Staphylococcus aureus causes numerous diseases in humans ranging from the mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may also be asymptomatically carried in the anterior nares, vagina or on the skin, which serve as reservoirs for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and a major cause of TSS. Our prior studies indicated that α-toxin was a major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. It also facilitated the penetration of TSS Toxin-1 (TSST-1) across vaginal mucosa. However, the majority of menstrual TSS isolates produce low α-toxin due to a nonsense point mutation at codon 113, designated hly, suggesting mucosal adaptation. The aim of this study was to characterize the differences between TSS USA200 strains [high (hla+) and low (hly+) α-toxin producers] in their abilities to infect and disrupt vaginal mucosal tissue. A mucosal model was developed using ex vivo porcine vaginal mucosa, LIVE/DEAD® staining and confocal microscropy to characterize biofilm formation and tissue viability of TSS USA 200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hly), MNPE (hla+) and MNPE isogenic hla knockout (hlaKO). All TSS strains grew to similar bacterial densities (1-5 x 108 CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587, MN8 (hly+), or MNPE hlaKO, formed biofilms and were less cytotoxic. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. Our studies suggest α-toxin affects S. aureus phenotypic growth on vaginal mucosa, by promoting tissue disruption and biofilm formation; and α–toxin mutants (hly) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV).

We investigated the effects of glycerol monolaurate (GML) on Lactobacillus , Candida , and Gardnerella vaginalis human vaginal microflora. Our previous work demonstrated that 6 months of GML treatment vaginally does not alter lactobacillus counts in monkeys. Candida and G. vaginalis are commonly associated with vaginal infections in women, many becoming chronic or recurrent. In vitro growth inhibition studies determined the effects of GML (0 to 500 μg/ml) against multiple Candida species and G. vaginalis . A randomized, double-blind study investigated the effects of GML on vaginal microflora Lactobacillus , Candida , and G. vaginalis in colonized or infected women ( n = 36). Women self-administered intravaginal gels containing 0% ( n = 14), 0.5% ( n = 13), or 5% ( n = 9) GML every 12 h for 2 days. Vaginal swabs were collected before and immediately after the first gel administration and 12 h after the final gel administration. Swabs were tested for Lactobacillus , Candida , G. vaginalis , and GML. In vitro GML concentrations of 500 μg/ml were candicidal for all species tested, while a concentration of 10 μg/ml was bactericidal for G. vaginalis . Control and GML gels applied vaginally in women did not alter vaginal pH or Lactobacillus counts. Control gels reduced G. vaginalis counts but not Candida counts, whereas GML gels reduced both Candida and G. vaginalis . No adverse events were reported by participating women. GML is antimicrobial for Candida and G. vaginalis in vitro . Vaginal GML gels in women do not affect Lactobacillus negatively but significantly reduce Candida and G. vaginalis .

Background. Kawasaki disease (KD) is characterized by systemic vasculitis of unknown etiology. Our previous studies showed expression ofCD40ligand on CD4+ T cells correlated to the coronary artery lesion (CAL) and disease progress in KD. Other studies from Japan suggested the role ofCD40Lin the pathogenesis of CAL, and this might help explain the excessive number of males affected with KD but cannot be reproduced by Taiwanese population. This study was conducted to investigate theCD40polymorphism in KD and CAL formation.Methods. A total of 950 subjects (381 KD patients and 569 controls) were investigated to identify 2 tagging single-nucleotide polymorphisms (tSNPs) ofCD40(rs4810485 and rs1535045) by using the TaqMan allelic discrimination assay.Results. A significant association was noted with regards toCD40tSNPs (rs1535045) between controls and KD patients (P=0.0405, dominant model). In KD patients, polymorphisms ofCD40(rs4810485) showed significant association with CAL formation (P=0.0436, recessive model). Haplotype analysis did not yield more significant results between polymorphisms ofCD40and susceptibility/disease activity of KD.Conclusions. This study showed for the first time that polymorphisms ofCD40are associated with susceptibility to KD and CAL formation, in the Taiwanese population.

Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. In addition, 50% of IE survivors develop strokes and metastatic abscesses due to the release of emboli from infected valves. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause IE and sepsis, and they may be categorized by pulsed-field gel electrophoresis, for example clonal types USA200, 300, and 400. We hypothesize that secreted virulence factors contribute to their differential ability to cause IE and/or sepsis. Rabbits are an excellent model for studying IE, which over the course of 4 days are monitored for development of vegetations (the hallmark signs of IE), and sepsis, as S. aureus are administered intravenously. Rabbit cardiac physiology is similar to humans, and rabbits exhibit susceptibility to superantigens and cytolysins produced by these clonal types of S. aureus. We examined the differential ability of community-associated MRSA and MSSA strains (5 USA200 or related strain FRI1169, 3 USA300, and 2 USA400) to cause vegetations versus lethal sepsis in rabbits. USA200 and related strain FRI1169 exhibited intermediate LD50s in sepsis (5x106-5x108) colony-forming units (CFUs), and 4/5 caused significant IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s of 1 x 106-5 x 107 CFUs) but were minimally capable of causing IE. USA300 variant strain Newman was not highly lethal (LD50 of 2 x 109 CFUs) but was highly effective in causing IE. USA400 strains were both highly lethal (LD50s of 1 x 107-5 x 107 CFUs) and highly effective causes of IE. Additional studies showed that phenol soluble modulins produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus have differential abilities to cause IE and lethal sepsis and suggest that secreted virulence factors, including superantigens and cytolysins, account for these differences.