The Human Microbiome: With Focus on Probiotics Haifa Khadour Isabelle Lam Fayyaz Samji

The Human Microbiome Introduction to Microbiome Consists of all of the microorganisms in the body; both on the surface and within the body. Majority are not harmful to their human host. Main sites: Skin Mouth Gut Conjunctiva Vagina Nasal / Respiratory tract 1013 germ and somatic cells 1014 microbiota

The Human Microbiome Project (HMP) Introduction to Microbiome The Human Microbiome Project (HMP) Objective: to characterize and identify the entire human microbiome Five-year project that commenced in 2008 HMP is not a single project; it is a collective initiative Initially, microorganisms were identified through cultivation methods However, there are limitations

HMP: Current methods Introduction to Microbiome PCR of 16S rRNA genes from bacterial communities Present in all bacteria It is vital as it codes for the small subunit of the ribosomal RNA complex which is necessary for protein synthesis Contains conserved regions, which are useful for broad-range PCR Metagenomic analysis – Examining the complexity of environmental samples by extracting DNA directly from mixed samples and creating sequenced genomic libraries – This serves well for comparison studies

Types of Microbiota: Bacteria Our Microbiota Types of Microbiota: Bacteria Play a normal and healthy role in human physiology However, in certain situations disease can result Bacterial numbers exceed their typical ranges Mutation (E. Coli 0157:H7) Bacteria colonize uncharacteristic areas Bacteria have the ability to digest certain nutrients The majority of these bacteria are anaerobes Normal bacterial flora can become opportunistic pathogens when the immune system is weakened

Our Microbiota Types of Microbiota Archaea Fungi Relative to bacteria, they are few in number Majority are methanogens Methanobrevibacter smithii, Methanosphaera stadtmanae There are currently no known archaeal pathogens Fungi Mainly yeast In particular, there is a lot of research done on Candida species

Skin Flora Our Microbiota Bacteria Fungi: Yeast Staphylococcus aureus Candida albicans Malassezia spp. Bacterial Phyla Percentage on Skin Actinobacteria 51.8% Firmicutes 24.4% Proteobacteria 16.5% Bacteroidetes 6.3%

Gut Flora Our Microbiota Most of the gut flora in the colon and large intestine are comprised of bacteria Escherichia coli in the colon Clostridium difficile in the large intestine Bifidobacterium species Bacterial microbes form 60% of the dry mass feces makes feces an ideal source for testing for gut flora (ie. HMP samples) Archaeal (methanogens) and Fungal microbes (mainly yeast) are also present in the gut but in limited numbers

Our Microbiota

Gut Flora Our Microbiota Functions of Bacteria in the gut: Breaking down unutilized carbohydrate substrates Gut flora have enzymes that humans lack Suppressing the growth of harmful microbes Competitive exclusion Early development of the immune system Mediate metabolic functions Synthesis of vitamins (ie. folate) Absorption of calcium, magnesium, and iron

Oral Cavity Flora Our Microbiota Bacteria: streptococci, lactobacilli, staphylococci, corynebacteria various anaerobes (ie. bacteroides) in the gingival crevice(supporting structures of the teeth) Lactobacillus species have been associated with dental caries Dental plaque is a biofilm and contains bacterial cells Streptococcus mutans Streptococcus sanguinis These microbes secrete metabolites, which can subject the teeth and gingival tissues to various dental diseases.

Vaginal Flora Our Microbiota Primarily Lactobacillus bacteria that produce lactic acid L. acidophilus and L. doderlein The vaginal flora facilitate in preventing bacterial vaginosis, yeast infections and other possible problems by maintaining an acidic pH such as Gardnerella vaginalis. In a healthy individual, the lactobacilli occupy areas that would otherwise be available for pathogenic organisms to occupy However, harmful pathogens or an imbalance in bacteria can lead to infection

Prebiotics Prebiotics Types: “A selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health” (Journal of Nutrition, 2007). Typically, they are carbohydrates Oligosaccharides and soluble fiber Types: – Short- chain (ie. Oligofructose) contain 2-8 links – Long-chain (ie. Inulin) contain 9- 64 links – Full-spectrum (ie. Oligofructose- Enriched Inulin) contain 2-64 links

Probiotics Probiotics “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (WHO). Main types of microbes: Lactic acid bacteria (LAB) Bifidobacteria Are commonly found in fermented foods Live cultures are usually added Yogurt, dietary supplements

Mechanisms The mechanism underlying the Variety of health benefits of probiotic remains poorly understood. Although these effect involve both micob-microb and microb-host interactions. Microbe-microbe interaction focuses on the inhibition of the pathogenic activity, this includes: Competition for nutrients. Antibacterial secretion. Whereas, Microbs-host interaction generally address divers aspect such as: Strengthening of intestinal barrier function, i.e adhesion to the most epithelium. Modulation of the innate and adaptive immune system.

