The World Within Microorganisms in the Digestive Tract

The World Within Micro-organisms in the Digestive Tract: Friends, Foes, and Visitors Janice M. Joneja, Ph. D 2002

The Internal Landscape 2

The Digestive Tract § Each site within the digestive tract is designed for optimal function: § Digestion of food § Protection against invading disease-causing microorganisms § Maintenance of healthy balance (homeostasis) § In the lower bowel, micro-organisms play an active role in all these functions § Sometimes, conditions favour colonisation by microorganisms; others are hostile to their survival § The proper functioning of the resident microflora is essential to the health of the body 3

Digestive Enzymes Mouth: Salivary -amylase Lingual lipase Stomach: Acid hydrolysis Gastric pepsins Small intestine: Pancreatic -amylase Lipase Colipase Trypsin Chymotrypsin Elastase Carboxypeptidases Large bowel Microbial metabolism Small intestine: Gall bladder: Bile salts Small intestine: Brush border: Lactase (ß galactosidase) Glucoamylase ( -glucosidase) Sucrase-isomaltase Amino-oligopeptidases Dipeptidyl-peptidase 4

Microbial Colonisation Mouth: Saliva Microbial colonisation Esophagus: Micro-organisms present Stomach High acidity Usually sterile Small intestine Neutral or slightly alkaline No resident microbial population Micro-organisms populate lower ileum Large bowel Dense microbial population Mostly anaerobic organisms Rectum Faeces Dense microbial population 5

Microbial Colonisation of the Digestive Tract § Factors allowing micro-organisms to live: § Body defences (immune system) § Determines who stays, who goes § Environment: § Acidity and alkalinity (p. H) § Level of oxygen present § Diet: § Provides nutrients for microbial growth and reproduction § Interactions between different types of microorganisms § Survival of the fittest 6

Colonisation by Microorganisms The Mouth § Micro-organisms enter through the mouth from the external environment § Nutrients and salivary secretions in the mouth allow colonisation: § Crevices around the teeth § Pockets in oral tissues § Bacterial plaque on the surface of teeth § Numbers and persistence of micro-organisms depends on: § Available nutrients § Hygiene § Speed of transit of contents 7

Micro-organisms in the Esophagus § Micro-organisms pass with the oral contents through the esophagus § The environment of the esophagus is the same as in the mouth, but it is a conduit, not a “vessel” § Material passes through, but does not remain in location, and therefore micro-organisms have no opportunity to colonise the area 8

Micro-organisms in the Stomach § In the healthy individual the stomach is sterile § The process of eating triggers release of gastric secretions and acid § After a meal the p. H can be as low as 3. 0 § Most micro-organisms cannot survive this § Gastric secretions and hydrochloric acid kill off most micro-organisms passing from the esophagus § Rate of flow of food through stomach also influences microbial survival 9

Micro-organisms in the Stomach § Low acidity (higher p. H) allows some microorganisms to survive § Conditions that may allow bacteria to live: § Achlorhydria (lack of gastric acid), especially in the very young, and the elderly § Neutralizing substances that reduce acidity of contents, e. g. : § sodium bicarbonate § other antacids § Most common pathogen: Helicobacter pylori 10

Survival of Micro-organisms in the Stomach § Rapid movement of food material through before p. H is low enough to kill them: § Before a meal, p. H of stomach is 4 -5 § Drops to p. H 3 while eating § Rate of flow of stomach contents influenced by: § Composition of meal: § Fat passes through slowly § Liquid passes through quickly § Micro-organisms that survive through the stomach pass into the small intestine 11

Micro-organisms in the Small Intestine § Very few micro-organisms live in the first part of the healthy small intestine § Numbers increase as the digesta passes into the terminal ileum § Conditions that influence microbial multiplication: § Rate of flow of digesta: § Flow rate greatest at the beginning § Slows as material reaches distal end § Normal length of time food material takes to transit small intestine: 3 -4 hours § Water is absorbed § Consistency is more solid and allows organisms to stay in place long enough to multiply 12

Micro-organisms in the Small Intestine § Under normal circumstances several processes inhibit adherence and colonization in the small intestine, and kill micro-organisms surviving from the stomach: § Mucus coats bacteria and disallows contact with the intestinal wall § Antibodies, especially secretory Ig. A, neutralize bacteria § Lysozyme in secretions is bactericidal (kills bacteria) § Bile salts are bactericidal 13

