Production Of Toxic Shock Syndrome Toxin Research Articles

Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.

Vaginal community state types (CSTs) alter environmental cues and production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1).

Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with the use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) superantigen is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota [herein referred to as community state types (CSTs)] and S. aureus MN8 depend on environmental cues and that these interactions subsequently affect TSST-1 production. Using S. aureus MN8 ΔccpA growing in various glucose concentrations, we demonstrate that the supernatants from different CSTs grown in vaginally defined medium (VDM) could significantly decrease tst expression. When co-culturing CST species with MN8 ∆ccpA, we show that Lactobacillus jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher interleukin 2 (IL-2) production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract and further indicate that the CSTs are likely important for the protection from mTSS.IMPORTANCEIn this study, we investigate the impact of the vaginal microbiota against Staphylococcus aureus in conditions mimicking the vaginal environment at various stages of the menstrual cycle. We demonstrate that Lactobacillus jensenii can inhibit toxic shock syndrome toxin-1 (TSST-1) production, suggesting the potential for probiotic activity in treating and preventing menstrual toxic shock syndrome (mTSS). On the other side of the spectrum, "unhealthy" or "transient" bacteria such as Gardnerella vaginalis and Lactobacillus iners support more TSST-1 production by S. aureus, suggesting that community state types are important in the development of mTSS. This study sets forward a model for examining contact-independent interactions between pathogenic bacteria and the vaginal microbiota. It also demonstrates the necessity of replicating the environment when studying one as dynamic as the vagina.

Inhibition of Toxic Shock Syndrome-Associated Staphylococcus aureus by Probiotic Lactobacilli.

Staphylococcus aureus is a human pathogen with many infections originating on mucosal surfaces. One common group of S. aureus is the USA200 (CC30) clonal group, which produces toxic shock syndrome toxin-1 (TSST-1). Many USA200 infections occur on mucosal surfaces, particularly in the vagina and gastrointestinal tract. This allows these organisms to cause cases of menstrual TSS and enterocolitis. The current study examined the ability of two lactobacilli, Lactobacillus acidophilus strain LA-14 and Lacticaseibacillus rhamnosus strain HN001, for their ability to inhibit the growth of TSST-1 positive S. aureus, the production of TSST-1, and the ability of TSST-1 to induce pro-inflammatory chemokines from human vaginal epithelial cells (HVECs). In competition growth experiments, L. rhamnosus did not affect the growth of TSS S. aureus but did inhibit the production of TSST-1; this effect was partially due to acidification of the growth medium. L. acidophilus was both bactericidal and prevented the production of TSST-1 by S. aureus. This effect appeared to be partially due to acidification of the growth medium, production of H2O2, and production of other antibacterial molecules. When both organisms were incubated with S. aureus, the effect of L. acidophilus LA-14 dominated. In in vitro experiments with HVECs, neither lactobacillus induced significant production of the chemokine interleukin-8, whereas TSST-1 did induce production of the chemokine. When the lactobacilli were incubated with HVECs in the presence of TSST-1, the lactobacilli reduced chemokine production. These data suggest that these two bacteria in probiotics could reduce the incidence of menstrual and enterocolitis-associated TSS. IMPORTANCE Toxic shock syndrome (TSS) Staphylococcus aureus commonly colonize mucosal surfaces, giving them the ability to cause TSS through the action of TSS toxin-1 (TSST-1). This study examined the ability of two probiotic lactobacilli to inhibit S. aureus growth and TSST-1 production, and the reduction of pro-inflammatory chemokine production by TSST-1. Lacticaseibacillus rhamnosus strain HN001 inhibited TSST-1 production due to acid production but did not affect S. aureus growth. Lactobacillus acidophilus strain LA-14 was bactericidal against S. aureus, partially due to acid and H2O2 production, and consequently also inhibited TSST-1 production. Neither lactobacillus induced the production of pro-inflammatory chemokines by human vaginal epithelial cells, and both inhibited chemokine production by TSST-1. These data suggest that the two probiotics could reduce the incidence of mucosa-associated TSS, including menstrual TSS and cases originating as enterocolitis.

