Real-time near-infrared imaging distinguishes nasopharyngeal colonization from aspiration of Streptococcus pneumoniae and identifies aspiration as a trigger of severe disease

Streptococcus pneumoniae asymptomatically colonizes the nasopharynx but can invade the lower respiratory tract to cause life-threatening disease, particularly in older adults. However, whether the initial site of bacterial deposition following intranasal inoculation determines disease progression has not been directly examined. Here, we developed a near-infrared (NIR) fluorescence imaging approach using indocyanine green (ICG)-labeled S. pneumoniae TIGR4 to visualize early bacterial distribution in real time. ICG labeling by simple mixing, without genetic or chemical modification, neither impaired bacterial growth at 33 or 37 °C, nor altered acid tolerance. Continuous video imaging during the first 10 min of infection resolved two distinct patterns: bacteria confined to the nasopharynx (colonization) and those aspirated into the lower respiratory tract (aspiration). Kaplan–Meier analysis revealed markedly higher mortality in the aspiration group in both young (hazard ratio = 7.9) and aged (hazard ratio = 8.4) mice, despite a 10-fold lower inoculum used for aged animals, with deaths beginning on day 3. Systemic profiling of blood at 24 h by RNA sequencing and plasma proteomics revealed that early aspiration in aged mice was associated with the activation of inflammatory and hematopoietic programs, enrichment of complement and coagulation cascades, and phagocytic pathways. Together, these findings establish aspiration into the lower respiratory tract as a trigger of severe pneumococcal disease and introduce real-time NIR imaging as a technique for linking early infection dynamics to systemic host responses.