WEEK 1: Microbiology of the gut

Question 1

Define the following terms:
Microbiome
Microbiota

Answer 1

Microbiome: The ecological community
(genetic information) of commensal,
symbiotic & microorganisms that
share our body space & have a role
in the hosts health & disease states.

Microbiota: Microorganisms constituting the
microbiome.

Question 2

Human adult comprises ~1013 cells
Vs.
~1014micro. in gastrointestinal tract

Bacteria predominant

Viruses, fungi & protozoa are a minor component in healthy individuals.

What influences Microorganisms acquired rapidly during & after birth?

Give example.

Answer 2

Microorganisms acquired rapidly during & after birth influenced by nutrition & environment.

E.g.
GIT of ‘breast-fed’ verses= Lactic acid streptococci & lactobacilli
‘bottle-fed babies’= Diversity of microorganisms

Question 3

The human oral microbiome.

Oral cavity - ‘major gateway to the human body’

Several microbial habitats, i.e. teeth, gingival sulcus, attached gingiva, tongue, cheek, lip, hard & soft palates.

Air passes through the nose & mouth on way to trachea & lungs.

Food enters its chewed, mixed with saliva on way to GIT.

Name the 6 major phylum of bacteria in the oral cavity.

Answer 3

Six major phyla (96%):
1. Firmicutes e.g. Streptococci & Staphylococci (Gram+ve cocci facultative anaerobes

2. Bacteroidetes e.g. Bacteroides (Gram+ve rods anerobes)
3. Proteobacteria e.g. Escherichia, Salmonella, Vibrio, (Gram-ve facultative or obligate anaerobes)
4. Actinobacteria e.g. Corynebacterium (Gram+ve rods facultative anaerobes)
5. Spirochaetes - Helical or spiral-shaped anaerobes
6. Fusobacteria e.g. Gram-ve rods anaerobes

Question 4

Discuss The oral microbiome.

Answer 4

1st yr (teeth absent) mainly facultative anaerobic bacteria
Streptococci & lactobacilli (phylum Firmicutes)

S. salivarius dominant until eruption of teeth

Formation of gingival crevice increases diversity of bacteria

Shift towards those adapted to teeth surfaces, crevices & small fissures i.e. S. sobrinus, S. sanguis & S. mutans

With increase in age, microorganisms become more varied, by puberty Bacteroides spp. colonies.

Question 5

Oral cavity microorganisms have been shown to cause oral infections.

State 2 micro-organisms that convert sucrose to lactic acid, which erodes teeth enamel leading to cavities.

State other oral cavity infections apart from tooth decay.

Answer 5

S. mutans & S. sobrinus

1. ABSCESSES
   These are localized collections of pus caused by bacterial infection.

In dental contexts, abscesses can occur either in the gums or at the root of a tooth. They often result from untreated dental cavities, trauma to the tooth, or gum disease.

Abscesses can be quite painful and may require drainage and antibiotic treatment.

2. GINGIVITIS:
   This is inflammation of the gums (gingiva) caused by bacterial plaque buildup along the gumline.

It’s a common and early stage of gum disease. Gingivitis is characterized by red, swollen gums that may bleed easily, especially during brushing or flossing.

Proper oral hygiene, including regular brushing, flossing, and professional dental cleanings, can usually reverse gingivitis.

3. Periodontal disease:

Also known as gum disease or periodontitis, this is a more advanced stage of gum disease that affects the tissues surrounding and supporting the teeth.

It’s caused by untreated gingivitis, allowing bacteria to penetrate deeper into the gums and form pockets between the teeth and gums.

Periodontal disease can lead to tooth loss if left untreated.

Treatment typically involves professional deep cleaning (scaling and root planning) and in severe cases, surgery.

Question 6

Discuss complications of oral bacteria in the CVD, stroke and pneumonia.

Answer 6

1. Cardiovascular Disease:

Some studies have found an association between gum disease and heart disease. The exact mechanism isn’t fully understood, but it’s believed that the inflammation and bacteria associated with gum disease may contribute to the inflammation of the arteries, which is a risk factor for heart disease.

