Bacterial population dynamics of pneumococcal colonization of the nasopharynx Open Access

Shak, Joshua (2013)

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Streptococcus pneumoniae (the pneumococcus) is a common commensal inhabitant of the nasopharynx and a frequent etiologic agent in diseases responsible for approximately 1.6 million deaths annually. Colonization of the nasopharynx by S. pneumoniae (Sp) is a necessary precursor to pneumonia, otitis media, bacteremia, and meningitis. Intraspecies and interspecies interactions influence pneumococcal carriage in important ways. Biofilms - multicellular, surface-associated communities - are an intraspecies interaction that is particularly critical for Sp nasopharyngeal colonization and pathogenesis. We found that pneumolysin (Ply) - the primary toxin of Sp and an important vaccine target - is vital to the assembly of pneumococcal biofilms. Ply was expressed during biofilm development and localized to cellular aggregates. Knockout mutants deficient in Ply produced significantly less biofilm mass than wild-type strains, both on polystyrene and on human respiratory epithelial cells. Immunogold transmission electron microscopy of biofilms demonstrated that Ply was present both on pneumococcal cell surfaces and in the extracellular biofilm matrix. Altogether, we demonstrate a novel role for pneumolysin in the assembly of biofilms that is likely important during both carriage and disease. To examine interspecies interactions we examined nasopharyngeal densities of Staphylococcus aureus (Sa), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mc) following controlled challenge of healthy human adults with Sp. Of the 52 subjects challenged with Sp, 33 (63%) carried Sp at 2, 7, or 14 days post-inoculation. While baseline presence and density of Sa, Hi, and Mc were not associated with likelihood of successful Sp colonization at a statistically significant level, trends in the data suggest a protective effect of baseline Sa colonization and a facilitative effect of Hi colonization. At 14 days post-challenge, the proportion carrying Sa was significantly lower among those colonized with Sp as compared to those not colonized with Sp (p = 0.008). These are the first data reported on bacterial densities in relation to experimental human pneumococcal colonization and provide estimated effect sizes that will be useful in designing future studies. Through investigation of both intraspecies and interspecies interactions, this dissertation highlights the importance of the nasopharyngeal bacterial ecosystem for understanding pneumococcal carriage.

Table of Contents

Chapter 1: Introduction 1

A brief history of the pneumococcus 1 Epidemiology of pneumococcal colonization 3 Molecular mechanisms of nasopharyngeal colonization 5 Role of biofilms during carriage 8 Other actors in the nasopharynx 10 Sequelae of colonization 12 Outline of the dissertation 14 References 15

Chapter 2: Influence of bacterial interactions on pneumococcal
colonization of the nasopharynx 29

Streptococcus pneumoniae: a commensal and a pathogen 30

Effects of co-colonization on serotype distribution, genetic
exchange, and density 31

Interactions between nasopharyngeal bacterial species 32

Pneumococcal biofilms in the nasopharynx 33

Concluding Remarks 34 References 34

Chapter 3: Novel Role for the Streptococcus pneumoniae toxin
pneumolysin in the assembly of biofilms 37

Abstract and Importance 38 Introduction 38 Results 39 Discussion 43

Materials and Methods 45

References 46

Supplemental Material 48

Addendum to Chapter 3 50

Chapter 4: Nasopharyngeal densities of common bacterial pathogens in
relation to experimental human pneumococcal carriage 54

Abstract 54 Introduction 55 Methods 57 Results 61 Discussion 67 References 71

Chapter 5: Summary and Outlook 75

References 80

Appendix: Other Published Work 81

Anemia and Helicobacter pylori seroreactivity in a rural Haitian
population 84

Aminoglycoside-resistant Aeromonas hydrophila as part of a
polymicrobial infection following a traumatic fall into freshwater 90

Characterization of Aeromonas hydrophila wound pathotypes from
comparative genomics and functional analysis of virulence genes 92

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