Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates.

Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections.

The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner.

UNLABELLED: The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery [corrected].

Efficacy of skin and nasal povidone-iodine preparation against mupirocin-resistant methicillin-resistant Staphylococcus aureus and S. aureus within the anterior nares.

Mupirocin decolonization of nasal Staphylococcus aureus prior to surgery decreases surgical-site infections; however, treatment requires 5 days, compliance is low, and resistance occurs. In 2010, 3M Company introduced povidone-iodine (PVP-I)-based skin and nasal antiseptic (Skin and Nasal Prep [SNP]). SNP has rapid, broad-spectrum antimicrobial activity. We tested SNP's efficacy using full-thickness tissue (porcine mucosal [PM] and human skin) explant models and human subjects. Prior to or following infection with methicillin-resistant Staphylococcus aureus (MRSA) (mupirocin sensitive and resistant), explants were treated with Betadine ophthalmic preparation (Bet), SNP, or mupirocin (Bactroban nasal ointment [BN]) or left untreated. One hour posttreatment, explants were washed with phosphate-buffered saline (PBS) plus 2% mucin. One, 6, or 12 h later, bacteria were recovered and enumerated. Alternatively, following baseline sampling, human subjects applied two consecutive applications of SNP or saline to their anterior nares. One, 6, and 12 h after application of the preparation (postprep), nasal swabs were obtained, and S. aureus was enumerated. We observed that treatment of infected PM or human skin explants with SNP resulted in >2.0 log10 CFU reduction in MRSA, regardless of mupirocin sensitivity, which was significantly different from the values for BN- and Bet-treated explants and untreated controls 1 h, 6 h, and 12 h after being washed with PBS plus mucin. Swabbing the anterior nares of human subjects with SNP significantly reduced resident S. aureus compared to saline 1, 6, and 12 h postprep. Finally, pretreatment of PM explants with SNP, followed by a mucin rinse prior to infection, completely prevented MRSA infection. We conclude that SNP may be an attractive alternative for reducing the bioburden of anterior nares prior to surgery.

A mucosal model to study microbial biofilm development and anti-biofilm therapeutics.

Biofilms are a sessile colony of bacteria which adhere to and persist on surfaces. The ability of bacteria to form biofilms is considered a virulence factor, and in fact is central to the pathogenesis of some organisms. Biofilms are inherently resistant to chemotherapy and host immune responses. Clinically, biofilms are considered a primary cause of a majority of infections, such as otitis media, pneumonia in cystic fibrosis patients and endocarditis. However, the vast majority of the data on biofilm formation comes from traditional microtiter-based or flow displacement assays with no consideration given to host factors. These assays, which have been a valuable tool in high-throughput screening for biofilm-related factors, do not mimic a host-pathogen interaction and may contribute to an inappropriate estimation of the role of some factors in clinical biofilm formation. We describe the development of a novel ex vivo model of biofilm formation on a mucosal surface by an important mucosal pathogen, methicillin resistant S. aureus (MRSA). This model is being used for the identification of microbial virulence factors important in mucosal biofilm formation and novel anti-biofilm therapies.

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 × 10(8) 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).

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.

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.

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.

Dietary polyunsaturated fatty acids modulate in vivo, antigen-driven CD4+ T-cell proliferation in mice.

Our objective was to determine whether dietary lipids affect in vivo, antigen-driven, proliferation of naïve CD4(+) T lymphocytes. To accomplish this, we adoptively transferred lymphocytes from T-cell receptor (TCR) transgenic DO11.10 (i.e., donor) mice into syngeneic, nontransgenic BALB/c (i.e., recipient) mice. Before adoptive transfer, recipient mice were fed for 4 wk AIN93G-type diets that differed only in fat source: lard, low in PUFA, fish oil, rich in (n-3) PUFA, and soybean oil, rich in (n-6) PUFA. One week after transfer, recipient mice were immunized with antigen (i.e., ovalbumin), and expansion of CD4(+) T(DO11.10) cells in the spleen and draining lymph nodes (LN) was measured by flow cytometry. Five days postimmunization (p.i.), at the peak of expansion, CD4(+) T(DO11.10) cells in the draining LN and spleen were 5- to 10-fold higher than in unimmunized mice, then quickly declined during the contraction phase (i.e., 7 and 10 d p.i.). Recipients fed the (n-6) PUFA rich diet had approximately 25% greater in vivo expansion of CD4(+) T(DO11.10) cells than lard- and fish oil-fed recipient mice at 5 d p.i. (P < 0.05). However, at 7 and 10 d p.i., CD4(+) T(DO11.10) cells in the draining lymph nodes did not differ between groups, nor in the spleen at 5, 7, and 10 d p.i. In summary, we are the first to demonstrate that dietary PUFAs affect antigen-driven expansion of naïve CD4(+) T cells in vivo. Surprisingly, (n-3) PUFA consumption did not reduce CD4(+) T-cell expansion.

Dietary fish oil impairs primary host resistance against Listeria monocytogenes more than the immunological memory response.

The primary objective of this study was to determine whether dietary (n-3) polyunsaturated fatty acids (PUFA) impair the ability of mice to generate an immunological memory response against the bacterial pathogen, Listeria monocytogenes. Weanling BALB/c female mice were fed for 28 d one of two semipurified high fat diets containing either lard or refined menhaden fish oil, rich in long-chain (n-3) PUFA. Mice were immunized with 10(4) or 10(3) colony forming units (cfu) bacteria. Thirty-five days later, these immune mice and age-matched naïve (i.e., unimmunized) mice were challenged with 10(5) cfu bacteria. Three days postchallenge, bacterial clearance was determined. Compared with lard-fed mice, naïve mice in the fish oil treatment group had higher bacterial loads in their liver and spleen (P < 0.001). When mice were immunized with 10(4) cfu bacteria before rechallenge with 10-fold more bacteria, both lard- and fish oil-fed mice had significantly lower bacterial loads in their liver and spleen (e.g., approximately 2 log(10); P < 0.001) compared with their naïve counterparts. However, when the immunization dose was reduced to 10(3) bacteria, a modest diet treatment effect was observed, such that compared with immune lard-fed mice, immune fish oil-fed mice had significantly greater bacterial loads in their liver and spleen (i.e., approximately 0.5 log(10); P < 0.01). These data demonstrate for the first time that although dietary (n-3) PUFA can significantly impair host resistance to a primary as well as a secondary L. monocytogenes infection, the impairment of the immunological memory response is much less severe.