Microbiome studies for microbial disease pathogenesis research Jennifer Myskiw Msc Student in Dr. Stephanie Booths Lab NML

What is a microbiome? Group of microorganisms or microbes that inhabit a particular environment May consist of bacteria, viruses, and/or fungi Can be symbiotic or pathogenic Play a significant role in human disease and health Gut Health at every age, Abbot Global, 2017 <http://www.nutritionnews.abbott/nutrition-as-medicine/gut-health-at-every-age-.html>

Human Microbiomes Extremely abundant and diverse Approximately 10-100 trillion microbial cells living within the human body The human gut houses the most bacteria in the body and varies from person to person The human gut plays a significant role to human health and has been linked to many diseases Unlocking the secrets of the microbiome, The New York Times, 2017 <https://www.nytimes.com/2017/11/06/well/live/unlocking-the-secrets-of-the-microbiome.html>

Microbiome Studies Microbiome papers published, NCBI, 2017 <https://www.thinkbiome.com/talkbiome/microbiome-bites-may-17th-meta-analysis-of-published-microbiome-studies-finds-disease-specific-microbiome-patterns> Due to advances in technology and appreciation of how human microbiomes play a role to human health, the amount of microbiome studies have drastically increased

Microbiome Study Challenges Reproducibility is key Meticulous documentation of how data was collected, processed, and analyzed is necessary Metadata is an important factor when planning and executing microbiome studies Confounding factors can skew data leading to incorrect assumptions Experimental design considerations for microbiome experiment. Retrieved from “Best practices for analysing microbiomes” by R.Knight et al. 2018, Nature, 16, 414.

Typical Experimental Workflow Typical experimental workflow with experimental variables. Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 119.  

Experimental Design – Human Studies Cross-sectional Studies Observational study at an individual time point Useful when wanting to find differences between human populations Longitudinal Studies Observational study that follows an individual over a span of time Possible to collect baseline sample Reduce variables found in cross-sectional studies Interventional Studies Experimental study where there is a test group and a compare group (baseline sample) Allows direct observation between microbiome effect on or from treatment/environment

Experimental Design – Animal Studies More easily controlled and manipulated compared to human studies Rodents are preferred based on their physiological similarities to humans and them being well defined Germ Free mice (GF) Born and raised without exposure to microorganisms Allow a direct link between specific bacterium and effect Humanized mice Microbiome composition very similar to donor human Limitations include strong species-associated microbiota within the gut Retrieved from <https://www.skedaddlewildlife.com/how-to-keep-mice-out-of-your-garden/>

Experimental Design – Animal Studies Caution must be used when using rodents for microbiome studies Research environment (cages) and breeding are confounding facts Caging of mice, diets of the mice and mothers, and stress factors must all be taken into consideration and documented Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 125.

Sample Collection and DNA extraction Human and mice samples often include feces collection or extraction of intestinal tissue DNA extraction methods may lead to biases, it is important that the same reagent kits be used for all samples within a study Blank controls should be used when using amplification kits to rule out any potential contamination DNA Precipitate, 2009, Retrieved from <https://www.sciencelearn.org.nz/resources/2036-dna-extraction

Sequencing Different sequencing and analysis methods for microbiome research. Retrieved from “Combing metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems level understanding of human microbiome” by S. Bikel et al. 2015, Computational and Structural Biotechnology Journal, 13, 390-401.  

Sequencing – Marker Gene analysis Sequencing with the use of primers that are specific to a gene region 16sRNA is used for bacteria and archaea and internal transcribed spacer (ITS) is used for fungi Well tested, relatively cheap, and fast Errors should be removed from sequencing through clustering OTUs or through oligotyping Taxonomic names may then be designated to microbial communities through databases

Sequencing – Metagenomic and Metatranscriptomic analysis Metagenomic sequencing DNA sequencing of all microbial genomes found in a sample Metatranscriptomic sequencing RNA sequencing in order to understand gene expression and functional activity Both of these methods allow for in depth understanding of the microbes collected Collected DNA or RNA sequences should be compared to reference data bases in order to allocate taxonomy Data may also be organized into short reads forming contigs, by doing so it is possible to explore data beyond taxonomic assingment

Case Study Davenport et al. used a GWAS to investigate genetic contribution to microbial composition within the gut of humans Worked with Hutterite communities in North America Looked for differences in gut microbiome composition in regards to age, sex, and genetic affects 184 stool samples collected over summer and winter Microbial composition was compared to genotype information from each participant Retrieved from <http://amishamerica.com/hutterite-clothing/> Found 8 bacterial taxa whose presence in gut was related to SNPs in the host genome One of these bacterial taxa was linked to obesity, suggesting some individuals may be predisposed to diseases such as obesity due to genetic factors

Significance Microbial communities within the gut have many functions such as synthesis of vitamins and amino acids, development of the immune system, and resistance to pathogens Defects in the host genome affecting microbial composition of the gut or disruption of homeostasis in the gut often lead to complex diseases By furthering our understanding of the relationship between the human body and the microbes that inhabit it, new treatments, therapies and preventatives to devastating human diseases are possible Main functions of bacteria in the gut. Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 118.  

Questions?