2. Stroke:
   Gum disease has been linked to an increased risk of stroke. Again, inflammation seems to be a key factor. The bacteria and inflammation associated with gum disease may contribute to the formation of blood clots, which can block blood flow to the brain and lead to a stroke.
3. Pneumonia:
   Oral bacteria can be aspirated into the lungs, particularly in people with poor oral hygiene or compromised immune systems. This can lead to infections such as pneumonia, especially in vulnerable populations such as the elderly or those with respiratory conditions.
4. Bacterial Endocarditis:
   This is an infection of the inner lining of the heart chambers or valves.

Certain oral bacteria, like Streptococci, can enter the bloodstream through wounds in the mouth (such as during dental procedures) and adhere to heart valves, potentially leading to bacterial endocarditis.

Question 7

Gastric microbiome

Stomach secretions highly acidic (chemical barrier)

State the pH of the stomach.

Which bacteria brave these harsh conditions?

Answer 7

pH 2

Dominating phyla include:
Proteobacteria, Bacteroidetes, Actinobacteria, Fusobacteria, Firmicutes & Prevotella.

Helicobacter pylori (Proteobacteria) most
common bacterium
>50% of adults harbor H. pylori.

Also commonly present: Neisseria, Haemophilus (Proteobacteria) & Porphyromonas species (Bacteroidetes)

Helicobacter greatly influences the gastric flora i.e. When H. pylori present as a commensal, there is gastric microbial diversity constituted by several phyla
Firmicutes
Streptococcus species

Actinobacteria

Prevotella
However,
If H. pylori is pathogenic, it dominates & gastric microbial diversity is dramatically reduced.

Question 8

Discuss Helicobacter pylori.

Answer 8

Helicobacter pylori
Gram-ve, helix shaped & microaerophilic

Burrow into mucosal lining to depth where pH essentially neutral.

Urease: converting urea produced by stomach into ammonia & CO2

NB. H. pylori causative agent of gastric ulcers ~20% of people colonized.

Question 9

Name the following:

1. Dominant in stomach
2. Dominant in distal esophagus, duodenum & jejunum

Answer 9

Helicobacter pylori dominant in stomach

Streptococcus spp. dominant in distal esophagus, duodenum & jejunum

Question 10

Why are the small intestines facing challenges in establishing dense populations of microorganisms compared environment of the large intestine (colon).

Answer 10

With food passing through the small intestine in approximately 3-5 hours, microbial colonization may indeed face challenges in establishing dense populations compared to the slower-moving environment of the large intestine (colon).

The conditions within the small intestine, such as the acidic environment of the stomach and the presence of bile acids and digestive enzymes, can further limit microbial survival and growth.

So, colonization may be difficult, limiting bacterial density in comparison to large intestine.

Question 11

Describe the 2 distinct parts of the small intestines.

Answer 11

Two distinct environments:
Duodenum vs Ileum

1. Duodenum next to stomach, fairly acidic & microbiota ~stomach
   10^3-10^5 microorganisms/ g
   Lactobacilli
   Streptococci
2. Ileum- pH less acidic, environment more anoxic & bacterial density increases
   
   *In medical contexts, “anoxic” may refer to a lack of oxygen supply.

10^8 microorganisms/ g
-Fusiform anaerobic bacteria typically present
-Enterobacteriaceae
-Bacteroides spp.
(In addition to some Lactobacilli & Streptococci)

Question 12

What are Enterobacteriaceae?

State the microorganisms.

Which part of the gut are they found?

Answer 12

*Enterobacteriaceae is a large family of gram-negative bacteria that belong to the phylum Proteobacteria.

These bacteria are commonly found in the environment, as well as in the gastrointestinal tracts of humans and animals.

EXAMPLES: Escherichia, Salmonella, Klebsiella, Enterobacter, Citrobacter, Proteus

Ileum

Question 13

Ileum empties into caecum & things slow down.

How long does food take to reach the colon?

Slower flow rate enables high proliferation of bacteria.

Bacteria packed into lumen account to how many % of colon contents & how many kg of total body weight in an adult?

