Ying Chi Lin

Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have emerged as serious health threats in the last 15 years. They are associated with large numbers of atopic dermatitis skin and soft tissue infections, but when they originate from skin and mucous membranes, have the capacity to produce sepsis and highly fatal pulmonary infections characterized as necrotizing pneumonia, purpura fulminans, and postviral toxic shock syndrome. This review is a discussion of the emergence of 3 major CA-MRSA organisms, designated CA-MRSA USA400, followed by USA300, and most recently USA200. CA-MRSA USA300 and USA400 isolates and their methicillin-sensitive counterparts (community-associated methicillin-sensitive S aureus) typically produce highly inflammatory cytolysins alpha-toxin, gamma-toxin, delta-toxin (as representative of the phenol soluble modulin family of cytolysins), and Panton Valentine leukocidin. USA300 isolates produce the superantigens enterotoxin-like Q and a highly pyrogenic deletion variant of toxic shock syndrome toxin 1 (TSST-1), whereas USA400 isolates produce the superantigens staphylococcal enterotoxin B or staphylococcal enterotoxin C. USA200 CA-MRSA isolates produce small amounts of cytolysins but produce high levels of TSST-1. In contrast, their methicillin-sensitive S aureus counterparts produce various cytolysins, apparently in part dependent on the niche occupied in the host and levels of TSST-1 expressed. Significant differences seen in production of secreted virulence factors by CA-MRSA versus hospital-associated methicillin-resistant S aureus and community-associated methicillin-sensitive S aureus strains appear to be a result of the need to specialize as the result of energy drains from both virulence factor production and methicillin resistance.

Immunity to Staphylococcus aureus secreted proteins protects rabbits from serious illnesses

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins β-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Alpha-Toxin Promotes Staphylococcus aureus Mucosal Biofilm Formation

Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla+) and low (hla−) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla−), MNPE (hla+), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1–5 × 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 (hla−), MN8 (hla−), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla−) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV).

Glycerol Monolaurate Inhibits Candida and Gardnerella vaginalis In Vitro and In Vivo but Not Lactobacillus

ABSTRACT 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.

Proinflammatory exoprotein characterization of toxic shock syndrome Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) clonal type USA200 is the most widely disseminated Staphylococcus aureus colonizer of the nose and is a major cause of toxic shock syndrome (TSS). Exoproteins derived from these organisms have been suggested to contribute to their colonization and causation of human diseases but have not been well-characterized. Two representative S. aureus USA200 isolates, MNPE (α-toxin positive) and CDC587 (α-toxin mutant), isolated from pulmonary post-influenza TSS and menstrual vaginal TSS, respectively, were evaluated. Biochemical, immunobiological, and cell-based assays, including mass spectrometry, were used to identify key exoproteins derived from the strains that are responsible for proinflammatory and cytotoxic activity on human vaginal epithelial cells. Exoproteins associated with virulence were produced by both strains, and cytolysins (α-toxin and γ-toxin), superantigens, and proteases were identified as the major exoproteins, which caused epithelial cell inflammation and cytotoxicity. Exoprotein fractions from MNPE were more proinflammatory and cytotoxic than those from CDC587 due to high concentrations of α-toxin. CDC587 produced a small amount of α-toxin, despite the presence of a stop codon (TAG) at codon 113. Additional exotoxin identification studies of USA200 strain [S. aureus MN8 (α-toxin mutant)] confirmed that MN8 also produced low levels of α-toxin despite the same stop codon. The differences observed in virulence factor profiles of two USA200 strains provide insight into environmental factors that select for specific virulence factors. Cytolysins, superantigens, and proteases were identified as potential targets, where toxin neutralization may prevent or diminish epithelial damage associated with S. aureus.

Efficacy of concurrent application of chlorhexidine gluconate and povidone iodine against six nosocomial pathogens

