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Gut Bacteriophage Alterations After Fecal Microbiota Transplantation for Recurrent Or Refractory Clostridioides Difficile Infection

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Gut Bacteriophage Alterations After Fecal Microbiota Transplantation for Recurrent Or Refractory Clostridioides Difficile Infection Gut Bacteriophage Alterations after Fecal Microbiota Transplantation for Recurrent or Refractory Clostridioides difficile Infection by Jessmyn A. Niergarth A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Bioinformatics Guelph, Ontario, Canada © Jessmyn A. Niergarth, September, 2020 ABSTRACT GUT BACTERIOPHAGE ALTERATIONS AFTER FECAL MICROBIOTA TRANSPLANTATION FOR RECURRENT OR REFRACTORY CLOSTRIDIOIDES DIFFICILE INFECTION Jessmyn A. Niergarth Advisors: University of Guelph, 2020 Dr. Zeny Feng Dr. Peter T. Kim Clostridioides difficile infection (CDI) is a concern for health care providers around the world because CDI can be acquired nosocomially, and has high rates of treatment failure and recurrence (rCDI). Alternative therapeutic options have been explored, including fecal microbiota transplantation (FMT). FMT is a promising alternative to antibiotics that has been shown to achieve high cure rates for rCDI. Its mechanism of success is not fully under- stood and could potentially involve gut bacteriophages (phages), so we investigated the gut phage changes after FMT treatment of recurrent or refractory CDI. To achieve this objective, we purified DNA phages from fecal samples of rCDI patients treated with FMT, and from FMT donor samples. We created an in-house bioinformatics pipeline to preprocess raw metagenomic shotgun sequencing phage reads and identify phages from the fecal samples. We proposed a three-step statistical analysis procedure to analyze the association between HRQoL measures and phage abundances. We also explored the transition patterns of phage communities in patients from pre-FMT to post-FMT. We found that the Shannon diversity and proportion of reads mapping to donor phage contigs increased, and the Caudovirales:Mi- croviridae ratio decreased in patients after FMT. These results suggest that donor phages were engrafted into or augmented in patients via FMT, and that these changes lead to a gut phageome more similar to that of a healthy individual. Our regularized mixed effects re- gression model for joint selection of dsDNA phages that were associated with Bodily Pain produced a final model that retained 36 phages as covariates. These phages are potential targets for future work and included five Leuconostoc phages, six Lactococcus phages, and one C. difficile phage. Leuconostoc and Lactococcus bacteria are associated with food, so our findings suggest that patient diet should be controlled for in future work. Within phages predicted to infect Proteobacteria, there was a relatively high abundance of phages predicted to infect Gammaproteobacteria, a bacterial class with a high proportion of pathogenic species. FMT donors were screened for pathogenic gut bacteria, but to further ensure the safety of FMTs, Gammaproteobacteria and their associated phages in FMTs could be studied further. iv ACKNOWLEDGEMENTS I would like to express my deep and sincere gratitude to my advisors Dr. Zeny Feng and Dr. Peter T. Kim for allowing me the opportunity to do this research and for providing invaluable guidance throughout my degree. I would also like to thank Dr. Christine Lee for guidance and for access to clinical data, Dr. Andrew Kropinski for expert advice on bacteriophages, Dr. Sarah Adamowicz for support with the administrative elements of my program, Dr. J. Scott Weese for advice on biological and computational elements of my work and for access to computational resources, MSc Emma J. Smith for imputed data, Dr. Diego Gomez-Nieto for introduction to laboratory protocols, Joyce Rousseau for laboratory research support, and finally I would like to thank the following people for their work processing samples in the laboratory: Cailin Harris, Augustine Wigle, Stephen Rush, Sarah Gordon, Susanna Hon, and Owen Krystia. v TABLE OF CONTENTS Abstract ii Acknowledgements iv Table of Contentsv List of Tables viii List of Figures ix List of Abbreviationsx List of Appendices xii Statement of Authorship xiii 1 Introduction1 1.1 Clostridioides difficile Infection (CDI)........................1 1.1.1 Clostridioides difficile ............................1 1.1.2 C. difficile Toxins...............................1 1.1.3 Global Significance of CDI..........................2 1.1.4 CDI and Antibiotics.............................3 1.1.5 Recurrent or Refractory CDI.........................4 1.2 Fecal Microbiota Transplantation...........................4 1.3 Phages, rCDI, and FMT................................5 1.4 Human Gut Microbiota and Pain...........................6 1.5 Human Gut Proteobacteria..............................6 1.6 Thesis Statement...................................7 2 Background8 2.1 Fecal Samples.....................................8 2.2 Demographic, Clinical, and HRQoL Data......................8 vi 3 Review of Literature 13 3.1 The Human Gut Microbiota.............................. 