Increased nasopharyngeal bacterial titers and local inflammation facilitate transmission of Streptococcus pneumoniae

Abstract

The transmission of the bacterium Streptococcus pneumoniae (the pneumococcus) marks the first step toward disease development. To date, our ability to prevent pneumococcal transmission has been limited by our lack of understanding regarding the factors which influence the spread of this pathogen. We have previously developed an infant mouse model of pneumococcal transmission which was strictly dependent on influenza A virus (IAV) coinfection of both the experimentally colonized "index mice" and the naive cohoused "contact mice." Here, we sought to use this model to further elucidate the factors which facilitate S. pneumoniae transmission. In the present report, we demonstrate that increasing the nasopharyngeal load of S. pneumoniae in the colonized index mice (via the depletion of neutrophils) and inducing a proinflammatory response in the naive cohoused contact mice (as demonstrated by cytokine production) facilitates S. pneumoniae transmission. Thus, these data provide the first insights into the factors that help mediate the spread of S. pneumoniae throughout the community.

Importance: Streptococcus pneumoniae (the pneumococcus) is a major cause of worldwide morbidity and mortality and is a leading cause of death among children under the age of five years. Transmission of S. pneumoniae marks the first step toward disease development. Therefore, understanding the factors that influence the spread of pneumococci throughout the community plays an essential role in preventing pneumococcal disease. We previously developed the first reproducible infant mouse model for pneumococcal transmission and showed that coinfection with influenza virus facilitates the spread of S. pneumoniae. Here, we show that increasing the bacterial load in the nasal cavity of colonized individuals as well as inducing an inflammatory response in naive "contact cases" facilitates the spread of pneumococci. Therefore, this study helps to identify the factors which must be inhibited in order to successfully prevent pneumococcal disease.

FIG 1

Multiple IAV (influenza A virus) strains facilitate pneumococcal transmission. (A and C) The experimental model used to assess pneumococcal transmission. In brief, at 5 days of age, pups were randomly distributed into two groups of equal size (± one mouse). One group, designated index mice, was colonized with S. pneumoniae (indicated by the black mice). The index mice were then returned to, and cohoused in the same cage as, the remaining, noncolonized pups (contact mice; indicated by the white mice). At 14 days of age, either only the index mice (A) or both the index and contact mice (C) were infected with IAV. Six days later, transmission of S. pneumoniae from the index to the contact mice was assessed. (B) Pneumococcal and IAV transmission following infection of index mice with different strains of IAV. (D) Pneumococcal and IAV transmission following infection of both index and contact mice with different strains of IAV. Data were pooled from a minimum of two independent experiments and represent numbers of infected contact mice/total numbers of contact mice.

FIG 2

The effect of pneumococcal load in the index mice on S. pneumoniae transmission. (A) Titers of S. pneumoniae (Sp) in the nasal cavity 6 days after mock infection, infection with IAV, or treatment with 1A8 or rat IgG. Statistical significance was determined using a Mann-Whitney U test accompanied by a Bonferroni posttest, and statistical significance is denoted by one asterisk (P < 0.05). Data represent the means ± standard errors of the means (SEM) and are pooled from a minimum of two independent experiments. ns, not statistically significant. (B) Titers of IAV in the nasal cavity 6 days postinfection. Data represent means ± SEM and were pooled from a minimum of two independent experiments. (C) Experimental design to assess the role of increased pneumococcal titers in the index mice in S. pneumoniae transmission. In brief, at 5 days of age, pups were randomly distributed into two groups of equal size (± one mouse). One group, designated index mice, was colonized with S. pneumoniae (indicated by the black mice). The index mice were then returned to, and cohoused in the same cage as, the remaining, noncolonized pups (contact mice; indicated by the white mice). At 14 to 19 days of age, the index mice were treated with rat IgG or the neutrophil-depleting MAb 1A8. At 14 days of age, the contact mice were infected with IAV (strain PR8/34). Six days later, transmission of S. pneumoniae from the index to the contact mice was assessed. (D) Pneumococcal and IAV transmission in mice 6 days postinfection/treatment. Statistical significance was determined using Fisher’s exact test, and statistical significance is denoted by two asterisks (P < 0.01). Data were pooled from a minimum of three independent experiments.

FIG 3

The effect of inflammation in the contact mice on pneumococcal transmission. (A) Experimental design to assess the neutrophil depletion in the contact mice in S. pneumoniae transmission. In brief, at 5 days of age, pups were randomly distributed into two groups of equal size (± one mouse). One group, designated index mice, was colonized with S. pneumoniae (indicated by the black mice). The index mice were then returned to, and cohoused in the same cage as, the remaining, noncolonized pups (contact mice; indicated by the white mice). At 14 to 19 days of age, the contact mice were treated with rat IgG or the neutrophil-depleting MAb 1A8. At 14 days of age, the index mice were infected with IAV (strain PR8/34). Six days later, transmission of S. pneumoniae from the index to the contact mice was assessed. (B and E) Pneumococcal and IAV transmission in mice 6 days postinfection/treatment. Statistical significance was determined using Fisher’s exact test, and statistical significance is denoted by two asterisks (P < 0.01). Data were pooled from a minimum of three independent experiments. N.S, not statistically significant. (C) Experimental design to assess the role of inflammation in the contact mice in S. pneumoniae transmission. In brief, at 5 days of age, mice were randomly distributed into two groups of equal size (± one mouse). One group, designated index mice, was colonized with S. pneumoniae (indicated by the black mice). The index mice were then returned to, and cohoused in the same cage as, the remaining, noncolonized mice (contact mice; indicated by the white mice). At 14 to 19 days of age, the contact mice were treated with PBS or LPS. At 14 days of age, the index mice were infected with IAV (strain PR8/34). Six days later, transmission of S. pneumoniae from the index to the contact mice was assessed. (D) Cytokine levels in the nasal cavity 6 days postinfection/treatment. These experiments were performed using mice not colonized with S. pneumoniae to reflect the inflammatory response that occurs in the contact mice. Therefore, 14-day-old mice were (i) infected with PR8/34, Udorn/72, or HKx31 or (ii) treated daily with LPS or PBS from day 14 to day 19. At day 20, mice were culled and cytokine levels were determined in clarified homogenates from nasal cavity tissues. Data shown represent means ± SEM. Statistical significance (relative to naive mice) was determined using a Mann-Whitney U test accompanied by a Bonferroni posttest, and statistical significance is denoted by one asterisk (P < 0.05), two asterisks (P < 0.01), or three asterisks (P < 0.001).