Antivirulence Bispecific Monoclonal Antibody-Mediated Protection against Pseudomonas aeruginosa Ventilator-Associated Pneumonia in a Rabbit Model.

Ventilator-associated pneumonia is an important clinical manifestation of the nosocomial pathogen Pseudomonas aeruginosa. We characterized the correlates of protection with MEDI3902, a bispecific human IgG1 monoclonal antibody that targets the P. aeruginosa type 3 secretion system PcrV protein and the Psl exopolysaccharide, in a rabbit model of ventilator-associated pneumonia using lung-protective, low-tidal-volume mechanical ventilation. Rabbits infused with MEDI3902 prophylactically were protected, whereas those pretreated with irrelevant isotype-matched control IgG (c-IgG) succumbed between 12 and 44 h postinfection (100% survival [8/8 rabbits] versus 0% survival [8/8 rabbits]; P < 0.01 by log rank test). Lungs from rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, had bilateral, multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, and diffuse fibrinous edema in alveolar spaces. All rabbits pretreated with c-IgG developed worsening bacteremia that peaked at the time of death, whereas only 38% of rabbits pretreated with MEDI3902 (3/8 rabbits) developed such high-grade bacteremia (two-sided Fisher's exact test, P = 0.026). Biomarkers associated with acute respiratory distress syndrome were evaluated longitudinally in blood samples collected every 2 to 4 h to assess systemic pathophysiological changes in rabbits pretreated with MEDI3902 or c-IgG. Biomarkers were sharply increased or decreased in rabbits pretreated with c-IgG but not those pretreated with MEDI3902, including the ratio of arterial oxygen partial pressure to the fraction of inspired oxygen of <300, hypercapnia or hypocapnia, severe lactic acidosis, leukopenia, and neutropenia. Cytokines and chemokines associated with acute respiratory distress syndrome were significantly downregulated in lungs from rabbits pretreated with MEDI3902, compared with c-IgG. These results suggest that MEDI3902 prophylaxis could have potential clinical utility for decreasing the severity of P. aeruginosa ventilator-associated pneumonia.

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.

Pseudomonas aeruginosa Ventilator-Associated Pneumonia Rabbit Model for Preclinical Drug Development.

Development and validation of large animal models of Pseudomonas aeruginosa ventilator-associated pneumonia are needed for testing new drug candidates in a manner that mimics how they will be used clinically. We developed a new model in which rabbits were ventilated with low tidal volume and challenged with P. aeruginosa to recapitulate hallmark clinical features of acute respiratory distress syndrome (ARDS): acute lung injury and inflammation, progressive decrease in arterial oxygen partial pressure to fractional inspired oxygen PaO2:FiO2, leukopenia, neutropenia, thrombocytopenia, hyperlactatemia, severe hypotension, bacterial dissemination from lung to other organs, multiorgan dysfunction, and ultimately death. We evaluated the predictive power of this rabbit model for antibiotic efficacy testing by determining whether a humanized dosing regimen of meropenem, a potent antipseudomonal ß-lactam antibiotic, when administered with or without intensive care unit (ICU)-supportive care (fluid challenge and norepinephrine), could halt or reverse natural disease progression. Our humanized meropenem dosing regimen produced a plasma concentration-time profile in the rabbit model similar to those reported in patients with ventilator-associated bacterial pneumonia. In this rabbit model, treatment with humanized meropenem and ICU-supportive care achieved the highest level of survival, halted the worsening of ARDS biomarkers, and reversed lethal hypotension, although treatment with humanized meropenem alone also conferred some protection compared to treatment with placebo (saline) alone or placebo plus ICU-supportive care. In conclusion, this rabbit model could help predict whether an antibiotic will be efficacious for the treatment of human ventilator-associated pneumonia.

Efficacy of Active Immunization With Attenuated α-Hemolysin and Panton-Valentine Leukocidin in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia.

Staphylococcus aureus is a common pathogen causing infections in humans with various degrees of severity, with pneumonia being one of the most severe infections. In as much as staphylococcal pneumonia is a disease driven in large part by α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL), we evaluated whether active immunization with attenuated forms of Hla (HlaH35L/H48L) alone, PVL components (LukS-PVT28F/K97A/S209A and LukF-PVK102A) alone, or combination of all 3 toxoids could prevent lethal challenge in a rabbit model of necrotizing pneumonia caused by the USA300 community-associated methicillin-resistant S. aureus (MRSA). Rabbits vaccinated with Hla toxoid alone or PVL components alone were only partially protected against lethal pneumonia, whereas those vaccinated with all 3 toxoids had 100% protection against lethality. Vaccine-mediated protection correlated with induction of polyclonal antibody response that neutralized not only α-hemolysin and PVL, but also other related toxins, produced by USA300 and other epidemic MRSA clones.

