others (Smith, 1979).[62] S. aureus secretes up to 30 different protein antigens, including at least eight T cell superantigens': toxic shock syndrome toxin-l (TSST-1) and staphylococcal enterotoxins A, B, C1, C2, C3, D, and E. [80] Superantigens differ from conventional antigens according to several criteria. Unlike conventional antigens, processing is not required for T-cell activation [120] Clinical isolates of Staphylococcus aureus can produce a spectrum of extracellular protein toxins and virulence factors which are thought to contribute to the pathogenicity of the organism. Designated Staphylococcal enterotoxin A (SEA), SEB, SEC, SED, and SEE. Minor epitope differences in the SEC group have resulted in a further subdivision into SEC1, SEC2, and SEC3 SEA, SED, and SEE share immunological determinants, as do SEB and SEC1 and streptococcal pyrogenic exotoxin A (SPEA). [30, 34, 48, 49] Toxic shock syndrome toxin 1 (TSST-1), plays a significant role in the pathogenesis of TSS. [39]. The etiology of TSS is based on a localized or, rarely, systemic infection with certain Staph. aureus strains that are capable of producing toxins, the most important one being TSST-1.[151] Numerous studies have shown that pyrogenic toxin superantigens PTSAgs are important determinants for TSS[124] Because Production by Staphylococcus aureus of toxic shock syndrome toxin 1 (TSST-1) is a major causative agent of toxic shock syndrome (TSS). [40, 41]. Toxic shock syndrome toxin 1 (TSST-1) is a 22-kDa exotoxin produced by Staphylococcus aureus .[120] . Toxic shock syndrome toxin-1 (TSST-1) and enterotoxins are the secretory products of Staphylococcus aureus that lead to TSS. [108] Tsst1 consists of a single chain lacking any half-cystine residues. TSST-1 is a very active molecule, and its mitogenic activity may plays an important role in toxic shock syndrome. The biologically active sites in the toxin molecule have not been fully elucidated, although in previous studies they have been associated with the 15-kDa cyanogen bromide-generated middle fragment.[87] This toxin has been implicated in the development of menstrually associated TSS. This protein has profound effects on murine and human immune systems, precipitating massive Tcell stimulation. In humans, this activation can lead to shock and in some cases death. It has been described, along with other staphylococcal enterotoxins, as a superantigen.[120] Most of the study has observation that these toxins are produced in sufficient concentrations to produce illness in the presence of certain tampons. This necessitates evaluating tampons, as well as wound dressings for their effects on S. aureus growth and toxin production.[142] TSS toxin 1 (TSST-1), is thought to play a central role in this disease.[123] Extensive investigations of the epidemiology of TSS and the chemical and biological features of TSST-1 resulted in the general impression that the pathogenesis of TSS was straightforward [91, 92] The monokine interleukin-1 (IL-1) is a primary mediator in host response to infection, injury, and inflammation. Although it is not the sole mediator, IL-1 has been shown to modulate many systemic aspects of the acute-phase response, including fever, systemic metabolic changes, and release of the acute-phase reactants. Although many of the properties of IL-1 are of potential benefit to the host, excessive release of IL-1 may play a role in the pathogenesis of certain disease processes, including chronic inflammation, rheumatoid arthritis, and fibrosis. The massive release of IL-1 has been implicated in mediating many of the symptoms of toxic shock syndrome (TSS) As little as 100 pg of TSST-1 per ml can induce the secretion of IL-1 by human monocytes[73] A hypothesis has been developed for the pathogenesis of menstrually related TSS. Certain tampon fibers that are highly absorbent for water are also ion exchangers for magnesium ions. The latter ions uniquely affect the production of TSS toxin 1 (TSST-1) by appropriate strains of Staphylococcus aureus, with a marked increase in the amount of toxin when magnesium concentrations are limiting and suppression of toxin production when magnesium is in excess. Many epidemiologic features of TSS could be explained by this hypothesis. The absorbability of highly absorptive fibers is not affected by the addition of small amounts of magnesium sufficient to suppress production of TSST-1; thus absorption is distinguishable from toxin production in vitro.