Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice

Abstract

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia. IMPORTANCE Pneumonia is one of the leading causes of death worldwide. Here, we compared the impact of bacterial- and viral-induced pneumonia on the respiratory and gastrointestinal microbiome. Using a meta-omics approach, we identified specific profiles that allow discrimination between bacterial and viral causative.

Keywords: 16S rRNA gene sequencing; Streptococcus pneumoniae; influenza A; meta-omics; metaproteomics; microbiome; pneumonia.

FIG 1

Bacterial and viral infections of C57BL/6J mice. (A) One group of female C57BL/6J mice was intranasally colonized with 1 × 10⁷ CFU S. pneumoniae 19F (19F_C), a second group was infected with 1 × 10⁸ CFU S. pneumoniae 19F (19F_P), and a third group was challenged with PBS as a control. Weight was monitored daily over 7 consecutive days. (B) CFU counts in nasal wash specimens (NAL) at the endpoint of the analyses. (C) C57BL/6J mice were intranasally inoculated with 1 × 10⁵ PFU of H1N1 or challenged with PBS, and weight was monitored daily over 5 consecutive days. (D) To exclude potential bacterial contamination, nasal wash specimens were plated on blood agar. Mean values ± standard deviations (SD) are displayed in panels A and C. Each dot represents one mouse, and horizontal lines display mean values (B and D). Two independent experiments with at least five mice per group were performed: total, for panels A and B, PBS, n = 10, 19F_C, n = 10, and 19F_P, n = 12; total for panels C and D, PBS, n = 6, and H1N1, n = 12. The level of significance between the groups was determined using Kruskal-Wallis test with Dunn’s multiple-comparison posttest.

FIG 2

(A) Impact on the taxonomic composition of the URT microbiome of C57BL/6J mice during S. pneumoniae 19F colonization and infection (PBS, n = 10; 19F_C, n = 10; 19F_P, n = 12). (B) Alterations in composition of the respiratory microbiome after IAV infection (PBS, n = 4; H1N1, n = 12). For better illustration, only high-abundance families (relative abundance > 0.01) with significant changes in relative abundance are shown (ANOVA; P = 0.05).

FIG 3

Effects on the taxonomic composition of the URT microbiome of S. pneumoniae 19F colonization (19F_C) or infection (19F_P) (A) and influence of viral pneumonia (B), illustrated in a PCA plot. Shown are results for PBS-treated controls (gray crosses and gray squares), S. pneumoniae-colonized (blue triangles) and -infected (orange circles) mice, and H1N1-infected mice (light orange diamonds).

FIG 4

Taxonomic profile of the murine gastrointestinal microbiome highlighting similar and countervailing trends, as well as specific changes due to S. pneumoniae 19F or H1N1 pneumonia, based on metaproteomic data. For better illustration, only higher-abundance families with significant changes are shown (ANOVA; P < 0.05). Color code: gray bars, PBS control groups; blue bars, 19F-colonized group; orange bars, 19F-infected group; and light orange, H1N1-infected group. For statistics, see Tables S6A and B.

FIG 5

PCA plot illustrating the effects of pneumococcal colonization and mild infection (A), as well as the influence of mild H1N1 infection (B) on the taxonomic composition of the gastrointestinal microbiome. PBS-treated control mice (gray crosses and gray squares), S. pneumoniae-colonized (blue triangles) and -infected (orange circles) mice, and H1N1-infected mice (light orange diamonds).

FIG 6

Voronoi treemaps illustrating changes in the functional composition of the murine gastrointestinal microbiome during pneumococcal colonization and infection, as well as H1N1 infection, based on the eggNOG database. The Voronoi treemap on the top left provides an overview of the corresponding functional category of the eggNOG database, to which the individual PGs have been assigned. On the top right, the influence on the gastrointestinal microbiome by pneumococcal colonization of the URT is shown. The Voronoi treemap on the bottom right monitors effects of severe pneumonia on the functional composition of the GIT microbiome, induced by S. pneumoniae infection. The influence of the H1N1 infection is illustrated in the Voronoi treemap on the bottom left. The size of the fields represents the average abundance of the identified PGs under all conditions. For comparison, the log₂ fold change (FC) (challenged versus healthy) was used. The two-sided gradient orange fields represent an increase in abundance (log₂ FC value of 1.5), the dark blue fields a decrease in abundance (log₂ FC value of −1.5), and the gray fields no change in abundance. Higher saturated color indicates more pronounced alterations in the functional composition of the gastrointestinal microbiome.

FIG 7

PCA plot illustrating the effects of bacterial and viral pneumonia on the functional assignment of the gastrointestinal microbiome. Shown are results for PBS-treated control mice (gray crosses and gray squares), S. pneumoniae-colonized (blue triangles) and -infected (orange circles) mice, and H1N1-infected mice (light orange diamonds).