Gut microbiota in health and disease

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

1. Gut Microbiota in Gastrointestinal Health and Disease 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 ” المعدة بيت الداء " طبيب العرب الحارث بن كلدة.. 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 genera Phyla Ruminococcus Clostridium Lactobacillus Enterococcus Firmicutes (60-80%) Bacteroides Prevotella Xylanibacter Bacteroidetes (20-30%) Bifidobacterium Actinobacteria (< 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.

11. Factors Affecting Gut Microbiota

12. 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

13. 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

14. 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.

15. 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.

16. Role of microbiota in Health

17. 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

18. G ut Mic r ob i ota i n He a l th- 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 resistanc e )

19. Produce pathogen-associate d molecular pattern s ( P AMPs) an d metabolic by-products and regulate intestina l immun e responses PAMPs are recognized by pattern recognition receptor (PRR)-bearing cells of the innate immune system and many epithelial cells G ut Mic r ob i ota i n He a l th- innate immunity

20. Gut Mic r obiota in Health: Adaptiv e immune system Microbiota stimulation lead s to B cell switch to IgA, regulatory T cell induction , T cell di f ferentiation to Th17

21. 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

22. 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

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

24. 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)

25. Role of microbiota in Disease

26. Dysbiosis and disease

27. Dysbiosis: Proposed mechanism/s leading to disease Genetic an d environmenta l factors induc e impaire d barrie r function Overgrowth of pathogenic bacteria; inhibition of protective bacteria T ranslocation o f bacteri a an d bacteria l produc t s Immune activatio n an d proinflammator y cytokine production Chroni c inflammatio n lead s to tissue destructio n an d complicatio n s Concept of leaky gut

28. 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

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 synd r ome Malignancies: l iver c ancer, 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

30. Malabsorption syndrome

31. 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.

32. Gut Microbiota and CDI

33. Gut Microbiota - CDI

34. GIT malignancies: Colorectal cancer

35. 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 transformatio n s 4- Microbial metabolism can produc e by-product s toxic to epithelium ; 5- Disproportionate pro-inflammator y signalling a t the GIT mucosa, lead s to increased sloughing an d repa i r o f epithelium , which can ultimatel y lead to neoplasia an d malignancy. 6- Certain microbial species can hav e direc t o r indirec t (through hos t cell activation ) cytotoxic e f fects on cells

36. 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.

37. Inflammatory Bowl Disease (IBD)

38. 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

39. IBD is driven by T cells mucosal homeostasis  cytokine production by regulatory (T Reg ) T cells supresses pro-inflammatory responses mucosal inflammation  increased production of pro-inflammatory cytokines by T helper (T H ) cells T H 1, T H 2, T H 17 T Reg TNF, IFN γ , IL-17  T Reg 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

40. Involvement of the microbiota in regulating the balance between T H and T Reg 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 T Reg T H

41. Obesity and metabolic synd r ome

42. Obesity and metabolic synd r ome 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.

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

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

45. 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.

46. Gut Microbiota and Obesity

47. Microbiota and Diabetes mellitus

48. Gut Microbiota and Cardiovascular Diseases

49. 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.

50. 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).

51. 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.

52. 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

53. 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

54. Methods of Microbiota Analysis

55. 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

56. 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

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

58. Fecal Microbiota Transplantation

59. 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]

60. History First documented in 4 th Century China as “Yellow Soup” In some countries, maternal feces is inserted into the newborn’s mouth to “jumpstart” the colon June 17 th , 2013: FDA approved the procedure for recurrent C. diff. 0 documented serious side effects 92% - 95% success rate

61. 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

62. 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

63. 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.

64. 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.

65. 67 Monkez M Yousif From Next time Don’t feel Lonely….cause you have some friends inside you to take care of yourself……