New therapeutic approaches are urgently needed to improve survival outcomes for patients with necrotizing pneumonia caused by Staphylococcus aureus One such approach is adjunctive treatment with intravenous immunoglobulin (IVIG), but clinical practice guidelines offer conflicting recommendations. In a preclinical rabbit model, prophylaxis with IVIG conferred protection against necrotizing pneumonia caused by five different epidemic strains of community-associated methicillin-resistant S. aureus (MRSA) as well as a widespread strain of hospital-associated MRSA. Treatment with IVIG, either alone or in combination with vancomycin or linezolid, improved survival outcomes in this rabbit model. Two specific IVIG antibodies that neutralized the toxic effects of α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL) conferred protection against necrotizing pneumonia in the rabbit model. This mechanism of action of IVIG was uncovered by analyzing loss-of-function mutant bacterial strains containing deletions in 17 genes encoding staphylococcal exotoxins, which revealed only Hla and PVL as having an impact on necrotizing pneumonia. These results demonstrate the potential clinical utility of IVIG in the treatment of severe pneumonia induced by S. aureus.

b 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.

g 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 (⌬hla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented ⌬hla 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-alphatoxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the ⌬hla 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.

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.

Staphylococcus aureus is a common cause of skin and soft tissue infections and invasive disease, such as bacteremia and endocarditis. Tedizolid, the microbiologically active moiety of the prodrug tedizolid phosphate (TZD), is a novel oxazolidinone approved for treatment of acute bacterial skin and skin structure infections caused by Gram-positive bacteria, including strains of methicillin-resistant S. aureus (MRSA). Tedizolid phosphate is rapidly cleaved in the bloodstream to yield the active component, tedizolid. It is approximately 4 times more active by weight than linezolid against S. aureus in vitro (1) and 16 times more active against cfr plasmid carrying linezolid-resistant staphylococci in vitro (2). Moreover, unlike the bacteriostatic activity of linezolid, tedizolid has been shown to have some bactericidal activity in a neutropenic mouse thigh model (3, 4). To assess further its potency and bactericidal activity in vivo, tedizolid was compared to vancomycin and daptomycin in a rabbit model of aortic valve endocarditis (AVE) caused by the MRSA strain COL. MATERIALS AND METHODSBacterial strains. S. aureus strain COL is a homogeneous, methicillinresistant strain. COL inoculum was prepared by diluting a frozen stock in 0.9% injectable sodium chloride. The frozen stock was prepared from an overnight culture grown in tryptic soy broth. Cells were washed and resuspended in phosphate-buffered saline with 10% glycerol and stored at Ϫ80°C.Susceptibility studies. Susceptibility studies were performed by broth dilution to determine the MICs using standard CLSI methods (5). Briefly, the bacteria and drugs were diluted in cation-adjusted Mueller-Hinton broth (CAMHB) and incubated at 37°C overnight. Ca 2ϩ supplementation was provided for daptomycin testing (6). The MIC was determined as the lowest concentration of drug that inhibited growth. Tedizolid and daptomycin standard powder for in vitro testing were provided by the manufacturers, and vancomycin was purchased from Sigma-Aldrich.Time-kill studies. Time-kill studies were conducted in duplicate at 37°C in 10 ml of CAMHB containing vancomycin at 5 g/ml (5ϫ MIC), daptomycin at 5 g/ml (5ϫ MIC), or tedizolid at 2 g/ml (16ϫ MIC) at a starting inoculum of 10 6 CFU/ml. Rabbit model of endocarditis. New Zealand White rabbits (2.5 to 3 kg) were used. Endocarditis was established by standard methods (7). Briefly, a cutdown was made over the right carotid artery. A polyethylene catheter was introduced via carotid arteriotomy, positioned into the left ventricle, and sutured in place. Forty-eight hours later, a 1-ml suspension of 10 7 to 10 8 CFU of S. aureus in 0.9% NaCl was injected intravenously (i.v.). On postinoculation day 1, approximately 16 to 18 h after infection, untreated control rabbits were sacrificed to determine pretreatment bacterial counts. The hearts, spleens, and kidneys were harvested. Aortic valves and endocardial vegetations and approximately 0.2-g samples of spleen and kidney were placed in 1.0 ml of 0.9% NaCl and homogenized with a tissue grinder. Ten-...