Staphylococcal Toxic Shock Syndrome Toxin-1 Research Articles

Comparison of Transcriptional Signatures of Three Staphylococcal Superantigenic Toxins in Human Melanocytes.

Staphylococcus aureus, a gram-positive bacterium, causes toxic shock through the production of superantigenic toxins (sAgs) known as Staphylococcal enterotoxins (SE), serotypes A-J (SEA, SEB, etc.), and toxic shock syndrome toxin-1 (TSST-1). The chronology of host transcriptomic events that characterizes the response to the pathogenesis of superantigenic toxicity remains uncertain. The focus of this study was to elucidate time-resolved host responses to three toxins of the superantigenic family, namely SEA, SEB, and TSST-1. Due to the evolving critical role of melanocytes in the host’s immune response against environmental harmful elements, we investigated herein the transcriptomic responses of melanocytes after treatment with 200 ng/mL of SEA, SEB, or TSST-1 for 0.5, 2, 6, 12, 24, or 48 h. Functional analysis indicated that each of these three toxins induced a specific transcriptional pattern. In particular, the time-resolved transcriptional modulations due to SEB exposure were very distinct from those induced by SEA and TSST-1. The three superantigens share some similarities in the mechanisms underlying apoptosis, innate immunity, and other biological processes. Superantigen-specific signatures were determined for the functional dynamics related to necrosis, cytokine production, and acute-phase response. These differentially regulated networks can be targeted for therapeutic intervention and marked as the distinguishing factors for the three sAgs.

Staphylococcal TSST-1 Association with Eczema Herpeticum in Humans.

ABSTRACTAtopic dermatitis (AD) is a condition affecting 30 million persons in the United States. AD patients are heavily infected with Staphylococcus aureus on the skin. A particularly severe form of AD is eczema herpeticum (ADEH), where the patients’ AD is complicated by S. aureus and herpes simplex virus (HSV) infection. This study examined the S. aureus strains from 15 ADEH patients, provided blinded, and showed a high association of ADEH with strains that produce toxic shock syndrome toxin-1 (TSST-1; 73%) compared to 10% production by typical AD isolates from patients without EH and those from another unrelated condition, cystic fibrosis. The ADEH isolates produced the superantigens associated with TSS (TSST-1 and staphylococcal enterotoxins A, B, and C). This association may in part explain the potential severity of ADEH. We also examined the effect of TSST-1 and HSV-1 on human epithelial cells and keratinocytes. TSST-1 used CD40 as its receptor on epithelial cells, and HSV-1 either directly or indirectly interacted with CD40. The consequence of these interactions was chemokine production, which is capable of causing harmful inflammation, with epidermal/keratinocyte barrier disruption. Human epithelial cells treated first with TSST-1 and then HSV-1 resulted in enhanced chemokine production. Finally, we showed that TSST-1 modestly increased HSV-1 replication but did not increase viral plaque size. Our data suggest that ADEH is associated with production of the major TSS-associated superantigens, together with HSV reactivation. The superantigens plus HSV may damage the skin barrier by causing harmful inflammation, thereby leading to increased symptoms.IMPORTANCE Atopic dermatitis (eczema, AD) with concurrent herpes simplex virus infection (eczema herpeticum, ADEH) is a severe form of AD. We show that ADEH patients are colonized with Staphylococcus aureus that primarily produces the superantigen toxic shock syndrome toxin-1 (TSST-1); however, significantly but to a lesser extent the superantigens staphylococcal enterotoxins A, B, and C are also represented in ADEH. Our studies showed that TSST-1 uses the immune costimulatory molecule CD40 as its epithelial cell receptor. Herpes simplex virus (HSV) also interacted directly or indirectly with CD40 on epithelial cells. Treatment of epithelial cells with TSST-1 and then HSV-1 resulted in enhanced chemokine production. We propose that this combination of exposures (TSST-1 and then HSV) leads to opening of epithelial and skin barriers to facilitate potentially serious ADEH.

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 and Their Association among Bacteremic and Infective Endocarditis Patients in Egypt.

Purpose Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus (S.) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE. Methods This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea, seb, sec, sed, see, and tsst-1, the major SAg coding genes, and the obtained findings were statistically analyzed. Results Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference (P value = 0.139, effect size = 0.572) indicating no specific association between any of the detected SAgs and IE. Conclusion There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs.

Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food.

Although coagulase-positive staphylococci are considered to be the main factor responsible for food poisoning, an increasing role for the coagulase-negative staphylococci in the production of enterotoxins has been observed in recent years. This study was conducted to assess the occurrence of genes responsible for the production of staphylococcal enterotoxins (SE), enterotoxin-like toxins (SEI) and toxic shock syndrome toxin-1 (TSST-1) in coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food from bars and restaurants. One hundred and eighteen CoNS strains were tested using polymerase chain reaction (PCR) to five superantigenic toxin genes, including five different types of classical enterotoxins (sea, seb, sec, sed and see) and the toxic shock syndrome toxin-1 (tsst-1) as well as to supertoxin-like genes. PCR-positive isolates were then tested using immunoenzymatic methods (SET-RPLA, Vidas SET 2) for toxin expression. Out of 118 CoNS strains, the presence of staphylococcal enterotoxins was confirmed in 72% of them. The most frequently found enterotoxin-like genotype was ser, selu. Two of the tested strains had up to ten different enterotoxin genes in the genome at the same time. Although no production of enterotoxins was detected in the CoNS, which means that their possible role in the epidemiology of food-borne diseases is minimal, the data demonstrated that the toxigenic capacity of the CoNS should not be ignored, and that this group of microorganisms should be continuously monitored in food.

Searching the Staphylococcal Toxic Shock Syndrome Toxin -1 in Septic Children with negative Cultures: A Comparative Study in Tehran, Iran

Bacteria induced sepsis is common in infants and children. Staphylococcus aureus produces numerous exotoxins, like staphylococcal Toxic shock syndrome toxin (TSST- 1), which stimulate the immune system by T cell activation and inflammation in various organs. Recent studies suggest that staphylococcal toxins, generally named super antigens (SAgs), may also have a significant role in the pathogenesis of some pediatric disorders especially in the clinical presentation of sepsis and septic shock. This study was carried out in order to compare staphylococcal TSST- 1 (SAgs) in children with sepsis symptoms (and septic shock) with negative blood culture versus a control group. This cross-sectional study was conducted during 2 years (2014 -2016) in two referral hospitals (Rasoul Akram and Bahrami hospitals) in Tehran, Iran. We selected 44 children) mean age of 4 years) who were admitted in pediatrics and PICUs wards with sepsis symptoms- /+septic shock. Forty-five children (mean age of 3.9 years) were selected as a control group. All cases with blood samples were examined for TSST-1 (SAgs) by polymerase chain reaction (PCR) method in both case and control groups and results were compared. Data were analyzed by SPSS-16software. Chi-square or Fisher test was used to compare the variables. P-value < 0.05 was considered as a valuable tool. Positive blood cultures with other bacteria, Streptococcus pneumonia, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, were detected in 5 cases with negative TSST-1 in blood samples. S.aureus isolated from blood culture was detected in 2 cases with positive TSST- 1.Positive TSST-1 (SAgs) was detected in 6 cases (14%) with negative blood culture for S.aureus; it was significantly higher in cases (14% vs. 2%; P value = 0.05). This study indicates the probable role of TSST-1(SAgs) in the progression of sepsis (and septic shock) in toxic children with negative blood culture for S.aureus. Anti-staphylococcal treatment is immediately required, especially in toxic children with related clinical presentations, even in cases with negative blood cultures. Indeed, the clinical use against SAgs suppressants of downstream cell-destructive events might be helpful.

Phenotypic and Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureus Isolated from Dairy and Meat Products in Iran

Background: Methicillin-Resistant Staphylococcus aureus (MRSA) plays an important role in gastrointestinal diseases. The goal of this research was to determine phenotypic and genotypic characteristics of MRSA isolated from dairy and meat products in Iran. Methods: Ninety-three S. aureus isolates were prepared which had been obtained in our previous study. Antimicrobial susceptibility testing was done using disk diffusion method. The isolates were further analyzed by mecA gene detection. Staphylococcal Enterotoxins (SEs) and Toxic Shock Syndrome Toxin 1 (TSST1) were screened. Biotyping and molecular typing were done by short sequence repeats of spa and coa genes. Results: Five out of 93 S. aureus isolates (5.37%) included mecA. All five MRSA isolates were sensitive to at least six tested antibiotics and none were resistant to vancomycin. Furthermore, two isolates were multidrug resistant. Four isolates produced SEs and TSST1. Three out of 5 isolates were related to human biotype and two belonged to non-host-specific biotype. Conclusion: Presence of MRSA in dairy and meat products may be an important hygienic risk for the Iranian consumers, especially for immunocompromised people.