BACKGROUND Chlorhexidine gluconate (CHG) and povidone iodine (PI) are rarely used concurrently despite a lack of evidence regarding functional incompatibility of these agents. METHODS CHG and PI, alone and combined, were evaluated against Staphylococcus aureus (methicillin-susceptible S aureus [MSSA] and methicillin-resistant S aureus [MRSA]), Staphylococcus epidermidis (MRSE), Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli using checkerboard microbroth dilution techniques. Minimum bactericidal concentration (MBC) was the concentration (percent wt/vol) that reduced bacterial burden ≥ 5-log(10) colony-forming units/mL at 2 hours when compared with bacterial densities in growth controls. Fractional bactericidal concentration indexes (FBCIs) were calculated to determine CHG and PI compatibility. Additionally, tissue plugs from freshly excised porcine vaginal mucosa were infected with S aureus (MSSA), treated for 2 hours with CHG 3%, PI 5%, or CHG 3% and PI 5% combined and then viable bacteria on the tissue plugs enumerated. RESULTS In broth, CHG demonstrated dose-dependent bactericidal activity, whereas PI activity was all-or-none. All isolates studied were similarly susceptible to CHG (MBCs: 0.0078% ± 0.0019%, 0.0069% ± 0.0026%, 0.0024% ± 0.0005%, 0.0024% ± 0.0005%, 0.0059% ± 0.0%, and 0.0029% ± 0.0%, respectively). The MBCs of PI were identical (0.625%) for all isolates. Overall, FBCI calculations showed indifference. Treatment of MSSA-infected porcine tissue for 2 hours demonstrated that the CHG-PI combination was superior to either antiseptic alone. CONCLUSION FBCIs, determined in broth culture, indicate that combining CHG and PI had no negative impact on antisepsis. Moreover, data from an ex vivo porcine mucosal infection model suggest a potential benefit when combining the 2 antiseptic agents.

Comparison of Staphylococcus aureus strains for ability to cause infective endocarditis and lethal sepsis in rabbits.

Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause these illnesses. Common S. aureus strains include pulsed-field gel electrophoresis (PFGE) types USA200, 300, and 400 types where we hypothesize that secreted virulence factors contribute to both IE and sepsis. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens (SAgs) and cytolysins. As such, rabbits are an excellent model for studying IE and sepsis, which over the course of four days develop IE vegetations and/or fatal septicemia. We examined the ability of MRSA and MSSA strains (4 USA200, 2 USA300, 2 USA400, and three additional common strains, FRI1169, Newman, and COL) to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1+ strains that produce only low amounts of α-toxin, exhibited modest LD50 in sepsis (1 × 108 – 5 × 108) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high-levels of α-toxin, was both highly lethal (LD50 5 × 106 CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s 1 × 106 and 5 × 107 CFUs) but were minimally capable of causing IE. Strain Newman, which is phylogenetically related to USA300 strains, was not highly lethal (LD50 of 2 × 109 CFUs) and was effective in causing IE. USA400 strains were both highly lethal (LD50s of 1 × 107 and 5 × 107 CFUs) and highly effective causes of IE. The menstrual TSS isolate FRI1169, that is TSST-1+, produces high-levels of α-toxin, but is not USA200, was both highly lethal and effective in causing IE. Additional studies showed that phenol soluble modulins (PSMs) produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus differ in abilities to cause IE and lethal sepsis and suggest that secreted virulence factors, including SAgs and cytolysins, account for some of these differences.

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 µg/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 3×108 CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-α (TNF-α) concentrations and mortality over 7 days. DDG (50 and 100 µg/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-α 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-α, 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.

Vaginal Staphylococcus aureus Superantigen Profile Shift from 1980 and 1981 to 2003, 2004, and 2005

ABSTRACT We determined vaginal Staphylococcus aureus superantigens. Staphylococci were quantified from tampons/diaphragms in 2003 to 2005, with counts compared to those determined in 1980 and 1981. In 2003 to 2005, more women were colonized than in 1980 and 1981 (23 versus 12%). Enterotoxins G and I and enterotoxin-like superantigens M and N declined, but enterotoxin-like superantigens K, L, and Q increased.

New insights into the prevention of staphylococcal infections and toxic shock syndrome

Staphylococcus aureus is a major human pathogen capable of causing various diseases, from skin infections to life-threatening pneumonia and toxic shock syndrome. S. aureus exoproteins, including superantigens, contribute significantly to the pathogenesis of this organism. Antibiotics inhibit growth, but often provide no protection from S. aureus exoproteins. With the emergence of antibiotic-resistant S. aureus, new therapeutic options to treat or prevent S. aureus-associated diseases are critical. Most S. aureus infections begin on the skin or mucosal surfaces from direct inflammatory or cytotoxic effects of exotoxins. Therefore, antitoxin therapies that prevent toxin production and prevent their effects on host cells are being researched. Current treatments for staphylococcal diseases and recent developments in antitoxin therapeutic agents and vaccines are reviewed.