Answer 13

24-48hrs for food to move in colon vs. 3-5hr in small intestine.

Slower flow rate enables high proliferation of bacteria.

Bacteria packed into lumen account 35-50% colon contents & 0.9kg of total body weight in an adult.

Question 14

Which bacteria are in our large intestine?

Answer 14

98% of gut microbiota fall into 4 bacterial phyla:

1. Firmicutes
2. Bacteroidetes
3. Proteobacteria
   In the gut it’s low density or absence + high abundance of signature genera i.e. Bacteroides, Prevotella & Ruminococcus suggests ‘a healthy’ intestinal microbiota.
4. Actinobacteria

Question 15

State the Colon predominant phyla.

Answer 15

Colon predominant phyla: Firmicutes & Bacteroidetes

Question 16

State the following in the Gut.

1. Predominant phyla include:
2. Phylum markedly low

Answer 16

In gut
Predominant phyla include: Firmicutes & Bacteroidetes

Phylum Proteobacteria markedly low

Question 17

Describe Large intestine microbiome.

Answer 17

Colon strictly anoxic & fermentation vessel

High density of ‘obligate’ anaerobes (1010-1011/g) including Bacteroides & Clostridium species.

Archaeal methanogen i.e. Methanobrevibacter smithii ~ 10% of all anaerobes in colons of healthy adults

Less common bacteria:
Proteobacteria

Include ‘pathogens’ but in low abundance (<0.1%) e.g. Campylobacter jejuni, Salmonella enterica, Vibrio cholera & Escherichia coli, Proteus, Enterobacter

(Facultative anaerobic bacteria consume O2, help make large intestine anoxic)

Question 18

What makes up ~ 10% of all anaerobes in colons of healthy adults?

Answer 18

Archaeal methanogen i.e. Methanobrevibacter smithii ~ 10% of all anaerobes in colons of healthy adults

Question 19

A healthy Gut microbiome associated with good health.

Defining a healthy Gut microbiome.

A landmark study by Chatelier et al., 2013. Nature: 500:541-546. Danish metagenomic study of 169 people with an obese phenotype & 123 with a non-obese phenotype revealed:

Based on composition & diversity of their microbial communities they could be grouped as:

High gene count (HGC) vs. Low gene count (LGC)

How many genes do ‘LGC & HGC’ individuals have?

Discuss the distinction between the categories.

Answer 19

‘LGC & HGC’ individuals had counts of 380,000 & 640,000 bacterial/ phage genes respectively.

Generally, HGC individuals had more diverse microbial metabolic functions & better overall health i.e. lower prevalence of metabolic disorders i.e. obesity & diabetes
**Thus, regarded as ‘healthy gut microbiome***

Question 20

Define a healthy gut.

Answer 20

So healthy gut microbiome can be defined by:
Richness & diverse beneficial bacterial metabolic activities

Question 21

Discuss HGC individuals.

Answer 21

Greater repertoire of microbial metabolic functions.

Higher proportion of certain ‘Firmicutes’ bacteria i.e. Bacteroides, Roseburia, Bifidobacterium, Fecalibacterium that carry out ‘beneficial metabolic activities:

Production of ‘Short chain fatty acids’ (SCFAs): butyrate, propionate & acetate which:

i) Rich sources of energy for the host
ii) Regulate intestinal physiology & immune function

Higher proportion of bacteria with higher propensity for H2 & methane production & reduced H2S production

Question 22

Discuss LGC individuals.

Answer 22

Generally had higher proportion of pro-­inflammatory bacteria e.g. Bacteroides & Ruminococcus gnavus assoc. with inflammatory bowel disorders

Question 23

What is the gut microbiota up to?

Answer 23

1. Metabolism of carbohydrates & proteins

Intestinal bacteria genome encodes for a greater no. of diff. ‘carbohydrate-active’ enzymes than human genome. Vital in metabolism of complex dietary carbohydrates

e.g. digestion of xyloglucans commonly found in vegetables i.e. lettuce & onion, linked to members of phylum Bacteroidetes.

