1. P4 Medicine: Genomics of Microbiota and
Probiotics
Prof. Samir K. Brahmachari
J.C. Bose National Fellow
Academy Professor, AcSIR
Founder Director CSIR-IGIB
Former Director General, CSIR and Secretary DGIR, GOI.
3rd December, 2016
8th India Probiotic Symposium, Chennai
2. 2014-Present
CSIR-IGIB, South Campus, New Delhi
J. C Bose National Fellow
Chief Mentor, OSDD
Open Source Drug Discovery
Systems Biology
Genome Informatics
Affordable Healthcare &
Wellness Genomics
Academy Professor, AcSIR
Life Time Honorary Professor,
Delhi University
Mentor Young Students
Chairman, West Bengal Education Commission
Education Roadmap and Vision Document 2020…2030
for 90 million people of the State (2014-15)
Mentoring Startups
Ethical, Actionable Clinical Genomics
Mentoring Industry
Big Data Analytics Health
Member, Advisory Board, ADP- UNDP, NY(2014- ).
Member, Advisory Board, NCBO, Stanford
University (2012- 2015)
Honorary Chaired Professor, Mayo Clinic,
USA
Member, International Scientific Advisory
Group, India TB Research Consortium (2016-)
Member, International Scientific Advisory Board,
Center for Research and Interdisciplinarity (CRI),
University of Paris V, Paris (2016-).
3. Started in 1990 by HUGO to sequence
3.3 billion base-pairs of the 24 Human
Chromosomes (1-22, X and Y)
The Human Genome Project:
Globally Distributed Effort to Understand Nature’s Blueprint
Global interest:
• To understand human disease &
evolution$3-Billion Project
US DoE and NIH
20 Sequencing Centers in
France, Germany, Great Britain, Japan,China and United States
(1, 6, 9, 10, 11, 13,
20, 22, Part of X)
3, 12, Part of X 2, 4 Y5, 16, 19
4. Human genome project - surprises
No such thing as THE human genome sequence
15 million SNPs, 1 million ins/del, 20,000 structural variants
Each person with
~250 -300 loss of function variants
~50 – 100 variants implicated in disorders
Between two individuals there are 1 million differences!!
More surprises !!!
Human and Chimpanzee differ by only 1%
We are born 100 % human but die as 10% human- 90 % cells
are bacterial in origin
6. 1- 5% of any population suffer from common diseases
Increased average life expectancy
Long term medications, side effects
Expensive interventions
Life time prevalence
Common Diseases: A Global Burden
7. The advent of next generation sequencing technologies
and other high throughput measurements of ‘omics’
data, along with clinical phenotype association studies,
have created a data deluge.
However, explosive growth in biomedical data
generation has not yet translated to proportionate
increases in clinical returns.
The Genomics Data Deluge – Clinical Returns Paradox
8. Y axis is on log scale
Genotype to Phenotype Gaps
9. • Extensive Genome Wide Studies (GWAS) have shown
that genetic contribution in chronic non-communicable
diseases mainly Cardiovascular disease, Asthma,
Irritable bowel syndrome, Metabolic disorders like
Diabetes and Obesity accounts for only a small
percentage.
• The ability to sequence gut microbiota and progressive
realization that these microbiomes play an important
role in pathogenesis of both intestinal and extra
intestinal disorders opens up a new way of treating the
disease and understanding of healthy microbiota.
Non-Communicable Diseases : Genomics and Microbiota
10. Need for Systems Medicine
• Systems Biology tools applied to questions of
medical consequence
– high-throughput / large-scale medical data
– high-definition visualization
– Computational modeling
Biology Medicine Systems Medicine
11. “Survival of the Sickest explores
earth, history, & the human
genome to discover how
environmental, cultural, & genetic
differences shaped us through
evolution & continue to play an
active role in our health today”
“Many of the conditions that we
think of as diseases today actually
gave our ancestors a leg up in the
survival sweepstakes -------”
Genes, Evolution & Diseases
12. Global prevalence of diseases may vary due to:
Cultural practices : Spread of lactose tolerance with dairy farming
or parasite load for e.g. malaria
13. P4 Medicine
The Evolving Future of Medicine
• The P4 medicine uses systems biology approaches and information technologies to
enhance wellness rather than just treat disease.
