Severe Pneumonia Caused by Coinfection With Influenza Virus Followed by Methicillin-Resistant Staphylococcus aureus Induces Higher Mortality in Mice

Abstract

Background: Coinfection with influenza virus and bacteria is a major cause of high mortality during flu pandemics. Understanding the mechanisms behind such coinfections is of utmost importance both for the clinical treatment of influenza and the prevention and control of epidemics. Methods: To investigate the cause of high mortality during flu pandemics, we performed coinfection experiments with H1N1 influenza virus and Staphylococcus aureus in which mice were infected with bacteria at time points ranging from 0 to 7 days after infection with influenza virus. Results: The mortality rates of mice infected with bacteria were highest 0-3 days after infection with influenza virus; lung tissues extracted from these co-infected mice showed higher infiltrating cells and thicker lung parenchyma than lung samples from coinfected mice in which influenza virus was introduced at other times and sequences. The levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-6 in the 0-3 day coinfected group were significantly higher than those in the other groups (p < 0.01), as were the mRNA levels of IFN-γ, IL-6, and TNF-α. Coinfection with influenza virus and S. aureus led to high mortality rates that are directly dependent on the sequence and timing of infection by both pathogens. Moreover, coinfection following this particular schedule induced severe pneumonia, leading to increased mortality. Conclusions: Our data suggest that prevention of bacterial co-infection in the early stage of influenza virus infection is critical to reducing the risk of clinical mortality.

Keywords: bacteria; coinfection; influenza; methicillin-resistant Staphylococcus aureus; pneumonia.

Figure 1

Mortality in mice coinfected with influenza virus and methicillin-resistant S. aureus differed with the sequence and timing of infection. (A) Survival rates in mice coinfected with influenza virus (PR8) and methicillin-resistant S. aureus (MRSA) were infection sequence- and time-dependent. In mice coinfected with equivalent doses, those first infected with PR8 followed by MRSA had higher mortality rates than those first infected with MRSA. (B) Mortality of mice coinfected using various infection sequences and timings (***p < 0.001; ****p < 0.0001, n = 6). (C) Body weight changes in coinfected mice. (D) The cumulative mortality rate after coinfection of mice in groups d to d-7 with PR8 shows that the coinfected mice had the highest mortality rate on the seventh day post-PR8 infection. (E) The cumulative mortality rate after groups d to d-7 were coinfected with MRSA; mortality rates rose in the first 5 days after MRSA infection.

Figure 2

Pathological differences in mice coinfected with influenza virus and methicillin-resistant S. aureus using different infection sequences and times. (A) Anatomy of the lung tissues of coinfected mice with different infection sequences and times. Lung tissue injury in groups d-0, d-1, d-2, and d-3 was significantly more severe than that in the other groups as denoted by the color change from pink to dark purple. (B) The lung indices of mice in groups d-0, d-1, d-2, and d-3 were significantly higher than in the other groups (**p < 0.01; ***p < 0.001, n = 6). (C) Lung pathological sections of coinfected mice with different infection sequences and times (15 × ). Lung injury in groups d-0, d-1, d-2, and d-3 on the fifth day after infection was significantly higher than in the other groups. (D) Local enlarged view of pathological sections of lungs showing severe lung with 100 × magnification. The alveoli showed extensive atrophy or did not dilate. Pulmonary consolidation, in which severe infiltration of inflammatory cells (lymphocytes and granulocytes), was observed. PR8, influenza A virus strain; MRSA, methicillin-resistant S. aureus; PBS, phosphate-buffered saline.

Figure 3

Over-proliferation of pulmonary pathogens in coinfected mice in group d-2. (A) PR8 in the lungs of group d-2 mice showed significant proliferation between day 1 and day 5 after coinfection compared to the control PR8 group and the group infected with a non-lethal dose of PR8 alone. (B) MRSA in the lungs of group d-2 mice showed significant proliferation between day 1 and day 5 after coinfection compared with the control PR8 group and the group infected with a non-lethal dose of MRSA alone. (C) Real-time fluorescence quantitative PCR was used to detect the mRNA levels of PR8 in lung homogenates from group d-2 mice acquired on the fifth day of coinfection. Compared with the control PBS group and the group infected with a non-lethal dose of PR8 alone, mRNA levels in group d-2 far exceeded those in the control groups (****p < 0.0001, n = 6). (D) Lung homogenate from mice in the d-2 group was obtained on the fifth day post-coinfection, and bacterial culture was performed. Compared with the control PBS group and the group infected with a non-lethal dose of PR8 alone, the MRSA bacterial load in the lungs of group d-2 mice far exceeded those of the control groups (****p < 0.0001, n = 6). PR8, influenza A virus strain; MRSA, methicillin-resistant S. aureus; PBS, phosphate-buffered saline.

Figure 4

Inflammatory factor levels in the BALF of mice coinfected with influenza virus and methicillin-resistant S. aureus. Levels of (A) IFN-γ, (B) TNF-α, (C) IL-6, and (D) IL-8 in the BALF of coinfected mice are shown at different infection sequences and times. For multiple comparisons, we used one-way analysis of variance with the Tukey post hoc test. Data are represented as mean ± standard error of the mean. (N.S., not significant; *p < 0.05; **p < 0.01; ***p < 0.001, n = 6). BALF, bronchioalveolar lavage fluid; PR8, influenza A virus strain; MRSA, methicillin-resistant S. aureus; PBS, phosphate-buffered saline. IFN, interferon; TNF, tumor necrosis factor; IL, interleukin.

Figure 5

Inflammatory cytokine mRNA levels in mice coinfected with influenza virus and methicillin-resistant S. aureus. mRNA levels of (A) IFN-γ, (B) TNF-α, (C) IL-6, and (D) IL-8 in the lung tissues of coinfected mice according to different infection sequences and times. For multiple comparisons, we used one-way analysis of variance with the Tukey post hoc test. Data are represented as mean ± standard error of the mean. (N.S., not significant; *p < 0.05; **p < 0.01; ***p < 0.001, n = 6). PR8, influenza A virus strain; MRSA, methicillin-resistant S. aureus; PBS, phosphate-buffered saline. IFN, interferon; TNF, tumor necrosis factor; IL, interleukin.