The Human Microbiome: With Focus on Probiotics Haifa Khadour Isabelle Lam Fayyaz Samji.
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Transcript of The Human Microbiome: With Focus on Probiotics Haifa Khadour Isabelle Lam Fayyaz Samji.
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The Human Microbiome:With Focus on
Probiotics
Haifa Khadour Isabelle Lam Fayyaz Samji
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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
Intr
oduc
tion
to M
icro
biom
e
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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
Intr
oduc
tion
to M
icro
biom
e
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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
trod
uctio
n to
Mic
robi
ome
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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
Our
Mic
robi
ota
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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
Our
Mic
robi
ota
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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%
Our
Mic
robi
ota
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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
Our
Mic
robi
ota
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Our
Mic
robi
ota
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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
Our
Mic
robi
ota
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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
Mic
robi
ota
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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
Our
Mic
robi
ota
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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
ebio
tics
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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
Prob
iotic
s
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Mechanisms
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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.
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Mechanisms
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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.
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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.
Mec
hani
sms
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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
Mec
hani
sms
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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.
Mec
hani
sms
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Restriction of usage
1. Major concerns that have been investigated:
a. Bacteraemia. b. Adverse immunological effects.c. Antibiotic-resistance transfer.
Rest
rictio
ns
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a. Potential transmigration, Bacteraemia:– Diagnosed with immunosuppression.– Prior prolonged hospitalization.– Prior surgical interventions.
b. Immunological effects– No clinical proof.
Rest
rictio
ns
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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.
Rest
rictio
ns
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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.
Rest
rictio
ns
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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
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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.
Bene
fits
of P
robi
otics
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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.
H. pylori infectionBe
nefit
s of
Pro
bioti
cs
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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
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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
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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
Bene
fits
of P
robi
otics
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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.
Bene
fits
of P
robi
otics
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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.
Bene
fits
of P
robi
otics
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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
Bene
fits
of P
robi
otics
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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.
Bene
fits
of P
robi
otics
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Prob
iotic
s in
Foo
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
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Prob
iotic
s in
Foo
dsReminder
1.Antimicrobial activity2.Enhance intestinal epithelial barrier
function
3.Modulate host immune system
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Prob
iotic
s in
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
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Prob
iotic
s in
Foo
dsTolerance of transit in upper gastrointestinal tract
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Prob
iotic
s in
Foo
dsCell adhesion capabilities
Variability of <1%-25%
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Prob
iotic
s in
Foo
dsEpithelial barrier function
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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
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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
In th
e Fu
ture
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In th
e Fu
ture
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
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Continued Development of Microbiome Sequencing
Progress of projects under Human Microbiome Project
In th
e Fu
ture
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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
In th
e Fu
ture
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Education and Awareness• Companies will soon be accountable to their claims about probiotics• Foods with natural probiotics
In th
e Fu
ture
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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
Que
stion
Per
iod