By

Dr Monkez M Yousif
Professor of Internal Medicine
Zagazig University
Member of AGA, Member of EASL
Member of ISC-Hepatitis Working Group
 AGENDA
• Overview of Gut Microbiota
• Composition of Gut Microbiota
• Factors and processes that influence community
assembly and composition
• Functions of Gut Microbiota (Symbiosis)
• Techniques of analysis
• Gut Microbiota and Disease
• Fecal Microbiota Transplantation
 Diseases of Civilization
Multiple lines of study have shown that the primary cause of this environmental associated
inflammation may be dysfunction of the "gut-brain axis.“ secondary to alteration of gut microbiota.

Gut Brain Axis, Integrative Psychiatry

https://www.integrativepsychiatry.net/gut_brain_dysfunction.html. Accessed May 1st 2017
 A short story
• One day the body parts were fighting with each
other as to who is the most important
• The brain said: I am the most important as I
control everything
• The heart said: I am the most important as my
beating keeps the body going
• The liver said: I am the most important as help in
metabolism, excretion, detoxication, etc
• Finally the GUT said: I am the most important, I
digest, absorb, protect, excrete, produce
hormones, reabsorb etc
• EVERYONE LAUGHED---HA HA HA
• The GUT got angry and shut down
• Within a few days all the other body systems
agreed, it was the most important!
 Gut Microbiome

“All disease begins in the gut”

Hippocrates 460 BC – 370BC

“Health is determined by the microbiota in our gut”

Hippocrates

" ‫” المعدة بيت الداء‬

..‫طبيب العرب الحارث بن كلدة‬
 We Are Really More Bug than Man.......

Humans as micro biomes:-

- 10-100 trillion microbes in human
intestine.
- 3 million genes (100X).
- 2 kg weight.
- 300-1000 species of bacteria.
- control almost all body functions.
Where on a healthy human is the
microbiome located?

Every human body surface which is exposed to

the environment and every body part with an
opening to the environment has a microbiome.
Sites that harbor a
normal flora:

−Skin and mucous membranes

−External ear canal
−Upper respiratory tract
−Gastrointestinal tract
−Outer opening of urethra
−External genitalia
−Vagina

−External eye (lids, conjunctiva)

Distribution of Gut Microbiota
Major Bacteria Phyla and Genera Predominating in Human Gut Microbiota

Phyla Representative genera

Firmicutes (60-80%) ‒ Ruminococcus
‒ Clostridium
‒ Lactobacillus
‒ Enterococcus
Bacteroidetes (20-30%) ‒ Bacteroides
‒ Prevotella
‒ Xylanibacter
Actinobacteria (< 10%) ‒ Bifidobacterium
Proteobacteria ((< 1%) ‒ Escherichia
‒ Enterobacteriaceae

Munoz-Garach A, Diaz-Perdigones C, Tinahones FJ.

Microbiota y diabetes mellitus tipo 2. Endocrinol Nutr. 2016;63:560---568.
Factors Affecting Gut Microbiota
Factors that can influence the composition and function of the
human gut microbiota

Quigley, E. M. M. (2017) Gut microbiome as a clinical tool in gastrointestinal disease management: are we there yet?
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2017.29
 The Microbiome: Who’s there?
• Early gut colonization has four phases
– Phase 1: Sterile gut
– Phase 2: Initial acquisition: vagina, feces, hospital
– Phase 3: Breast feeding or bottle-feeding (different)
• Breast fed more bifidobacteria (up to 90% of flora)
• Bottle fed more diverse; more Bacteroides , and
Clostridial species
– Phase 4: Start of solids; move to adult flora (Fermicutes
and bacteriotedes)
• Bifidobacteria remain key flora into adulthood
Ley, Peterson, Gordon. Cell 2006 ;124:837
Ley, et al. PNAS. 2005, 102: 11070
Edwards, et al. Br J Nutr. 2002
Gut Microbiota and Geographic Location