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

018 TSST-1+ Staphylococcus aureus in bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease that is treated with high dose immunosuppression due to lack of specific targets. Staphylococcus aureus is a commensal bacterium implicated inflammatory and autoimmune disorders because of its secretion of toxins with superantigen effects. We prospectively evaluated S. aureus colonization and its production of toxic shock syndrome toxin-1 (TSST-1) in 28 new onset BP patients. Inclusion criteria were active blistering and linear basement membrane IgG/C3 or a serum ELISA >14 for BP180 IgG. Bacterial swabs were obtained from the lesion interior, nares and unaffected skin of BP patients and nares and anatomically matched skin of 28 age- and sex-matched controls. Staphylococcal growth was assessed on blood agar, and TSST-1 production by cultured S. aureus isolates and in blister fluid was evaluated by immunoblot. S. aureus colonization of BP lesions was 3-6-fold higher than the nares or unaffected skin from the same patients (p≤0.300) and 6-fold higher than control nares or skin (p≤0.0015). Evaluation of superantigen gene profiles using PCR indicated that 96% of BP patients are colonized with a clonal strain of TSST-1+ S. aureus. Colonization and circulating levels of TSST-1 neutralizing antibodies, measured by ELISA, did not correlate. Interestingly, S. aureus colonization was not observed in patients who had received prior antibiotics. In colonized patients with severe disease, addition of anti-staphylococcal antibiotics resulted in clinical improvement and eliminated lesional colonization. This study shows that BP lesions harbor a clonal strain of TSST-1 producing S. aureus that is not evident in the general elderly population. Thus, immunosuppressive therapies should be balanced with the knowledge that S. aureus is likely present in BP lesions and the knowledge that antibiotics may play an important therapeutic role through bacterial clearance.

Vaginal Tampon Colonization by Staphylococcus aureus in Healthy Women.

Tampons recovered from a cohort of 737 healthy women (median age, 32 years) were analyzed for the presence of Staphylococcus aureus A total of 198 tampons (27%) were colonized by S. aureus, 28 (4%) by a strain producing toxic shock syndrome toxin 1 (TSST-1). S. aureus was detected more frequently in tampons that did not require an applicator for their insertion (74/233 [32%] versus 90/381 [24%]; odds ratio [OR] = 1.51 [95% confidence interval, 1.04 to 2.17]) and in women who used an intrauterine device for contraception (53/155 [34%] versus 145/572 [27%]; OR = 1.53 [95% confidence interval, 1.05 to 2.24]). The S. aureus strains isolated from tampons belonged to 22 different clonal complexes (CCs). The most prevalent CC was CC398 agr1 (n = 57 [27%]), a clone that does not produce superantigenic toxins, followed by CC30 agr3 (n = 27, 13%), producing TSST-1 (24/27 [89%]), the principal clone of S. aureus involved in menstrual toxic shock syndrome (MTSS).IMPORTANCE Menstrual toxic shock syndrome (MTSS) is an uncommon severe acute disease that occurs in healthy menstruating women colonized by TSST-1-producing S. aureus who use intravaginal protection, such as tampons and menstrual cups. The catamenial product collected by the protection serves as a growth medium for S. aureus and allows TSST-1 production. Previous studies evaluated the prevalence of genital colonization by S. aureus by vaginal swabbing, but they did not examine tampon colonization. This study demonstrated a high prevalence of tampon colonization by S. aureus and the presence of the CC30 TSST-1 S. aureus clone responsible for MTSS in tampons from healthy women. The results support the vaginal carriage of this lineage in healthy women. In addition, the higher prevalence of S. aureus within tampons that do not require an applicator indicates a crucial role for handwashing before tampon handling to decrease the risk of tampon contamination.

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Impact of Currently Marketed Tampons and Menstrual Cups on Staphylococcus aureus Growth and Toxic Shock Syndrome Toxin 1 Production In Vitro.

