Populational dynamics and pharmacodynamics of bacterial colonies in physically structured habitats Pubblico

Abstract

Bacterial infections are very common in human society. Thus extensive research has been conducted to reveal the molecular mechanisms of the pathogenesis and to evaluate the antibiotics' efficacy against bacteria. Little is known, however, about the population dynamics of bacterial populations and their interactions with the host's immune system. In this dissertation, a stochatic model is developed featuring stochastic phenotypic switching of bacterial individuals to explain the single-variant bottleneck discovered in multi strain bacterial infections. I explored early events in a bacterial infection establishment using classical experiments of Moxon and Murphy on neonatal rats. I showed that the minimal model and its simple variants do not work. I proposed modifications to the model that could explain the data quantitatively. The bacterial infections are also commonly established in physical structures, as biofilms or 3-d colonies. In contrast, most research on antibiotic treatment of bacterial infections has been conducted in well-mixed liquid cultures. I explored the efficacy of antibiotics to treat such bacterial colonies, a broadly applicable method is designed and evaluated where discrete bacterial colonies on 2-d surfaces were exposed to antibiotics. I discuss possible explanations and hypotheses for the experimental results. To verify these hypotheses, we investigated the dynamics of bacterial population as 3-d colonies. We showed that a minimal mathematical model of bacterial colony growth in 3-d was able to account for the experimentally observed presence of a diffusion-limited regime. The model further revealed highly loose packing of the cells in 3-d colonies and smaller cell sizes in colonies than plancktonic cells in corresponding liquid culture. Further experimental tests of the model predictions have revealed that the ratio of the cell size in liquid culture to that in colony cultures was consistent with the model prediction, that the dead cells emerged randomly in a colony, and that the cells packed heterogeneously in the outer part of a colony, possibly explaining the loose packing.

Table of Contents

DEDICATION. iii

ACKNOWLEDGEMENTS. iv

LIST OF TABLES. vii

LIST OF FIGURES. viii

I INTRODUCTION. 1

II SINGLE VARIANT BOTTLENECK IN THE EARLY DYNAMICS OF H. INFLUENZAE BACTEREMIA IN NEONATAL RATS QUESTIONS THE THEORY OF INDEPENDENT ACTION. 7

2.1 Introduction. 7

2.2 Hypothesis and Model. 11

2.3 Results. 13

2.3.1 The colloquial model. 13

2.3.2 Modications to the colloquial model. 17

2.3.3 Beyond the independent action model. 21

2.4 Discussion. 23

III ANTIBIOTIC SUSCEPTIBILITY OF BACTERIAL COLONIES: AN ASSAY AND EXPERIMENTS WITH STAPHYLOCOCCUS AUREUS. 27

3.1 Introduction. 27

3.2 Materials and Methods. 29

3.2.1 Bacteria. 29

3.2.2 Media. 29

3.2.3 MIC determination. 29

3.2.4 Procedure for the colony assay of antibiotic ecacy and liquid culture controls. 29

3.3 Results. 33

3.3.1 Resource saturation of S. aureus Newman in liquid cultures and as colonies. 33

3.3.2 The eect of physiological states of cells grown as colonies on the susceptibility of these cells to antibiotics. 34

3.3.3 The eect of the physical structure of bacterial colonies on the susceptibility of these cells to antibiotics. 37

3.4 Discussion. 42

IV EXPERIMENTAL STUDY AND MODELING OF THE POPULATION DYNAMICS OF NUTRIENT-LIMITED THREE-DIMENSIONAL BACTERIAL COLONY GROWTH. 46

4.1 Introduction. 46

4.2 Materials and Methods. 48

4.2.1 Bacteria. 48

4.2.2 Medium. 48

4.2.3 Imaging. 49

4.2.4 Image analysis. 51

4.2.5 Numerical solution of the model. 51

4.2.6 Model tting and condence intervals estimation. 52

4.3 Results. 53

4.3.1 The experimental results of bacterial growth in liquid cultures and as 3-d colonies. 53

4.3.2 Minimal model of resource-limited bacterial growth. 54

4.3.3 Parameter optimization of the minimal model of bacterial growth. 58

4.3.4 Experimental tests of the model's predictions and observations beyond the model. 66

4.4 Discussion. 70

V SUMMARY AND OUTLOOK. 75

APPENDIX A | ANTIBIOTICS IN RICHER MEDIUM. 78

APPENDIX B |SUPPLEMENTAL INFORMATION ON PARAMETER FITTING OF 3-D COLONY MODEL. 80

REFERENCES. 82

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biophysics, General Biology, Microbiology Physics, Theory
Parola chiave	bacterial colonies pharmacodynamics mathematical modeling cell size
Committee Chair / Thesis Advisor	Nemenman, Ilya, Emory University Levin, Bruce, Emory University
Committee Members	Weeks, Eric, Emory University Mugler, Andrew, Purdue University Boettcher, Stefan, Emory University

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