The Human Microbiome:With Focus on

Probiotics

Haifa Khadour Isabelle Lam Fayyaz Samji

The Human 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

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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

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HMP: Current methods• 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 studiesIn

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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

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Types of MicrobiotaArchaea• 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

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Skin Flora• Bacteria– Staphylococcus aureus

• Fungi: Yeast– Candida albicans– Malassezia spp.

Bacterial Phyla Percentage on Skin

Actinobacteria 51.8%Firmicutes 24.4%

Proteobacteria 16.5%

Bacteroidetes 6.3%

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Gut Flora• 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

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Gut Flora

• 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

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Oral Cavity Flora

• 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.Our

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Vaginal Flora

• 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

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Prebiotics• “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 linksPr

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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

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Mechanisms

Mechanisms

Microbe-microbe interaction • 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

Mechanisms

Microbe-host interaction

• 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

• 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.

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Cell service in host interaction

• The diversity of cell wall composition and structure provides species and strain specific properties that are likely involved in specific host interaction

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Microbe-host interaction

• 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.

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Restriction of usage

1. Major concerns that have been investigated:

a. Bacteraemia. b. Adverse immunological effects.c. Antibiotic-resistance transfer.

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a. Potential transmigration, Bacteraemia:– Diagnosed with immunosuppression.– Prior prolonged hospitalization.– Prior surgical interventions.

b. Immunological effects– No clinical proof.

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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.

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Who to Watch for

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.

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Benefits of Probiotics

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

Infection control

• 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.

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• 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.

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Clinical studies showed the use of Lactobacillussalivarious:– 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.

Benefits of Probiotics

Immune Stimulation

In human trail, 24 subjects fed 450 g ofyoghurt 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

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Cancer suppression

• 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.

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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.

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Diarrhea

• 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

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Lactose Malabsorption

• 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.

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Prob

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dsConsumed 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

Prob

iotic

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Foo

dsReminder

1.Antimicrobial activity2.Enhance intestinal epithelial barrier

function

3.Modulate host immune system

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iotic

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Foo

dsProbiotic Ability

Study by Jensen, Grimmer, Naterstad and Axelsson (2012)

1. Tolerance of transit in upper gastrointestinal tract

2. Cell adhesion capabilities

3. Epithelial barrier function

- Measured transepithelial electrical resistance (TER)

Human isolated strains:Lactobacillus reuteri DSM20016DSM 17938 mm4-1afj1

Prob

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dsTolerance of transit in upper gastrointestinal tract

Prob

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Variability of <1%-25%

Prob

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dsEpithelial barrier function

Prob

iotic

s in

Foo

dsWhat does it all mean?

• 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

An Expanding Field• 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

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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

Continued Development of Microbiome Sequencing

Progress of projects under Human Microbiome Project

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FDA Regulation• 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

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

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As a Thank YouFlorastor, 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 bacteriaCourtesy of the Globe and Mail

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