Profiles of Enterotoxin Genes and Antimicrobial Resistance in &lt;i&gt;Staphylococcus pseudintermedius&lt;/i&gt; Strains Isolated from Livestock and Companion Animals

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and is recognized as a zoonotic pathogen causing public health concern. Although canine-associated S. pseudintermedius has mainly been recognized for its antimicrobial resistance and ability to cause skin infections in dogs, information on antimicrobial resistance profiles and enterotoxigenicity of S. pseudintermedius in livestock is very limited. In this study, we investigated the prevalence of 18 different staphylococcal enterotoxin (SE) genes and toxic shock syndrome toxin gene (tst-1) in S. pseudintermedius strains isolated from dogs, pigs, and beef cattle. Moreover, antimicrobial resistance profiles of the strains were determined along with the presence of mecA and SCCmec types. Except for one bovine isolate, all S. pseudintermedius isolates from dogs and pigs were resistant to multiple drugs (≥ 4 different drugs). Four out of six canine isolates were methicillin resistant and carried SCCmec type V. In addition, 11 different SE genes (seb, sec, see, seg, sei, sej, sel, seo, sep, seq, and seu) and tst-1 were identified in S. pseudintermedius isolates from dogs, pigs, and beef cattle. Most S. pseudintermedius isolates (83%) harbored multiple SE genes, and sel (42%) and sep (42%) were most frequently detected in the isolates. Our results suggested that S. pseudintermedius isolates from livestock and companion animals may serve as a reservoir for SE genes and antimicrobial resistance.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates from rural community settings in Trinidad and Tobago.

To determine the virulence and antimicrobial resistant genes in methicillin resistant Staphylococcus aureus isolates recovered from patients attending two rural health centers in Trinidad and Tobago. Cross-sectional observational analysis of patients from two local health centers located in communities in northern region of the country. Nasal and wound swabs from 300 patients were analyzed using standard and molecular techniques. Multiplex polymerase chain reaction was used to detect 16S rRNA, mec A, Staphylococcal chromosomal cassette SCC mec types, pvl, alpha hemolysin (hla), and Toxic Shock Syndrome Toxin 1 (tst 1) genes. S. aureus ATCC 33591 and Staphylococcus epidermidis ATCC 12228 were used for quality control, respectively. Over a quarter (26.7%, 80/300) of the surveyed patient's samples grew bacterial isolates of which 45% (36/80) were S. aureus and 44.4% (16/36) were mecA-positive. Majority (62.5%, 10/16) possessed the pvl gene, whereas 25% (4/16) possessed the alpha hemolysin (hla) gene. None of the methicillin-resistant Staphylococcus aureus (MRSA) isolates possessed the tst 1 gene. Also, 18.8% (3/16) isolates possessed both virulence genes, pvl and hla. Although the SCCmec types IV and V were detected, but none of the SCCmec I, II, and III were harbored by the isolates. SCCmec type IV and the pvl genes were common among the MRSA isolates from the community. The hla gene was found infrequently, but none of the isolates possessed the tst 1 gene. Knowledge of this is important for robust surveillance of such cases from the community in the country.

HLA-DR polymorphisms influence in vivo responses to staphylococcal toxic shock syndrome toxin-1 in a transgenic mouse model.