Fifteen currently marketed intravaginal protection products (11 types of tampon and 4 types of menstrual cup) were tested by the modified tampon sac method to determine their effect on Staphylococcus aureus growth and toxic shock syndrome toxin 1 (TSST-1) production. Most tampons reduced S. aureus growth and TSST-1 production, with differences based on brand and composition, and the level of S. aureus growth was higher in destructured than in unaltered tampons. We observed higher levels of S. aureus growth and toxin production in menstrual cups than in tampons, potentially due to the additional air introduced into the bag by cups, with differences based on cup composition and size.IMPORTANCE Menstrual toxic shock syndrome is a rare but severe disease. It occurs in healthy women vaginally colonized by Staphylococcus aureus producing toxic shock syndrome toxin 1 using intravaginal protection, such as tampons or menstrual cups. Intravaginal protection induces TSS by the collection of catamenial products, which act as a growth medium for S. aureus Previous studies evaluated the impact of tampon composition on S. aureus producing toxic shock syndrome toxin 1, but they are not recent and did not include menstrual cups. This study demonstrates that highly reproducible results for S. aureus growth and TSST-1 production can be obtained by using a simple protocol that reproduces the physiological conditions of tampon and cup usage as closely as possible, providing recommendations for tampon or cup use to both manufacturers and consumers. Notably, our results do not show that menstrual cups are safer than tampons and suggest that they require similar precautions.

Effects of Iclaprim and Trimethoprim on Exotoxin Production by Methicillin-resistant Staphylococcus aureus

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes serious, often life-threatening, infections. Exotoxins such as alpha-hemolysin (AH), Panton Valentine leukocidin (PVL), and Toxic shock syndrome toxin 1 (TSST-1) mediate pathogenesis and inhibition of toxin production is an important consideration in choosing appropriate treatments. Vancomycin is recommended for severe MRSA infections; however, increasing vancomycin resistance, poor clinical outcomes and nephrotoxicity are serious concerns. Thus newer agents are needed, including those that block bacterial toxin production. In the current study, we compared the effects of sub-inhibitory doses (sub-MIC) of two folic acid inhibitor antibiotics (iclaprim, trimethoprim) with cell wall-active agents (nafcillin, vancomycin) on transcription and translation of AH, PVL and TSST-1 in two clinical MRSA isolates.MethodsCommunity-acquired MRSA strains 1560 (a USA400 strain; AH+, TSST-1+, PVL-) and 04014 (CDC strain 368–04; AH+, TSST-1-, PVL+) were studied. MICs were determined by standard microbroth dilution. Gene expression was studied by northern blotting and/or qRT-PCR; toxins were quantitated by ELISA (PVL and TSST-1) and rabbit erythrocyte lysate assay (AH).ResultsIn agreement with our previous findings, nafcillin increased production of AH, TSST-1, and PVL compared with untreated control cultures. In both MRSA strains, iclaprim and trimethoprim delayed the onset of mRNA production and shifted its peak production to later time points. Both iclaprim and trimethoprim suppressed AH production in both strains of MRSA and delayed, but did not reduce, maximal TSST-1 production in MRSA1560. Trimethoprim significantly increased maximal PVL production over both untreated and iclaprim-treated cultures.ConclusionThe folic acid antagonist antibiotics, iclaprim and trimethoprim, altered both mRNA synthesis dynamics and protein toxin production in MRSA at concentrations below those that inhibit bacterial growth. These results, plus the fact that iclaprim is 15-fold more active than trimethoprim (MICs = 0.13 and 2.0 ug/ml, respectively), provide additional rationale for the use of iclaprim to treat complicated MRSA infections.Disclosures A. Bryant, Motif Biosciences: Grant Investigator, Research grant; D. Huang, Motif Bio: Employee, Salary; D. Stevens, Motif Biosciences: Grant Investigator, Research grant

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.

Impact of the Regulators SigB, Rot, SarA and sarS on the Toxic Shock Tst Promoter and TSST-1 Expression in Staphylococcus aureus.

