Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

BACKGROUND:Glycerol monolaurate (GML), a 12 carbon fatty acid monoester, inhibits Staphylococcus aureus growth and exotoxin production, but is degraded by S. aureus lipase. Therefore, dodecylglycerol (DDG), a 12 carbon fatty acid monoether, was compared in vitro and in vivo to GML for its effects on S. aureus growth, exotoxin production, and stability. METHODOLOGY/PRINCIPAL FINDINGS:Antimicrobial effects of GML and DDG (0 to 500 microg/ml) on 54 clinical isolates of S. aureus, including pulsed-field gel electrophoresis (PFGE) types USA200, USA300, and USA400, were determined in vitro. A rabbit Wiffle ball infection model assessed GML and DDG (1 mg/ml instilled into the Wiffle ball every other day) effects on S. aureus (MN8) growth (inoculum 3x10(8) CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-alpha (TNF-alpha) concentrations and mortality over 7 days. DDG (50 and 100 microg/ml) inhibited S. aureus growth in vitro more effectively than GML (p<0.01) and was stable to lipase degradation. Unlike GML, DDG inhibition of TSST-1 was dependent on S. aureus growth. GML-treated (4 of 5; 80%) and DDG-treated rabbits (2 of 5; 40%) survived after 7 days. Control rabbits (5 of 5; 100%) succumbed by day 4. GML suppressed TNF-alpha at the infection site on day 7; however, DDG did not (<10 ng/ml versus 80 ng/ml, respectively). CONCLUSIONS/SIGNIFICANCE:These data suggest that DDG was stable to S. aureus lipase and inhibited S. aureus growth at lower concentrations than GML in vitro. However, in vivo GML was more effective than DDG by reducing mortality, and suppressing TNF-alpha, S. aureus growth and exotoxin production, which may reduce toxic shock syndrome. GML is proposed as a more effective anti-staphylococcal topical anti-infective candidate than DDG, despite its potential degradation by S. aureus lipase

Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

Background: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS + E. faecalis endocarditis. Methodology/Principal Findings: When challenged with AS + E. faecalis, 10 rabbits actively immunized against AS + E. faecalis developed more significant vegetations than 9 animals immunized against AS 2 E. faecalis, and 9/10 succumbed compared to 2/9 (p,0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS + E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9610 6 versus 2.0610 5, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fab

Staphylococcal Toxic Shock Syndrome 2000–2006: Epidemiology, Clinical Features, and Molecular Characteristics

Circulating strains of Staphylococcus aureus (SA) have changed in the last 30 years including the emergence of community-associated methicillin-resistant SA (MRSA). A report suggested staphylococcal toxic shock syndrome (TSS) was increasing over 2000-2003. The last population-based assessment of TSS was 1986.Population-based active surveillance for TSS meeting the CDC definition using ICD-9 codes was conducted in the Minneapolis-St. Paul area (population 2,642,056) from 2000-2006. Medical records of potential cases were reviewed for case criteria, antimicrobial susceptibility, risk factors, and outcome. Superantigen PCR testing and PFGE were performed on available isolates from probable and confirmed cases.Of 7,491 hospitalizations that received one of the ICD-9 study codes, 61 TSS cases (33 menstrual, 28 non-menstrual) were identified. The average annual incidence per 100,000 of all, menstrual, and non-menstrual TSS was 0.52 (95% CI, 0.32-0.77), 0.69 (0.39-1.16), and 0.32 (0.12-0.67), respectively. Women 13-24 years had the highest incidence at 1.41 (0.63-2.61). No increase in incidence was observed from 2000-2006. MRSA was isolated in 1 menstrual and 3 non-menstrual cases (7% of TSS cases); 1 isolate was USA400. The superantigen gene tst-1 was identified in 20 (80%) of isolates and was more common in menstrual compared to non-menstrual isolates (89% vs. 50%, p = 0.07). Superantigen genes sea, seb and sec were found more frequently among non-menstrual compared to menstrual isolates [100% vs 25% (p = 0.4), 60% vs 0% (p<0.01), and 25% vs 13% (p = 0.5), respectively].TSS incidence remained stable across our surveillance period of 2000-2006 and compared to past population-based estimates in the 1980s. MRSA accounted for a small percentage of TSS cases. tst-1 continues to be the superantigen associated with the majority of menstrual cases. The CDC case definition identifies the most severe cases and has been consistently used but likely results in a substantial underestimation of the total TSS disease burden