Toxic shock syndrome toxin-1 (TSST-1) is a potent superantigen produced by Staphylococcus aureus. In addition to menstrual and nonmenstrual toxic shock syndromes, TSST-1 is also implicated in the immunopathogenesis of pneumonia, infective endocarditis, neonatal exanthematous disease, and atopic dermatitis among others. Superantigens first bind to major histocompatibility complex (MHC) class II molecules and then activate a large proportion of T cells by cross-linking their T cell receptor. As binding to MHC class II molecules is a critical step in the robust activation of the immune system by TSST-1 and other superantigens, polymorphic variations between different HLA-DR alleles could potentially influence the magnitude of immune activation and immunopathology caused by TSST-1. As TSST-1 is highly toxic to humans and given that multiple variations of alleles of HLA-DR and HLA-DQ are expressed in each individual, it is difficult to determine how HLA-DR polymorphisms quantitatively and qualitatively impact immune activation caused by TSST-1 in humans. However, such investigations can be conducted on transgenic mice lacking all endogenous MHC class II molecules and expressing specific HLA class II alleles. Therefore, transgenic mice expressing different HLA-DRB1 alleles (HLA-DRB1*15:01, HLA-DRB1*15:02, HLA-DRB1*03:01, HLA-DRB1*04:01), and sharing HLA-A1*01:01 chain, were systemically challenged with purified TSST-1 and multiple immune parameters were assessed. Among the HLA-DR alleles, mice expressing HLA-DRB1*15:01 allele elicited a significantly higher serum cytokine/chemokine response; greater splenic T cell expansion and most severe organ pathology. Our study highlights the potential utility of human leukocyte antigen (HLA) transgenic mice in understanding the impact of HLA polymorphisms on the outcomes of diseases caused by TSST-1 and other superantigens.

Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates in Fishery Products in Iran.

Staphylococcus aureus is one of the most common causes of seafood-borne diseases worldwide, which are attributable to the contamination of food by preformed enterotoxins. In this study, a total of 206 (34.3%) Staphylococcus aureus strains were obtained from 600 fish and shrimp samples and were tested for their antimicrobial susceptibility. We assessed the prevalence of the genes responsible for the staphylococcal enterotoxins (SEA, SEB) and toxic shock syndrome toxin 1 (TSST-1) genes. The results indicated that 34% of aqua food samples were contaminated with S. aureus, and 23.8% of these isolates were mec-A-positive. Sixty-four percent of the strains isolated from contaminated seafood was enterotoxigenic S. aureus, and 28.2% of SEs were MRSA-positive. The most prevalent genotype was characterized by the presence of the sea gene (45.2%), followed by the seb gene (18.5%), and the tst gene encoding TSST-1 was found in eight strains (3.9%). Of the 206 S. aureus isolates, 189 strains (84.9%) were resistant to at least one antibiotic. Given the frequent outbreaks of enterotoxigenic MRSA, it is necessary to make revisions to mandatory programmes to facilitate improved hygiene practices during fishing, aquaculture, processing, and sales to prevent the contamination of fishery products in Iran.

Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype.

Lactobacilli require physical contact to reduce staphylococcal TSST-1 secretion and vaginal epithelial inflammatory response.

ITALIC! Staphylococcus aureusbiofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species ITALIC! Lactobacilluscan alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is hypothesized that these effects of lactobacilli require direct physical contact between lactobacilli, staphylococci and the epithelium. Indeed, lactobacilli only reduced ITALIC! S. aureus-induced inflammatory cytokine expression when allowed physical contact with vaginal epithelial cells. Furthermore, a reduction in toxic shock syndrome toxin-1 secretion only occurred when a probiotic ITALIC! Lactobacillusstrain was allowed contact, but not when being physically separated from ITALIC! S. aureus Bacterial-probe atomic force microscopy demonstrated that lactobacilli and staphylococci strongly adhere to epithelial cells, while lactobacilli adhere stronger to staphylococci than staphylococci to each other, giving lactobacilli opportunity to penetrate and reside in staphylococcal biofilms, as visualized using confocal laser scanning microscopy with fluorescence ITALIC! in situhybridization probes. These results identify that physical contact and biochemical signaling by lactobacilli are intrinsically linked mechanisms that reduce virulence of ITALIC! S. aureusbiofilm.

Do Staphylococcus aureus superantigens play a role in the pathogenesis of otitis media with effusion in children?