2. Production of SCFAs e.g. butyrate, propionate & acetate produced by bacteria that ferment undigestible from fiber e.g. from fiber-rich foods e.g. legumes, fruits, vegetables.

SCFAs are essential for nutrition for colon cells; energy sources for host & critical in modulating intestinal immune responses.

3. Amino acid metabolism: gut microbiota has efficient proteinases & peptidases that catabolize proteins to amino acids.

Several amino acid (aa) transporters are on bacterial cell wall. These transport aa’s from intestinal lumen into bacteria.

Aa’s then metabolized into.

1. Signaling molecules & antimicrobial peptides.

e.g. conversion of L-­histidine to histamine by bacterial enzyme histamine decarboxylase.

glutamate to Gamma-­Amino-Butyric-Acid (GABA) by bacterial glutamate decarboxylases

Mere presence of normal gut microbiota reduces proportion of pathogens.

4. Xenobiotic & drug metabolism

Question 24

State the functions of factors & metabolites that secreted by gut microbiota.

Answer 24

-Modulate intestinal permeability, epithelial cell function & intestinal motility

-Regulate pro- & anti-inflammatory mechanisms of intestinal mucosal immune system

Question 25

Unhealthy Gut microbiome?
Consequences of Gut microbiota dysbiosis.

There is research evidence that associates ‘dysbiosis’ with higher disease risk.

What is dysbiosis?

Give examples.

Answer 25

1. Disruption of gut microbiota.
2. Dysbiosis refers to an imbalance or disruption in the microbial community that inhabits a particular environment, such as the human gut.

Examples:
-Individuals with symptoms of metabolic disorders tend to have less diverse fecal microbiota than healthy individuals.

-Colitis sufferers, esp. those with ileal Crohn’s disease, tend to have severely reduced no’s of butyrate-producingFirmicutesspp.

However, the ‘causal’ link between microbiota composition & diseases remains undetermined.

Question 26

Good bacteria in gut help to digest food, absorb nutrients & keep check on ‘bad bacteria’. Uncontrolled growth of bad bacteria can cause a range of diseases.

Outline causes of dysbiosis.

Answer 26

1. Associated with some diseases i.e. inflammatory bowel diseases; irritable bowel syndrome; allergies; metabolic diseases e.g. obesity & diabetes.
2. Antibiotic use may inhibit growth of antibiotic-susceptible normal flora

Certain antibiotics e.g. clindamycin & 3rd generation cephalosporins most commonly implicated.

3. Resistant microorganisms i.e. Clostridium difficile proliferate, causing infections e.g. colitis, diarrhoea

Question 27

Outline the good bacteria in the small intestines.

Answer 27

Bifidobacteria
Lactobacilli
E. coli

Question 28

Outline the bad bacteria in the small intestine.

Answer 28

Clostridium difficile
Campylobacter jejuni
Campylobacter coli
Enterococcus faecalis

Question 29

Discuss the pathogenesis of Clostridium difficile diarrhea..

Answer 29

1.Clostridium difficile produces toxins A and B and hydrolytic enzymes.

2. Toxins A and B results in the release of Tissue necrosis factor alpha and pro-inflammatory interleukins.
3. There is increased vascular permeability, neutrophil and monocyte recruitment.
4. There is opening of epithelial cell junction.
5. There is apoptosis of epithelial cells.
6. Hydrolytic enzymes result into connective tissue degeneration, leading to colitis, pseudo membrane formation.
7. Watery diarrhea forms

Question 30

Outline Several factors can influence the Gut taxonomic diversity.

Answer 30

1. Mode of foetus delivery
2. Geographic origin
3. Host genome
4. Diet
5. Antibiotics
6. Probiotics
7. Age
8. Stress.

Question 31

Discuss ways of Re-establishing Gut microbiota.

Answer 31

1. Cessation of antibiotic treatment enables relatively quick, re-colonization of normal microflora.
2. Ingestion of probiotics &/or prebiotics to facilitate establishment of desired species.
3. Fecal Microbiota Transplant (FMT):
   Fecal matter, collected from healthy donor, mixed with saline or other solution, strained & placed in patient by colonoscopy, endoscopy, sigmoidoscopy or enema.