Micro-organisms in the Small Intestine § Micro-organisms can colonise the small intestine and cause infection if they can adhere to the intestinal wall § Usually, contents pass through too rapidly to allow this § Some situations may predispose to colonization: § Motility disorders that interfere with the normal passage of material through § Material becomes lodged within tissue pockets (diverticulae) 14

The Large Bowel § Most of the micro-organisms that colonise the human body live and thrive in the large intestine § Digesta from small intestine enters the caecum where microbial activity begins in earnest § As the contents pass from the caecum to the rectum, microbial numbers increase dramatically § Adult eating typical Western diet: § Total contents: 220 grams dry weight § Bacterial dry matter: 18 grams 15

Micro-organisms in the Large Bowel § Contents of the large bowel pass from the body as faeces § Micro-organisms in faeces same as in terminal part of large bowel § Bacteria in faeces: § Approximately a trillion per gram dry weight § The longer the material remains in the colon, the greater the number of micro-organisms § Several hundred different microbial species § About 99% of these belong to only 30 -40 species 16

Micro-organisms in the Large Bowel § Food material remains in the colon approximately 70 hours § Inter-individual variation: 20 - 120 hours § Many species multiply rapidly: some double every twenty minutes § Type and species of micro-organisms is surprisingly stable for each individual § Even when infection changes the nature of the species, after pathogens are removed, microflora tends to revert to its original composition 17

Micro-organisms in the Large Bowel § Conditions that influence type and numbers of micro-organisms: § Amount of oxygen available (many are strict anaerobes and are killed by exposure to oxygen) § Competition for nutrients § Type of nutrients available § Type of micro-organisms present: § Organisms that can break down food material and use nutrients fastest will multiply fastest § Confined space § Organisms that multiply fastest, crowd out others 18

Micro-organisms in the Large Bowel Inter-Species Competition § Space and nutrients are limited § Species that break down and use available nutrients most efficiently achieve the highest numbers § Advantage to species that can: § Use substrates most other species cannot process § Use waste products of other species, e. g. § Hydrogen sulphide § Organic acids 19

Source of Nutrients in the Large Bowel § Material that has not been completely digested and absorbed in the small intestine: § Food matter consumed in diet § Cells and tissues sloughed off from digestive tract § Enzymes and other material from body processes such as: § saliva § intestinal secretions such as mucin § blood cells § Dead micro-organisms 20

Micro-organisms in the Large Bowel Nutrient Substrates § Most important nutrient substrates are: § Carbohydrates § Starch § Plant storage material § Non-starch polysaccharides (dietary fibre) § Plant structural material § Oligosaccharides (long chain sugars) § From partial digestion of carbohydrates § Sometimes disaccharides (sugars) § Most are broken down in small intestine § Proteins § Diet § Body secretions, including digestive enzymes § Dead micro-organisms 21

Microbial Use of Material in the Large Bowel: Carbohydrates § Majority of bacterial species in the large bowel act on carbohydrates § Carbohydrates entering the colon of the average adult eating a Western diet per day include: § Dietary fibre…………… 12 grams § Undigested starch………………… 30 -40 grams § Material from the digestive tract (mucins, enzymes and dead microorganisms)…………………. . . ………. 3 -4 grams 22

Carbohydrates § Dietary fibre § Structural parts of plants § Have beta-glycosidic linkages between molecules § Indigestible by human enzymes § Includes: § § § Pectin Cellulose Gums Beta-glucans Fructans 23

Dietary Fibre § Usually separated into two types depending how it interacts with water: § Soluble fibre: § Forms gel or gum § Insoluble fibre: § Remains unchanged in water § Both types present in plants, e. g in legumes: § Hard outer skin is insoluble type § Inner “pulse” higher in soluble type § Cooking and processing does not change the nature of fibre 24

Carbohydrates § Starches § Previously thought all starch was digested and absorbed in the small intestine § Enzymes break alpha-glycosidic linkages between molecules § Recent research shows 15%-20% of dietary starch passes undigested into the colon from high starch foods such as: § potato pasta § rice banana § grains (wheat, corn) 25

Starch § Undigested starch is called “resistant” starch § Starch that is readily digested and absorbed in the small intestine is called “non-resistant” § Resistant starch is resistant to digestive enzymes § Passes into the colon where it is fermented by gut microflora § Unlike fibre, resistant starch is affected by cooking and processing 26

Resistant Starch § Process of digestion in the small intestine can be speeded up by cooking - starch is gelatinized § Cooling causes a process of crystallization (retrogradation) that renders the molecules nondigestible by enzymes § Undigested material passes into the large bowel § Freezing and drying can also cause changes in starch that makes it resistant to digestion § Research on contents of ileostomy bag 27

