Staphylococcus aureus-specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis-like flares in mice.

BACKGROUND: The contribution of Staphylococcus aureus to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. OBJECTIVES: This study sought to reappraise the main bacterial factors and underlying immune mechanisms by which S aureus triggers AD-like inflammation. METHODS: This study capitalized on a preclinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. RESULTS: The development of S aureus-induced dermatitis depended on the nature of the S aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and nonsecreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor apoptosis-associated speck-like protein containing a CARD domain- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S aureus, and an accumulation of S aureus-specific γδ and CD4+ tissue resident memory T cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis on new bacteria exposures, but also, protected the mice from persistent bacterial colonization. CONCLUSIONS: These data highlight the induction of unique AD-like inflammation, with the generation of proinflammatory but protective tissue resident memory T cells in a context of natural exposure to pathogenic S aureus strains.

Structural Variations in the Central Heterocyclic Scaffold of Tripartite 2,6-Difluorobenzamides: Influence on Their Antibacterial Activity against MDR Staphylococcus aureus.

Five series of heterocyclic tripartite 2,6-difluorobenzamides, namely 1,2,3-triazoles, 1,2,4- and 1,3,4-oxadiazoles, analogs of reported model anti-staphylococcal compounds, were prepared. The purpose was to investigate the influence of the nature of the heterocyclic central scaffold on the biological activity against three strains of S. aureus, including two drug-resistant ones. Among the 15 compounds of the new collection, a 3-(4-tert-butylphenyl)-1,2,4-oxadiazole linked via a methylene group with a 2,6-difluorobenzamide moiety (II.c) exhibited a minimal inhibitory concentration between 0.5 and 1 µg/mL according to the strain. Subsequent studies on II.c demonstrated no human cytotoxicity, while targeting the bacterial divisome.

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.

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.

Clindamycin suppresses virulence expression in inducible clindamycin-resistant Staphylococcus aureus strains.

Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus. Recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections. Clindamycin modulates virulence expression at sub-inhibitory concentrations (sub-MICs) in clindamycin-susceptible S. aureus strains but previous report shown that this effect was supressed for constitutive clindamycin resistant strains. However, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we show that sub-MICs of clindamycin decrease Panton-Valentine leucocidin, toxic-shock-staphylococcal toxin (TSST-1) and alpha-haemolysin (Hla) expression in six inducible clindamycin-resistant isolates cultivated in vitro in CCY medium. These results suggest that the clindamycin anti-toxin effect is retained for inducible clindamycin-resistant S. aureus isolates; therefore, its usage should be considered within the treatment regimen of toxin related infections for inducible clindamycin-resistant S. aureus.

Effects of Tedizolid Phosphate on Survival Outcomes and Suppression of Production of Staphylococcal Toxins in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia.

The protective efficacy of tedizolid phosphate, a novel oxazolidinone that potently inhibits bacterial protein synthesis, was compared to those of linezolid, vancomycin, and saline in a rabbit model of Staphylococcus aureus necrotizing pneumonia. Tedizolid phosphate was administered to rabbits at 6 mg/kg of body weight intravenously twice daily, which yielded values of the 24-h area under the concentration-time curve approximating those found in humans. The overall survival rate was 83% for rabbits treated with 6 mg/kg tedizolid phosphate twice daily and 83% for those treated with 50 mg/kg linezolid thrice daily (P = 0.66 by the log-rank test versus the results obtained with tedizolid phosphate). These survival rates were significantly greater than the survival rates of 17% for rabbits treated with 30 mg/kg vancomycin twice daily (P = 0.003) and 17% for rabbits treated with saline (P = 0.002). The bacterial count in the lungs of rabbits treated with tedizolid phosphate was significantly decreased compared to that in the lungs of rabbits treated with saline, although it was not significantly different from that in the lungs of rabbits treated with vancomycin or linezolid. The in vivo bacterial production of alpha-toxin and Panton-Valentine leukocidin, two key S. aureus-secreted toxins that play critical roles in the pathogenesis of necrotizing pneumonia, in the lungs of rabbits treated with tedizolid phosphate and linezolid was significantly inhibited compared to that in the lungs of rabbits treated with vancomycin or saline. Taken together, these results indicate that tedizolid phosphate is superior to vancomycin for the treatment of S. aureus necrotizing pneumonia because it inhibits the bacterial production of lung-damaging toxins at the site of infection.