• The microbiotas of European and African

children have completely different
compositions.
– African children have greater proportions of
Bacteroidetes and Gram-positive organisms in
their bowels,
– while a Western lifestyle appears to promote
increases in Firmicutes and Gram-negative
organisms.
Gut Microbiota and Modern Lifestyle
• Changes in human ecology have affected the
composition of microbiota during human evolution, but
a more radical change has occurred in recent decades.
• One of the most significant findings is that in developed
countries there has been a loss of certain species that
colonized our bowel some decades ago, with the
resultant loss of biodiversity of our microbiota.
• Factors that have influenced this change in microbiota
include
– water sanitation,
– increased performance of cesarean sections,
– more frequent use of antibiotics in preterm newborns,
– decreased breast-feeding,
– increased hygiene, or
– widespread use of antibacterial soaps.
Role of microbiota
in Health
 Role of microbiota in Health
• Symbiotic relationship: (Interaction
between two different organisms living
in close physical association, typically to
the advantage of both).
• Shaping and maintaining immunity:
– Innate immunity
– Adaptive immunity
Gut Microbiota in Health- symbiosis
Provides:
• Nutrients
• Shelter

Human (host) Microbiome

• Increases the metabolic capacity of the

host -digest plant carbohydrates, milk
products (glycans) - ~10% of the calories
from diet
• Provide vitamins (e.g. B2, B12, K and
folic acid)
• Protect from colonization with pathogenic
bacteria (Colonization resistance)
 Gut Microbiota in Health- innate immunity
• Produce pathogen-
associated molecular
patterns (PAMPs) and
metabolic by-
products and
regulate intestinal
immune responses
• PAMPs are
recognized by
pattern recognition
receptor (PRR)-
bearing cells of the
innate immune
system and many
epithelial cells
Gut Microbiota in Health: Adaptive immune system

• Microbiota stimulation leads to B cell switch to IgA,

regulatory T cell induction, T cell differentiation to Th17
- Commensal bacteria
induce CD4+T cell
differentiation.

- Naïve CD4+T cells can

differentiate into four
major cell types: Th1,
Th2, Tregs and Th17.

- Once differentiated,
each lineage secretes a
special (set of) cytokine
and perform specific
functions
Protective function (barrier effect)

 Compete and adhere to the attachment sites in

the brush border of intestinal epithelial.
 Compete for available nutrients.
 Produce antimicrobial (bacteriocins).

All of this will prevent attachment and

subsequent entry of pathogenic bacteria into
the epithelial cells
 The Intestinal Barrier Importance
normal development of immune system

2 3

5 1
7
 The microbiome–gut–brain axis

Modified, with permission, from Elsevier © Collins, S. M. & Bercik P. Gastroenterology 136, 2003–2014 (2009)

Quigley, E. M. M. (2017) Gut microbiome as a clinical tool in gastrointestinal disease management: are we there yet?
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2017.29
Role of microbiota in
Disease
Dysbiosis and disease
Dysbiosis: Proposed mechanism/s leading to disease

• Genetic and environmental factors induce impaired

barrier function
• Overgrowth of pathogenic bacteria; inhibition of
protective bacteria
• Translocation of bacteria and bacterial products
• Immune activation and proinflammatory cytokine
production
• Chronic inflammation leads to tissue destruction and
complications
• Concept of leaky gut
The 'leaky gut' hypothesis

Quigley, E. M. M. (2017) Gut microbiome as a clinical tool in gastrointestinal disease management: are we there yet?
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2017.29
Dysbiosis and diseases
• Diseases of the GUT
– Malabsorption syndrome
– Malignancies: Colorectal cancer
– Inflammatory Bowl disease (IBD)
– Irritable Bowl syndrome
– Diarrheal diseases
– Clostridium Difficile Infection (CDI)

• Non-mucosal diseases
– Obesity and metabolic syndrome
– Malignancies: liver cancer, breast cancer
– Complications of liver cirrhosis
– Allergic conditions
– Autoimmune disorders (T1DM, arthritis etc)
– Abnormalities of the gut-brain axis- Autism and
other neurological disorders
– Obesity and other metabolic disorders
– Chronic fatigue syndrome
– Periodontal diseases
Malabsorption
syndrome
 Malabsorption syndrome
• In health: The bacterial growth is restricted in the upper
small bowel under the influence of acid and motility.
• Factors predisposing to bacterial overgrowth:
Surgical, anatomical, motor (scleroderma & DM), hypochlohydra
(atrophic gastritis, post gastrectomy)
• Consequences of bacterial overgrowth:
– Fat malabsorption due to:
• Bacteria deconjugation of bile acids which allows free bile acids to be
reabsorped decrease luminal bile acid concentration ---limit micelle
formation.
• Patchy mucosal damage by bacteria or toxic effects of FFA
– CHO and protein malabsorption due to mucosal damage or
bacterial metabolism of these nutrients.
– B12 malabsorption: B12 is utilized by the bacteria, in contrast
bacteria produces folic acid.
Gut Microbiota and CDI
Gut Microbiota - CDI
GIT malignancies:
Colorectal cancer
 GIT malignancies: Colorectal cancer
Colonic bacteria may initiate cancer through:
1- Production of carcinogens from diets rich in meat and fat
(nitroso compounds).
2- Elevation in damage to DNA of colonic cells by dietary
carcinogens (heterocyclic aromatic amines found in cooked
meat).
3- Abnormalities in repair can lead to neoplastic transformations
4- Microbial metabolism can produce by-products toxic to
epithelium;
5- Disproportionate pro-inflammatory signalling at the GIT
mucosa, leads to increased sloughing and repair of epithelium,
which can ultimately lead to neoplasia and malignancy.
6-Certain microbial species can have direct or indirect (through
host cell activation) cytotoxic effects on cells
• High risk of colon cancer was associated with presence of
– Bacteroides vulgatus and Bacteroides stercoris
• low risk was associated with presence of
– Lactobacillus acidiphilus, Eubacterium aerofaciens.

