A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection.

Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Monoclonal antibodies neutralizing alpha-hemolysin, bicomponent leukocidins, and clumping factor A protected against Staphylococcus aureus-induced acute circulatory failure in a mechanically ventilated rabbit model of hyperdynamic septic shock.

Background: Patients with septic shock caused by Staphylococcus aureus have mortality rates exceeding 50%, despite appropriate antibiotic therapy. Our objectives were to establish a rabbit model of S. aureus septic shock and to determine whether a novel immunotherapy can prevent or halt its natural disease progression. Methods: Anesthetized rabbits were ventilated with lung-protective low-tidal volume, instrumented for advanced hemodynamic monitoring, and characterized for longitudinal changes in acute myocardial dysfunction by echocardiography and sepsis-associated biomarkers after S. aureus intravenous challenge. To demonstrate the potential utility of this hyperdynamic septic shock model for preclinical drug development, rabbits were randomized for prophylaxis with anti-Hla/Luk/ClfA monoclonal antibody combination that neutralizes alpha-hemolysin (Hla), the bicomponent pore-forming leukocidins (Luk) including Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysin, and clumping factor A (ClfA), or an irrelevant isotype-matched control IgG (c-IgG), and then challenged with S. aureus. Results: Rabbits challenged with S. aureus, but not those with saline, developed a hyperdynamic state of septic shock characterized by elevated cardiac output (CO), increased stroke volume (SV) and reduced systemic vascular resistance (SVR), which was followed by a lethal hypodynamic state characterized by rapid decline in mean arterial pressure (MAP), increased central venous pressure, reduced CO, reduced SV, elevated SVR, and reduced left-ventricular ejection fraction, thereby reproducing the hallmark clinical features of human staphylococcal septic shock. In this model, rabbits pretreated with anti-Hla/Luk/ClfA mAb combination had 69% reduction in mortality when compared to those pretreated with c-IgG (P<0.001). USA300-induced acute circulatory failure-defined as >70% decreased in MAP from pre-infection baseline-occurred in only 20% (2/10) of rabbits pretreated with anti-Hla/Luk/ClfA mAb combination compared to 100% (9/9) of those pretreated with c-IgG. Prophylaxis with anti-Hla/Luk/ClfA mAb combination halted progression to lethal hypodynamic shock, as evidenced by significant protection against the development of hyperlactatemia, hypocapnia, hyperkalemia, leukopenia, neutropenia, monocytopenia, lymphopenia, as well as biomarkers associated with acute myocardial injury. Conclusion: These results demonstrate the potential utility of a mechanically ventilated rabbit model that reproduced hallmark clinical features of hyperdynamic septic shock and the translational potential of immunotherapy targeting S. aureus virulence factors for the prevention of staphylococcal septic shock.

Postoperative Staphylococcus aureus Infections in Patients With and Without Preoperative Colonization.

Importance: Staphylococcus aureus surgical site infections (SSIs) and bloodstream infections (BSIs) are important complications of surgical procedures for which prevention remains suboptimal. Contemporary data on the incidence of and etiologic factors for these infections are needed to support the development of improved preventive strategies. Objectives: To assess the occurrence of postoperative S aureus SSIs and BSIs and quantify its association with patient-related and contextual factors. Design, Setting, and Participants: This multicenter cohort study assessed surgical patients at 33 hospitals in 10 European countries who were recruited between December 16, 2016, and September 30, 2019 (follow-up through December 30, 2019). Enrolled patients were actively followed up for up to 90 days after surgery to assess the occurrence of S aureus SSIs and BSIs. Data analysis was performed between November 20, 2020, and April 21, 2022. All patients were 18 years or older and had undergone 11 different types of surgical procedures. They were screened for S aureus colonization in the nose, throat, and perineum within 30 days before surgery (source population). Both S aureus carriers and noncarriers were subsequently enrolled in a 2:1 ratio. Exposure: Preoperative S aureus colonization. Main Outcomes and Measures: The main outcome was cumulative incidence of S aureus SSIs and BSIs estimated for the source population, using weighted incidence calculation. The independent association of candidate variables was estimated using multivariable Cox proportional hazards regression models. Results: In total, 5004 patients (median [IQR] age, 66 [56-72] years; 2510 [50.2%] female) were enrolled in the study cohort; 3369 (67.3%) were S aureus carriers. One hundred patients developed S aureus SSIs or BSIs within 90 days after surgery. The weighted cumulative incidence of S aureus SSIs or BSIs was 2.55% (95% CI, 2.05%-3.12%) for carriers and 0.52% (95% CI, 0.22%-0.91%) for noncarriers. Preoperative S aureus colonization (adjusted hazard ratio [AHR], 4.38; 95% CI, 2.19-8.76), having nonremovable implants (AHR, 2.00; 95% CI, 1.15-3.49), undergoing mastectomy (AHR, 5.13; 95% CI, 1.87-14.08) or neurosurgery (AHR, 2.47; 95% CI, 1.09-5.61) (compared with orthopedic surgery), and body mass index (AHR, 1.05; 95% CI, 1.01-1.08 per unit increase) were independently associated with S aureus SSIs and BSIs. Conclusions and Relevance: In this cohort study of surgical patients, S aureus carriage was associated with an increased risk of developing S aureus SSIs and BSIs. Both modifiable and nonmodifiable etiologic factors were associated with this risk and should be addressed in those at increased S aureus SSI and BSI risk.