[157] The toxic shock syndrome toxin I (TSST-1) is an extracellular single chain protein. [85, 86, 12] TSST-1 has a wide variety of biological effects, including T-lymphocyte mitogenicity induction of interleukin-1 and tumor necrosis factor release from human monocytes, suppression of immunoglobulin M response against sheep erythrocytes, stimulation of lymphokine release by T lymphocytes, and enhancement of host susceptibility to lethal endotoxic shock. These properties are shared by a group of related exoproteins, staphylococcal enterotoxin A (SEA),SEB, SEC, SED, and SEE [123]. All toxins thus far identified share a number of important Properties. [44, 81, 82, 83, 84] Three SAgs are most associated with production of TSS: 1) menstrual TSS caused nearly exclusively by TSST-1; and 2) non-menstrual TSS caused by TSST-1, SEB, and SEC14, 22-25. These three SAgs typically are produced in the highest concentrations (of the SAgs) by S. aureus strains as tested in vitro, with amounts being produced in broth (planktonic) cultures often in the 5-50 μg/ml range. Other SAgs typically are produced in vitro in amounts 104 to 106 lower than these three SAgs. These differences are important since it has been shown that SAg amounts as low as 0.1μg/human are able to cause TSS symptoms2 [47] The recovery of S. aureus strains that produce TSST-1 or staphylococcal enterotoxins or both from TSS patients was discussed in several papers, but only minimal attention was given to the coagulase-negative Staphylococci and their ability to produce these toxins. In previous study it has been showed that coagulase-negative Staphylococci that produce TSST-1, one or more of the enterotoxins, or both, are important in TSS.[156] Comparison of the tertiary structures of TSST-1 with those of SEA, SEB, and SEC reveal that despite only 20 to 30% primary sequence identity, these toxins have nearly identical folds. TSST-1 is folded into two domains, domain A and domain B. Domain B has five b strands folded into a barrel. Domain A contains the central a helix resting against a five-strand b sheet. Above this central a helix is the amino-terminal a helix, and on the sides are two loops which in TSST-1 extend just up to the axis of the central a helix. Together, these loops and the amino-terminal a helix define the walls of grooves that give access to the central a helix. [133] The SEs are generally small (25-30 kD), basic, heat- and acid-stable, single-chain molecules containing a short centrally located disulfide loop [140] Schlievert et al. (1981) found the production of a toxic protein called exotoxin C in 100% of the S. aureus strains isolated from patients with TSS. [97] Thus, Staphylococcal exotoxins (SE) are potent activators of the immune system and cause a variety of diseases in humans, including food poisoning, toxic shock, and autoimmune diseases. Their interactions with cells of the immune system result in massive production of proinflammatory cytokines and chemokines. The cytokines tumor necrosis factor alpha (TNF-_), interleukin-1 (IL-1), and gamma interferon (IFN-_) are key mediators in superantigen-induced toxic shock (1, 21). Both TNF-_ and IL-1 have potent immunostimulating activities and act synergistically with IFN-_ to enhance immune reactions and promote tissue injury. Consequently, these cytokines are pathogenic at high concentrations in vivo and are responsible for fever and toxic shock induced by SE [141] The properties of SEB-producing strains (SEB+) are well documented; Asheshov and co-workers demonstrated that the strains in the 94/96 phage complex were very often SEB+; Melconian and colleagues observed that these strains were lysed mainly by phages of groups II and V; and Dornbusch and Hallander have demonstrated a relationship between oxacillin resistance and toxin production. Recently, Lee and colleagues have shown that TS-associated strains produced either TSST-1, enterotoxin B or, exceptionally, both; they have also shown that SEB+ strains belonged to the same zymotype, suggesting a clonal origin.[131] MHC class I1 receptor sites on antigen presenting cells, thereby preventing the activation of T-cells by the unmodified toxin. In addition, the mutant toxin binds MHC class I 1 molecules equally well as the native or wild-type toxin (Cullen et al., 1995).[146] It is clear that antigens such as the toxic shock syndrome toxin (TSST-1) must be able to cross the human vaginal mucosa since the clinical features of toxic shock syndrome are systemic and affect many organs. On the other hand, the presence of toxin-producing organisms is not inevitably associated with clinical disease, suggesting that the vagina can exert a barrier function. The permeability barrier in vagina, like that of other non-keratinized mucosal tissues, consists of organized neutral lipids located in the superficial intercellular spaces of the epithelium. The toxin reached a steady state flux after 3-4 hrs and could be localized in the epithelium and connective tissue. In previous study it has been suggested that TSST-1 can penetrate the permeability barrier in vaginal mucosa under in-vitro conditions but this does not result in marked, direct, tissue damage. It is likely that this requires other components, possibly endotoxin, and a cellular infiltrate in the mucosal tissue, which is absent in-vitro [134] Extensive damage to the epithelial cells of mucosal surfaces is frequently observed in TSS,and recent studies have demonstrated that human epithelial cells rapidly internalize TSST-1 via receptormediated endocytosis (RME), which suggests that this route of cell entry may have pathogenetic implications. [12, 14] Experimentally, a striking feature of TSST-1. is its ability to sensitize the host to lethal injury by small amounts of endotoxin.