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.

FDA Public Workshop Summary: Advancing Animal Models for Antibacterial Drug Development.

The U.S. Food and Drug Administration (FDA) hosted a public workshop entitled "Advancing Animal Models for Antibacterial Drug Development" on 5 March 2020. The workshop mainly focused on models of pneumonia caused by Pseudomonas aeruginosa and Acinetobacter baumannii The program included discussions from academic investigators, industry, and U.S. government scientists. The potential use of mouse, rabbit, and pig models for antibacterial drug development was presented and discussed.

Necrotizing Soft Tissue Infection Staphylococcus aureus but not S. pyogenes Isolates Display High Rates of Internalization and Cytotoxicity Toward Human Myoblasts.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) caused by group A Streptococcus (GAS) and occasionally by Staphylococcus aureus (SA) frequently involve the deep fascia and often lead to muscle necrosis. METHODS: To assess the pathogenicity of GAS and S. aureus for muscles in comparison to keratinocytes, adhesion and invasion of NSTI-GAS and NSTI-SA isolates were assessed in these cells. Bloodstream infections (BSI-SA) and noninvasive coagulase-negative staphylococci (CNS) isolates were used as controls. RESULTS: NSTI-SA and BSI-SA exhibited stronger internalization into human keratinocytes and myoblasts than NSTI-GAS or CNS. S. aureus internalization reached over 30% in human myoblasts due to a higher percentage of infected myoblasts (>11%) as compared to keratinocytes (<3%). Higher cytotoxicity for myoblasts of NSTI-SA as compared to BSI-SA was attributed to higher levels of psmα and RNAIII transcripts in NSTI-SA. However, the 2 groups were not discriminated at the genomic level. The cellular basis of high internalization rate in myoblasts was attributed to higher expression of α5ß1 integrin in myoblasts. Major contribution of FnbpAB-integrin α5ß1 pathway to internalization was confirmed by isogenic mutants. CONCLUSIONS: Our findings suggest a factor in NSTI-SA severity is the strong invasiveness of S. aureus in muscle cells, a property not shared by NSTI-GAS isolates.

Treatment Efficacy of MEDI3902 in Pseudomonas aeruginosa Bloodstream Infection and Acute Pneumonia Rabbit Models.

Pseudomonas aeruginosa is a challenge for clinicians due to increasing drug resistance and dwindling treatment options. We report on the activity of MEDI3902, an antibody targeting type 3 secretion protein PcrV and Psl exopolysaccharide, in rabbit bloodstream and lung infection models. MEDI3902 prophylaxis or treatment was protective in both acute models and exhibited enhanced activity when combined with a subtherapeutic dose of meropenem. These findings further support MEDI3902 for the prevention or treatment of serious P. aeruginosa infections.

The Role of Antibiotics in Modulating Virulence in Staphylococcus aureus.

Staphylococcus aureus is often involved in severe infections, in which the effects of bacterial virulence factors have great importance. Antistaphylococcal regimens should take into account the different effects of antibacterial agents on the expression of virulence factors and on the host's immune response. A PubMed literature search was performed to select relevant articles on the effects of antibiotics on staphylococcal toxin production and on the host immune response. Information was sorted according to the methods used for data acquisition (bacterial strains, growth models, and antibiotic concentrations) and the assays used for readout generation. The reported mechanisms underlying S. aureus virulence modulation by antibiotics were reviewed. The relevance of in vitro observations is discussed in relation to animal model data and to clinical evidence extracted from case reports and recommendations on the management of toxin-related staphylococcal diseases. Most in vitro data point to a decreased level of virulence expression upon treatment with ribosomally active antibiotics (linezolid and clindamycin), while cell wall-active antibiotics (beta-lactams) mainly increase exotoxin production. In vivo studies confirmed the suppressive effect of clindamycin and linezolid on virulence expression, supporting their utilization as a valuable management strategy to improve patient outcomes in cases of toxin-associated staphylococcal disease.

MEDI3902 Correlates of Protection against Severe Pseudomonas aeruginosa Pneumonia in a Rabbit Acute Pneumonia Model.