ObjectivesStaphylococcal enterotoxins (SEs), acting as superantigens, have been reported to be involved in the pathogenesis of chronic inflammatory diseases of the upper and lower airway. There has been no previous study investigating the role of SEs in otitis media with effusion (OME). Therefore, this study was designed to analyze middle ear aspirates from children with and without OME for the presence of SEs. MethodsMiddle ear aspirates were obtained from 24 patients and 24 controls. All samples were processed for bacterial culture and detection of five staphylococcal SEs (SEA, SEB, SEC and SED) and toxic shock syndrome toxin-1 using the Rapid Latex Agglutination Test. ResultsIn bacterial culture assays, six samples (25%) of the study group and five samples (20.8%) of the control group showed bacterial growth. At least one SE was demonstrated in 6 of 24 patients and in 3 of 24 controls. There was no statistically significant difference between the two groups with respect to the presence of SEs. ConclusionAlthough there is evidence that SEs have a potential role in the pathogenesis of chronic inflammatory diseases, there is no evidence that the inflammation process is initiated by SEs in patients with OME.

English

The aim of this study was to determine the prevalence of nasal carriage of Staphylococcus aureus among patients and personnel of private centers at Casablanca, and to determine the resistance pattern of isolates. The carriage of virulence toxin genes by the methicillin resistant strains was also investigated. This study was conducted from November 2008 to February 2009. Nasal swabs were taken from 145 and 42 patients and personnel respectively. The susceptibility testing to 16 antibiotics was performed using the agar disc diffusion method. Minimum inhibitory concentrations (MICs) of oxacillin were determined by the agar dilution method for all strains demonstrating resistance to cefoxitin. In addition, resistant isolates were examined for the existence of the mecA gene by polymerase chain reaction (PCR). Furthermore, the carriage of 22 virulence toxin genes among strains showing resistance to cefoxitin was investigated by PCR Multiplex. The prevalence of nasal carriage of S. aureus was 32.4% (n=47) and 38.1% (n=16) in patients and personnel, respectively. Patients’ strains showed 16 resistance patterns against only 4 in personnel strains. No S. aureus isolates were found to be resistant to lincomycin, pristinamycin, gentamicin, chloramphenicol, trimethoprim-sulfamethoxazole, rifampicin and vancomycin, while over 90% (n = 59) were resistant to penicillin G. For the other antibiotics, the percentage of resistance varied between 2.63 and 18.75%. One S. aureus (1.6%) was methicillin resistant by possession of mecA gene. This isolate harboured the staphylococcal enterotoxin genes sec, sed, sell, selm, selo, ser and toxic shock syndrome toxin gene (tst). Investigation of S. aureus nasal carriage and characterization of isolates among patients undergoing hemodialysis is important to develop infection prevention and to limit the spread of methicillin resistant S. aureus (MRSA) strains. Key words: Hemodialysis centers, patients, personnel, Staphylococcus aureus, nasal carriage, antibiotic susceptibility, methicillin resistance, toxin genes.

A diversity profile from the staphylococcal community on atopic dermatitis skin: a molecular approach

The aim of this study was to determine the biodiversity of the skin staphylococcal community from patients with atopic dermatitis (AD) and superantigen (SAg) detection from Staphylococcus aureus isolates. In this study, we developed a novel multiplex PCR that allows the identification and discrimination of bacteria belonging to the Staphylococcus genus both Staph.aureus and coagulase-negative Staphylococcus - Staph.capitis, Staph.epidermidis, Staph.haemolyticus and Staph.hominis isolated from the skin of patients with AD. In addition, a multiplex PCR assay that allows the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs), SE-like toxins and toxic shock syndrome toxin-1 was also performed and applied in Staph.aureus isolates. The microflora of the skin of patients with AD was dominated by Staph.aureus (69 isolates, 35·6%) followed by Staph.epidermidis (59 isolates, 30·4%) species. The SElM and SElN genes were the most frequently detected in our study (15 isolates, 71·4%), followed by SEG and SElO (14 isolates, 66·7%). Our molecular-based approach successfully identified the staphylococcal microflora that was relatively specific to patients with AD. Considering skin colonization and expression of virulence factors, the Staph.aureus may play a relevant role in AD pathophysiology. This ability to classify disease-related microbial species provides new insights into the relevance of those microbes in human disorders.