• Although the evidence is not conclusive, colonic flora seem to be a major

environmental factor that modulates risk of colonic cancer in human beings.
Inflammatory Bowl
Disease (IBD)
 Inflammatory Bowl Disease (IBD)
A group of inflammatory and autoimmune conditions that affect
the colon and small intestine, typically resulting in severe
abdominal pain, weight loss, vomiting and diarrhea.

• Affects all layers of the

bowel wall
• Granuloma formation in up
to 60% of patients

• Affects superficial mucosal

layers
 IBD is driven by T cells
mucosal homeostasis
 cytokine production by regulatory (TReg) T cells supresses pro-
inflammatory
T 1, responses
H
TH2, TRe
TH17 g

mucosal inflammation
 increased production of pro-inflammatory cytokines by T
helper (TH) cells
TNF, IFNγ, IL-17
 TReg transfer can
prevent the induction of
experimental colitis

adapted from Bouma and Strober, Nat rev Immunol., 2003 and Vignali et
al., Nat rev Immunol., 2008
Involvement of the microbiota in regulating the balance
between TH and TReg cell subsets in the gut

TH TReg

 Intestinal bacteria direct the differentiation of both pro- and anti-

inflammatory T cell populations and may therefore play a crucial role in IBD
Obesity and metabolic
syndrome
 Obesity and metabolic syndrome
•Over the past 25 years, the prevalence of obesity has
risen dramatically in several developed and developing
countries.
•Recent evidence suggests that the gut microbiota plays
a role in energy harvest, storage, and expenditure.
•Germ-free mice are protected against obesity and that
the transfer of gut microbes from conventionally raised
obese animals results in dramatic increases in body fat
content and insulin resistance.
•The composition of the gut microbiota is shown to differ
in lean and obese humans and animals and to change
rapidly in response to dietary factors.
• Hence, recent research has implicated
the gut microbiota as a critical
determinant of nutrient uptake, energy
regulation, and ultimately, weight and
metabolic disorders
Study: Different gut microbial community
structure in obese mice
% Sequences

Firmicutes

Bacteroidetes

Ley et al., PNAS 102: 11070-5 (2006)

 Study: Microbiota fecal transplantation

• Microbiotas were taken

from the fat mice,
transferred to gf
recipients, and those
that received the
microbiotas from the
obese donor gained
more weight, even
though they didn’t eat
more.
• Shifting the relative
abundances of the
microbiota is changing
the function of the
community in a way that
has an impact on the
host.
Gut Microbiota
and Obesity
Microbiota and Diabetes mellitus
Gut Microbiota and Cardiovascular
Diseases
 Gut Microbiota and Cardiovascular Diseases

Tang WH, Hazen SL.: The contributory role of gut microbiota in cardiovascular disease.
J Clin Invest. 2014 Oct;124(10):4204-11. doi: 10.1172/JCI72331. Epub 2014 Oct 1.
 Neonatal gut microbiota induces lung immunity
against pneumonia
• Streptococcus pneumoniae, Escherichia coli or Candida
albicans were inoculated into newborn mice delivered from
dams that were either untreated or treated 5 days prepartum
with an antibiotic cocktail (ampicillin, gentamicin and
vancomycin).
• Maternal antibiotic exposure reduced the total commensal
bacterial load in the gut of the pups, but also increased the
proliferation of S. pneumonia in the lung.
• Oral inoculation of microbiota from antibiotic-free mice to
pups born from antibiotic-exposed dams restored their
resistance to pneumonia, suggesting that the presence of gut
commensal bacteria is critical for immune responses in the
lung. Gray, J. et al. Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of
newborn mice to infection. Sci. Transl Med. 9, eaaf9412 (2017).
• Disruption of neonatal gut microbiota can
induce inflammatory responses in the lung.