Staphylococcus aureus is an important pathogen manifesting virulence through diverse disease forms, ranging from acute skin infections to life-threatening bacteremia or systemic toxic shock syndromes. In the latter case, the prototypical superantigen is TSST-1 (Toxic Shock Syndrome Toxin 1), encoded by tst(H), and carried on a mobile genetic element that is not present in all S. aureus strains. Transcriptional regulation of tst is only partially understood. In this study, we dissected the role of sarA, sarS (sarH1), RNAIII, rot, and the alternative stress sigma factor sigB (σB). By examining tst promoter regulation predominantly in the context of its native sequence within the SaPI1 pathogenicity island of strain RN4282, we discovered that σB emerged as a particularly important tst regulator. We did not detect a consensus σB site within the tst promoter, and thus the effect of σB is likely indirect. We found that σB strongly repressed the expression of the toxin via at least two distinct regulatory pathways dependent upon sarA and agr. Furthermore rot, a member of SarA family, was shown to repress tst expression when overexpressed, although its deletion had no consistent measurable effect. We could not find any detectable effect of sarS, either by deletion or overexpression, suggesting that this regulator plays a minimal role in TSST-1 expression except when combined with disruption of sarA. Collectively, our results extend our understanding of complex multifactorial regulation of tst, revealing several layers of negative regulation. In addition to environmental stimuli thought to impact TSST-1 production, these findings support a model whereby sporadic mutation in a few key negative regulators can profoundly affect and enhance TSST-1 expression.

Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR

To minimize specimen volume, handling and testing time, we have developed two TaqMan® multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8–20 cfu per rtPCR for the clinical stool matrix.

Influence of Subinhibitory Concentrations of Honey on Toxic Shock Syndrome Toxin -1 (TSST-1) Production by Two Strains of Staphylococcus Aureus

Antibiotic resistant bacteria are a worldwide health concern and it is essential to develop new antimicrobial agents to kill these bacteria and to reduce the use of antibiotics. Staphyloccus aureus (S.aureus) an important medical pathogen is responsible for many wound infections and up to 25% of all strains produce the toxic shock syndrome toxin (TSST-1) which stimulates the release of inflammatory cytokines which cause fever and shock. Here we report on the inhibition of two penicillin resistant TSST-1 producing strains of S.aureus by seven different honeys. Bacterial growth was reduced after 24 hours at 37oC, from 10.0 log 10 in the TSB growth control to less than 1.0 log 10 in Highland, Chilean and Manuka honey. TSST-1 production was reduced from 256ng/ml in the TSB growth control to less than 30 ng/ml in sub inhibitory concentrations of all honeys.

Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production and Lactobacillus species growth in a defined medium simulating vaginal secretions

Lactobacillus species are commensal with the healthy vaginal environment and inhibit the growth of many pathogenic bacteria in the vaginal tract by a variety of mechanisms, such as the production of hydrogen peroxide, organic acids, and antimicrobial substances. Simulation of the vaginal environment is crucial for proper investigation of the effects of Lactobacillus species on pathogenic bacteria. In this study, we modified a medium used to simulate vaginal secretions to improve the growth of toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus clinical strains and Lactobacillus species so that interactions between these bacteria may be examined. A medium consisting of basal salts, vitamins, albumin, glycogen, mucin, urea, sodium bicarbonate, polyoxyethylene sorbitan monolaurate, and amino acids supported the growth of S. aureus and the production of TSST-1 as determined by Western analysis. Improved growth of the Lactobacillus species was seen when this same medium was supplemented with manganese chloride, sodium acetate, and an increase in glucose concentration. However, growth of S. aureus in the supplemented medium resulted in reduced levels of TSST-1. Production of TSST-1 was not detected in a medium routinely used for the growth of Lactobacillus species although S. aureus growth was not inhibited. The development of an improved genital tract secretion medium provides a more authentic environment in which to study the interactions of Lactobacillus species and vaginal pathogens, such as S. aureus.

TSST-1, enterotoxin and bacteriocin-like substance production by Staphylococcus aureus isolated from foods

The production of Toxic Shock Syndrome Toxin-1 (TSST-1), enterotoxins and bacteriocin-like substances was evaluated in 95 strains of Staphylococcus aureus recovered from raw bovine milk (n=31) and from food samples involved in staphylococcal food poisoning (n=64). Enterotoxigenicity tests with the membrane over agar associated to optimal sensibility plate assays were performed and showed that 96.77% of strains recovered from milk and 95.31% from food samples produced enterotoxins A, B, C, D or TSST-1. Reference strains S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis and Bacteroides fragilis were used as indicator bacteria in the antagonistic assays, the first five being sensitive to antagonistic substances. Brain heart infusion agar, in pH values ranging from 5.0 to 7.0 in aerobic atmosphere showed to be the optimum condition for antagonistic activity as evaluated with the best producer strains against the most sensitive indicator bacterium, L. monocytogenes. Sensitivity to enzymes confirmed the proteinaceous nature of these substances. Neither bacteriophage activity nor fatty acids were detected and the antagonistic activity was not due to residual chloroform. Results did not establish a positive correlation between the bacteriocinogenic profile and toxigenicity in the tested S. aureus strains.