Microbe-microbe interaction Mechanisms Competition for nutrients: Pathogens vs. probiotics. Endogenous microbs vs. probiotic. Biochemical effects: Secrete antibacterial substances: Non-pH related effect. pH related effect. Adhesion: carbohydrate-binding specificities. steric hindrance. proteinaceous components Binding to toxins.

Mechanisms

Microbe-host interaction Mechanisms Intestinal barrier: Induction of mucin secretion. Reinforcement of tight junction function. Modulation of apoptosis of epithelial cells. p75 and p40 secretion by L. rhamnosus GG. In Vivo vs. in vitro.

Microbe-host interaction Mechanisms Intestinal barrier: Induction of mucin secretion. Reinforcement of tight junction function. Modulation of apoptosis of epithelial cells. p75 and p40 secretion by L. rhamnosus GG. In Vivo vs. in vitro.

Cell service in host interaction Mechanisms The diversity of cell wall composition and structure provides species and strain specific properties that are likely involved in specific host interaction Schematic representation of the Gram-positive cell wall. Several of the constituents of these macromolecular structures have been proposed to be directly involved in interactions with mammalian cells and thus may play a role in (health-promoting) modulation of the physiological status of the host. Specific components of the cell wall are indicated, including the peptidoglycan layer, wall- and lipoteichoic acids (WTA and LTA), exopolysaccharides (EPS), and proteins that can be presented to the cell surface in a variety of ways, including secretion to the environment (SP), membrane proteins (MP), cell-wall-associated proteins (CWP), sortase-dependent and peptidoglycan-anchored proteins (SDP), lipoproteins (LPP), membrane-anchored proteins (MAP), and surface layer proteins (SLP).

Microbe-host interaction Mechanisms Immunomodulation: regulate natural and acquired immune mucosa-associated lymphoid tissues. Binding to receptor induce immune response by: production of defensive molecules such as mucins. Repair factors such as short-chain fatty acids. Enhance signaling in the host. Switching in immune response in allergic reactions. Reduce the production of inflammatory substances.

Restriction of usage Restrictions 1. Major concerns that have been investigated: a. Bacteraemia. b. Adverse immunological effects. c. Antibiotic-resistance transfer.

Restrictions a. Potential transmigration, Bacteraemia: Diagnosed with immunosuppression. Prior prolonged hospitalization. Prior surgical interventions. b. Immunological effects No clinical proof.

Restrictions c. Antibiotic resistance transfer: Plasmid in Lactic acid bacteria. possibility of transfer of a tetracycline resistance plasmid, 14 strains were tested: Only 7 transferred resistance to Enterococcus, 2 transferred resistance to Lactococcus lactis, and non were able to transfer resistance to Staphylococcus aureus.

Who to Watch for Restrictions Use of probiotics generally safe but these patients should be monitored when using probiotics: Immuno-compromised. Premature infants. Patients suffering short bowel syndrome. Those with central venous catheters. Elderly and patients with cardiac valve disease.

Benefits of Probiotics Gut microbial balance: Lactobacilli and bifidobacteria vs. pathogenic microorganism. Predisposition to clinical diseases such as: cancer, inflammatory diseases, and infection by transient entrophathogens like salmonella, campylobacter, and E.Coli

Benefits of Probiotics Infection control Benefits of Probiotics Why probiotics for infection treatment? New problems created by antibiotics. disruption the protective flora. antibiotic resistance due to the abuse and over use of antibiotic prescriptions. Running out of antibiotics. Interest in the usage of ecological methods instead.

H. pylori infection Benefits of Probiotics Gram-negative, spiral shaped, micro-aerophilic rod, which colonizes the human gastric muscosa. Urease is an important product by this organism. Increase pH in the stomach Treatment involves anti-biotic and acid suppression drugs.

Benefits of Probiotics Clinical studies showed the use of Lactobacillus salivarious: Decreased the side effects of antibiotics. Improved patient compliance with taking the prescribed therapy. Increased the rate at which H. pylori were eradicated. No studies have showed direct eradication, but long term usage of probiotic showed positive effects.

Benefits of Probiotics Mechanisms used may involve: Competition for nutrients. Secretion of antimicrobial substances. Blocking of adhesion sites. Immunomodulation. Alteration of pH level though SCFA. Short chain fatty acids, product of bacterial fermentation in the colon. Blocking of toxin receptor sites. Attenuation of virulence.

Immune Stimulation Benefits of Probiotics In human trail, 24 subjects fed 450 g of yoghurt per day for 4 months showed: Increase the levels of -interferon; macrophage activating factors. Increase the concentration of natural killer cells. Stimulate production of antibodies (local and systematic). Enhance the activity of macrophages

Benefits of Probiotics Cancer suppression Benefits of Probiotics colonic microbiota may be involved in the etiology of CRC. Strategies might include: Suppression of carcinogen/ procarcingens by binding, blocking, or removal. Suppression of bacterial enzymes that promotes the conversion of procarcingens to carcinogens. L. acidophilus. Reducing intestinal pH. Stimulation of the immune system. Animal studies vs. Human studies.