Neutralizing Staphylococcus aureus Virulence with AZD6389, a Three mAb Combination, Accelerates Closure of a Diabetic Polymicrobial Wound.

Nonhealing diabetic foot ulcers (DFU), a major complication of diabetes, are associated with high morbidity and mortality despite current standard of care. Since Staphylococcus aureus is the most common pathogen isolated from nonhealing and infected DFU, we hypothesized that S. aureus virulence factors would damage tissue, promote immune evasion and alter the microbiome, leading to bacterial persistence and delayed wound healing. In a diabetic mouse polymicrobial wound model with S. aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes, we report a rapid bacterial proliferation, prolonged pro-inflammatory response and large necrotic lesions unclosed for up to 40 days. Treatment with AZD6389, a three-monoclonal antibody combination targeting S. aureus alpha toxin, 4 secreted leukotoxins, and fibrinogen binding cell-surface adhesin clumping factor A resulted in full skin re-epithelization 21 days after inoculation. By neutralizing multiple virulence factors, AZD6389 effectively blocked bacterial agglutination and S. aureus-mediated cell killing, abrogated S. aureus-mediated immune evasion and targeted the bacteria for opsonophagocytic killing. Neutralizing S. aureus virulence not only facilitated S. aureus clearance in lesions, but also reduced S. pyogenes and P. aeruginosa numbers, damaging inflammatory mediators and markers for neutrophil extracellular trap formation 14 days post initiation. Collectively, our data suggest that AZD6389 holds promise as an immunotherapeutic approach against DFU complications. IMPORTANCE Diabetic foot ulcers (DFU) represent a major complication of diabetes and are associated with poor quality of life and increased morbidity and mortality despite standard of care. They have a complex pathogenesis starting with superficial skin lesions, which often progress to deeper tissue structures up to the bone and ultimately require limb amputation. The skin microbiome of diabetic patients has emerged as having an impact on DFU occurrence and chronicity. DFU are mostly polymicrobial, and the Gram-positive bacterium Staphylococcus aureus detected in more than 95% of cases. S. aureus possess a collection of virulence factors which participate in disease progression and may facilitate growth of other pathogens. Here we show in a diabetic mouse wound model that targeting some specific S. aureus virulence factors with a multimechanistic antibody combination accelerated wound closure and promoted full skin re-epithelization. This work opens promising new avenues for the treatment of DFU.

Multimechanistic Monoclonal Antibody Combination Targeting Key Staphylococcus aureus Virulence Determinants in a Rabbit Model of Prosthetic Joint Infection.

In a rabbit model of methicillin-resistant Staphylococcus aureus prosthetic joint infection (PJI), prophylaxis with AZD6389*-a combination of three monoclonal antibodies targeting alpha-hemolysin, bicomponent cytotoxins (LukSF/LukED/HlgAB/HlgCB), and clumping factor A-resulted in significant reductions in joint swelling, erythema, intra-articular pus, and bacterial burden in synovial tissues and biofilm-associated prosthetic implants compared with isotype-matched control IgG. Targeting specific staphylococcal virulence factors may thus have potential clinical utility for prevention of PJI.

Protective Efficacy of Monoclonal Antibodies Neutralizing Alpha-Hemolysin and Bicomponent Leukocidins in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia.

Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins. We found that passive immunization with either a monoclonal antibody (MAb) neutralizing alpha-hemolysin or a broadly cross-reactive MAb neutralizing Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB conferred only partial protection, whereas the combination of those two MAbs conferred significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 methicillin-resistant S. aureus epidemic clone.

Associations of pathogen-specific and host-specific characteristics with disease outcome in patients with Staphylococcus aureus bacteremic pneumonia.