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

Microbicide excipients can greatly increase susceptibility to genital herpes transmission in the mouse

<p>Abstract</p> <p>Background</p> <p>Several active ingredients proposed as vaginal microbicides have been shown paradoxically to <it>increase </it>susceptibility to infection in mouse genital herpes (HSV-2) vaginal susceptibility models and in clinical trials. In addition, "inactive ingredients" (or excipients) used in topical products to formulate and deliver the active ingredient might also cause epithelial toxicities that increase viral susceptibility. However, excipients have not previously been tested in susceptibility models.</p> <p>Methods</p> <p>Excipients commonly used in topical products were formulated in a non-toxic vehicle (the "HEC universal placebo"), or other formulations as specified. Twelve hours after exposure to the excipient or a control treatment, mice were challenged with a vaginal dose of HSV-2, and three days later were assessed for infection by vaginal lavage culture to assess susceptibility.</p> <p>Results</p> <p>The following excipients markedly increased susceptibility to HSV-2 after a single exposure: 5% glycerol monolaurate (GML) formulated in K-Y^®Warming Jelly, 5% GML as a colloidal suspension in phosphate buffered saline, K-Y Warming Jelly alone, and both of its humectant/solvent ingredients (neat propylene glycol and neat PEG-8). For excipients formulated in the HEC vehicle, 30% glycerin significantly increased susceptibility, and a trend toward increased HSV-2 susceptibility was observed after 10% glycerin, and 0.1% disodium EDTA, but not after 0.0186% disodium EDTA. The following excipients did not increase susceptibility: 10% propylene glycol, 0.18%, methylparaben plus 0.02% propylparaben, and 1% benzyl alcohol.</p> <p>Conclusions</p> <p>As reported with other surfactants, the surfactant/emulsifier GML markedly increased susceptibility to HSV-2. Glycerin at 30% significantly increased susceptibility, and, undiluted propylene glycol and PEG-8 greatly increased susceptibility.</p

Glycerol monolaurate prevents mucosal SIV transmission

Although there has been great progress in treating human immunodeficiency virus 1 (HIV-1) infection1, preventing transmission has thus far proven an elusive goal. Indeed, recent trials of a candidate vaccine and microbicide have been disappointing, both for want of efficacy and concerns about increased rates of transmission2–4. Nonetheless, studies of vaginal transmission in the simian immunodeficiency virus (SIV)–rhesus macaque (Macacca mulatta) model point to opportunities at the earliest stages of infection in which a vaccine or microbicide might be protective, by limiting the expansion of infected founder populations at the portal of entry5,6. Here we show in this SIV–macaque model, that an outside-in endocervical mucosal signalling system, involving MIP-3α (also known as CCL20), plasmacytoid dendritic cells and CCR5+ cell-attracting chemokines produced by these cells, in combination with the innate immune and inflammatory responses to infection in both cervix and vagina, recruits CD4+ T cells to fuel this obligate expansion. We then show that glycerol monolaurate—a widely used antimicrobial compound7with inhibitory activity against the production of MIP-3α and other proinflammatory cytokines8—can inhibit mucosal signalling and the innate and inflammatory response to HIV-1 and SIV in vitro, and in vivo it can protect rhesus macaques from acute infection despite repeated intra-vaginal exposure to high doses of SIV. This new approach, plausibly linked to interfering with innate host responses that recruit the target cells necessary to establish systemic infection, opens a promising new avenue for the development of effective interventions to blockHIV-1 mucosal transmission