Differential Interaction of the Staphylococcal Toxins Panton-Valentine Leukocidin and γ-Hemolysin CB with Human C5a Receptors.

Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton-Valentine leukocidin LukSF-PV (PVL) and γ-hemolysin CB (HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the apparent redundancy of both toxins cannot be adequately addressed in experimental models of infection because mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity toward neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Overexpression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity toward neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that the toxicity of PVL, but not of HlgCB, is neutralized by various C5aR1 antagonists. This study offers directions for the development of improved preclinical models for infection, as well as for the design of drugs antagonizing leukocidin toxicity.

In vivo efficacy of ceftaroline fosamil in a methicillin-resistant panton-valentine leukocidin-producing Staphylococcus aureus rabbit pneumonia model.

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with broad-spectrum in vitro activity against Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae (MDRSP), and common Gram-negative pathogens. This study investigated the in vivo activity of ceftaroline fosamil compared with clindamycin, linezolid, and vancomycin in a severe pneumonia model due to MRSA-producing Panton-Valentine leukocidin (PVL). A USA300 PVL-positive clone was used to induce pneumonia in rabbits. Infected rabbits were randomly assigned to no treatment or simulated human-equivalent dosing with ceftaroline fosamil, clindamycin, linezolid, or vancomycin. Residual bacterial concentrations in the lungs and spleen were assessed after 48 h of treatment. PVL expression was measured using a specific enzyme-linked immunosorbent assay (ELISA). Ceftaroline, clindamycin, and linezolid considerably reduced mortality rates compared with the control, whereas vancomycin did not. Pulmonary and splenic bacterial titers and PVL concentrations were greatly reduced by ceftaroline, clindamycin, and linezolid. Ceftaroline, clindamycin, and linezolid were associated with reduced pulmonary tissue damage based on significantly lower macroscopic scores. Ceftaroline fosamil, clindamycin, and, to a lesser extent, linezolid were efficient in reducing bacterial titers in both the lungs and spleen and decreasing macroscopic scores and PVL production compared with the control.

Effects of linezolid on suppressing in vivo production of staphylococcal toxins and improving survival outcomes in a rabbit model of methicillin-resistant Staphylococcus aureus necrotizing pneumonia.

BACKGROUND: Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking. METHODS: A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production. RESULTS: Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs. CONCLUSIONS: Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

Effects of subinhibitory concentrations of antibiotics on virulence factor expression by community-acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: To examine the effect of subinhibitory concentrations (sub-MICs) of antistaphylococcal drugs on Panton-Valentine leucocidin (PVL), α-haemolysin (Hla) and protein A (SpA) expression by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). METHODS: Five clinical isolates representing the main worldwide CA-MRSA clones were grown with sub-MICs (1/8, 1/4 and 1/2 MIC) of five antibiotics (clindamycin, daptomycin, linezolid, tigecycline and vancomycin). After 4 and 6 h of incubation, culture pellets were used for relative quantitative RT-PCR with primers specific for pvl, hla, spa and gyrB. The PVL, Hla and SpA concentrations were measured in the supernatant (for PVL and Hla) and in the cell pellet (for SpA) using specific ELISAs. RESULTS: For all strains tested, clindamycin and linezolid dramatically reduced mRNA levels of PVL and SpA. Tigecycline also decreased the PVL and SpA mRNA levels of 3/5 and 4/5 strains tested, respectively, whereas daptomycin and vancomycin had no significant effect. PVL and SpA quantification confirmed the concentration-dependent inhibition of PVL and SpA production by clindamycin and, to a lesser extent, by linezolid and tigecycline. Only clindamycin decreased Hla mRNA expression, whereas linezolid, tigecycline and daptomycin showed heterogeneous strain-dependent results, and vancomycin had no significant effect. Analysis of the Hla level revealed a stronger concentration-dependent inhibition of Hla release by clindamycin than by linezolid. CONCLUSIONS: The effect of sub-MICs on virulence expression depended on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppressed the expression of different virulence factors by CA-MRSA, whereas tigecycline specifically suppressed PVL expression. Daptomycin and vancomycin seem to have no significant effects at these concentrations.