Surface Coatings that Promote Rapid Release of Peptide‐Based AgrC Inhibitors for Attenuation of Quorum Sensing in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen responsible for a variety of life-threatening infections. The pathogenicity of this organism is attributed to its ability to produce a range of virulence factors and toxins, including the superantigen toxic shock syndrome toxin-1 (TSST-1). While many S. aureus infections can be treated using conventional antibiotics, strains resistant to these bactericidal agents have emerged. Approaches that suppress pathogenicity through mechanisms that are nonbactericidal (i.e., antivirulence approaches) could provide new options for treating infections, including those caused by resistant strains. Here, we report a nonbactericidal approach to suppressing pathogenicity based on the release of macrocyclic peptides (1 and 2) that inhibit the agr quorum sensing (QS) circuit in group-III S. aureus. It is demonstrated that these peptides can be immobilized on planar and complex objects (on glass slides, nonwoven meshes, or within absorbent tampons) using the rapidly dissolving polymer carboxymethylcellulose (CMC). Peptide-loaded CMC films released peptide rapidly (<5 min) and promoted strong (>95%) inhibition of the agr QS circuit without inducing cell death when incubated in the presence of a group-III S. aureus gfp-reporter strain. Peptide 1 is among the most potent inhibitors of QS in S. aureus reported to date, and the group-III QS circuit regulates production of TSST-1, the primary cause of toxic shock syndrome (TSS). These results thus suggest approaches to treat the outer covers of tampons, wound dressings, or other objects to suppress toxin production and reduce the severity of TSS in clinical and personal care contexts. Because peptide 1 also inhibits QS in S. aureus groups-I, -II, and -IV, this approach could also provide a pathway for attenuation of QS and associated virulence phenotypes in a broader range of contexts.

Expression of toxic shock syndrome toxin-1 by epidemic meticillin-resistant Staphylococcus aureus 16

BackgroundStaphylococcal toxic shock syndrome (TSS) is a fatal illness attributed to Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1), encoded by the tst gene. No epidemiological data on TSS exist from the UK. Strains of S aureus may carry the tst gene but not cause TSS, such as the UK strain epidemic meticillin-resistant SA-16 (EMRSA-16). The aim of this study was to determine the epidemiology of TSS in the UK and investigate the expression of TSST-1 by EMRSA-16 and tst+ meticillin-sensitive S aureus (MSSA). MethodsCases of TSS were identified at the UK Health Protection Agency (HPA) and epidemiological features analysed. A selection of clonal complex (CC)-30 EMRSA-16 and TSS-associated MSSA strains was chosen for investigation. The tst gene and promoter region were sequenced in all strains. The staphylococcal pathogenicity island (SaPI) harbouring tst was determined by PCR. Growth of strains in brain heart infusion broth was monitored by optical density (OD600). TSST-1 in supernatants was quantified by western blot using densitometry according to a TSST-1 standard curve. RNA was isolated for tst transcript analysis by quantitative reverse-transcription-PCR (qRT-PCR). Whole genome sequencing was performed on all strains to identify potential mutations in known regulators of toxin synthesis. Findings150 cases of TSS were reported to the HPA over a period of 4 years. Six cases (4%) were attributable to MRSA and the remainder to MSSA. There was no difference in the tst gene or promoter sequence among the strains and tst was carried by SaPI2. TSST-1 production by EMRSA-16 strains commenced earlier in growth than did MSSA strains and was greater in TSS-associated MSSA than in EMRSA-16 strains throughout growth. These results were compared with tst transcription by MSSA and EMRSA16 using qRT-PCR. InterpretationEMRSA-16 produces less TSST-1 than does MSSA of the same lineage; this may be due to differences in global gene regulators, perhaps as a result of changes in PBP2a expression, or the carriage of a large SCCmec element by EMRSA-16. This difference in production may explain the paucity of TSS cases caused by MRSA. FundingUK National Centre for Infection Prevention and Management.