Pseudomonas aeruginosa is among the most formidable antibiotic-resistant pathogens and is a leading cause of hospital-associated infections. With dwindling options for antibiotic-resistant infections, a new paradigm for treatment and disease resolution is required. MEDI3902, a bispecific antibody targeting the P. aeruginosa type III secretion (T3S) protein PcrV and Psl exopolysaccharide, was previously shown to mediate potent protective activity in murine infection models. With the current challenges associated with the clinical development of narrow-spectrum agents, robust preclinical efficacy data in multiple animal species are desirable. Here, we sought to develop a rabbit P. aeruginosa acute pneumonia model to further evaluate the activity of MEDI3902 intervention. In the rabbit model of acute pneumonia, prophylaxis with MEDI3902 exhibited potent dose-dependent protection, whereas those receiving control IgG developed fatal hemorrhagic necrotizing pneumonia between 12 and 54 h after infection. Blood biomarkers (e.g., partial pressure of oxygen [pO2], partial pressure of carbon dioxide [pCO2], base excess, lactate, and creatinine) were grossly deranged for the vast majority of control IgG-treated animals but remained within normal limits for MEDI3902-treated animals. In addition, MEDI3902-treated animals exhibited a profound reduction in P. aeruginosa organ burden and a marked reduction in the expression of proinflammatory mediators from lung tissue, which correlated with reduced lung histopathology. These results confirm that targeting PcrV and Psl via MEDI3902 is a promising candidate for immunotherapy against P. aeruginosa pneumonia.

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.

Targeting Alpha Toxin To Mitigate Its Lethal Toxicity in Ferret and Rabbit Models of Staphylococcus aureus Necrotizing Pneumonia.

The role broad-spectrum antibiotics play in the spread of antimicrobial resistance, coupled with their effect on the healthy microbiome, has led to advances in pathogen-specific approaches for the prevention or treatment of serious bacterial infections. One approach in clinical testing is passive immunization with a monoclonal antibody (MAb) targeting alpha toxin for the prevention or treatment of Staphylococcus aureus pneumonia. Passive immunization with the human anti-alpha toxin MAb, MEDI4893*, has been shown to improve disease outcome in murine S. aureus pneumonia models. The species specificity of some S. aureus toxins necessitates testing anti-S. aureus therapeutics in alternate species. We developed a necrotizing pneumonia model in ferrets and utilized an existing rabbit pneumonia model to characterize MEDI4893* protective activity in species other than mice. MEDI4893* prophylaxis reduced disease severity in ferret and rabbit pneumonia models against both community-associated methicillin-resistant S. aureus (MRSA) and hospital-associated MRSA strains. In addition, adjunctive treatment of MEDI4893* with either vancomycin or linezolid provided enhanced protection in rabbits relative to the antibiotics alone. These results confirm that MEDI4893 is a promising candidate for immunotherapy against S. aureus pneumonia.

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches.

Critical Role of Alpha-Toxin and Protective Effects of Its Neutralization by a Human Antibody in Acute Bacterial Skin and Skin Structure Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility to S. aureus superantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhla strains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhla mutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.

Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins.

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Identifying potential therapeutic targets of methicillin-resistant Staphylococcus aureus through in vivo proteomic analysis.

BACKGROUND: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.

Global gene expression of methicillin-resistant Staphylococcus aureus USA300 during human and mouse infection.

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.

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.

Clonal composition and community clustering of drug-susceptible and -resistant Escherichia coli isolates from bloodstream infections.

Multidrug-resistant Escherichia coli strains belonging to a single lineage frequently account for a large proportion of extraintestinal E. coli infections in many parts of the world. However, limited information exists on the community prevalence and clonal composition of drug-susceptible E. coli strains. Between July 2007 and September 2010, we analyzed all consecutively collected Gram-negative bacterial isolates from patients with bloodstream infection (BSI) admitted to a public hospital in San Francisco for drug susceptibility and associated drug resistance genes. The E. coli isolates were genotyped for fimH single nucleotide polymorphisms (SNPs) and multilocus sequence types (MLSTs). Among 539 isolates, E. coli accounted for 249 (46%); 74 (30%) of them were susceptible to all tested drugs, and 129 (52%) were multidrug resistant (MDR). Only five MLST genotypes accounted for two-thirds of the E. coli isolates; the most common were ST131 (23%) and ST95 (18%). Forty-seven (92%) of 51 ST131 isolates, as opposed to only 8 (20%) of 40 ST95 isolates, were MDR (P < 0.0001). The Simpson's diversity index for drug-susceptible ST genotypes was 87%, while the index for MDR ST genotypes was 81%. ST95 strains were comprised of four fimH types, and one of these (f-6) accounted for 67% of the 21 susceptible isolates (P < 0.003). A large proportion (>70%) of both MDR and susceptible E. coli BSI isolates represented community-onset infections. These observations show that factors other than the selective pressures of antimicrobial agents used in hospitals contribute to community-onset extraintestinal infections caused by clonal groups of E. coli regardless of their drug resistance.

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.