Interkingdom Signaling Induces Streptococcus pneumoniae Biofilm Dispersion and Transition from Asymptomatic Colonization to Disease

Dispersion of biofilm bacteria by virus infection and host factors. (A) The ATP concentrations in the supernatant of HRECs infected with influenza A virus (IAV) or in the nasopharyngeal lavage fluid recovered from mice 24 h after IAV infection were determined. (B) The impact of IAV infection on the ratio of bacteria in the supernatant (dispersed) to epithelium-associated bacteria (biofilm) 48 h after biofilm transplant to live HRECs was measured. (C) The change in the optical density at 600 nm (ΔOD₆₀₀) of biofilm supernatant was measured 2 h after application of the indicated stimuli (NE = norepinephrine). The change in optical density correlated well with the number of CFU obtained from bacterial growth of the same supernatants. For each analysis, three independent assays were performed in duplicate. Statistical analysis was performed using Student’s t test (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).

Characterization of pneumococcal populations for gene regulation, adherence, invasion, toxicity, and cytokine induction from exposed HRECs. Biofilm and dispersed and planktonic pneumococcal populations show distinct gene expression profiles of key virulence genes (P < 0.05 for all comparisons using 2-way analysis of variance [ANOVA]) (A) and differences in transparent- and opaque-phase states (*** = P < 0.001 using 1-way ANOVA). (C to F) The populations further differed in their ability to adhere to (C), invade (D), and kill (E) HRECs (** = P < 0.01 for all comparisons using 1-way ANOVA) and induce different levels of key cytokines involved in proinflammatory responses from the exposed HRECs (* = P < 0.05 using 2-way ANOVA) (F). Each experiment was repeated at least three times in duplicate. ns, nonsignificant.

Colonization and dissemination of pneumococcal populations after intranasal inoculation, aspiration inoculation, or intraperitoneal inoculation. (A and B) Individual 6-week-old female BALB/cByJ mice were inoculated with biofilm (designated “B”) or dispersed (designated “D”) or broth-grown, planktonic (designated “P”) populations of EF3030 (A) and D39 (B) pneumococci intranasally without anesthesia to determine nasopharyngeal colonization and dissemination from the nasopharynx to the lungs and middle ears. Additionally, the bacterial burden in nasopharyngeal (NP) lavage fluid was determined. (C and D) Mice were furthermore inoculated with EF3030 (C) and D39 (D) pneumococci intranasally after anesthesia in an aspiration pneumonia model, and the bacterial burden in the lung tissue and dissemination to the bloodstream were determined by plate counts. (E and F) Finally, each bacterial population of EF3030 (E) and D39 (F) pneumococci was injected intraperitoneally to determine the respective levels of virulence upon reaching the vascular compartment. For both nonanesthetized and anesthetized intranasal challenges, nasal lavage fluids and all tissues were collected at 48 h postinfection. For intraperitoneal challenge, samples were collected at 24 h postinfection. Each dot in the graphs represents an individual mouse. An “X” represents a mouse that became moribund and required euthanasia before the end of the experiment. Each experiment included at least 6 mice. Statistical analysis was performed using one-way ANOVA (* = P < 0.05, ** = P < 0.01, and *** = P < 0.001, indicating significant differences between the populations).

Histological representation of mouse tissues infected with various pneumococcal populations. Representative histological images of nasal epithelium, middle ear space, and lungs 7 days following nonanesthetized intranasal challenge (rows 1 to 3) with PBS (mock-infected control), biofilm, or dispersed or planktonic populations. Row 4 shows histological sections of the lungs 48 h following intratracheal aspiration of these same bacterial populations. Tissues were stained with hematoxylin and eosin and examined microscopically at magnifications of ×400 for row 1 and ×200 for rows 2 to 4. The sections are labeled with relevant structures (LI, leukocyte infiltrate; TM, tympanic membrane; SM, stapes muscle; ME, middle ear cavity; Spn, S. pneumoniae).

Bacterial dissemination of mice stably colonized with pneumococci and challenged with influenza A virus. Bacterial burden in tissues and nasopharyngeal lavage fluid was measured for individual 6-week-old female BALB/cByJ mice colonized intranasally without anesthesia with EF3030 (A and C) or D39 (B and D) biofilm bacteria at 1 (A and B) or 5 (C and D) days postinfection with IAV. Each dot represents an individual mouse. Statistical analysis was performed using Student’s t test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.