Irritable Bowel Syndrome (IBS) Benefits of Probiotics Irritable Bowel Syndrome (IBS) Characteristics: Abdominal pain. Bloating Change in bowel habit. absence of any clear mucosal abnormality. L . rhamnousus in infants. probiotics remains a promising treatment in IBS.

Benefits of Probiotics Diarrhea Benefits of Probiotics Lactobacillus GG, Lactobacillus reuteri, and Lactobacillus casri. Rehydration combined with probiotics strains shorten the duration. Reduction of acute diarrhea in children by 1 day. Prevention of diarrhea induced by enteropathogens such as E. coli, shigella, and salmonella. diarrhea in hospitalized children. treating radiation-induced diarrhea in cancer patients and antibiotic associated diarrhea

Lactose Malabsorption Benefits of Probiotics Insufficient activity of lactase in human gut. Abdominal distension, excessive flatulence, and diarrhea. Lactose administrated in yoghurt vs. in untreated milk. improve tolerance to lactose with -galactosidase.

Consumed probiotics vs. endogenous strains Probiotics in Foods Consumed probiotics vs. endogenous strains Probiotics we consume can be much different from probiotics we have There are 3 functions probiotics should have in order to confer mentioned benefits

Reminder Probiotics in Foods Antimicrobial activity Enhance intestinal epithelial barrier function Modulate host immune system

Probiotic Ability Probiotics in Foods Study by Jensen, Grimmer, Naterstad and Axelsson (2012) Tolerance of transit in upper gastrointestinal tract Cell adhesion capabilities Epithelial barrier function Measured transepithelial electrical resistance (TER) Human isolated strains: Lactobacillus reuteri DSM20016 DSM 17938 mm4-1a fj1

Probiotics in Foods Tolerance of transit in upper gastrointestinal tract

Probiotics in Foods Cell adhesion capabilities Variability of <1%-25%

Probiotics in Foods Epithelial barrier function

What does it all mean? Probiotics in Foods All probiotics have their benefits Depends on many factors: The host – are they responsive? The strain Capacity of the probiotic itself How they are consumed Study by Tompkins, Mainville & Arcand (2011) Better consumed BEFORE a meal Capacity: Example our own E. coli cannot produce enough Vit. K for whole body How strong is probiotics ability to promote immunotolerance dependent on priming dendritic cells to stimulate regulatory T-cell differentiation - Eating yogurt alone? Is it enough to buffer the system

An Expanding Field In the Future Relatively new field compared to others with many applications and new studies - metagenomics Need to expand menu of prebiotics, probiotics and antibiotics Focus on education and regulation Prebiotics – being discovered from human saliva and breast milk - Plant derived oligosaccharides and polysaccharides, some bacteria produce amino acids that have protective effects against gastro intestinal injuries, prebiotics can stimulate growth of lactate producing bacteria leading to production of fuel sources for epithelial cells

Designer Strains and GMOs In the Future Designer Strains and GMOs Made possible by recombinant DNA technology Bacteria being manipulated to express favorable genes Example: Reengineered Lactobacillus paracasei 338 to modulate fat cells to reduce obesity Engineer strains to help treatment of malnutrition – global applications Can manipulate genes and DNA to make mutant strains with specific abilities Now with research into the genes and the function they produce

Continued Development of Microbiome Sequencing In the Future Continued Development of Microbiome Sequencing Progress of projects under Human Microbiome Project

FDA Regulation In the Future MANY foods with fake probiotic claims Food labels often omit strains or advertise the wrong one Example: FDA had U.S. Marshals takedown UAS Laboratories Inc. for marketing their probiotics as disease curing drugs

Education and Awareness In the Future Education and Awareness Companies will soon be accountable to their claims about probiotics Foods with natural probiotics

As a Thank You Question Period Florastor, Medical Futures Inc.: Treats and prevents diarrhea Fem-Dophilus, Jarrow Formulas, and RepHresh Pro-B, Lil Drug Store : Prevents and treats urogenital infections Activia yoghurt, Dannon: Improves gastrointestinal transit time DanActive fermented milk drink, Dannon: Reduces duration of colds and helps prevent diarrhea Bio-K Plus, Bio-K Pharma: Reduces antibiotic-associated diarrhea VSL#3, Ferring Pharmaceuticals: Prevents pouchitis, used to treat ulcerative colitis Mutaflor, Medical Futures Inc.: Benefits patients with ulcerative colitis and moderate distal activity TuZen, Ferring Pharmaceuticals: Relieves abdominal pain and bloating in patients with irritable bowel syndrome CulturedCare Probiotic Chewing Gum: Fights bad breath Yoptimal fermented milk, Yoplait: Used when antibiotics are prescribed for Helicobacter pylori stomach infection Advanced 4-strain Probiotic, Jamieson: For general replenishment of beneficial bacteria Courtesy of the Globe and Mail