Single Cell Bottlenecks in the Pathogenesis of Streptococcus pneumoniae

Abstract

Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion.

Fig 1. Infant mouse model for co-infection of S.pneumoniae and influenza virus.

A. Schematic of the experimental schedule. On day 4 of age, pups were intranasally inoculated with a serotype 6A clinical isolate of S.pneumoniae. At age 8 days, the pups were inoculated with influenza A virus (IAV strain x31) or PBS. On day 12 of age, the pups were sacrificed and samples listed (NL, nasal lavage. MEL, middle ear lavage. Lung, lung homogenate) were collected. The following changes to the schematic described above were made. For the transmission experiments, one pup (index) was infected with S.pneumoniae and returned to the littermates (contacts). For the sepsis experiments, pups were not sacrificed at day of age 12 and monitored until showing sign of sepsis or euthanized at day of age 18 to obtain blood cultures. For the shedding experiments, daily cultures of secretions were obtained from day of age 8–12. B, C, E, F. Comparisons between the S.pneumoniae (Sp) and IAV co-infection group (black circle) and the S. pneumoniae plus PBS control infection group (open circle) and in each infection model. Each dot represents a single mouse. Mann-Whitney U test was used for the statistical analyses. *p<0.05 and **p<0.01 respectively. The dashed line indicates the detection limit. B. Colonization assessed by the density of pneumococci in nasal lavages. C. Otitis media assessed by the density of pneumococci in middle ear lavages. D. Time course of septic or sustained bacteremic infection. Pups were colonized with S. pneumoniae at age 4 days with (solid line, triangle, n = 8) or without IAV (dashed line, circle, n = 7) or at age 8 days without IAV (dotted line, square, n = 6). E. Transmission assessed by the density of pneumococci in nasal lavages of contact mice. F. Shedding assessed by the number of bacteria in secretions on the day of age indicated.

Fig 2. Steps in pneumococcal pathogenesis without a tight population bottleneck.

See Table 1 for summary of data. A. Nasal colonization to the middle ear cavity from eight representative animals. Pups were infected with an equal mixture of three marked mutants on day 4 and IAV on day 8 of age. Colonization density for each mutant (shown as a different colors) was determined in nasal lavages (above) and middle ear exudate from the corresponding pup (below). Middle ear lavages of right side with each vertical tick mark on the x-axis representing one pup. B. In nasal secretions. Pups were infected with three mutants on day 4 and IAV on day 8 of age. B1. Daily number of shed bacteria at the age indicated for each of the three mutant strains ± S.D. B2. All three strains were detected in nasal lavages (above) and in the shedding assay in the corresponding pups (below) on day 12 of age. Each vertical tick mark on the x-axis represents one pup. C. In bacteremia after IP challenge. The nasopharynx was bypassed and at age day 13 pups were challenged IP with an equal inoculum of all three mutants. After 14 hours, all pups showed signs of sepsis and blood was obtained for quantitative culture.

Fig 3. Analysis of tight population bottlenecks.

A. A tight population bottleneck exists in bacteremia after IN challenge. Pups were inoculated with three mutant mixture on day 4 and IAV (above) or PBS (below) on day 8 of age. Data with IAV is from three representative experiments. Each vertical tick mark on the x-axis represents results of blood cultures obtained when the pups showed signs of sepsis from one pup. B. Most transmission events originate from a single organism. Two representative litters out of five are shown. One pup was inoculated with each of three marked mutants on day 4 of age (index mice) and returned to the littermates (contacts). C. Multiple entry events may occur during transmission. Three pups were each inoculated with one of the three marked mutants on day 4 of age (index mice) and returned to the littermates (contacts). B, C. On day 8, all pups were infected with IAV. The bacterial density in the nasal lavage of each mouse on day of age 12 is shown. Each vertical tick mark on the x-axis represents results of cultures nasal lavages from a single pup.

Fig 4. Increased pneumococcal shedding among tlr2 ^-/- mice or by daily treatment with Toll-like receptor (TLR) agonists.

A. Wildtype (black circle) or tlr2 ^-/- pups (black triangle) were infected with strain P1547 on day 4, and IAV on day 8 of age as indicated. Daily nasal secretions were collected between day 8–12 of age. Each dot represents a single mouse. Statistical differences between the two groups in each day were evaluated by using Mann-Whitney U test. * p<0.05 and **p<0.01. B, C, D. Wildtype pups were infected with strain P1547 on day 4, and given a daily IN dose of PBS, Pam3Cys, poly-ICLC or LPS between days 8 and 12. Statistical analyses between four groups were performed by Kruskal-Wallis test (Dunn’s multiple comparison test). **p<0.01, *** p<0.001, **** p<0.0001 and n.s. not significant. B. Shedding (combined values from days 9 through12) was compared by quantitative culture of secretions. C. Colonization was compared by quantitative culture of nasal lavages obtained at day 12 of age. D. The number of neutrophils (PMNs) in the nasal lavages were counted by flow cytometry by gating on CD11b⁺, Ly6G⁺ events.