A postinfluenza model of Staphylococcus aureus pneumonia

Abstract

Background: Postinfluenza Staphylococcus aureus pneumonias are increasingly recognized as a major form of life-threatening infections.

Methods: A mouse model of postinfluenza S. aureus pneumonia was developed. Mice were intranasally infected with bacteria alone or bacteria plus virus. Infection was assessed by mouse survival, lung histopathology, bacterial density in the lungs, and cellular response to infection.

Results: Mice infected with both influenza virus and S. aureus showed higher mortality, greater lung parenchymal damage, and greater bacterial density at metastatic tissue sites than mice infected with only S. aureus. At 4 h, more polymorphonuclear leukocytes and fewer CD11c(+) cells were found in lung samples from mice infected with virus and bacteria than in those from mice infected with bacteria. alpha-Hemolysin and protein A were maximally expressed 4 h after infection, and Panton-Valentine leukocidin was maximally expressed 72 h after infection, with higher levels of alpha-hemolysin expression in mice infected with bacteria alone. Interferon gamma expression was higher in tissue collected from mice infected with virus plus bacteria than in those from bacteria-infected mice.

Conclusions: The results from this model demonstrate diverse effects caused by antecedent influenza virus infection, which have a profound influence on the morbidity and mortality associated with S. aureus pneumonia.

Figure 1

Survival assay with mice infected with different inocula of influenza virus (V, low dose; V“, high dose) and Staphylococcus aureus (B, low dose; B”, high dose). Mice were infected as outlined in Methods, with bacterial challenge 72 h after viral inoculation.

Figure 2

Histopathology of mouse lungs infected with low viral and bacterial doses. A, Lung tissue after instillation of phosphate-buffered saline showing unremarkable bronchiole and lung parenchyma without acute or chronic inflammation. B, Lung tissue 72 h after virus instillation and 4 h after bacterial infection showing acute suppurative bronchiolitis with extension into alveoli adjacent to the airway. Inset, Tissue Gram stain showing gram-positive cocci (arrow) in areas of suppurative inflammation (original magnification, ×600). C, Presence of acute pneumonia with neutrophils in the distal bronchiole and in alveolar spaces 24 h after bacterial instillation. Mild chronic inflammation is also present in the interstitium and in perivascular locations. D, Reduced acute inflammation 72 h after bacterial instillation. Inflammation is still seen as neutrophils and neutrophil debris throughout the lung interstitium with congestion and intraalveolar fluid. E, Section of trachea after vehicle instillation showing normal pseudostratified ciliated epithelium and lamina propria accessory glands. F, Sections of trachea 72 h after viral instillation showing disarray of epithelium consistent with regeneration and large reactive basal epithelial cells with loss of ciliated cell polarity. A–F, hematoxylineosin stain; original magnification, ×200.

Figure 3

Bacterial density in the lung, blood (spleen), and other tissue sites. A, Lung bacterial burden in mice infected with low-dose bacteria at different time points with or without antecedent low dose of virus. B, Bacterial counts in the livers, kidneys, and spleens of mice after infection with low doses of either virus or bacteria. B, bacteria; CFU, colony-forming units; VB, virus plus bacteria.

Figure 4

Flow cytometry and enzyme-linked immunosorbent assay measurement of cellular responses in mice infected with influenza virus plus Staphylococcus aureus or S. aureus only. A, Polymorphonuclear (PMN) leukocytes (Ly6G/CD45⁺). B, Dendritic cells and alveolar macrophages (CD11c⁺/CD45⁺). Data are percentages of total CD45⁺ population. C, Levels of keratinocyte-derived chemokine (KC) in bronchoalveolar lavage (BAL) fluid of infected mice. B, bacteria only; NS, not significant; VB, virus followed by bacteria.