Lecture 8: human microbiome

Question 1

There are several reasons why bacteria can reside in our gut and why there able to grow there, especially in our colon. Name these reasons.

Answer 1

• Suitable temperature
• Nutrients in high concentrations due to resorption of water in the colon.
• Food/nutrients resides in the colon for 24-48 hours → optimal uptake
• high pH
• Microbes are inhibited by their own metabolites → the colon resorpes these metabolite, so growth is not inhibited.

Question 2

Bacteria in the gut are (in general) beneficial for our body. Name reasons why bacteria are beneficial.

Answer 2

• Humans lack enzymes for degradation of fibers and carbohydrates. Bacteria break down our food for us (and for themselves).
• Colonization resistance → due to the colonization of bacteria in the gut, other pathogens can’t colonize. Bacteria are thus tolerated, so that pathogens can’t.
• Humans can’t produce vitamin K, bacteria do.
• Brain-gut axis
• Bacteria educate our adaptive immune system (→ 1000 days from the moment of conception)

Question 3

Fill in:

In earlier days, it was thought that there were … (1) times more bacteria than human cells. Now we know the ratio of human and bacterial cells in our body is … : … (2).

Answer 3

In earlier days, it was thought that there were 10 times more bacteria than human cells. Now we know the ratio of human and bacterial cells in our body is 1 : 1.

Question 4

But while the ratio of bacterial to human cells is 1 : 1, bacteria are much more efficient compared to human cells. Why could you consider bacteria to be more efficient?

Answer 4

Humans have up to 20.000-25.000 genes and bacteria have about 3.3 million genes. 2% of human genes are protein encoding, while in bacteria almost all genes are protein encoding.

Question 5

What’s the definition of the following:

1. The microbiota
2. The metagenome
3. The microbiome

Answer 5

1. Microbiota → the ecological community of commensal, symbiotic, and pathogenic microorganisms that share our body space.
2. Metagenome → the collection of genes that are encoded by the members of a microbiota.
3. Microbiome → the entire habitat, including the microorganisms, their genomes and the surrounding environmental conditions.

Question 6

In earlier days, microbiome research was based on culturing bacteria and using radiation probes. What development resulted in advancement of microbiome research?

Answer 6

The development of DNA sequencing technology, e.g. Sanger dideoxy sequencing and later the developent of Next-Generation Sequencing techniques like Illumina.

Question 7

How can microbiota be identified?

Answer 7

Microbiota have conserved and variable regions in their genome. Microbiota with the same conserved regions are closely related and thus share the same genus. By looking at the variable regions of these same genus bacteria, you can identify the microbe at species level. This can only be done when there’s a database with known/identified microbiota.

Question 8

Name two techniques that can be used to identify microbiota (in regard to the conserved and variable regions).

Answer 8

1. Metagenomic sequencing → sequencing of variable regions with the use of 16s rRNA.
2. Tagged PCR → using primers that bind to variable regions

Question 9

What’s a limitation of DNA sequencing (e.g. tagged PCR or 16s rRNA sequencing) that isn’t a limitation in culturing bacteria?

Answer 9

With DNA sequencing, there’s no clue to whether the sequenced DNA originates from dead or live bacteria. In culture, you can bypass this by selective culturing with the use of e.g. antibiotics.

Question 10

What’s another limitation of identification of bacteria by DNA/RNA sequencing?

Answer 10

DNA/RNA sequencing has a certain resolution, where in some cases a bacteria can only be identified on species or genus level.

Only knowing that the bacteria that is found is identified in the E. coli genus, isn’t enough. Only the strain will tell you whether the strain is harmfull or harmless. For example, E. coli strain K12 is commensal while E. coli strain O157:H7 is pathogenic.

Question 11

What can be identified with 16s rRNA sequencing?

Answer 11

• Species
• Relative abundance of species within a sample.

Question 12

Total microbiome DNA sequence is more demanding since you need a database to compare results with. But it generates lots of information about microbes. What can be identified with total microbiome DNA sequencing?

Answer 12

• Species
• Relative abundance of species within sample
• Genes
• Variants
• Polymorphisms
• Functional information

Question 13

What is the definition of Operational Taxonomic Unit (OTU)?

Answer 13

In metagenomics, OTU is used to classify bacteria based on sequence similarity of the 16s marker gene. An OTU consists of a group of bacteria whose 16S marker gene shows a sequence identity of 97 percent and above. It is used to classify bacteria at the genus level.

Question 14

What is seen here?

Answer 14

The surface of the colon, with epithelial cells in blue, mucus in black and bacteria in green. Bacteria reside on the shedding surface of epithelial cells. This prevents bacteria from colonizing for a long time and overgrowing and they also use the shedding and mucus as metabolites.

Question 15

Why is it extremely difficult to cultivate gut microbes?

Answer 15

Because the gut is anaerobic and only obligate anaerobic microbes can grow there. To cultivate these microbes, they have to live anaerobically in vitro.

Question 16

What microbe is responsible for stomach ulcers?

Answer 16

H. pylori

Question 17

What bacteria dominate the gut?

Answer 17

Firmicutes and bacteriodes

Question 18

What’s an enterotype?

Answer 18

Here, an organism is classified bases on the bacteriological composition of their gut microbiota.

Question 19

It was previously thought that there are 3 different enterotypes in humans. What enterotypes are these?

Answer 19

1. Enterotype with abundance of Bacteroides
2. Enterotype with abundance of Prevotella
3. Enterotype with abundance of Ruminococcus

Question 20

Why isn’t it entirely true that there are 3 different enterotypes for humans?

