Recently, we have demonstrated that the pathogenesis of Streptococcus pneumoniae bacteraemia contains a concomitant phase of intracellular replication within splenic macrophages in mice (Ercoli et al. Nat Micro. 2018). In the present study, we aimed to determine if intracellular replication of pneumococci may play a role in the pathogenesis of sepsis in humans, using two innovative approaches. We used a model (Chung et al. ALTREX.2018) involving ex vivo perfusion of human spleens from elective splenectomy patients (REC reference: 18/EM/0057). Organs were infected with 6.5×107 c.f.u. of pneumococci, and serial biopsies and ‘blood’ samples were taken at predetermined times. Samples were analysed by colony counts, confocal microscopy and flow cytometry. Additionally, infected tissue samples were taken for preparation of organotypic slice culture time courses. Bacteria injected into the perfusion circuit were rapidly cleared at early time points post-infection, recapitulating what is observed in experimental murine sepsis. Bacterial counts in the spleen increased, providing initial evidence of intracellular bacterial persistence. Microscopy analysis indicated that bacteria could be localised to splenic macrophages, with the size of infectious foci increased over time. Z-stack microscopy localised bacteria within cell membranes, indicating the infection was predominantly intracellular. In ex vivo slice cultures increasingly large numbers of pneumococci were cultured over time, further indicating intracellular replication. In conclusion, we provide evidence for a role of intracellular replication of pneumococci in human splenic macrophages in the pathogenesis of sepsis.

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