Download Diet Rapidly and Reproducibly Alters the Human Gut Microbiome

Diet Rapidly and Reproducibly
Alters the Human Gut Microbiome
Lawrence A. David et al.
Presented by Xiao Liu and Joshua Chevez
Microbiota and the Microbiome
Microbiota
The collection of microorganisms
present in certain habitat.
Microbiome
The set of genes associated with
this collection of microbes.
This includes viruses, archaea, fungi,
and bacteria.
Environment and the Microbiota
In prenatal stages of development, the gastrointestinal tract of a human fetus
is sterile.
The types microbes that we acquire is dependent on our interactions with the environment.
A good example of environment dependent acquisition of microbes is that a
newborn’s microbiota depends on the mode of delivery.
The microbiota of infants born normally resemble the mother’s vaginal microbiota.
The microbiota of infants born via Cesarean section resemble microbes found on skin.
Scale and Diversity of the Microbiome
Metagenomic methods in previous studies report a collection of 3.3 million
unique genes present in the human microbiome.
This compared to the estimated 22,000 genes in humans gives some insight into the just how
massive the human microbiome is in its diversity of genes.
Remarkably, the microbiota has been shown to be about 80-90% different
between any two individuals.
Our interactions with the environment plays a large role of which diet is a major component.
A Symbiotic Relationship
The human microbiota plays a major role in the digestion and acquisition of
nutrients.
There are certain food components which humans are not capable of breaking down with the
collection of enzymes available.
The microbial community, with a variety of genes at their disposal can digest these substrates.
One example is how a rare Bacteroides species can digest Xyloglugans, a
family of dietary fibre.
Even if these species are rare, they are still present in about 92% of individuals.
This exemplifies just how beneficial this relationship is to humans.
Significance
Changes in a person’s microbiota influenced by the environment may
contribute to increased risk of certain chronic illnesses.
Obesity
Inflammatory Bowel Disease
Other enteric diseases
Studies of how certain diets may affect the promotion or inhibition of growth in
certain species of microbes may give insight into what diets are to be
avoided, especially if a specific species is linked to chronic illnesses.
Previous Studies
Previous studies have shown that the abundance of certain species in the
microbiota can fluctuate depending on the type of diet.
Experiments in mice showed significant changes in their microbiota within one day.
But experiments with human subjects tracked these changes on the scale of weeks to months.
David et al., seeing that microbial communities in mice could significantly
change within the course of one day asked how quickly the human
microbiota can respond to dietary changes and if these changes in diversity
can be linked to enteric diseases.
Experimental Design
•
6 males and 4 females were used in this study to track the effect of two separate diets over the
course of 15 days for each diet.
-Plant-based diet: Grains, legumes, fruits, and vegetables.
-Animal-based diet: Meats, eggs, and cheeses.
•
The 15 days were broken into three segments.
-A 4-day baseline to record normal food intake and their normal microbiota.
-5 days of the animal or plant-based diet.
-A 6-day washout period in which subjects revert to normal eating habits
•
Sampling took place through faecal samples once per day and food dyes were given at the start
and end of the diet to track when the food reached the distal gut.
Methods/Sampling
•
16s rRNA sequencing
-PCR amplified the rRNA and used
Illumina sequencing with barcoding.
-Highly conserved (identifying diversity of
microbes present)
•
ITS sequencing
-Used to identify any fungi present in the
gut.
•
RNA-seq
-Used to measure any significant
changes in microbial gene
expression.
Methods/Sampling
•
SCFAs (Short-Chain Fatty Acids)
-Analyzed from faecal samples to analyze shifts
in microbial metabolic activity.
•
Bile Acids
-Helps with digestion in the gut.
-Primary: Produced by liver.
-Secondary: Produced by microbes in the gut.
(Bile Acid Profiles)
•
Microbial Culturing
-Done before and after animal diet to test
bacteria in fermented food was present
after the diet.