Influence of the Vaginal Microbiota on Toxic Shock Syndrome Toxin 1 Production by Staphylococcus aureus

Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of premenopausal age worldwide and arises from vaginal infection by Staphylococcus aureus and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies have illustrated the capacity of lactobacilli to reduce S. aureus virulence, including the capacity to suppress TSST-1. We hypothesized that an aberrant microbiota characteristic of pathogenic bacteria would induce the increased production of TSST-1 and that this might represent a risk factor for the development of TSS. A S. aureus TSST-1 reporter strain was grown in the presence of vaginal swab contents collected from women with a clinically healthy vaginal status, women with an intermediate status, and those diagnosed with bacterial vaginosis (BV). Bacterial supernatant challenge assays were also performed to test the effects of aerobic vaginitis (AV)-associated pathogens toward TSST-1 production. While clinical samples from healthy and BV women suppressed toxin production, in vitro studies demonstrated that Streptococcus agalactiae and Enterococcus spp. significantly induced TSST-1 production, while some Lactobacillus spp. suppressed it. The findings suggest that women colonized by S. aureus and with AV, but not BV, may be more susceptible to menstrual TSS and would most benefit from prophylactic treatment.

Chocs toxiques dans les infections à cocci à Gram positif

Although Gram-positive cocci are implicated in half of the documented causes of sepsis, toxic shock syndrome (TSS) due to these organisms is rare. By definition, TSS, whether related to staphylococcus (staph-TSS) or streptococcus (strep-TSS), is characterized by the production of superantigen exotoxins, alone or in association with other virulence factors, inducing an excessive production of pro-inflammatory cytokines. Staph-TSS may be menstrual (colonisation of an absorbent tampon or intrauterine device [IUD]: production of TSS toxin-1 (TSST-1), negative blood culture, low mortality) or non-menstrual (usually postsurgical colonisation or local infection: production of TSST-1 or enterotoxin, positive blood culture in 50% of the cases, 20-25% mortality rate). Strep-TSS is usually associated with necrosing fasciitis (60% positive blood culture, mortality up to 80% in the case with associated myositis). Organ failure is managed like septic shock. Mechanical eradication of the toxin source is mandatory (removal of tampon or IUD in menstrual staph-TSS, surgical revision even without signs of local inflammation in post-surgical non-menstrual staph-TSS, and aggressive surgery in strep-TSS with necrosing fasciitis). Antibiotic treatment should be bactericidal (high-dose β-lactam). In addition, it is recommended to add an antimicrobial with antitoxinic activity such as clindamycin (a large majority of Staphylococcus aureus and Streptococcus pyogenes strains implicated in TSS in France are susceptible to clindamycin) or polyvalent immunoglobulins, especially in case of shock with refractory hypotension.

Phenotype characterization of Staphylococcus species strains isolated from buffalo (Bubalus bubalis) milk

The objective of the current study was to isolate and identify phenotypes of Staphylococcus spp. strains derived from buffalo (Bubalus bubalis) milk. A total of 548 milk samples from 137 buffalo were cultured in Columbia agar enriched with 5% defibrinated sheep blood. Determination of the capacity of Staphylococcus aureus to produce enterotoxins A-D and toxic shock syndrome toxin-1 (TSST-1) was achieved by reverse passive latex agglutination (RPLA). Antimicrobial sensitivity of S. aureus strains was evaluated using the disk diffusion technique, and β-lactamase detection was achieved using the chromogenic test with paper discs impregnated with nitrocefin. From all the mammary quarters examined, 36 (10.8%) were positive for Staphylococcus spp., 83.3% were coagulase-negative staphylococcus (CNS), 11.1% were coagulase-positive staphylococcus (CPS), and 5.6% were of CPS+CNS positive. All isolates of S. aureus produced at least 1 toxin and 5 out of 6 isolates (83.0%) produced β-lactamase. One hundred percent of S. aureus isolates were sensitive to methicillin and amoxicillin + clavulanic acid, and resistant to ampicillin, penicillin, and oxacillin. Analysis of the results obtained in the current study highlight the epidemiologic importance of buffalo milk regarding the production of enterotoxins and TSST-1 and the potential risk to public health.