Comparison of Dietary Starch A comparison of dietary starch: a) Fed b) Recovered after digestion in the small intestine 28

Factors Affecting Amount of Starch in the Colon § Physical accessibility § § Cell walls of plant cells entrap starch Prevents its swelling and dispersion Delays or prevents digestion by enzymes Includes whole grains, nuts, seeds: § vegetables with “skins”: sweet corn, peas, beans § partly milled grains and seeds: “whole grain” breads and cereals § If the rigid structures of the plant are physically removed, more of the alpha-glycosidic bonds of the starch are exposed to the action of enzymes in the small intestine 29

Factors Affecting Amount of Starch in the Colon § Cooking § Disrupts starch granules § Facilitates digestion by enzymes in saliva and the small intestine § When foods with a high level of resistant starch are eaten raw, more undigested starch passes into the colon § e. g. Banana § Retrograded starch increases on cooling: eat foods with high level of resistant starch when it is hot 30

Factors Affecting Amount of Starch in the Colon § Chewing § Amylase (ptyalin) in saliva is first enzyme to start process of starch digestion § The more the food is chewed, the greater the exposure of the starch to enzymes in the mouth and the small intestine § Speed of transit of food § The faster the food transits the small intestine, the less exposure to enzymes § High fat slows transit § High fluid (water with the meal) speeds the transit 31

Oligosaccharides § § Polymers of glucose 3 - 8 hexose units in length Exist in plant materials as oligosaccharides Or are derived from partial digestion of starches Trisaccharides are most “notorious” § Raffinose § Stachyose § Principally in legumes such as dried peas, beans, lentils § Proficient in generating excessive amounts of intestinal gas and flatus 32

Oligosaccharides § Fructo-oligosaccharides § Polymers of fructose - called inulins § Made by plants such as: § § § § onions garlic artichokes chicory Appearing as “health foods” Resist human digestive enzymes Promote growth of Bifidobacteria in the large bowel Tend to reduce growth of “undesirable” bacteria 33

Fructo-oligosaccharides and Bifidobacteria § Bifidobacteria are beneficial because they: § § Stimulate immune function Enhance synthesis of B vitamins Restore normal microbial flora after antibiotic therapy Prevent colonization by potential pathogens, especially Clostridia § Fructo-oligosacchardies: § Reduce triglyceride and cholesterol levels in rats and diabetic humans 34

Disaccharides § Principally: § Lactose; sucrose; maltose § Usually broken down to monosaccharides (“single sugars”) and absorbed in the small intestine § When enzymes deficient, disaccharides pass undigested into the colon § Have several effects: § Change osmotic pressure § Act as substrate for microbial fermentation § Results in symptoms typical of lactose intolerance; § § Diarrhea Abdominal bloating Gas Pain 35

Products of Microbial Fermentation of Carbohydrates § Any carbohydrate entering the colon acts as substrate (nutrient) for microbial fermentation § Principal products are short-chain fatty acids (SCFAs): § Acetic acid § Propionic acid § Butyric acid § These three account for 85 -95% of SCFAs in the colon 36

Other Sources of SCFAs § A smaller percentage of SCFAs come from proteins § Up to 40% of SCFAs are derived from protein, depending on the diet § Branched chain amino acids are converted to branched chain fatty acids § Contribute to the total SCFAs in the colon resulting from microbial activity 37

Products of Microbial Fermentation of Carbohydrates § In converting the carbohydrates to these SCFAs intermediate products are formed: § Lactate § Succinate § Ethanol § Most do not accumulate, but are converted to SCFAs in the colon § However, occasionally ethanol may accumulate: § Results in “autobrewery syndrome” resembling alcohol intoxication 38

Function of SCFAs § SCFAs absorbed into the body through the colonic membrane (wall), and can be measured in blood § SCFAs serve a variety of functions in the colon: Provide source of energy Preserve the integrity of the colonic mucosa (lining) Stimulate absorption of water and sodium Reduce intestinal p. H Aid in protection against bacterial infection Butyrate thought to be particularly important in protection against colon cancer § May also protect against inflammatory bowel diseases such as ulcerative colitis § § § 39

Proteins in the Colon § 12 – 13 grams of protein enter the large bowel each day § Material comes from: § § Diet (even a vegan diet) Secretions from the digestive tract Dead bacteria Tissue cells § Much of the material is digested by pancreatic enzymes in the small intestine 40

Proteins in the Colon § Pancreatic enzymes continue digestion in the large bowel as they pass in with the digesta from the small intestine § Bacterial enzymes actively attack the undigested proteins § Bacterial species Bacteroides are particularly active in this process § These species are also the most active degraders of fibre in the colon 41

