Biomedical presentation describing updates in link between gut microbiota and human body in health and disease

1. By
Dr Monkez M Yousif
Professor of Internal Medicine
Zagazig University
Member of AGA, Member of EASL
Member of ISC-Hepatitis Working Group

2. 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

3. Gut Brain Axis, Integrative Psychiatry
https://www.integrativepsychiatry.net/gut_brain_dysfunction.html. Accessed May 1st 2017
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.

4. 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!

5. “All disease begins in the gut”
Hippocrates 460 BC – 370BC
“Health is determined by the microbiota in our gut”
Hippocrates
”‫الداء‬ ‫بيت‬ ‫المعدة‬"
‫كلدة‬ ‫بن‬ ‫الحارث‬ ‫العرب‬ ‫طبيب‬..
Gut Microbiome

6. 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.
We Are Really More Bug than Man.......

7. 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.

8. 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)

9. Distribution of Gut Microbiota

10. Major Bacteria Phyla and Genera Predominating in Human Gut Microbiota
Representative generaPhyla
‒ Ruminococcus
‒ Clostridium
‒ Lactobacillus
‒ Enterococcus
Firmicutes (60-80%)
‒ Bacteroides
‒ Prevotella
‒ Xylanibacter
Bacteroidetes (20-30%)
‒ BifidobacteriumActinobacteria (< 10%)
‒ Escherichia
‒ Enterobacteriaceae
Proteobacteria ((< 1%)
Munoz-Garach A, Diaz-Perdigones C, Tinahones FJ.
Microbiota y diabetes mellitus tipo 2. Endocrinol Nutr. 2016;63:560---568.

12. Factors Affecting Gut Microbiota

13. 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

14. 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

15. 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.

16. 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.

17. Role of microbiota
in Health

18. 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

19. Gut Microbiota in Health- symbiosis
Human (host) Microbiome
Provides:
• Nutrients
• Shelter
• 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)

20. • 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- innate immunity

21. 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

22. - 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

23. 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

24. The Intestinal Barrier Importance
normal development of immune system
1
2 3
4
5
6
7

25. The microbiome–gut–brain axis
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
Modified, with permission, from Elsevier © Collins, S. M. & Bercik P. Gastroenterology 136, 2003–2014 (2009)

26. Role of microbiota in
Disease

27. Dysbiosis and disease

28. 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

29. 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

30. 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

31. Malabsorption
syndrome

32. 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.

33. Gut Microbiota and CDI

34. Gut Microbiota - CDI

35. GIT malignancies:
Colorectal cancer

36. 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

37. • 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.

38. Inflammatory Bowl
Disease (IBD)

39. 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

40. IBD is driven by T cells
mucosal homeostasis
 cytokine production by regulatory (TReg) T cells supresses pro-
inflammatory responses
mucosal inflammation
 increased production of pro-inflammatory cytokines by T
helper (TH) cells
TH1,
TH2,
TH17
TRe
g
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

41. Involvement of the microbiota in regulating the balance
between TH and TReg cell subsets in the gut
 Intestinal bacteria direct the differentiation of both pro- and anti-
inflammatory T cell populations and may therefore play a crucial role in IBD
TReg
TH

42. Obesity and metabolic
syndrome

43. 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.

44. • Hence, recent research has implicated
the gut microbiota as a critical
determinant of nutrient uptake, energy
regulation, and ultimately, weight and
metabolic disorders

45. Study: Different gut microbial community
structure in obese mice
Firmicutes
Bacteroidetes
%Sequences
Ley et al., PNAS 102: 11070-5 (2006)

46. 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.

47. Gut Microbiota
and Obesity

48. Microbiota and Diabetes mellitus

50. Gut Microbiota and Cardiovascular
Diseases

51. 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.

52. 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).

53. • 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.

54. Intestinal colonization by commensals drives lung
immunity defense in newborn mice
Tamburini, S. & Clemente, J. C. (2017) Neonatal gut microbiota induces lung immunity against pneumonia
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2017.34
GUT-LUNG
AXIS

55. 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

56. Methods of Microbiota Analysis

57. Microbiota studies: Overview of techniques to
characterize the gut microbiota
Faecal sample FISH
DNA isolation
Amplification of 16s
rRNA gene by PCR
Separation of 16srRNA
(DNA Finger printing)
Band
resolution
Microbiome
shotgun
sequencing
Direct sequencing of 16S
rRNA amplicons
Sequencing of cloned 16s
rRNA amplicons
Quantitative PCR
DNA microarrays
Band excision and
sequencing
Probe hybridization
Culture

58. Molecular assessment of 16s rRNA
• The 16s rRNA gene constitute the
fundamental basis of molecular studies of
microbial communities
50S
30S
23S
5S
16s
Plus 32
proteins
Plus 21 proteins
70S RNA

59. Whole Metagenome
Shotgun Sequencing
A DNA sequencing method that enables
comprehensive sampling of all genes in
all organisms in a given complex
microbial sample.

60. Fecal Microbiota Transplantation

61. 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]

62. 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

63. 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

64. 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

65. 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.

66. 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.

67. 67
Monkez M Yousif
From Next time Don’t feel Lonely….cause you have
some friends inside you to take care of yourself……