Answer 20

Because in humans it’s more a continuous flow of different bacteria. For example, diet can be a major influence on the gut microbiota; if you eat a lot of carbohydrates (like in South-Asia or US), the gut is dominated by Prevotella. While in the Netherlands the gut is dominated by Bacteroides, due to our milk consumption.

Question 21

Besides diet throughout our life, what else is important in determining what bacteria dominates our gut?

Answer 21

The way babies are born (ceasarian or natural childbirth), are fed (breast or bottle fed) or when they eat solid food.

Question 22

What component in breast milk is important for the development of certain bacteria, like the bifidobacteria? And what do they do to solve this issue in babies that are bottle fed?

Answer 22

Oligosaccharides are important for the development of certain bacteria. Because of this, oligosaccharides are often added to milk formulas.

Question 23

There are four postulates (Koch’s postulates) that determine whether a microbe is a pathogen. Name these postulates.

Answer 23

1. Micro-organisms must be found in abundance in all organisms suffering from disease, but should not be found in healthy organisms.
2. Micro-organism must be isolated from a diseased organism and grown in pure culture.
3. Cultured micro-organism should cause disease when introduced in a healthy organism.
4. Micro-organism must be resiolated from the inoculated, diseased (experimental) host and identified as being identifical to the original specific causative agent.

Question 24

What is the role of gut microbiota in obesity in mouse models (compared to healthy mice)?

Answer 24

• Eubiosis (optimal balance of microbiota in the gut) in mice ensures an efficient way to gain energy from food (efficient energy metabolism). Mice stay lean with eubiosis.
• When the gut of mice is in dysbiosis, the mice will lack a hormone that inhibits the sensation of hunger. With this, more energy is obtained from the same diet and mice will get obese (inefficient energy metabolism).

Question 25

What is a germ free mouse?

Answer 25

A mouse that is born in a sterile environment and kept sterile. It is kept sterile so that it can be controlled what microbiome is added, the result is a probiotic mouse with known microbiota.

Question 26

Certain human gut microbes are associated with obesity. What happens when someone develops obesity?

Answer 26

The ratio Firmicutes and Bacteriodetes changes. In obese people there are more Firmicutes. Also seen in the picture.

Question 27

The number of (bacterial) genes (low gene count (LGC) or high gene count (HGC)) influences Body Mass Index (BMI) and adiposity. Does a HGC or LGC correlate with a higher BMI and more adiposity?

• One place in the body is an exception to this. Name this bodypart.

Answer 27

A LGC correlates with an increased BMI and adiposity. And vice versa a HGC correlates with health.

• In the vagina this is the other way around, where HGC is associated with disease and LGC with health.

Question 28

Fill in:

Inflammatory Bowel Disease (IBD) patients have a reduced functional capacity of the gut microbiome. This is due to a *increased/decreased* diversity of the gut microbiome.

Answer 28

Inflammatory Bowel Disease (IBD) patients have a reduced functional capacity of the gut microbiome. This is due to a *decreased* diversity of the gut microbiome.

Question 29

That health is associated with a low gene count/low diversity holds up for both the vagina as the urethra. But for the vagina, besides the importance of a low diversity, something else is also important for health. What is this?

Answer 29

It’s important that besides a low diversity, that there’s a dominant type of bacteria. This is seen in the picture, where red implies a dominating type in the vagina.

Question 30

Explain why a high diversity of microbes in the vagina is associated with a less protective environment.

Answer 30

The vagina is protected by its’ acidic environment. Vaginal bacteria can convert carbohydrates into lactic acid, making the environment of the vagina acidic. But increased diversity can slow down this process, making the vagina less acidic and therefore less protected.

Study this picture.

Question 31

What bacteria usually dominate the vagina in healthy conditions? And what bacteria is associated with an elevated pH of the vagina?

Answer 31

• Lactobacillales* and L. crispatus.
• Gardnerella vaginalis* is associated with an elevated pH of the vagina.

Question 32

This is a normal cell in the vagina. Describe what you see and explain what happens upon dysbiosis of the vagina.

Answer 32

The normal cell has very few bacteria, also these bacteria are mostly from the same species/strain. Upon dysbiosis, overgrowth can be seen with high cell numbers and high variety.

Question 33

The human skin is also a great source of bacteria. The place and type of the skin determines what bacteria can reside. Describe where on the skin Staphylococcus, Propionibacterium and Corynebacterium can be found.

Answer 33

• Staphylococcus → moist skin, under feet or armpits.
• Propionibacterium → high lipid concentration, on face.
• Corynebacterium → dry skin, on scalp or back.

Question 34

Antibiotics can form serious health problems due to the effect they can have on the gut microbiome. What can be the result of overusing antibiotics?

Answer 34

Some bacteria can be resistant to antibiotics and some bacteria are not. C. difficile is an example of bacteria that are resistant to antibiotics. C. difficile is able to form spores, these spores are completely resistant to antibiotics and can survive harsh environments. C. difficile is able to (over)grow because of this, while a big part of the bacterial diversity is lost due to antibiotics.

Question 35

What’s a solution/treatment for the relapse of C. difficile as a consequence of antiobiotic use?

Answer 35

When there’s C. difficile overgrowth due to use of antibiotics, a nasal tube to the stomach can be inserted where pro-biotics are added. Pro-biotics are bacteria from a stool donor that are able to restore the balance of the gut microbiome. This resulted in great recovery compared to other treatments used for the dysbiosis in the gut after use of antibiotics