Investigating the relationship between Streptococcus pneumoniae biofilm and the human host

Abstract

Streptococcus pneumoniae (pneumococcus) is a leading cause of worldwide morbidity and mortality. Recent studies have demonstrated that S. pneumoniae may colonise the adenoidal mucosal epithelium in the human nasopharynx as aggregated and surface-attached bacteria, known as a biofilm. Biofilms are clinically important due to greater recalcitrance to antibiotics and the immune system than free-living bacteria.

Pneumococcal biofilms have also been found in the middle ear. Adenoidectomy is effective in treating chronic middle ear (otitis media) infections in some children, suggesting an etiological role of nasopharyngeal biofilms in the pathogenesis of otitis media, which remains the most common infection in young children and the primary cause of antibiotic prescriptions in paediatrics. However, studies directly investigating the interaction between pneumococcal biofilms and the host are lacking.

In this study, the interaction between pneumococcus and host cells in adenoid tissue was examined. Specifically, to find evidence of pneumococcal biofilm formation, and whether aggregates of pneumococcus co-localised with macrophages and neutrophils (phagocytic cells). Immunohistochemistry was used to stain for a serotype 14 (S14) pneumococcal strain, which is prevalent in pneumococcal disease, using pure cultures and spiked adenoids. Optimisation was achieved by using different dilutions of a polyclonal rabbit anti-S. pneumoniae antibody, alongside various antigen retrieval techniques and different counterstains. Aggregates of pneumococcus were found in each spiked adenoid that was examined. However, evidence of co-localisation with phagocytes was inconclusive with immunohistochemistry and sequential section staining. To obtain results more representative of the in vivo host-pathogen environment, pneumococcus was stained in non-spiked patient adenoids with an alternative antibody reactive against over 90 different serotypes. Optimisation was achieved through different antibody dilutions and staining pure cultures of pneumococcus using immunofluorescence. Although individual pneumococcal cells only were found in two of the four patient adenoids examined, macrophages were in close proximity, and the established methods in this project demonstrated that they are suitable for staining pneumococcus in adenoid tissue and for further examination of assessing co-localisation between pneumococcal biofilms and immune cells in patient adenoids.

Since pneumococcus is predominantly an asymptomatic coloniser, the biofilm phenotype is reasoned to account for persistence in the upper respiratory tract. To date, in vitro studies recapitulating pneumococcal biofilms have been developed either on abiotic surfaces, on epithelial cells with a short contact time of up to 4 hours, on non-viable epithelial cells, or are transplanted from prefixed epithelia onto live epithelial cells. In this project, pneumococcal biofilms successfully grew on a confluent and viable bronchial epithelial cell line for up to 12 hours of culture with different pneumococcal loads (multiplicities of infection). Evidenced by scanning electron microscopy, nascent biofilms formed on viable epithelial monolayers using transwell membrane inserts, with morphological structure and topology reflective of mucosal epithelial samples found in ex vivo, with in vitro models and in vivo in mice. Epithelial cell integrity and viability was confirmed through transepithelial resistance, lactate dehydrogenase production and photomicrographic images. This more biologically representative model can be used for advanced modelling of the in vivo relationship between pneumococcal biofilms and the host.

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Identifiers

ORCID for Saul Faust: orcid.org/0000-0003-3410-7642

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