Browne, HP (2018) Development of anaerobic culturing methods to study the evolution and ...

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Browne, HP;(2018)Developmentofanaerobicculturingmethodstostudytheevolutionandtransmis-
sion of the human intestinal microbiota. PhD thesis, London School of Hygiene & Tropical Medicine.
DOI: https://doi.org/10.17037/PUBS.04647551
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DOI: https://doi.org/10.17037/PUBS.04647551
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honline.lshtm.ac.ukhttps://researc
Development of anaerobic culturing methods to study the evolution
and transmission of the human intestinal microbiota

Hilary Patrick Browne

Thesis submitted in accordance with the requirements for the degree of
Doctor of Philosophy of the

University of London
March 2018
Department of Pathogen Molecular Biology
Faculty of Infectious and Tropical Diseases
LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE

Funded by the Medical Research Council.
Research group affiliation: Lawley Faculty. Host-Microbiota Interactions
Laboratory. Wellcome Trust Sanger Institute

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Declaration
The work presented in this thesis is my own unless otherwise stated. It was carried
out under the supervision of Dr. Trevor Lawley of the Wellcome Trust Sanger
Institute (WTSI), Hinxton, Cambridgeshire, UK and Prof. Brendan Wren of the
London School of Hygiene and Tropical Medicine, London, UK. The work was
carried out at the Wellcome Trust Sanger Institute.
All experiments and analysis were carried out by me apart from the following:
Taxonomic classification of metagenomic sequence reads using Kraken, calculation
of relative abundance of metagenomic sequence reads and generation of the
sporulation signature gene list was carried out by Dr. Sam Forster (WTSI). David
Goulding (WTSI) created the transmission electron microscopy images from the
bacterial cultures provided. Library creation and DNA sequencing was performed by
the DNA pipelines group (WTSI). Assembly and annotation of whole-genome
sequences was performed by in-house pipelines developed by the Pathogen
Informatics group (WTSI). Figures taken from the review article ‘Transmission of
the gut microbiota: spreading of health’ published in Nature Reviews Microbiology,
(Reference [1]), were drawn by a Nature Review Microbiology illustrator but were
conceived and created by me in collaboration with Dr. Anne Neville, Dr. Sam
Forster and Dr. Trevor Lawley.
This thesis does not exceed 100,000 words in length, including tables, figures and
footnotes in accordance with the school regulations.

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Abstract
The human gastrointestinal tract is colonised by a diverse range of health-associated
bacteria, in addition to other microorganisms, termed the intestinal microbiota.
Sequence-based, culture-independent approaches have revolutionised this field of
study, however, due to the perception that these bacteria are largely unculturable, in
vitro phenotypic analysis has been hindered. In this study, an anaerobic culturing
workflow was developed which revealed that the majority of these bacteria can be
cultured using one growth medium. In total, 137 characterised and novel bacterial
species were isolated and whole-genome sequenced. Inter-host transmission of the
intestinal microbiota may represent a means to maintain a diverse assortment of
commensal bacteria within individuals, yet it remains a poorly understood process.
Some anaerobic pathogens utilise resilient aero-tolerant spores to survive externally
and to facilitate transmission to new hosts. To investigate if commensal spore-
formers utilise similar mechanisms, a phenotypic screen was incorporated into the
culturing workflow to target spore-forming bacteria. This resulted in the isolation of
66 phylogenetically diverse, spore-forming species which, through subsequent
phenotypic characterisation are shown to be specialised for host-to-host transmission
and intestinal colonisation. Further phylogenetic and evolutionary analysis revealed
body-site associated, loss of sporulation has occurred in different taxa. Also, loss of
sporulation is associated with features of host-adaptation that are not present in
spore-formers such as a smaller genome size and loss of genetic redundancy. This
suggests that the human intestinal microbiota is populated by commensal bacteria
that have evolved to engage in opposing lifestyles, either orientated towards inter-
host dispersal or within-host adaptation.
This study demonstrates the intestinal microbiota is not unculturable. In addition,
commensal microbial transmission may be more prevalent than once thought as a
significant proportion of these bacteria can survive outside of a host through the use
of spores that are intrinsically resistant to environmental stresses.

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Acknowledgements
I would like to thank my supervisors Trevor Lawley and Brendan Wren for
guidance, encouragement and advice and for providing me with the opportunity to
undertake this study. Thanks also to those at the Wellcome Trust Sanger Institute
who I have been fortunate to work with and who have contributed through
discussion, advice and training- members of the Host-Microbiota Interactions
Laboratory, in particular Anne Neville, Sam Forster, Nitin Kumar, Mark Stares and
Blessing Anonye and also, Sally Kay and Dave Goulding. Finally, thanks to Ann and
Cillian for their support and motivation.

