The University of Southampton
University of Southampton Institutional Repository

Investigating the relationship between Streptococcus pneumoniae biofilm and the human host

Investigating the relationship between Streptococcus pneumoniae biofilm and the human host
Investigating the relationship between Streptococcus pneumoniae biofilm and the human host
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.
University of Southampton
Wilkins, Matthew
05332538-6764-4211-8c43-0c7c809f3833
Wilkins, Matthew
05332538-6764-4211-8c43-0c7c809f3833
Faust, Saul
f97df780-9f9b-418e-b349-7adf63e150c1
Allan, Raymond
390a7d0a-38e1-410a-8dfe-c8ef8408f5e1
Hall-stoodley, Luanne
9dd7a299-a529-401c-9ae8-63a4807c2807

Wilkins, Matthew (2017) Investigating the relationship between Streptococcus pneumoniae biofilm and the human host. University of Southampton, Doctoral Thesis, 258pp.

Record type: Thesis (Doctoral)

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.

Text
Matt Wilkins Thesis - Version of Record
Available under License University of Southampton Thesis Licence.
Download (8MB)

More information

Published date: October 2017

Identifiers

Local EPrints ID: 435458
URI: http://eprints.soton.ac.uk/id/eprint/435458
PURE UUID: 7e2e6bc0-92ba-4c12-a20a-0a992bf0efc0
ORCID for Saul Faust: ORCID iD orcid.org/0000-0003-3410-7642

Catalogue record

Date deposited: 07 Nov 2019 17:30
Last modified: 23 Jul 2022 04:46

Export record

Contributors

Author: Matthew Wilkins
Thesis advisor: Saul Faust ORCID iD
Thesis advisor: Raymond Allan
Thesis advisor: Luanne Hall-stoodley

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×