• Another study identified IL-22 as the

'messenger' between intestinal bacteria and
pathogen resistance in the lung, preventing
pneumonial infection in mice.
Intestinal colonization by commensals drives lung
immunity defense in newborn mice

GUT-LUNG
AXIS

Tamburini, S. & Clemente, J. C. (2017) Neonatal gut microbiota induces lung immunity against pneumonia
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2017.34
 How do we suspect dysbiosis?
• Frequent gas or bloating
• Cramping, urgency, and/or mucus in faeces
• Brain fog, anxiety, or depression
• Food sensitivities
• Chronic bad breath
• Loose stool, diarrhea, constipation, or a combination
• Irritable Bowel Syndrome (IBS)
• History of prolonged antibiotics
• Carbohydrate intolerance, particularly after eating fiber and/or beans
• Fatigue or low energy
• Use of anti-acids for heartburn, reflux, or hiatal hernia?
• Autoimmunity, or an autoimmune condition such as Hashimoto’s
thyroiditis, psoriasis, or multiple sclerosis
• Sinus congestion

If five or more of these symptoms are present, then we may be

suffering from dysbiosis
Methods of Microbiota Analysis
Microbiota studies: Overview of techniques to
characterize the gut microbiota

Culture Faecal sample FISH

Microbiome
shotgun DNA isolation Quantitative PCR
sequencing

Direct sequencing of 16S Amplification of 16s

DNA microarrays
rRNA amplicons rRNA gene by PCR

Sequencing of cloned 16s Separation of 16srRNA

rRNA amplicons (DNA Finger printing)

Band
resolution

Band excision and

Probe hybridization
sequencing
 Molecular assessment of 16s rRNA

• The 16s rRNA gene constitute the

fundamental basis of molecular studies of
microbial communities
23S
50S Plus 32
proteins
5S
30S

16s Plus 21 proteins

70S RNA
Whole Metagenome
Shotgun Sequencing

A DNA sequencing method that enables

comprehensive sampling of all genes in
all organisms in a given complex
microbial sample.
Fecal Microbiota Transplantation
 Definition
• Fecal microbiota transplantation (FMT) is the
administration of a solution of fecal matter from a
donor into the intestinal tract of a recipient in order to
directly change the recipient’s gut microbial
composition and confer a health benefit.

[Bakken et al. 2011; Smits et al. 2013]

 History
• First documented in 4th Century China as
“Yellow Soup”
• In some countries, maternal feces is inserted
into the newborn’s mouth to “jumpstart” the
colon
• June 17th, 2013: FDA approved the procedure
for recurrent C. diff.
• 0 documented serious side effects
• 92% - 95% success rate
 Potential Indications
• GI Disorders:
– recurrent Clostridium difficile infection (RCDI).
– inflammatory bowel disease (IBD),
– irritable bowel syndrome (IBS), and
– chronic constipation

• Non-GI disorders:
– Obesity
– Chronic Fatigue Syndrome
– Autism
 The Procedure
• Carefully screened donor stool is mixed with a
saline solution
• The solution is introduced into the GI tract via
a NG tube, fecal enema, oral capsules, or
during a colonoscopy
• The “good” bacteria multiply and help flush
out the C. diff. bacteria
• 92% - 95% success rate
 Conclusions
• The human microbiome and the Microbiome
Project: research just beginning…

• The complexity of the fecal microbiota is actively

being defined and recent studies have shown that
the pathogenesis of many diseases result from
microbiota-related dysregulation.

• Manipulation of gut flora may be an integral part

of weight loss programs and different disease
treatments in the future.
 Conclusions
• FMT re-establishes a balanced intestinal
microbiota and results in impressive cure rates in
patients with recurrent CDI.

• Standardization of FMT protocols and a

randomized controlled trial are needed.

• FMT is likely to achieve widespread therapeutic

benefit for a variety of diseases in the future.
 From Next time Don’t feel Lonely….cause you have
some friends inside you to take care of yourself……

Monkez M Yousif
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