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Contents
Declaration ................................................................................................................... 2
Abstract ........................................................................................................................ 3
Acknowledgements ...................................................................................................... 4
Contents ....................................................................................................................... 5
List of Figures ............................................................................................................ 10
List of Tables.............................................................................................................. 13
Glossary ..................................................................................................................... 14
Chapter 1 Introduction ............................................................................................... 17
1.1 The human intestinal microbiota ...................................................................... 17
1.1.1 The microbiota of humans ......................................................................... 17
1.1.2 The human intestinal microbiota ............................................................... 20
1.1.3 Defining a healthy intestinal microbiota .................................................... 20
1.1.4 Host selection of commensal intestinal bacteria ........................................ 21
1.2 The host-microbiota interactions of other animals ........................................... 23
1.2.1 The rumen microbiota ................................................................................ 24
1.2.2 Extreme host adaptation- the insect microbiota ......................................... 25
1.2.3 Genome reduction of extremely host-adapted bacteria ............................. 28
1.3 Studying the intestinal microbiota .................................................................... 30
1.3.1 Culturing and sequence-based approaches ................................................ 30
1.3.2 The benefits of bacterial culturing ............................................................. 32
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1.4 Spore-forming bacteria within the intestinal microbiota .................................. 34
1.4.1 Sporulation and germination cycle and genetics ....................................... 34
1.4.2 Clostridium difficile- the model enteric spore-former ............................... 39
1.5 Transmission of commensal bacteria ............................................................... 42
1.5.1 Shared transmission routes of commensal and pathogenic intestinal
bacteria ................................................................................................................ 43
1.5.2 Distinguishing the routes of transmission of commensal and pathogenic
intestinal bacteria ................................................................................................ 46
1.5.3 Survival in the environment ....................................................................... 47
1.5.4 Reservoirs of commensal bacteria ............................................................. 52
1.6 Microbiota perturbation and restoration ........................................................... 57
1.6.1 Microbiota perturbation ............................................................................. 57
1.6.2 Microbiota restoration................................................................................ 59
1.6.3 Understanding microbiota perturbation through ecological theory ........... 60
1.7 Therapeutics based on the intestinal microbiota .............................................. 61
1.8 Thesis aims ....................................................................................................... 63
Chapter 2 Materials and Methods .............................................................................. 65
2.1 Culturing of bacteria from the human intestinal microbiota ............................ 65
2.1.1 Acquisition of donor faecal samples .......................................................... 65
2.1.2 Anaerobic culturing ................................................................................... 65
2.1.3 Microbiota profiling by 16S rRNA gene sequencing ................................ 68
2.1.4. Submission of cultured bacteria to public repositories ............................. 69
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2.2 Generation of whole-genome, metagenomic and 16S rRNA gene amplicon
sequence data .......................................................................................................... 70
2.2.1 DNA extraction .......................................................................................... 70
2.2.2 Generation of whole-genome and metagenomic sequence data ................ 71
2.2.3 Generation of 16S rRNA gene amplicon sequence data............................ 72
2.3 Analysis of sequence data generated through anaerobic culturing .................. 72
2.3.1 Assembly and annotation of whole-genome sequence data ...................... 72
2.3.2 Analysis of metagenomic sequence data ................................................... 73
2.3.3 Analysis of 16S rRNA gene amplicon sequence data ............................... 74
2.3.4 Comparison of culturing studies ................................................................ 74
2.3.5 Gene sporulation signature ........................................................................ 75
2.3.6 Comparison of abundance of bacteria in faecal samples and on culture
plates ................................................................................................................... 76
2.3.7 Diversity analysis of spore-forming and non-spore-forming bacteria ....... 76
2.4 Phylogenetic analysis ....................................................................................... 77
2.4.1 16S rRNA gene sequence phylogeny of cultured bacteria ........................ 77
2.4.2 Phylogeny of putative novel bacterial family ............................................ 77
2.4.3 Core gene phylogeny of Firmicutes ........................................................... 78
2.5 Phenotypic analysis of spore-forming bacteria ................................................ 79
2.5.1 Oxygen sensitivity assay ............................................................................ 79
2.5.2 Germination response to intestinal bile acids assay................................... 79
2.5.3 Transmission electron microscopy ............................................................ 80
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2.6 Functional redundancy analysis ....................................................................... 81
2.6.1 Ortholog analysis ....................................................................................... 81
2.6.2 Gene per genome analysis: ........................................................................ 83
2.6.3 Presence and absence of sporulation associated genes .............................. 83
2.6.4 Functional annotation of unique genes and genes in gene per genome
analysis................................................................................................................ 84
Chapter 3 Culturing of the human intestinal microbiota ............................................ 85
3.1 Introduction ...................................................................................................... 85
3.2 Results .............................................................................................................. 88
3.2.1 Establishment and assessment of culture process ...................................... 88
3.2.2 Extensive culturing and isolation of characterised and novel bacteria ...... 93
3.2.3 Phylogenetic analysis of an isolate from a putative novel family ........... 102
3.2.4 Comparison with other culturing studies ................................................. 104
3.3 Discussion ...................................................................................................... 106
Chapter 4 Characterisation of intestinal spore-forming bacteria in the context of
inter-host transmission ............................................................................................. 110
4.1 Introduction .................................................................................................... 110
4.2 Results ............................................................................................................ 111
4.2.1 Phylogenetic analysis of cultured ethanol-resistant spore-forming bacteria
.......................................................................................................................... 111
4.2.2 Phenotypic characterisation of intestinal spore-forming bacteria............ 114
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4.2.3 Genomic analysis of intestinal spore-forming bacteria and sporulation
dynamics ........................................................................................................... 120
4.3 Discussion ...................................................................................................... 128
Chapter 5 Host-adaptation of commensal bacteria through loss of sporulation and
reductive genome evolution ..................................................................................... 132
5.1 Introduction .................................................................................................... 132
5.2 Results ............................................................................................................ 133
5.2.1 Large and small scale loss of sporulation within the Firmicutes ............. 133
5.2.2 Genetic features of host-adaptation in non-spore-forming bacteria ........ 143
5.3 Discussion ...................................................................................................... 153
Chapter 6 Summary and future directions ............................................................... 157
References ................................................................................................................ 169
Appendix 1 Cultured Isolates ................................................................................... 189
Appendix 2 Isolates deposited in public culture collections .................................... 206
Appendix 3 Validation of the sporulation signature ................................................ 216
Appendix 4 Publications from this thesis ................................................................ 237

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