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.

Preventive effect of the phage VB-SavM-JYL01 on rabbit necrotizing pneumonia caused by Staphylococcus aureus.

Staphylococcus aureus is one of the most important pathogens causing rabbit necrotizing pneumonia and brings huge economic losses to rabbit production. This study investigated the preventive effect of a phage on rabbit necrotizing pneumonia caused by S. aureus. S. aureus S6 was isolated from the lungs of rabbits suffering necrotizing pneumonia and identified. A novel phage named VB-SavM-JYL01 was isolated by using S. aureus S6 as a host and showed a broader host range than the phages GH15 and K. The genome of VB-SavM-JYL01 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. A single intranasal administration of VB-SavM-JYL01 (3 × 109 PFU) could effectively improve the survival rate at 48 h to 90% (9/10) compared with the survival rate of 10% and 80% observed with the PBS or linezolid treatment, respectively. The bacterial count in the lungs of rabbits treated with the phage VB-SavM-JYL01 was 4.18 × 104 CFU/g at 24 h, which was significantly decreased compared to that of rabbits treated with PBS (7.38 × 107 CFU/g) or linezolid (3.12 × 105 CFU/g). The phage treatment significantly alleviated lung tissue damage. The levels of total proteins, Panton-Valentine leukocidin (PVL), alpha-toxin (Hla) and cytokines in the lungs of the rabbits treated with the phage were significantly lower than those of the rabbits treated with PBS and similar to those of the rabbits treated with linezolid. These data demonstrate the potential utility of phage as an alternative for preventing rabbit necrotizing pneumonia caused by S. aureus.

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.