13 3.2 The Human Gut Phageota............................... 14 3.2.1 Human Gut Virota.............................. 14 3.2.2 Human Gut Phageota............................. 14 3.2.3 Phage Taxonomy............................... 15 3.2.3.1 Phage Taxa............................. 16 3.2.4 Phage Life Cycles.............................. 19 3.2.4.1 Lytic and Lysogenic Life Cycles................. 19 3.2.4.2 Pseudolysogeny.......................... 19 3.2.4.3 Chronic Infection by Filamentous Phages............. 20 3.2.4.4 Carrier State Life Cycle...................... 20 3.2.4.5 Phage Life Cycle Characterization................ 20 3.2.5 Properties of Phage Genomes........................ 21 3.2.6 Effects of Phages on Bacteria in the Gut................... 21 3.2.7 Phage-Host Dynamics and Coevolution................... 24 3.2.7.1 Lotka-Volterra........................... 25 3.2.7.2 Kill the Winner.......................... 25 3.2.7.3 Piggyback-the-Winner....................... 26 3.2.7.4 Fluctuating Selection....................... 26 3.2.7.5 Community Shuffling....................... 26 3.2.7.6 Bacterial Interspecies Competition................ 26 3.2.8 Effects of Gut Phages on the Human Host.................. 26 3.2.8.1 Indirect Effects.......................... 27 3.2.8.2 Direct Effects........................... 28 3.2.9 Phage Metagenomics............................. 29 3.2.9.1 Importance of Metagenomics in Virome Studies......... 29 3.2.9.2 Viral Dark Matter and Database-Independent Metagenomic Methods 30 3.2.10 Interpersonal Gut Phage Diversity...................... 32 3.2.11 C. difficile Phages............................... 32 3.2.12 CRISPR in C. difficile ............................ 33 3.3 rCDI and Fecal Microbiota Transplantation..................... 34 3.3.1 Success Rates................................. 34 3.3.2 FMT and Phages............................... 34 4 Gut Bacteriophage Dynamics, Fecal Microbiota Transplantation, and Recurrent Clostridioides difficile Infection 36 4.1 Appendix A...................................... 57 4.2 Appendix B...................................... 61 4.3 Appendix C...................................... 64 vii 5 Investigating the Gut Bacteriophages Associated with Bodily Pain in Recurrent and Refractory Clostridioides difficile Infection Patients 65 6 Changes in Proteobacteria and Phages Predicted to Infect Proteobacteria After Fecal Microbiota Transplantation 107 7 Conclusions 155 7.1 Overall Phageome Alterations After FMT...................... 155 7.2 Alterations in dsDNA and ssDNA Phages After FMT................ 156 7.3 Alterations in Specific Phages After FMT...................... 158 7.4 Phage Community Phylogenetic Diversity...................... 159 7.5 Potential Phage-Dysbiosis Links........................... 159 7.5.1 Proteobacteria Phages............................ 160 8 Future Directions 161 8.1 Amplification Alternatives.............................. 161 8.2 Pipeline Benchmarking................................ 162 8.3 Integrative Model of Patient Pain........................... 164 8.4 Gnotobiotic Mouse Experiment............................ 165 Bibliography 166 Appendix 200 viii LIST OF TABLES 2.1 Demographic and Clinical Data of Patients...................... 10 2.2 SF-36 Bodily Pain Data of Patients.......................... 12 3.1 Bacteriophage Families................................ 18 7.1 Changes in Caudovirales Phages and Bodily Pain.................. 157 ix LIST OF FIGURES 3.1 Phage Genome Lengths................................ 22 7.1 Virus Taxon Abundances by Sample......................... 157 x LIST OF ABBREVIATIONS bp Base pair CDI Clostridioides difficile infection contig Contiguous sequence D10 10 days post-FMT Dnr Donor dsDNA Double-stranded DNA FMT Fecal microbiota transplantation FVT Fecal virome transplantation GIT Gastrointestinal tract GBA Gut-brain axis GC-content Guanine-cytosine content HBT Human biotherapy (synonym for fecal microbiota transplantation) HGT Horizontal gene transfer HRQoL Health-related quality of life xi IBD Inflammatory bowel disease ICTV International Committee on Taxonomy of Viruses KtW Kill the winner MDA Multiple displacement amplification NGS Next generation sequencing nt Nucleotide PCR Polymerase chain reaction PD Phylogenetic diversity phage Bacteriophage Pre Pre-FMT PtW Piggyback the winner rCDI Recurrent or refractory Clostridioides difficile infection SF-36 36-Item Short Form Health Survey [1] ssDNA Single-stranded DNA VLP Virus-like particle W05 Five weeks post-FMT W13 13 weeks post-FMT xii LIST OF APPENDICES Regularized Mixed Effects Regression Model . 200 xiii STATEMENT
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