OBJECTIVE: To understand the relationships of Staphylococcus aureus (SA) bacteremic pneumonia (SABP) outcome with patient-specific and SA-specific variables. METHODS: We analysed SA bloodstream isolates and matching sera in SABP patients by sequencing SA isolates (n = 50) and measuring in vitro AT production, haemolytic activity and expression of ClfA and ClfB. Controls were sera from gram-negative bacteremia patients with or without pneumonia and uninfected subjects. Levels of IgGs, IgMs and neutralizing antibodies (NAbs) against SA antigens were quantified and analysed by one-way ANOVA. Associations of patient outcomes with patient variables, antibody levels and isolate characteristics were evaluated by univariate and multivariate logistic regression analyses. RESULTS: SABP patients had higher levels of IgGs against eight virulence factors and anti-alpha toxin (AT) NAbs than uninfected controls. Levels of IgG against AT and IgMs against ClfA, FnbpA and SdrC were higher in clinically cured SABP patients than in clinical failures. Anti-LukAB NAb levels were elevated in all cohorts. Increased odds of cure correlated with higher haemolytic activity of SA strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM). DISCUSSION: Limitations included the cross-sectional observational nature of the study, small sample size and inability to measure antibody levels against all SA virulence factors. CONCLUSION: Our results suggest that SABP patients may benefit from immunotherapy targeting multiple SA antigens.

Candida albicans Augments Staphylococcus aureus Virulence by Engaging the Staphylococcal agr Quorum Sensing System.

Candida albicans and Staphylococcus aureus are among the most prevalent nosocomial pathogens that are responsible for severe morbidity and mortality, even with appropriate treatment. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with these pathogens results in synergistic lethality that is partially dependent on exacerbated prostaglandin signaling, while monomicrobial infection is nonlethal. Therefore, the objective of this study was to identify staphylococcal virulence determinants that drive lethal synergism during polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that coinfection with C. albicans led to a striking 80 to 100% mortality rate within 20 h postinoculation (p.i.) while monomicrobial infections were nonlethal. Use of a green fluorescent protein (GFP)-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by quantitative real-time PCR (qPCR), Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted alpha- and delta-toxins. C. albicans-mediated elevated toxin production and hemolytic activity were determined to be agrA dependent, and genetic knockout and complementation of hla identified alpha-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infections 8 h p.i. demonstrated equivalent bacterial burdens in the peritoneal cavity but significantly elevated levels of alpha-toxin (3-fold) and the eicosanoid prostaglandin E2 (PGE2) (4-fold) during coinfection. Importantly, prophylactic passive immunization using the monoclonal anti-alpha-toxin antibody MEDI4893* led to significantly improved survival rates compared to those following treatment with isotype control antibody. Collectively, these results define alpha-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human-pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivoIMPORTANCE Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so about how microbial "cross talk" shapes human health and disease. Candida albicans (a fungus) and Staphylococcus aureus (a bacterium) are formidable human nosocomial pathogens, causing severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. In this work, we have shown that coinfection with C. albicans and S. aureus is highly lethal, leading to >80% mortality by day 1 postinfection, whereas monoinfection with C. albicans or S. aureus does not cause mortality. This infectious synergism is dependent on the expression of staphylococcal alpha-toxin, and secretion of this potent virulence factor is actually augmented by C. albicans via an agr-dependent mechanism. Moreover, prophylactic neutralization of alpha-toxin with a monoclonal antibody is sufficient to elicit protection during coinfection. Therefore, we have demonstrated that a pathogenic fungus can enhance virulence determinants of a bacterium in vivo with devastating consequences to the host. These results have important implications in the surveillance and treatment of polymicrobial disease and highlight the dynamic intersection of environment, pathogens, and host.

Efficacy of a Multimechanistic Monoclonal Antibody Combination against Staphylococcus aureus Surgical Site Infections in Mice.

Surgical site infections (SSIs) are commonly caused by Staphylococcus aureus We report that a combination of three monoclonal antibodies (MEDI6389) that neutralize S. aureus alpha-toxin, clumping factor A, and four leukocidins (LukSF, LukED, HlgAB, and HlgCB) plus vancomycin had enhanced efficacy compared with control antibody plus vancomycin in two mouse models of S. aureus SSI. Therefore, monoclonal antibody-based neutralization of multiple S. aureus virulence factors may provide an adjunctive perioperative approach to combat S. aureus SSIs.

Mouse model of Gram-negative prosthetic joint infection reveals therapeutic targets.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

α-Toxin Induces Platelet Aggregation and Liver Injury during Staphylococcus aureus Sepsis.

During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.

Molecularly specific detection of bacterial lipoteichoic acid for diagnosis of prosthetic joint infection of the bone.