• Its four components include predictive, preventive, personalized, and participatory
medicine.
• To address chronic diseases globally and to reduce their burden and societal impact,
the current medicine has to evolve from a reactive to a proactive system.
IT, SOCIAL
NETWORKING&
DIGITAL
REVOLUTION
CONSUMER
DRIVEN
HEALTHCARE
SYSTEMS
BIOLOGY AND
MEDICINE
P4 MEDICINE
14. Normal
Pre-disease
Disease onset
Progress
Complications
& side effects
Prevention
Diagnosis &
screening
Customization of therapy
drug, diet,probiotic & life style
Maintain Health
Maintain Quality of life
Aim of Personalised Medicine in genomics era
Prognosis
Monitoring
15. The Next Frontier of Science
• Medicine will be changed
profoundly and forever
• Data-driven decision
making will supplant
groups of respectable wise
men making guidelines
• Every decision and every
outcome will be data for
the next decision
• Changing mindsets is the
main thing required. Data
is often already there
16. Wearable Technology…The New Frontier of Healthcare
Smart Contact Lens
Measure Glucose levels in tears
to manage diabetes
Electronic
Sensor Tattoos
Monitor skin
hydration,
temperature &
electric signals
from brain
activity
Fitness Bands
Measure and monitor physical
activity and hours of sleep
Smart Socks
Monitor heart rate and coach
on running techniques in real
time to prevent injuries
Smart Watches
Monitor heartbeat, read
pulse, all-day calories
burnt
Pain Relief Patches
Track pain for pain management
17. Reactive Medicine Proactive P4 Medicine
Reactive
Symptoms based response
Proactive and preventive
Pre-symptomatic biomarker response
Cross-sectional Disease Management Lifespan Health Management
Few measurements, limited diagnostic
and prognostic value
Many measurements, high resolution
diagnostic and prognostic value
Organ-centric Systems-Biology
Disease-centric Person-centric Based on needs,
personal requirements and biological
variability
Symptom focused therapy Disease mechanism focused
therapy/interventions
Top-Down Individual and health professional as a
team
Paradigm Shifts from Reactive to Proactive Medicine
19. Iterative mathematical modeling to increase knowledge on NCDs.
Bousquet J…..Brahmachari Samir, et.al
Genome Med. 2011; 3(7): 43.
20. •242 healthy adults sampled
•15 or 18 body sites up to
three times
•5,177 microbial taxonomic
profiles from 16S ribosomal
RNA genes
•3.5 terabases of
metagenomic sequence
•~ 800 reference strains
isolated from the human
body
Human Microbiome Project Consortium
2008, 115 million USD
Genus- and phylum-level classification of bacteria colonizing a composite
subject
Host – Microbiome distribution ???
22. Bacteroides sp. Prevotella sp. Ruminococcus sp.
Gram-negative, obligate
anaerobic bacteria
Gram-negative, anaerobic
bacteria
Gram-positive, anaerobic
bacteria
a symbiotic host-bacterial
relationship with humans.
host-associated bacteria
colonizing the human
mouth cavity.
allow their hosts to digest
cellulose and fiber
degradation.
They help in fermentation
of carbohydrates, utilization
of nitrogenous substances,
biotransformation of bile
acids and other steroids.
Found in people consuming
protein and fat rich diet.
They colonize by binding or
attaching to other bacteria
in addition to epithelial
cells.
common in people taking
more fiber rich food.
They help in breaking down
of cellulose that comes
through the digestive
system of the hostorganism.
They are capable of
fermenting glucose and
xylose.