Protein breakdown in the Colon § Proteins are first broken down to polypeptides § Some bacteria use these directly as nutrients § Other bacteria produce enzymes to break down the polypeptides into dipeptides § Dipeptides are then broken down further into single amino acids § 20 amino acids make up all dietary proteins 42

Amino Acids in the Colon § Bacteria utilize the amino acids in a variety of ways: § Deamination to ammonia § Decarboxylation to amines and carbon dioxide § Both systems are important in maintaining a healthy colon 43

Ammonia in the Colon Large quantities almost always present in the colon High levels can be toxic Can be a risk factor in the development of colon cancer Colon bacteria use ammonia as a source of nitrogen in their metabolism § These strains are important to maintain a healthy colon § These bacteria use carbohydrate, and especially fibre as a course of energy § Fibre in the diet thus aids in growth of the ammoniautilizing bacteria, which is thought to reduce the risk of colon cancer § § 44

Biogenic Amines in the Colon § Sometimes the amines are detrimental to a person’s health, e. g. § Histamine: § Migraine headaches § Symptoms resembling allergy § § § Hives Tissue swelling (angioedema) Rhinitis(“stuffy nose”) Itching Reddening and flushing Increased heart rate 45

Biogenic Amines in the Colon § Tyramine § Migraine headaches § Hypertensive crisis § § § Serotonin Piperidine Pyrrolidine Cadaverine Purescine § Have adverse effects only in excess and in sensitive individuals 46

Fate of Microbial Products § Most microbial products enter circulation by being absorbed through the colon wall § Taken to the liver § Cleared and excreted in the urine § Examples: § § Phenol and p-cresol from amino acid tyrosine in proteins 50 -100 mg per day in the healthy adult urine Level increases with increase in protein in the diet Decreases when bran added to the diet – bran acts as energy source for bacteria that use tyrosine to build bacterial protein 47

Fate of Microbial Products § Products of microbial activity normally cleared in the liver and excreted in the urine without adverse effects § Scientific data about the fate of many byproducts of microbial metabolism is presently lacking in many cases § There is suspicion that in sensitive individuals some “psychological disturbances” following ingestion of certain food materials might be caused by these microbial by-products 48

Protection Against Invading Pathogens § Because of its ideal environment, the large bowel may be the site of invasion by disease-causing microorganisms § Various factors protect against this: § Resident microflora protect their own space § SCFAs act as antagonists to many pathogenic microorganisms: § § Salmonella Shigella (dysentery) Vibrio (cholera) E. coli (enteritis) 49

Invading Pathogens § Antibiotics taken by mouth kill off many of the resident species § Less SCFAs are produced § p. H rises § Pathogens can now invade and colonize more readily § Takes time for the resident micro-flora to reestablish § Symptoms of irritable bowel syndrome not uncommon following enteric infections 50

Protection Against Invading Pathogens § Diarrheal diseases also decrease SCFAs § Microbial infection § Lactose intolerance § Magnesium-based laxatives § Fibre increases level of SCFAs because bacteria that produce them also use fibre as a substrate, which increases bacterial numbers 51

GAS § Fermentation always leads to production of various types of gases § 80% of the gas from fermentation is released as flatus § 20% is absorbed into the body and excreted in breath § Volume of gas depends on composition of diet: from 0. 5 to 4 litres per day in the adult human 52

Gas § Healthy people pass flatus an average of 14 times per day § 25 – 100 ml on each occasion § Can rise to 168 ml per hour when >50% of the diet is in the form of non-starch fibres and nonabsorbable sugars: § § Beans Whole grains Some vegetables Some fruits 53

Gases in Breath § Principal gases in breath are: § Hydrogen § Carbon dioxide § Small quantities: § § Methanediol Ethanediol Ammonia Hydrogen sulphide § Occasionally § Methane § Type of gas depends on the presence of the specific bacteria capable of producing it 54

Colonic Gases § Some bacteria use gases for their metabolism: § Hydrogen metabolized to: § Methane § Hydrogen sulphide § Acetate § These may be: § utilized by micro-organisms § excreted as flatus § passed into circulation and breath § Amount of hydrogen even from the same amount of substrate is not constant: it depends on: § Type of micro-organisms present § Speed of fermentation § Utilization by other micro-organisms 55