Discriminating sterile inflammation from infection, especially in cases of aseptic loosening versus an actual prosthetic joint infection, is challenging and has significant treatment implications. Our goal was to evaluate a novel human monoclonal antibody (mAb) probe directed against the Gram-positive bacterial surface molecule lipoteichoic acid (LTA). Specificity and affinity were assessed in vitro. We then radiolabeled the anti-LTA mAb and evaluated its effectiveness as a diagnostic imaging tool for detecting infection via immunoPET imaging in an in vivo mouse model of prosthetic joint infection (PJI). In vitro and ex vivo binding of the anti-LTA mAb to pathogenic bacteria was measured with Octet, ELISA, and flow cytometry. The in vivo PJI mouse model was assessed using traditional imaging modalities, including positron emission tomography (PET) with [18F]FDG and [18F]NaF as well as X-ray computed tomography (CT), before being evaluated with the zirconium-89-labeled antibody specific for LTA ([89Zr]SAC55). The anti-LTA mAb exhibited specific binding in vitro to LTA-expressing bacteria. Results from imaging showed that our model could reliably simulate infection at the surgical site by bioluminescent imaging, conventional PET tracer imaging, and bone morphological changes by CT. One day following injection of both the radiolabeled anti-LTA and isotype control antibodies, the anti-LTA antibody demonstrated significantly greater (P < 0.05) uptake at S. aureus-infected prosthesis sites over either the same antibody at sterile prosthesis sites or of control non-specific antibody at infected prosthesis sites. Taken together, the radiolabeled anti-LTA mAb, [89Zr]SAC55, may serve as a valuable diagnostic molecular imaging probe to help distinguish between sterile inflammation and infection in the setting of PJI. Future studies are needed to determine whether these findings will translate to human PJI.

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.

S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking.

Clinical severity of Staphylococcus aureus respiratory infection correlates with alpha toxin (AT) expression. AT activates the NLRP3 inflammasome; deletion of Nlrp3, or AT neutralization, protects mice from lethal S. aureus pneumonia. We tested the hypothesis that this protection is not due to a reduction in inflammasome-dependent cytokines (IL-1ß/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1ß/IL-18 did not. Primary human monocytes were used in vitro to determine the mechanism through which NLRP3 alters bacterial killing. In cells treated with small interfering RNA (siRNA) targeting NLRP3 or infected with AT-null S. aureus, mitochondria co-localize with bacterial-containing phagosomes. Mitochondrial engagement activates caspase-1, a process dependent on complex II of the electron transport chain, near the phagosome, promoting its acidification. These data demonstrate a mechanism utilized by S. aureus to sequester itself from antimicrobial processes within the cell.

Characterisation of anti-alpha toxin antibody levels and colonisation status after administration of an investigational human monoclonal antibody, MEDI4893, against Staphylococcus aureus alpha toxin.

Objectives: MEDI4893 is a novel, long-acting human monoclonal antibody targeting Staphylococcus aureus (SA) alpha toxin (AT). This report presents the results of the exploratory analyses from a randomised phase 1 dose-escalation study in healthy human subjects receiving single intravenous MEDI4893 doses or placebo. Methods: Anti-AT antibodies and AT expression were measured as described previously. Nasal swabs were analysed by culture and PCR. Data were summarised by treatment groups and visits by using SAS System Version 9.3. Results: Subjects receiving 2250 or 5000 mg of MEDI4893 had the highest serum anti-AT neutralising antibody (NAb) levels: approximately 180- to 240-, 70- to 100- and sevenfold to 10-fold higher than respective baseline levels at peak, 30 and 360 days, respectively. In these subjects, levels of serum anti-AT NAbs were >3.2 International Units (IU) mL-1 for at least 211 days. In the upper respiratory tract, anti-AT NAb levels increased with MEDI4893 dose. No apparent effect of MEDI4893 on SA nasal colonisation, hla gene sequence or AT expression was observed. Five AT variants were detected, their lytic activity was fully neutralised by MEDI4893. Discussion: Our results indicate that (1) MEDI4893 administration at 2250 and 5000 mg would provide effective immunoprophylaxis against systemic SA disease; (2) MEDI4983 distributes to the upper respiratory tract and retains neutralising activity against AT; and (3) potential for emergence of MEDI4893 resistance is low. Conclusion: Intravenous administration of MEDI4893 maintained levels of anti-AT NAbs in serum and nasal mucosa that may provide effective immunoprophylaxis against SA disease and support continued clinical development of MEDI4893.

Neutralizing Alpha-Toxin Accelerates Healing of Staphylococcus aureus-Infected Wounds in Nondiabetic and Diabetic Mice.

Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.

Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets.

Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against Saureus hematogenous implant-related infections.