[13,14] It has been hypothesized that TSST-1 in concert with low levels of endotoxin or lipopolysaccharide (LPS) may act synergistically and that this interaction may be important in the pathogenesis of TSS.1[149] Results of various studies suggest that vaginal epithelial cells are components of the innate immune system.[18] Direct effects of TSST-1 on neutrophils are difficult to rationalize, as no surface structures that can act as targets for these superantigens have been identified on freshly isolated neutrophils. Recently, neutrophils have been shown to express MHC class II molecules, but only after culture for a day or more with IFN-g, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-3, but this does not explain the rapid (10 min)effects of TSST-1 on freshly isolated neutrophils. Neutrophils possess a very short half-life in the circulation because they constitutively undergo apoptosis.[148] Superantigens (SAgs) are bacterial and viral proteins that share the ability to activate a large fraction of T-lymphocytes (Marrack and Kappler, 1990). The staphylococcal enterotoxins are the best characterized of the SAgs. They have been shown to bind as unprocessed proteins to major histocompatibility complex (MHC) class II molecules on antigen presenting cells, and subsequently activate T-cells through interaction with the variable region of the T-cell receptor a-chain (TCR-Vp) encoded by certain families of TCR-Vf genes (Marrack and Kappler, 1990). This results in the activation of between 2 and 15% of all T-cells ultimately leading to proliferation, production of a variety of cytokines as well as expression of cytotoxic activity [128] Peripheral T cells are divided into two major subsets, CD4' and CD8+ T cells. Whereas peptides bound to MHC class I and class II molecules are recognized by antigen-specific CD8+ cells and CD4+ cells, respectively, the binding of superantigens to MHC class II on accessory cells is sufficient for activation of CD4' and CD8+ T cells.9 Stimulation of T cells with TSST-1 results in activation of both CD4+ and CD8+ cells.8'0l"[155] It has been observed that Staph. aureus which cause TSS are usually methicillin resistant. First identified in the 1960s, methicillin-resistant Staphylococcus aureus (MRSA) was initially considered a nosocomial pathogen. However, in recent years, increasing numbers of MRSA strains have been isolated worldwide from patients with community-acquired infections. [50, 51, 52, 53, 54, 55, 56, 57, 58, 59] The potential disease spectrum coupled with the emergence of drug resistance and its formidable arsenal of strategies to counter the host immune response makes S. aureus one of the leading causes of infectious disease morbidity and mortality worldwide [127] Numerous investigations have attempted to identify phenotypic characters marking TSS-associated strains. As a group, these strains more frequently exhibit proteolytic activity in vitro.[129] Because superantigens may be involved in diseases in which intravenous IgG (IVIgG) is used as therapy An study showed that pooled IgG contains high concentrations of Staph-toxin-specific antibodies which are highly inhibitory to the in vitro activation of T cells by the Staph-toxins.[102] Despite this uncertainty, TSST-1 is generally accepted as being a marker, at least, for potentially pathogenic staphylococci. A quantitative assay for TSST-1 is essential for studying the factors that influence TSST-1 production and the biological effects of TSST-1 in a variety of experimental systems. [126]The relationship of Staphylococcus aureus growth to toxin production within the vaginal environment is not well understood. This association is important and complex in the case of menstrual toxic shock syndrome (mTSS). Toxic Shock Syndrome Toxin-1 (TSST-1) is expressed in vitro by S. aureus under specific conditions of nutrients, aeration (gas levels), temperature and pH. Additionally, supporting surfaces and mode of growth may impact toxin production. S. aureus carrying tst gene which is responsible for the production of TSST-1 were detected based on fluorescence resonance energy transfer (FRET) occurring between CdSe/ZnS QD donors and black hole quencher (BHQ) acceptors. [43] For epidemiological surveillance, the methods most frequently used for the detection of staphylococcal toxins are immunodiffusion, agglutination, radioimmunoassay, and enzyme-linked immunosorbent assay. Among the techniques used to identify toxin genotypes, DNA-DNA hybridization and PCR have been reported to be very successful and reliable, and our laboratory previously designed specific primers for the successful and reliable detection of SEs, TSST-1,and ETs by PCR.