A randomised study published in New England Medical Journal, Jan. 2013 reported 94% cure rate of C. difficile colitis with FMT vs. only 31% with vancomycin

Due to outstanding results, study prematurely stopped as it was considered unethical not to offer FMT to all study participants.

Unfortunately, FMT not yet FDA approved.

Question 32

Interesting tit bits:Gut microbiome & obesity?

Discuss Role of Gut microbiome in obesity?

Answer 32

Gut microbiomes have essential role in breakdown of food & convert it into energy.

Metagenomic sequencing of distal gut microbiome in healthy & diseased states showed that gut microbiome primarily:

1. Phylum Firmicutes
   largely Clostridium & Lactobacillus species
2. Phylum Bacteroidetes
   largely Bacteroides or Prevotella species

Question 33

The mice study.

Initial evidence from studies comparing normal mice & germ-free (gnotobiotic) mice fed with same food rations

Normal mice had 40% more total body fat vs. those raised in germ-free environments

Germ-free mice then given faecal microbiota transplants from normal mice BUT no changes in food intake

Germ-free mice developed gut microflora & their total body fat increased……….Why?…….

Answer 33

1. Initial Difference in Total Body Fat:

The observation that normal mice had 40% more total body fat compared to germ-free mice suggests that the presence of gut microbiota influences fat accumulation in the body. This initial difference highlights the importance of gut microbiota in regulating host metabolism and energy balance.

2. Fecal Microbiota Transplantation (FMT) from Normal Mice:

When germ-free mice were given FMT from normal mice, they acquired a gut microbial community resembling that of the donor mice. Despite receiving the same food rations as before, the germ-free mice experienced an increase in total body fat.

3. Potential Mechanisms for Increased Body Fat:

Several factors may contribute to the observed increase in total body fat in germ-free mice following FMT:

a. Altered Energy Metabolism: The gut microbiota play a crucial role in regulating host energy metabolism.

Changes in gut microbial composition can influence energy extraction from the diet, nutrient absorption, and energy storage, leading to changes in body fat accumulation.

b. Enhanced Nutrient Absorption: The presence of gut microbiota can enhance the absorption of nutrients, including dietary fats, by facilitating their breakdown and absorption in the intestines.

Changes in gut microbial composition may promote more efficient nutrient absorption, contributing to increased fat deposition.

c. Regulation of Host Metabolism: Gut microbiota can interact with host metabolic pathways, hormones, and signaling molecules involved in lipid metabolism and fat storage.

Alterations in gut microbial composition may dysregulate these metabolic pathways, leading to increased fat accumulation.

d. Influence on Appetite and Satiety: Gut microbiota can influence host appetite and satiety through the production of signaling molecules and metabolites that interact with the central nervous system.

Changes in gut microbial composition may affect host feeding behavior and energy intake, indirectly influencing fat deposition.

Answer 34

Mice that were genetically obese had increase in phylum Firmicutes; 50% less Bacteroidetes & greater no. methanogens.

Hypothesis:
1. M. smithii scavenges H2 from other microbes, using it to produce methane.

2. H2 removal stimulates fermentation by neighboring H2 producing bacteria.
3. These H2-producing bacteria are often involved in the breakdown of complex carbohydrates and fibers from the diet.
4. These thrive & produce Volatile fatty acids (VFA) & extract nutrients from food more efficiently.
5. Essentially allowing host to harvest more calories from food & this may contribute to weight gain.

In obese phenotype cases ratio of Firmicutes: Bacteroidetes elevated
Significant decrease in Bacteroidetes in obese individuals vs. non-obese individuals- …but this is debatable.

Also, abundance of certain spp. i.e. Methanobrevibacter smithii, Bacteroides thetaiotamicron & assoc. with weight gain.

Although exact cause of obesity still to be fully understood

Gut microbiome may play a role in the ability of host to harvest ‘more’ organic nutrients from their diet

This may also offer a non-genetic explanation for obesity & possibly a resolution:
…………..………..Faecal microbiota transplant is the future?