Bacteroides acidifaciens,
B. gracilis, B. fragilis
Prevotella albensis,P.
brvantii, P. melaninogenica
Ruminococcus albus, R.
bromii, R. flavefaciens
Enterotypes found in Gut Ecosystem
The Gut ecosystem predominantly includes the following Enterotypes:
Bacteroides, Prevotella, Ruminococcus sp.
23. Gut microbiota plays a major role in following human metabolic
functions:
• Synthesize essential amino acids and vitamins.
• Facilitate degradation of indigestible food compounds by
glycosidehydrolases and polysaccharide lysases.
• Fermentation of saccharides to provide energy for intestinal
epithelial cells.
• Conversion of complex carbohydrates and proteins into
simple compounds which are further fermented into short-
chain fatty acids (SCFAs) as well as to carbon dioxide and
molecular hydrogen.
• Improve the absorption of calcium, magnesium, and
phosphorus in the intestine
Microbial-Host Metabolism
24. • Microbiota in the large intestine helps in fermentation of the
soluble dietary fibers leading to production of Short Chain
Fatty Acids (SCFA).
• SCFAs have beneficial effects on intestinal epithelium and the
gut immune system.
• High Fat Diet intake and inflammation mechanisms alter the
microbial community resulting in dysbiosis.
• A dysbiotic state of the gut microbiota is considered as an
environmental factor that interacts with a host’s
metabolism and function
• Such dysbiosis-associated metabolites can be implicated
in obesity, systemic metabolic disorders as well as
gastrointestinal disorders
• But the specific contribution of the gut microbiota to these
diseases are needed to be explored.
Function of Gut Microbiome in Human Health
and Disease
Prof BS Ramakrishna, CMC Vellore
25. The role and influence of gut Microbiota in pathogenesis
and management of obesity and metabolic syndrome
Parekh et al (2014).
Doi: 10.3389/fendo.2014.00047
26. Predominant Bacterial Species in Different Disease
Conditions
Disease Name of prevalent bacteria
Type 2
Diabetes
Akkermansia muciniphila
Bacteroides intestinalis
Bacteroides sp. 20_3
Clostridium bolteae
Clostridium ramosum
Clostridium sp. HGF2
Clostridium symbiosum
Colstridium hathewayi
Desulfovibrio sp. 3_1_syn3
Eggerthella lenta
Escherichia coli
Disease Name of prevalent bacteria
Obesity/
IBD/CD
Acidimicrobidae ellin 7143
Actinobacterium GWS-BW-H99
Actinomyces oxydans
Bacillus licheniformis
Drinking water bacterium Y7
Gamma proteobacterium
DD103
Nocardioides sp. NS/27
Novosphingobium sp. K39
Pseudomonas straminea
Sphingomonas sp. AO1
IBD-Irritable Bowel Disease; CD- Crohn’s disease
Mandal RS et al. Genomics Proteomics Bioinformatics 13 (2015) 148–158
27. ARTICLE
Persistent microbiome alterations modulate
the rate of post-dieting weight regain
Thaiss, C. et al. Nature (2016).10.1038/nature20796
The weight reduction strategies cause dieting individuals undergo
excessive weight regain cycles instead of retaining their reduced body
weight.
An intestinal microbiome signature has been identified that persists
after successful dieting of obese mice, which on re-exposure to
obesity-promoting conditions, results in faster weight regain and
metabolic aberrations. This may act as a predisposition factor and
transmit the post dieting weight regain phenotype.
A machine-learning algorithm could enable personalized microbiome-
based prediction of the extent of post-dieting weight regain.
Also, post diet reduced flavonoid levels and energy consumption may
be associated with the excessive secondary weight gain.
Microbiome-targeting approaches may help to diagnose and treat this
common disorder.
28. Probiotic and Dairy products:
Metagenomes unveiled using 3rd
Generation sequencing
29. Common Probiotic Lactobacillus sp. and
Bifidobacterium sp.