Cross-talk between Staphylococcus aureus leukocidins-intoxicated macrophages and lung epithelial cells triggers chemokine secretion in an inflammasome-dependent manner.

Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1ß release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1ß release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and ß-haemolysin synergize with PVL to amplify IL-1ß release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1ß release in human alveolar macrophages. Furthermore, IL-1ß released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury.

Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.

Simulated antibiotic exposures in an in vitro hollow-fiber infection model influence toxin gene expression and production in community-associated methicillin-resistant Staphylococcus aureus strain MW2.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain MW2 harbors a plethora of toxins to mediate its virulence. However, toxin expression and regulation with simulated clinical antimicrobial exposures are unclear. This study evaluated these relationships using an in vitro pharmacodynamic hollow-fiber infection model. Clinical doses of clindamycin, linezolid, minocycline, trimethoprim-sulfamethoxazole (SXT), and vancomycin were simulated over 72 h against MW2 in the hollow fiber model. Expression levels of lukSF-PV and enterotoxin genes sec4, sek, seq, and sel2 were quantified by real-time PCR. Panton-Valentine leukocidin (PVL) was quantified by enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was determined on polymorphonuclear cells (PMNs). Vancomycin produced the maximum MW2 killing (2.53 log(10) CFU/ml) after the first dose, but the greatest sustained killing over 72 h occurred with linezolid and clindamycin. Vancomycin and minocycline induced gene upregulation from 0 to 8 h, followed by downregulation for the remaining simulation period. Clindamycin decreased gene expression in the first 24 h, followed by moderate increases (2.5-fold) thereafter. Linezolid increased gene expression 11.4- to 200.4-fold but inhibited PVL production (0.6 ± 0.3 versus 5.9 ± 0.2 µg/ml, linezolid versus control at 72 h; P < 0.05). Similar effects on PVL production occurred with clindamycin and minocycline. SXT increased PVL production at 48 h (2.8-fold) and 72 h (4.9-fold) of treatment (P < 0.05), resulting in increased PVL cytotoxicity on PMNs. Linezolid, clindamycin, and minocycline were the most effective agents on decreasing the virulence potential in CA-MRSA, notably after 8 h of treatment. SXT had minimal effects on toxin gene regulation, but it increased production and cytotoxicity of PVL toxin in the model and may enhance virulence when it is used to treat severe infections.

Targeted Proteomics Analysis of Staphylococcal Superantigenic Toxins in Menstrual Fluid from Women with Menstrual Toxic Shock Syndrome (mTSS).

Menstrual toxic shock syndrome (mTSS) is a rare life-threatening febrile illness that occurs in women using intravaginal menstrual protection. It is caused by toxic shock syndrome toxin 1 (TSST-1) produced by Staphylococcus aureus, triggering a sudden onset of rash and hypotension, subsequently leading to multiple organ failure. Detecting TSST-1 and S. aureus virulence factors in menstrual fluid could accelerate the diagnosis and improve therapeutic management of mTSS. However, menstrual fluid is a highly complex matrix, making detection of bacterial toxins challenging. Here, we present a mass-spectrometry-based proteomics workflow for the targeted, quantitative analysis of four S. aureus superantigenic toxins in menstrual fluids (TSST-1, SEA, SEC, and SED). This method was applied to characterize toxin levels in menstrual fluids collected from patients with mTSS and healthy women. Toxins were detectable in samples from patients with mTSS and one healthy donor at concentrations ranging from 0 to 0.46 µg/mL for TSST-1, and 0 to 1.07 µg/mL for SEC. SEA and SED were never detected in clinical specimens, even though many S. aureus strains were positive for the corresponding genes. The method presented here could be used to explore toxin production in vivo in users of intravaginal devices to improve the diagnosis, understanding, and prevention of mTSS.