[135]The Staphylococcal PTSAgs cause or have been implicated in the pathogenesis of several acute or chronic human disease states. [44, 45, 46] Many features of the disease are as yet unexplained, and a better understanding of the mechanisms involved in the response to TSST-1 would allow us to test the efficacy of therapeutic modalities. [85] For epidemiological surveillance, the methods most frequently used for the detection of staphylococcal toxins are immunodiffusion, agglutination, radioimmunoassay, and enzyme-linked immunosorbent assay. Among the techniques used to identify toxin genotypes, DNA-DNA hybridization and PCR have been reported to be very successful and reliable, and our laboratory previously designed specific primers for the successful and reliable detection of SEs, TSST-1, and ETs by PCR. [135] Several sensitive methods for the identification of TSST-1-producing S. aureus have been developed. TSST-1 can be detected in culture supernatants either by enzyme-linked immunosorbent assays (ELISA) or by latex agglutination. TSST-1 can also be detected directly from S. aureus colonies by a colony immunoblot assay.[125] It is very important to know that the TSST-1 purified from human-associated TSS strains has an isoelectric point (pI) of 7.0 to 7.2. [104, 105, 106] However, variations in the molecular sizes (20 to 24 kDa), isoelectric points (6.8 to 7.2),Amino acid compositions, and biologic activities of different TSST-1 preparations have been reported[145] Ho et al. also showed that TSST-1 and its variant produced by ovine. S. aureus isolates can be distinguished by their different isoelectric points (pI). [138] TSST-1 contains a high percentage of hydrophobic amino acids, yet it is highly soluble in water. There are no cysteine residues, and the toxin is generally resistant to heat and proteolysis. For example, TSST-1 can be boiled for more than 1 h without detectable loss of biological activity, and it is not cleaved after prolonged exposure to trypsin [44] TSST-1 contains a high percentage of hydrophobic amino acids, yet it is highly soluble in water. There are no cysteine residues, and the toxin is generally resistant to heat and proteolysis. For example, TSST-1 can be boiled for more than 1 h without detectable loss of biological activity, and it is not cleaved after prolonged exposure to trypsin [44] Schlievert and Blomster observed that toxin is produced during the logarithmic phase, and maximal production occurs just before the stationary phase. [40,107]. As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier.[147] Robbins et al. developed a disk membrane- agar method for the testing of materials and unrolled tampons for the production of TSST-1 by S. aureus. The latter method has the unique advantage that materials may 14e tested prior to being processed into a tampon. A disadvantage is that it is difficult to limit the air to that available from the material being tested.[110] TSS has been difficult to establish. Antibody directed against TSST-1 is usually absent or minimal in sera of patients with TSS, in contrast to age-matched controls from the population at large. The absence of antibody in cases of TSS is circumstantial evidence for a pathogenic role for TSST-1 in this disease.[152] S aureus is usually readily isolated from the site of infection (and sometimes from the blood), and toxin production can then be confirmed. In most cases the cause is the toxic shock syndrome toxin-I (TSST-1), though other staphylococcal toxins may also cause the same pattern of illness.[71] Various complex and synthetic bacterial growth media have been used to study the growth of Staphylococcus aureus and the production of toxic shock syndrome toxin 1 (TSST-1) under certain in vitro culture conditions. Because of the biochemical and nutritional differences between these media and human menses, a program was designed to determine the growth and metabolism of S. aureus under conditions that more closely approximate in vivo conditions. Human menses, an artificial menses developed to match human menses, whole human blood, and complex bacterial culture media (with and without added whole human blood) were tested for the ability to support the growth of S. aureus and the production of TSST-1 in vitro. In addition, the impact of other organisms, commonly isolated from the human vagina, on the growth of S. aureus and the production of TSST-1 was evaluated.