Identification of Toxic Shock Syndrome Toxin-1 (TSST-1) gene in Staphylococcus aureus isolated from bovine mastitis milk

Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis of dairy domestic ruminants. This agent produces a variety of extracellular toxins and virulence factors including Toxic Shock Syndrome Toxin-1 (TSST-1) which is the major cause of Toxic Shock Syndrome (TSS). In this study 58 S. aureus isolates obtained from 9 dairy herds in East and West Azerbaijan provinces of Iran. The tested isolates were identified on the basis of the cultural and biochemical properties as well as by amplification of the aroA gene, specific to S. aureus. Isolates were also analyzed for the presence of the TSST-1 encoding gene (tst) using Polymerase Chain Reaction (PCR). tst gene 350 bp was detected in 9 (15.5%) of the total number of 58 isolates. The present study revealed that the PCR amplification of the aroA gene could be used as a powerful tool for identification of S. aureus from the cases of bovine mastitis. Results also showed that the strain of S. aureus which caused mastitis can potentially produce staphylococcal toxic shock syndrome toxin-1. Overall, our results suggest that it is of special importance to follow the presence of TSST-1 producing S. aureus in foodstuffs, especially for protecting the consumers from toxic shock syndrome.

Vaccination with Plasmid DNA Encoding a Mutant Toxic Shock Syndrome Toxin-1 Ameliorates Toxin-induced Lethal Shock in Mice

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

Epithelial Proinflammatory Response and Curcumin-Mediated Protection from Staphylococcal Toxic Shock Syndrome Toxin-1

Staphylococcus aureus initiates infections and produces virulence factors, including superantigens (SAgs), at mucosal surfaces. The SAg, Toxic Shock Syndrome Toxin-1 (TSST-1) induces cytokine secretion from epithelial cells, antigen presenting cells (APCs) and T lymphocytes, and causes toxic shock syndrome (TSS). This study investigated the mechanism of TSST-1-induced secretion of proinflammatory cytokines from human vaginal epithelial cells (HVECs) and determined if curcumin, an anti-inflammatory agent, could reduce TSST-1-mediated pathology in a rabbit vaginal model of TSS. TSST-1 caused a significant increase in NF-κB-dependent transcription in HVECs that was associated with increased expression of TNF- α, MIP-3α, IL-6 and IL-8. Curcumin, an antagonist of NF-κB-dependent transcription, inhibited IL-8 production from ex vivo porcine vaginal explants at nontoxic doses. In a rabbit model of TSS, co-administration of curcumin with TSST-1 intravaginally reduced lethality by 60% relative to 100% lethality in rabbits receiving TSST-1 alone. In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin treated rabbits that survived. These data suggest that the inflammatory response induced at the mucosal surface by TSST-1 is NF-κB dependent. In addition, the ability of curcumin to prevent TSS in vivo by co-administration with TSST-1 intravaginally suggests that the vaginal mucosal proinflammatory response to TSST-1 is important in the progression of mTSS.

Lactobacillus reuteri -produced cyclic dipeptides quench agr -mediated expression of toxic shock syndrome toxin-1 in staphylococci

The production of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus has been associated with essentially all cases of menstruation-associated toxic shock syndrome (TSS). In this work, we show that the human vaginal isolate Lactobacillus reuteri RC-14 produces small signaling molecules that are able to interfere with the staphylococcal quorum-sensing system agr, a key regulator of virulence genes, and repress the expression of TSST-1 in S. aureus MN8, a prototype of menstrual TSS S. aureus strains. Quantitative real-time PCR data showed that transcription from the Ptst promoter, as well as the P2 and P3 promoters of the agr system from all four agr subgroups of S. aureus, was strongly inhibited in response to growth with L. reuteri RC-14 cultural supernatant. Alterations in the transcriptional levels of two other virulence-associated regulators sarA and saeRS were also observed, indicating a potential overall influence of L. reuteri RC-14 signals on the production of virulence factors in S. aureus. S. aureus promoter-lux reporter strains were used to screen biochemically fractionated L. reuteri RC-14 supernatant, and the cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Tyr-L-Pro) were identified as the signaling molecules. The results from this work contribute to a better understanding of interspecies cell-to-cell communication between Lactobacillus and Staphylococcus, and provide a unique mechanism by which endogenous or probiotic strains may attenuate virulence factor production by bacterial pathogens.