Synthesis and Biological Evaluation of Benzo[b]thiophene Acylhydrazones as Antimicrobial Agents against Multidrug-Resistant Staphylococcus aureus.

The benzo[b]thiophene nucleus and the acylhydrazone functional group were combined to prepare three new series of compounds for screening against Staphylococcus aureus. The reaction of substituted benzo[b]thiophene-2-carboxylic hydrazide and various aromatic or heteroaromatic aldehydes led to a collection of 26 final products with extensive structural diversification on the aromatic ring and on position 6 of the benzo[b]thiophene nucleus. The screening lead to the identification of eight hits, including (E)-6-chloro-N'-(pyridin-2-ylmethylene)benzo[b]thiophene-2-carbohydrazide (II.b), a non-cytotoxic derivative showing a minimal inhibitory concentration of 4 µg/mL on three S. aureus strains, among which were a reference classical strain and two clinically isolated strains resistant to methicillin and daptomycin, respectively.

Beta-lactams interfering with PBP1 induce Panton-Valentine leukocidin expression by triggering sarA and rot global regulators of Staphylococcus aureus.

Previous articles reported that beta-lactam antibiotics increase the expression of Staphylococcus aureus Panton-Valentine leukocidin (PVL) by activating its transcription. We investigated the mechanisms underlying the inductor effect of beta-lactams on PVL expression by determining targets and regulatory pathways possibly implicated in this process. We measured PVL production in the presence of oxacillin (nonselective), imipenem (penicillin-binding protein 1 [PBP1] selective), cefotaxime (PBP2 selective), cefaclore (PBP3 selective), and cefoxitin (PBP4 selective). In vitro, we observed increased PVL production consistent with luk-PV mRNA levels that were 20 to 25 times higher for community-acquired methicillin-resistant S. aureus (CA-MRSA) cultures treated with PBP1-binding oxacillin and imipenem than for cultures treated with other beta-lactams or no antibiotic at all. This effect was also observed in vivo, with increased PVL mRNA levels in lung tissues from CA-MRSA-infected mice treated with imipenem but not cefoxitin. To confirm the involvement of PBP1 inhibition in this pathway, PBP1 depletion by use of an inducible pbp1 antisense RNA showed a dose-dependent relationship between the level of pbp1 antisense RNA and the luk-PV mRNA level. Upon imipenem treatment of exponential-phase cultures, we observed an increased sarA mRNA level after 30 min of incubation followed by a decreased rot mRNA level after 1 to 4 h of incubation. Unlike the agr and saeRS positive regulators, which were nonessential for PVL induction by beta-lactams, the sarA (positive) and rot (negative) PVL regulators were necessary for PVL induction by imipenem. Our results suggest that antibiotics binding to PBP1 increase PVL expression by modulating sarA and rot, which are essential mediators of the inductor effect of beta-lactams on PVL expression.

Reductive evolution of virulence repertoire to drive the divergence between community- and hospital-associated methicillin-resistant Staphylococcus aureus of the ST1 lineage.

