Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection

Abstract

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Figure 1:. SARS-CoV-2-pneumococcal coinfection in K18-hACE2 mice.

Kaplan-Meier survival curves (A), percent weight loss (B), cumulative clinical score (C), and temperature (D) of mice infected with SARS-CoV-2 (250 PFU; white circles, solid lines) followed by infection with 10³ CFU D39 at 3 d (yellow diamonds, dotted lines), 5 d (magenta squares, dashed lines), or 7 d (cyan triangles, dash-dotted lines) pvi. Data are shown as the mean ± standard deviation (SD) and significant differences are indicated by *, P < 0.05; **, P < 0.01 for comparisons between SARS-CoV-2 infection and SARS-CoV-2-pneumococcal coinfection.

Figure 2:. Dynamics of pathogen loads during SARS-CoV-2 infection and pneumococcal coinfection.

Lung bacterial loads (CFU/lung) (A), blood bacterial loads (B), and lung viral loads (PFU/lung) (C) in female (circles) and male (triangles) mice infected with SARS-CoV-2 (250 PFU; white) followed by infection with 10³ CFU D39 at 3 d (yellow), 5 d (magenta), or 7 d (cyan) pvi. Each symbol represents a single mouse and the mean ± standard deviation (SD) are for combined male and female groups. Significant differences are indicated by ns, not significant; *, P < 0.05; ***, P < 0.0001. For bacterial titers, comparison was with the inoculum (dotted line). (D-E) Representative immunohistochemical staining of SARS-CoV-2 nucleocapsid protein in the lungs of mice 24 h after they were infected with SARS-CoV-2 (250 PFU) ± 10³ CFU D39 at 3 d (D) or at 5 d (E) pvi.

Figure 3:. Immune cell dynamics during SARS-CoV-2 infection and pneumococcal coinfection.

Total neutrophils (A), F4/80^midCD11c^midCD11b⁺ monocytes/macrophages (B), inflammatory macrophages (iMΦ) (F4/80^hiCD11c^hiCD11b⁺) (C), alveolar macrophages (AMΦ) (F4/80^hiCD11c^hiCD11b⁻MHC-II^low/−) (D), CD19⁺ B cells (E), CD4⁺ T cells (F), and CD8⁺ T cells (G) in the lungs of female (circles) and male (triangles) mice infected with SARS-CoV-2 (250 PFU; open symbols) followed by infection with 10³ CFU D39 at 3 d (yellow), 5 d (magenta), or 7 d (cyan) pvi. Each symbol represents a single mouse and the mean ± standard deviation (SD) are for combined male and female groups. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons between indicated groups and by †, P < 0.05 for differences between males and 422742MCfemales within a group or between coinfection times within 17 d group.

Figure 4:. Pulmonary cytokines and chemokines during SARS-CoV-2 infection and SARS-CoV-2-pneumococcal coinfection.

Total IL-6 (A), IL-18 (B), LIF (C), IL-15 (D), CXCL10 (E), RANTES (F), IL-3 (G), IL-22 (H), IL-28 (I), and MIP-2α (J) in the lungs of female (circles) and male (triangle) mice infected with SARS-CoV-2 (250 PFU; white) followed by infection with 10³ CFU D39 at 3 d (yellow), 5 d (magenta), or 7 d (cyan) pvi. Each symbol represents a single mouse and the mean ± standard deviation (SD) are for combined male and female groups. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons between indicated groups. Plots depicting additional cytokine and chemokine quantities (absolute log₁₀ picograms) are in Figure S5 and a heatmap representing the normalized quantity (average log₂ change over naïve) is in Figures S6.

Figure 5:. Lung pathology during SARS-CoV-2 infection and pneumococcal coinfection.

Average endothelial hypertrophy (A), peribronchiolar/perivascular lymphoid cells (B), interstitial inflammation/septal thickening (C), alveolar inflammation (D), extent of alveolar involvement (E), and consolidation (F) in lungs of mice infected with SARS-CoV-2 (250 PFU; open bars) followed by 10³ CFU D39 at 3 or 5 d pvi (filled bars). Plots represent the mean ± standard deviation (SD) bars for combined male and female groups.