[143] Preliminary work from laboratory indicated that production of TSST-1 in vitro was greatly enhanced by cultivation of S. aureus in Mg2 -deficient medium. Moreover, certain fibers used in the production of tampons were found to bind Mg2+, potentially reducing the amount available to the organisms. The mechanism by which Mg2+ influences production of this toxin requires systematic investigation. The present study was undertaken, as the first step in this objective, to characterize in detail the effect of Mg2+ on the production of TSST-1 by S. aureus. Studies by Ingham et al. implicated a complex interaction of staphylococci with tampon materials under in vitro conditions whereby the metabolic activities of the microorganism are altered. For instance, the production of extracellular products of S. aureus such as hemolysin is decreased when the bacteria are grown in broth in the presence of tampon fibers. Holland et al. have reported the effect of solidphase material such as chemically modified cotton on the secretion ofextracellular enzymes by a TSS strain. Likewise, selective surface proteins of S. aureus appeared to be expressed or enhanced in production when grown on a solid surface as compared to those grown in a liquid medium . Thus, the microenvironment available to the bacteria is an important determinant in modifying the mode of the metabolic machinery of the microorganism.[101] Studies of TSST-1 at the molecular level were initiated by Kreiswirth et al. with the isolation and expression of the toxin structural gene in Escherichia coli. This enabled the prediction of the amino acid sequence of the entire polypeptide (22 kilodaltons [kDa] in molecular size) from the nucleotide sequence.[144] Women who use tampons during menstruation and are either infected or colonized by Staphylococcus aureus in the vagina are the most likely to develop TSS [4] Synthetic tampons and toxic shock syndrome toxin-one (TSST-1)-producing strains of Staphylococcus aureus have been linked to an increased incidence of toxic shock syndrome (TSS). [109] Wagner et al. demonstrated that the insertion of a tampon into the vagina raised the oxygen level to that of the atmosphere while decreasing initially the carbon dioxide level. Slow infusion of fluid into the tampon as well as the cutting off of atmospheric oxygen are essential to understanding how and why the tampon plays a role in menstrually related TSS cases. [110, 111] There are various conditions which are involve in TSS such as the concentrations of O2 and CO2, two components important to microbial growth, have been measured in the vagina before and after tampon insertion. In vitro studies indicate that a number of environmental factors affect production of TSST-1, including magnesium concentration,oxygen concentration, growth rate, temperature, and pH. [9, 112, 113, 114, 115, 116,117] There are several factors known to regulate TSST-1 production by S. aureus. In vitro studies demonstrate that O2 and CO2, iron, pH, glucose, and temperature are factors that can alter the concentration of TSST-1 produced by S. aureus. Recent in vitro studies have shown that both alpha and beta-globin chains of human hemoglobin can inhibit the production of TSST-1 without impacting growth of the organism.Given that menstrual blood is rich in hemoglobin, the study suggests that toxigenic S. aureus present on tampons might produce TSST-1 only in areas that lack menstrual blood.[100] Numerous studies have demonstrated the importance of oxygen tension in the regulation of TSST-1 production by S. aureus. Excess aeration of cultures, as well as complete anaerobiosis, resulted in repression of TSST-1 production, while microaerobic environments appeared to stimulate toxin expression. It has been suggested that elevated vaginal oxygen levels associated with the insertion of a tampon stimulate the production of TSST-1. Wagner et al. demonstrated that the vaginal environment is normally anaerobic and that insertion of a tampon dramatically increased the oxygen level on the vaginal mucosal surface. Following tampon insertion, oxygen levels slowly declined throughout the observation period of 8 h.[136] Four factors are thought to be required for the development of the mTSS: (1) vaginal colonization with a toxigenic strain of S. aureus; (2) production of TSST-1;(3) penetration of a sufficient concentration of TSST-1 across the epithelium to cause the disease; and (4) absence or insufficient titers of neutralizing antibody to the toxin.[137]The aim of this was to evaluate the presence of TSST1 gene in young female in Karachi, Pakistan

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