ITS (Internal Transcribed Spacer) Sequencing
•
This ITS structure is unique to
eukaryotes (18S/5.8S/28S as opposed
to 16S/23S)
Primers can be made to specificly
target ITS1 and/or ITS2
•
David et al. focused on sequencing
ITS1 using barcodes, PCR to amplify,
and Illumina sequencing
NTS: Nontranscribed Spacer
ETS: External Transcribed Spacer
ITS: Internal Transcribed Spacer
Hypothesis
Primary: The human microbiota is capable of rapid diet-induced changes that
may contribute to the development of certain illnesses such as inflammatory
bowel disease.
Secondary: Microbes from fermented foods (animal diet) as well as plant
pathogens are capable of surviving transit through the gut, potentially influencing
the microbiota.
Results
•
Shows the changes in macronutrient
intake.
-Fibre Intake
-Fat Intake
-Protein Intake
•
Plant-based Diet
-Increase in Fibre
-Decrease in Fat and Protein
•
Animal Diet
-Increase in Fat and Protein
-Nearly zero Fibre intake
Results
Alpha (𝛂) Diversity: The species diversity
found within one sample or habitat.
Beta (𝛃) Diversity: The difference in the
diversity between two samples or
habitats.
Diversity of the diversity between
two samples.
Comparing the diversity of samples
during diet to the subject’s
baseline diversity.
Results
•
The log2 fold change in microbial
species clusters compared to
baseline.
-Clusters of species formed by
their similarity in “dynamics”.
-Green: 3 clusters significantly
different in Plant-based Diet.
-Red: 22 clusters significantly
different in Animal-based Diet.
•
An Animal-based diet results in more
changes in the microbiota of
subjects than the plant-based diet.
Results
•
Hierarchical clustering of the
microbiome based on gene expression
profiles
-Analyzed on day 3 and 4 of both diets
-Clustered based on diets
Results
Diet alters microbial metabolic activity
•
Diet affects fermentation products
•
Gene expression associated with animal-based
diet:
-Gln amidotransferase-producing vitamin
biosynthesis
-Methyltransferase- degradation of
carcinogenic compound produced during the
charring of meat
-B- Lactamase
Results
Are foodborne microbes able to
colonize in the gut?
Animal based diet1. Determined the abundance of
bacteria and fungi in the meal given
to the volunteers
Results
Foodborne microbes are able to
transiently colonize in the gut!
2. RNA sequence fecal samples-
-increased gene expression of bacteria
found previously in cured meats and
cheese (b-e)
-fungal concentration increased on
animal based diet (f)
-rubus chlorotic mottle virus colonization
found only in plant-based diet (g)
Results
Are foodborne microbes able
to colonize in the gut?
Plant-based diet
1. Determined the abundance
of bacteria and fungi in the
meals
2. Candida genus of fungi is
significantly increased
Animal-based diet -> changes in microbiota -> human intestinal disease
• Animal-based diets were high in fat:
increases bile acid concentration (a, c)
Increases bacterial genes coding for
bile salt hydrolases (b)
High fat + high bile acid concentration=
• Increase in B. wadsworthia- sulfite
reducing bacteria (d, e)
Conclusion
1. Alpha diversity in either diet did not change significantly.
2. Beta diversity changed significantly in the animal-based diet and not in the
plant-based diet.
3. Foodborne microbes- including bacteria, fungi, and viruses can survive
through the digestive system and may be active in the gut.
4. An animal-based diet leads to an increase in concentration of secondary bile
acids which disrupt bacterial growth and can lead to intestinal diseases.
Critiques
- Sample size is too small at 9 total people subjected to both diets
- There was one vegetarian that could have skewed the average data
- Subjects’ BMIs ranged from 19 to 32 Kg m-2
- A person considered obese if BMI is >30Kg m-2
- Might affect “average” baseline data
- “Subjects could eat unlimited amounts of the provided foods”
- Contradictory to: “ Differential weight loss between the two diets cannot be explained simply
by energy intake, as subjects consumed equal numbers of calories on the plant- and animalbased diets”
Further Readings
http://stm.sciencemag.org/content/1/6/6ra14.full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5277010/
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Questions?