Methicillin-resistant Staphylococcus aureus (MRSA) of the ST1-SCCmecIV lineage has been associated with community-acquired (CA) infections in North America and Australia. In Brazil, multi-drug resistant ST1-SCCmecIV MRSA has emerged in hospital-associated (HA) diseases in Rio de Janeiro. To understand these epidemiological differences, genomic and phylogenetic analyses were performed. In addition, virulence assays were done for representative CA - and HA-MRSA strains. Despite the conservation of the virulence repertoire, some genes were missing in Brazilian ST1-SCCmecIV including lukSF-PV, fnbB, and several superantigen-encoded genes. Additionally, CA-MRSA lost the splDE while HA-MRSA strains conserved the complete operon. Most of these variable genes were located in mobile genetic elements (MGE). However, conservation and maintenance of MGEs were often observed despite the absence of their associated virulence markers. A Bayesian phylogenetic tree revealed the occurrence of more than one entrance of ST1 strains in Rio de Janeiro. The tree shape and chronology allowed us to infer that the hospital-associated ST1-SCCmecIV from Brazil and the community-acquired USA400 from North America are not closely related and that they might have originated from different MSSA strains that independently acquired SCCmecIV cassettes. As expected, representatives of ST1 strains from Brazil showed lower cytotoxicity and a greater ability to survive inside human host cells. We suggest that Brazilian ST1-SCCmecIV strains have adapted to the hospital setting by reducing virulence and gaining the ability to persist and survive inside host cells. Possibly, these evolutionary strategies may balance the biologic cost of retaining multiple antibiotic resistance genes.

A histidine-to-arginine substitution in Panton-Valentine leukocidin from USA300 community-acquired methicillin-resistant Staphylococcus aureus does not impair its leukotoxicity.

Panton-Valentine leukocidin (PVL) is a synergohymenotropic toxin (SHT) produced by Staphylococcus aureus. At present, there are conflicting reports on the leukotoxic activity of PVL and its consequent role as a virulence factor in USA300. In this work, we compared the cytolytic effects induced by wild-type PVL and those of PVL harboring a histidine-to-arginine substitution at amino acid 176 in the S. aureus USA300 strain. We also investigated the capacity of wild-type and H176R LukS-PV to recruit and form pores with the F components of other SHTs. For this purpose, we assayed polymorphonuclear neutrophils for leukotoxicity after incubation with either culture supernatants from strains bearing different PVL haplotypes or recombinant toxins from different types of SHT. We show here that the H176R variation in the PVL sequence causes no change in leukotoxicity and that the R variant is as efficient as wild-type PVL at inducing pore formation in leukocytes.

Immunogenicity of toxins during Staphylococcus aureus infection.

BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects were analyzed for immunoglobulin (Ig) G binding to 20 toxins, using flow-cytometry based technology. Antibody levels were associated with polymerase chain reaction-defined presence of toxin genes in homologous S. aureus isolates. RESULTS: IgG levels directed to exfoliative toxin (ET) A, ETB, gamma hemolysin B (HlgB), leukocidin (Luk) D, LukE, LukS, staphylococcal enterotoxin (SE) A, SEE, SEH, SEI, and SElM were higher in S. aureus-infected patients than in control subjects (P < .05). Furthermore, in the S. aureus-infected patient group, IgG levels were higher if genes encoding ETA, ETB, SEA, SEC, SEH, SElQ, toxic shock syndrome toxin-1 (TSST-1), or Panton-Valentine leukocidin (PVL) were present in the infectious isolate (P< .05). Levels of anti-SEA IgG increased during infections with sea-positive (median fluorescence intensity from 11,555 to 12,388; P<.05) but not sea-negative strains. In addition, anti-LukS IgG levels increased during skin and soft-tissue infections with luk-PV-positive (median fluorescence intensity from 15,231 to 15,911; P<.05) but not luk-PV-negative strains. Bacteremia was associated with sea (odds ratio, 3.4; 95% confidence interval, 1.2-10.0) and tst (odds ratio, 5.7; 95% confidence interval, 1.6-20.8). Skin and soft-tissue infections and bone and joint infections were associated with luk-PV (odds ratio, 2.5; 95% confidence interval, 1.2-5.2). CONCLUSIONS: Many toxins are expressed in vivo and recognized by the immune system during staphylococcal infections, suggesting their involvement in S. aureus pathogenesis.

Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests.

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus (lukS-PV and lukF-PV) and the mecA gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL(+)); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL(+) strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL(+) strains ranged from 0 to 399 microg/ml by ELISA. By the use of 0.015 microg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.