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone

Topical microbicides to prevent the transmission of HIV: formulation gaps and challenges

The efforts of the topical microbicide field to identify a safe and effective topical microbicide were realized in July of 2010 with the reporting of the results of the Centre for the AIDS Programme of Research in South Africa 004 trial. In this trial, a 1% tenofovir gel was found to reduce women’s risk for HIV acquisition by 39% compared to placebo. To understand the impact of this trial on future microbicide development, we must view it from the historical perspective of previous phases 2 and 3 clinical trials with detergents and sulfated polyanions. This knowledge and emerging information must then be parlayed into the next steps needed to create a safe, effective, and acceptable topical microbicide. This review will look at the lessons learned from preclinical and clinical development of topical microbicides, focusing on two significant future challenges: (1) topical microbicide formulation safety and (2) the critical role that adherence to product use has in determining safety and efficacy in clinical trials and ultimately commercial viability of the licensed product. In addition to framing these issues within our current understanding of formulation and prevention of HIV acquisition, recent advances in our understanding of the mechanism of HIV transmission and how it informs on future formulation strategies will be briefly discussed

Ecto-5′-Nucleotidase: A Candidate Virulence Factor in Streptococcus sanguinis Experimental Endocarditis

Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5′-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P = 0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log10CFU, P = 0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE

A murine model of ulcerative colitis: induced with sinusitis-derived superantigen and food allergen

BACKGROUND: The etiology of ulcerative colitis (UC) is to be understood. The basic pathological feature of UC is intestinal chronic inflammation. Superantigen, such as Staphylococcus enterotoxin B (SEB), is reported to compromise intestinal barrier function by increasing epithelial permeability and initiate inflammation in the intestinal mucosa. Inasmuch as anatomic position of the sinus, chronic sinusitis-derived SEB may follow the secretion and to be swallowed down to the gastrointestinal tract and induce lesions to the intestinal mucosa. METHODS: Sinus wash fluid (SWF, containing SEB) was collected from a group of patients with both chronic sinusitis (CS) and UC. A group of mice were sensitized to ovalbumin (OVA) in the presence of SWF. The sensitized mice were challenged with the specific antigen OVA. The inflammatory status of the colonic tissue was determined with histology, serology and electron microscopy. Using horseradish peroxidase (HRP) as a tracer, another group of mice was stimulated with SWF for 2 hours. The HRP activity was detected in the colonic tissue with enzymatic approaches and electron microscopy. RESULTS: Epithelial hyperpermeability in colonic epithelium was induced by stimulating with SWF. The HRP activity in the colonic mucosa was almost 11 times more in the SWF treated group (3.2 ± 0.6 μg/g tissue) than the control group (0.3 ± 0.1 μg/g tissue). Mice were sensitized using a mixture of SWF and OVA (serum OVA-specific IgE was detected with a highest titer as 1:64). Challenge with OVA induced extensive inflammation in the colonic mucosa by showing (1) marked degranulation in mast cells (MC, 46.3 ± 4.5%) and eosinophils (Eo, 55.7 ± 4.2%); (2) inflammatory cell infiltration (MC = 145.2 ± 11.4; Eo = 215.8 ± 12.5; mononuclear cell = 258.4 ± 15.3/mm(2 )tissue); (3) increased MPO activity (12.9 ± 3.2 U/g tissue) and inflammatory scores (1.8 ± 0.3); (4) mucosal surface ulcers; (5) edema in the lamina propria; (6) bacterial translocation and abscess formation in the subepithelial region. CONCLUSION: Introducing Sinusitis-derived SEB-containing SWF to the gastrointestinal tract compromised colonic mucosal barrier function increasing epithelial permeability to luminal macromolecular protein in mice. The SWF facilitated colonic mucosal sensitization to luminal antigen. Multiple challenging the sensitized colonic mucosa with specific antigen OVA induced inflammation, induced a condition similar to human ulcerative colitis