Hydrogen Breath Test for Lactose Intolerance § Results of hydrogen breath test used in the diagnosis of lactose intolerance varies depending on type of micro-organisms in the bowel § Rationale for test: § If lactose is not digested by brush-border lactase, it passes into the large bowel § Here it will be fermented by the resident microorganisms, with the production of hydrogen § The hydrogen is absorbed, taken in blood to the lungs where it is excreted § Amount of hydrogen collected from breath is measured and used as an indication of the degree of lactase deficiency 56

Methane § Methane-producing bacteria convert hydrogen to methane § 30 -50% of healthy adults have methane-producing bacteria in their colon § Gas is excreted in the breath § Not detectable in children under the age of two years § In methane-producers, adult level of methane reached by age 10 years § Tends to be familial § Methane production does not vary with diet § May be associated with: § large bowel cancer § intestinal polyps § ulcerative colitis 57

Hydrogen Sulphide § Sulphate-reducing bacteria in the colon convert hydrogen to hydrogen sulphide § Methane-producing and sulphide-producing bacteria compete for hydrogen in the colon § When the diet is high in foods that contain sulphates, hydrogen-sulphide producing bacteria have an advantage § Another source of sulphate is body secretions such as mucins that contain sulphated glycoproteins 58

Sulphate-Containing Foods § Sulphates may occur naturally: § Some fruits § Some vegetables § Sulphates may be used as clarifying agents and stabilizers in manufactured foods, such as: § § § § § Cheeses Egg products Pickles Candied and glazed fruit Flours; breads; cereals; pastas Sugars Wine; beers Nutritional supplements Laxatives; homeopathic remedies; medications 59

Acetate § If methane-producing and sulphide-producing bacteria are absent, bacteria may convert hydrogen and carbon dioxide to acetate § The extent to which this occurs is unknown § Acetate may be used by the body as a source of energy in certain metabolic processes § The type of gases excreted as flatus or in breath depends more on the species of micro-organisms colonising the bowel than on the composition of the diet § Components of the diet determine the amount of gas produced 60

Vitamins Produced by Bacteria § Bacteria not only break down food material (catabolism), they synthesise nutrients (anabolism) from these building blocks § Vitamin K § Required in blood clotting § Menaquinone component of the vitamin is derived from bacterial action on vegetable material mostly in the ileum from where it is absorbed § Taken to the liver, where it is complexed with prothrombin 61

Vitamins Produced by Bacteria § Vitamin B 12 § Made solely by micro-organisms in ruminant digestive tract § Absorbed through small intestine § Passes into meat and milk of the animals § Human bacteria (Pseudomonas and Klebsiella) also synthesise B 12 § 5 mcg excreted in feces daily § Site of synthesis in humans is large bowel but absorption from here is poor § Some people have micro-organisms capable of synthesising B 12 in the small intestine 62

Vitamins Produced by Bacteria § Biotin § Synthesised by bacteria in animals and humans § Absorbed in lower ileum § Antibiotics can reduce biotin levels in urine, indicating significant reduction in biotin synthesis when bacteria are killed § Folic acid § Thiamine § Produced by bacteria, especially in the large bowel § Amount absorbed is inadequate alone, and the vitamins must be provided in food to avoid deficiency 63

Changing the Microbial Flora of the Bowel § Diet has very little influence on the types of micro -organisms that colonise the digestive tract § Attempts to alter the gut microflora by direct dietary manipulation tend to be frustrating § Differences in types and numbers in the bowel of one individual compared to another in the same community, eating the same diet § Microflora can be changed by use of oral antibiotics § Microflora tends to return to pre-antibiotic types over time 64

Probiotics § Food supplement containing live bacterial culture § Trials in disease situations such as : § Diarrheal diseases § Re-establishment of normal flora after antibiotic therapy § Inflammatory bowel diseases § Fungal disease (e. g. candidiasis) § Cancers § Cholesterol lowering 65

Probiotics § Examples of bacteria: § Lactobacilli § Bifidobacteria § Examples of food supplements containing live culture: § § § § Yogurts Fermented milks Fortified fruit juice Powders Capsules Tablets Sprays 66

Prebiotics § Non-digestible food ingredients that selectively stimulate a limited number of bacteria, to improve health § Examples: § Fructo-oligosaccharides § Lactulose § Galacto-oligosaccharides § Provided in: § § Beverages and fermented milks Health drinks and spreads Cereals, confectionery, cakes Food supplements 67

Synbiotics § Combine prebiotics and probiotics § Prebiotic substrate should enhance survival of probiotic bacteria § Example: § Bifidobacteria + fructo-oligosaccharide § In order to establish the new species, need to continue to provide live culture, and appropriate substrate 68