A mouse model of postinfluenza CA-MRSA pneumonia was developed for this study. Our findings highlight a number of differences in the respiratory infection and its outcome on the basis of whether there was antecedent influenza virus infection. Most noteworthy was the increased mortality in mice infected with virus plus bacteria. This increased mortality was associated with evidence of greater pathological damage to the lung parenchyma, a higher-grade bacteremia, increased metastatic seeding to the liver and kidney, and an altered cellular response to infection in the infected mice. In contrast there were minimal differences in virulence between different strains of CA-MRSA. Of interest, the pattern of PVL expression was different from those of both protein A and α-hemolysin, regardless of time point or whether there was antecedent viral infection.
The importance of postinfluenza bacterial pneumonias as a primary cause of death in the 1918 influenza pandemic has recently been reemphasized [9
]. Commenting on the mortality associated with the 1918 pandemic, investigators noted that most influenza pneumonias were self-limited and that the supervening bacterial superinfections accounted for most of the fatalities. This interpretation is supported by an analysis of the timeline of infections and reviews of firsthand accounts as well as the histopathological material available from the epidemic [9
]. This concern regarding the primary cause of death has taken on increased importance with the emergence of the highly pathogenic H5N1 influenza strain that appears genetically similar to the H1N1 1918 strain, as well as the recently detected “swine flu” H1N1 strain [20
Investigators have recently described several models of CA-MRSA pneumonia [22
]. These studies examined the virulence of different strains of CA-MRSA. However, the role of antecedent viral infections in these different models was not examined. Montgomery et al [24
] reported that USA300 isolates were more virulent than USA400 strains in a rat model of pneumonia. These investigators also examined the in vitro expression of S. aureus
virulence determinants, including PVL and α
-hemolysin, finding higher levels of virulence gene expression in the USA300 strains. A subsequent study using the same model failed to identify differences in the host inflammatory and cytokine response in mice infected with the PVL isogenic pair [25
]. In contrast, Labandeira-Rey et al [22
] reported that the PVL toxin was primarily responsible for the parenchymal damage. Other investigators have failed to confirm these findings and presented data suggesting that α
-hemolysin was the primary factor [26
]. Our studies failed to find a difference in virulence between the isogenic strains USA300 and USA400 [17
]. The role of PVL in CA-MRSA pneumonia therefore remains uncertain, perhaps in part because of the use of different strains and/or different animal species. In our study, PVL levels were low overall and maximally expressed only 72 h after infection; in contrast, for α
-hemolysin higher levels were observed 4 h after infection, perhaps suggesting a lesser role for PVL.
Our histopathological findings demonstrated (as have findings of others) that influenza virus infection alone primarily damages the tracheobronchial epithelium [28
]. This damage may contribute to the inability to clear secretions efficiently and may lead to increased accumulation of debris in the alveoli. Damage at the 4-h time point was increased in mice previously infected with influenza virus, with a pronounced inflammatory response and extensive parenchymal infiltration. This difference was not evident at the later time points.
In addition to the pathological changes that undoubtedly contributed to the severity of these infections, immunological effects caused by the viral infection contributed to the progression of subsequent bacterial infections. Several studies have recently demonstrated a role for the IFN-γ
signaling pathway as a mediator of the inflammatory—and more specifically neutrophil—response to S. aureus
]. Shornick et al [31
] reported that the innate immune response due to activation of the IFN signaling protein Stat1 is impaired during influenza virus infection. This may result from the effects of the influenza nonstructural protein NS1 [32
]. More recently, Sun and Metzger [33
] and Shahangian et al [14
] reported an impaired antibacterial response to S. pneumoniae
infection mediated by the IFN-γ
produced during influenza virus infection. S. aureus
was also demonstrated by Martin et al [34
] to induce type I IFN. These observations are further supported by our results showing higher levels of expression of IFN-γ
, as well as an increased PMN cellular response in mice infected with both virus and bacteria compared with those infected with bacteria alone. The failure to contain the infection in the lungs, as evidenced by seeding of other tissues, also suggests an impaired host response, possibly caused by the defects in alveolar macrophage function described elsewhere or the demonstrated parenchymal damage. These results highlight the potential importance of the impaired immune response in the observed pathological damage and the resultant inability to contain the bacterial infection.
The in vivo RNA levels were higher for α
-hemolysin in the bacteria-infected group than in the mice infected with bacteria plus influenza virus. The basis for this observation, though uncertain, is of interest. The finding suggests that influenza virus may cause alterations in the level of selected bacterial gene expression, perhaps via effects on staphylococcal regulatory genes such as agr
, which up-regulates both α
-hemolysin and PVL expression [35
]. Though a preliminary observation, it is worth further investigation.
This study has a number of limitations. The mouse model, though in many ways reflecting the clinical setting of necrotizing CA-MRSA pneumonia, does not necessarily reflect the host response in humans. Specifically, mouse white blood cells appear to be less susceptible to staphylococcal leukotoxins than human cells [36
]. This effect may have diminished the pathological findings in the lungs. Only a limited number of S. aureus
isolates were used in this study. Results may vary when additional strains are used. Despite the limitations, these findings demonstrate the importance of antecedent influenza infections in the outcome of possible subsequent S. aureus
Our model not only demonstrated the differences between bacterial and dual viral and bacterial respiratory tract infection, but it also provides a possible rationale for the enhanced morbidity and mortality encountered in these dual infections. Future studies can now address the pathogenesis and therapy of these life-threatening infections in greater detail.