DETERMINING THE VARIABILITY IN THE LUNG MICROBIOME THROUGHOUT THE COURSE OF MYCOBACTERIUM TUBERCULOSIS INFECTION by MacKenzie Marie Bryant BS, Alfred State College - SUNY College of Technology, 2015 Submitted to the Graduate Faculty of Infectious Diseases and Microbiology Graduate School of Public Health in partial fulfillment of the requirements for the degree of Master of Science University of Pittsburgh 2017 UNIVERSITY OF PITTSBURGH Graduate School of Public Health This thesis was presented by MacKenzie Marie Bryant It was defended on April 11th, 2017 and approved by Thesis Advisor: JoAnne L. Flynn, PhD, Professor, Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh Committee Member: Joshua T. Mattila, PhD, Assistant Professor, Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh Committee Member: Robbie B. Mailliard, PhD, Assistant Professor, Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh ii Copyright © by MacKenzie Marie Bryant 2017 iii JoAnne L. Flynn, PhD DETERMINING THE VARIABILITY IN THE LUNG MICROBIOME THROUGHOUT THE COURSE OF MYCOBACTERIUM TUBERCULOSIS INFECTION MacKenzie Bryant, MS University of Pittsburgh, 2017 ABSTRACT Tuberculosis remains a major health threat throughout the world, despite having a vaccine and treatments. Mycobacterium tuberculosis (Mtb) infects alveolar macrophages in the lung and inflammation occurs after infection. The lung microbiome in regards to Mtb infection is poorly understood, and whether inflammation from infection affects the lung microbiome is unknown. The goal of our study is to determine whether Mtb induces a significant and durable change in the lung microbiome of cynomolgus macaques. We investigated and compared the community clusters between the lung and oral cavity, assessed how the diversity of the lung microbiota changes throughout infection, and associated these changes in the lung with inflammation. Bronchoalveolar lavage (BAL) was obtained pre-infection and at several time points post-Mtb infection, as well as oral wash and saline bronchoscope control samples from respective macaques. Operational taxonomic units (OTUs) were generated after 16s rRNA sequencing was performed once DNA was extracted from collected samples. We profiled microbial communities to see the community structure differences between oral and lung environment and show how the microbiome changes throughout infection. PET/CT imaging was used to visualize and quantify inflammation over the course of infection by using FDG avidity (total PET HOT). Our results show that the oral and lung compartments are distinct with regard to community iv structure, distinct bacterial taxa are more relatively abundant in certain lobes, and lung inflammation and lung microbiome changes are variable within macaques and between macaques. Analysis of the first cohort of macaques (N=10) did not reveal correlations between lung inflammation and relative abundance or alpha-diversity, but our data is preliminary and based on small sample size. Our sample size will greatly increase after the second cohort of macaques are fully sequenced and analyzed. These changes and disruptions in the lung microbiome may have public health relevance in regards to overall lung health and may also play a role in the outcome of Mtb infection. v TABLE OF CONTENTS PREFACE .................................................................................................................................. XII 1.0 INTRODUCTION ........................................................................................................ 1 1.1 TUBERCULOSIS ................................................................................................ 1 1.1.1 Epidemiology and Clinical Definitions........................................................ 1 1.1.2 The Spectrum of M. Tuberculosis Infection ................................................ 3 1.1.3 Transmission and Immunology ................................................................... 4 1.1.4 The Granuloma ............................................................................................. 5 1.1.5 The Use of PET/CT Imaging to Track Granulomas.................................. 6 1.2 THE LUNG MICROBIOME ............................................................................. 9 1.2.1 Obstacles in Studying the Lung Microbiome ............................................. 9 1.2.2 Contamination Controversies and Issues ................................................. 12 1.2.3 Lung Disease, Dysbiosis, and Inflammation ............................................. 14 1.2.4 The Lung Microbiome and M. tuberculosis Infection ............................. 16 1.3 THE MACAQUE MODEL AND ITS IMPORTANCE IN TUBERCULOSIS AND MICROBIOME STUDIES ...................................................................................... 18 1.3.1 The NHP Model and Tuberculosis ............................................................ 18 1.3.2 The NHP Model and the Lung Microbiome ............................................. 19 2.0 STATEMENT OF INTENT, HYPOTHESIS, AND SPECIFIC AIMS ................ 21 2.1 SPECIFIC AIM 1: IDENTIFY AND COMPARE COMMUNITY CLUSTERS OF THE LUNG AND ORAL CAVITY IN MACAQUES. ....................... 22 vi 2.2 SPECIFIC AIM 2: INVESTIGATE THE COMMUNITY STRUCTURE AND STABILITY OF THE LUNG MICROBIOME THROUGHOUT THE COURSE OF M. TUBERCULOSIS INFECTION IN MACAQUES. ............................................. 22 2.3 SPECIFIC AIM 3: ASSOCIATE MICROBIOTA VARIABILITY IN THE LUNG WITH INFLAMMATION. ................................................................................... 23 3.0 METHODS ................................................................................................................. 24 3.1 INFECTION AND NHP MODEL ................................................................... 24 3.2 BRONCHOALVEOLAR LAVAGE COLLECTION .................................... 24 3.3 DNA EXTRACTION/ISOLATION ................................................................. 25 3.4 16S RRNA SEQUENCING AND DATA ANALYSIS ................................... 26 3.5 PET/CT IMAGING ........................................................................................... 27 3.6 STATISTICAL ANALYSIS ............................................................................. 27 4.0 RESULTS ................................................................................................................... 29 4.1 EXPERIMENTAL OVERVIEW ..................................................................... 29 4.2 THE ORAL CAVITY AND LUNG COMMUNITY STRUCTURES ARE DISTINCT ........................................................................................................................... 30 4.3 LUNG MICROBIOTA ARE VARIABLE THROUGHOUT INFECTION 35 4.4 INFLAMMATION IS VARIABLE THROUGHOUT INFECTION ........... 45 5.0 DISCUSSION ............................................................................................................. 49 5.1 POSSIBLE MECHANISM ............................................................................... 50 5.2 ISSUES................................................................................................................ 51 5.3 FUTURE DIRECTIONS................................................................................... 52 5.4 PUBLIC HEALTH SIGNIFICANCE AND FINAL THOUGHTS............... 53 vii BIBLIOGRAPHY ....................................................................................................................... 55 viii LIST OF TABLES Table 1. Previous lung microbiome and TB studies ..................................................................... 17 Table 2. Sample distribution of cohorts ........................................................................................ 30 Table 3. Involved vs uninvolved lobes, OTU relative abundance p-values ................................. 38 Table 4. Spearman’s correlation values and associated p-values in involved lobes ................. 47 ix LIST OF FIGURES Figure 1. The spectrum of Mtb infection ........................................................................................ 3 Figure 2. Structure and organization in a TB granuloma................................................................ 6 Figure 3. Hypothetical PET/CT image showing granuloma dynamism throughout time .............. 7 Figure 4. The respiratory tract ...................................................................................................... 11 Figure 5. Potential sites for contamination while sampling the respiratory tract ......................... 13 Figure 6. The inflammation-dysbiosis cycle ................................................................................. 15 Figure 7. Experimental layout of sample collection sites and timeline ........................................ 29 Figure 8. Principle coordinate analysis comparing the genetic diversity between oral wash and lung BAL samples ................................................................................................................................. 31 Figure 9. Relative abundance of top 10 generated OTUs between oral wash, involved lobe, and uninvolved lobe samples ............................................................................................................... 33 Figure 10. Lung microbiome diversity over the course of Mtb infection. ...................................