We have demonstrated here that intranasal infection with sublethal doses of IAV into mice resulted in lethal infection after further intranasal infection with sublethal doses of GAS strains (Fig. and Table ). The majority of the superinfected mice died within 4 or 5 days of the GAS infection. The mortality rate of mice superinfected with TSLS-GAS strains was higher than that of mice superinfected with non-TSLS-GAS strains (Table ). In addition, almost 10% of the dead mice had developed necrotizing fasciitis. It is not easy to elucidate the similarities and differences of influenza virus infection in mice and in humans from the results of the present study because the method used was not the same as infection via the upper respiratory tract in humans. However, since influenza virus infection leads both mice and humans to acute respiratory inflammation, we believe that the pathogenicity of influenza virus infection in mice and humans may be similar.
The most intriguing result from the present mouse model was the appearance of necrotizing fasciitis at the forelimb and hind leg but not the primary infection sites such as the upper respiratory tract and skin (Fig. and Table ). Mice with necrotizing fasciitis carried greater numbers of GAS organisms in their blood and internal organs compared to those that died without necrotizing fasciitis (Fig. ). Furthermore, aggregated clusters of GAS organisms were frequently seen in blood vessels near the affected sites (Fig. ). These results suggest that the outbreak of necrotizing fasciitis is induced by GAS that has spread via sepsis from the original infection site.
GAS bacteria were most frequently recovered from the lungs and other organs of dead mice after the superinfection, where IAV coexisted with GAS for up to 24 h after the GAS infection of IAV-infected mice (Fig. ). Thus, there is the possibility that IAV-infected alveolar epithelial cells that express HA promote the internalization of GAS into these cells (Fig. ). HA, a 75-kDa protein expressed on the capsule of the virus (26
), participates in the fusion between the virus envelopes and endosomes of virus-infected cells prior to viral invasion of the cytoplasm (4
). Recently, HA has been shown to bind to sialic acid present in the capsule of group B streptococcus (15
). We observed with an electron microscope that IAV also binds to GAS directly (Fig. ). Furthermore, we determined the quantitative direct binding of GAS to IAV by counting the GAS organisms bound to IAV coated on the plate. In the experiment, the number of bacteria bound to the plate coated with 4 μg of IAV/ml was 10 times higher than the number of bacteria bound to the plate not coated with IAV (unpublished data). These results suggest that binding of GAS and IAV by HA expression might contribute to the enhanced pathogenicity of invasive GAS infection during dual infection. The organisms of GAS from TSLS and non-TSLS origins were found to possess only trace amounts of sialic acid. Pretreatment of the GAS with sialidase did not affect internalization of the bacterial cells to the IAV-infected epithelial cells (data not shown). Therefore, the ligand to HA remains to be determined.
It should be noted that prior nonlethal IAV infection was critically important for bacteria to accomplish an invasive type of infection in the lungs. In fact, GAS infection prior to IAV infection or simultaneous infection with GAS and IAV did not increase mouse mortality (Fig. ). HA was expressed on the alveolar epithelial cells within 24 h after IAV infection (Fig. ), which then promoted GAS internalization. However, treatment with anti-HA MAb suppressed this internalization and therefore suppressed the outbreak of the invasive GAS infection (Fig. and ).
Dallaire et al. (7
) demonstrated that the induction of severe pneumonia by infection with Streptococcus pneumoniae
was associated with an increment of proinflammatory cytokine productions. In the present study, we found that the secretion levels of IL-6 and TNF-α in the BAL fluid of superinfected mice were significantly higher than in the BAL fluid from IAV- or GAS-infected mice (Fig. ). The highest levels of these cytokines were detected 2 to 3 days after GAS infection. Taken together, an incremental increase of proinflammatory cytokines is followed by an enhanced internalization of GAS in the lungs caused by the superinfection, which results in severe pneumonia.
Several investigators have reported that the expression of SPEA in GAS might be associated with the outbreak of invasive GAS infection (6
). In this regard, Zhang et al. (39
) found that mice given nonlethal doses of IAV and SEB died within 4 days of the SEB exposure, which suggests that streptococcal superantigen, mitogenic factor, SPEA, and SPEC play important roles in the induction of the lethal synergism. However, no correlation between the expression of the superantigen genes of GAS and mortality in superinfected mice was found in the present study (Table ). Therefore, superantigens did not significantly affect the pathogenesis of the experimental invasive diseases in mice with superinfection.
In the northern hemisphere, seasonal peaks of infection by both IAV and GAS occur from October to April (3
). Indeed, more than 80% of the invasive GAS infection of patients in Canada in 1990 to 1991 were found to occur in winter (8
). A recent investigation revealed that more than 90% of those who died from lethal Spanish influenza virus (type A, H1N1) infections exhibited various symptoms of severe bacterial pneumonia (37
). The prevalence of GAS-dependent necrotizing fasciitis was also shown in the period when Spanish influenza virus infections prevailed (25
). Taken together, these findings suggest that some patients with invasive GAS infection may also be infected with IAV. Therefore, the correlation between the outbreak of disease and the mixed infection requires clarification in humans. In addition, Locci (23
) suggested that secondary bacterial infections, e.g., Staphylococcus aureus
, GAS, group B streptococcus, Streptococcus pneumoniae
, and Haemophilus influenzae
, are closely associated with complications in influenza. Some groups of researchers have also found that infection with bacteria after IAV infection leads to the death of mice (11
). In the present study, all mice infected with both IAV and GAS developed severe pneumonia (Fig. ), which we determined to be due to the GAS infection. This fact raises the possibility that HA expressed on the virus-infected alveolar epithelial cells after IAV infection may lead to different kinds of bacterial infections in the lungs.
In the present study, we have established a novel and unique mouse model for the induction of invasive GAS infection in order to mimic human TSLS. The induction of invasive diseases in this model system is mainly due to the existence of IAV HA on the epithelial surfaces of lung tissues. Therefore, it is quite possible that a mixed infection with the influenza virus and GAS is one of the most essential factors causing outbreaks of invasive GAS diseases.