Transcript Topic 7: The Human Microbiome

The Human Microbiome:
With Focus on
Probiotics
Haifa Khadour Isabelle Lam Fayyaz Samji
Introduction to Microbiome
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
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
Introduction to Microbiome
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 broadrange 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
• 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
Types of Microbiota
Archaea
• Relative to bacteria, they are few in number
• Majority are methanogens
– Methanobrevibacter smithii, Methanosphaera stadtmanae
Our Microbiota
• There are currently no known archaeal pathogens
Fungi
• Mainly yeast
– In particular, there is a lot of research done on Candida
species
Skin Flora
• Bacteria
Our Microbiota
– 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%
Gut Flora
• Most of the gut flora in the colon and large intestine
are comprised of bacteria
Our Microbiota
– 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
• Functions of Bacteria in the gut:
– Breaking down unutilized carbohydrate substrates
• Gut flora have enzymes that humans lack
Our Microbiota
– 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
• Primarily Lactobacillus bacteria that produce lactic acid
– L. acidophilus and L. doderlein
Our Microbiota
• 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
• “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:
Prebiotics
– Short- chain (ie. Oligofructose)
contain 2-8 links
– Long-chain (ie. Inulin) contain 964 links
– Full-spectrum (ie. OligofructoseEnriched Inulin) contain 2-64
links
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
Probiotics
• Are commonly found in fermented foods
– Live cultures are usually added
– Yogurt, dietary supplements
Mechanisms
Microbe-microbe interaction
• Competition for nutrients:
– Pathogens vs. probiotics.
– Endogenous microbs vs. probiotic.
• Biochemical effects:
Mechanisms
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
• Intestinal barrier:
Mechanisms
– 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:
Mechanisms
– 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
Microbe-host interaction
• Immunomodulation:
– regulate natural and acquired immune
– mucosa-associated lymphoid tissues.
Mechanisms
• 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
1. Major concerns that have been
investigated:
Restrictions
a. Bacteraemia.
b. Adverse immunological effects.
c. Antibiotic-resistance transfer.
a. Potential transmigration, Bacteraemia:
Restrictions
– Diagnosed with immunosuppression.
– Prior prolonged hospitalization.
– Prior surgical interventions.
b. Immunological effects
– No clinical proof.
c. Antibiotic resistance transfer:
Restrictions
– 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
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
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.
Benefits of Probiotics
H. pylori infection
• 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.
Benefits of Probiotics
Immune Stimulation
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
• 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
• 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
• 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
Benefits of Probiotics
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.
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
1.Antimicrobial activity
2.Enhance intestinal epithelial
barrier function
3.Modulate host immune system
Probiotic Ability
Study by Jensen, Grimmer, Naterstad and Axelsson (2012)
Probiotics in Foods
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
DSM20016
DSM 17938
mm4-1a
fj1
Probiotics in Foods
Tolerance of transit in upper
gastrointestinal tract
Cell adhesion capabilities
Probiotics in Foods
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
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
Designer Strains and GMOs
In the Future
• 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
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
• Companies will soon be accountable to their claims about probiotics
• Foods with natural probiotics
As a Thank You
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
Question Period
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