Probiotic Species Genome Sequence
(strain designation) Reference
( Accession Number)
Lactobacillus
L. acidophilus (NCFM, La-1)
L. casei (BL23)
L. johnsonii (NCC 533)
L. Plantarum (JDM1)
L. reuteri (SD2112)
L. rhamnosus (GG)
L. Salivarius (UCC118)
L. bulgaricus (ATCC11842)
Bifidobacterium
B. animalis subsp.lactis (B1-04)
B. breve (UCC2003)
B. longum (NCC2705)
Antonie van Leeuwenhoek (2014) 106:141–156.DOI 10.1007/s10482-014-0171-y
30. Objective
Developing a fast and multiplexed Microbiome Assay of
dairy products, probiotics and fermented drinks for
quality control using the portable and simple Nanopore
sequencing Technology.
31. Nanopore Sequencing Technology
Nanopore technology
A nanopore is a nano-scale pore.
• In its devices, Oxford Nanopore passes an
ionic current through nanopores and
measures the changes in current as
biological molecules pass through the
nanopore or near it.
• The information about the change in current
can be used to identify that molecule.
We & Genotypic in collaboration with Oxford Nanopore sequencing
technologies are developing applications around their revolutionary –
portable – 3rd Generation Nanopore Technology .
32. Advantages of Nanopore Sequencing
Technology
Microorganisms can be sequenced directly from
fermented food products or preferably after isolation of
single colonies.
• Whole genome can be assembled in a single day.
• Can be used in complementation with illumina
(short reads) or stand alone.
• Enzyme manufacturers are already using Nanopore
sequencing to sequence their favorite stains.
34. Metagenomics using Nanopore:
Workflow (4-6 hrs)
DNA Extraction
Barcoding
Library Preparation
Nanopore
Sequencing Run
Analysis of Reads for
Microbial Diversity
35. Barcoding and Library Preparation for Running
Multiple Samples (12 samples in one flow cell)
Extraction & QC
of DNA from milk
products and the
probiotic
capsules
Time taken: 2 hours
Time taken: 1.5 hours
Time taken: 1.5 hours
39. Species Reads Classification_Score
Lactobacillus
rhamnosus GG 30 0.0519
Lactobacillus
rhamnosus LOCK900 11 0.0252
Lactobacillus reuteri
SD2112 10 0.0825
Lactobacillus casei ATCC
334 9 0.0089
Lactobacillus plantarum 5 0.2624
Bifidobacterium
animalis subsp. lactis 5 0.0684
Lactobacillus plantarum
JDM1 4 0.0355
Lactobacillus casei 4 0.039
Enterococcus faecium 4 0.0622
Lactobacillus paracasei
subsp. paracasei 8700:2 3 0.1623
Lactobacillus
rhamnosus 2 0.049
Lactobacillus casei
LOCK919 2 0.0095
Lactobacillus casei
group 2 0.0585
Lactobacillus casei W56 1 0.01
Lactobacillus 1 0.036
Enterococcus faecium
NRRL B-2354 1 0.012
Enterococcus faecium
Aus0004 1 0.008
Carnobacterium
maltaromaticum
LMA28 1 0.124
Commercially Available Probiotic Capsule
(Claims 5 different bugs – we picked up all with ~100 reads)
Taxonomic
tree
Donut Chart
40. How Unique is Nanopore Sequencing?
Microbiological analysis WGS Metagenome
Nanopore
Time: Long incubations(days/weeks) 4-6 hrs
Detection: Cultured microorganisms Includes Non-
culturable also
Effort: Media for different organisms Native sample
sequenced
Validation: Molecular biology/PCR methods Validated results
Cost: Comparable
**Unlike 16S metagenome our method can also identify virus, fungus and phages
41. Interpretation
From 12 different fermented milk products the microbial
composition could be obtained in 4-6 hrs.
• Lactobacillus casei is the major component as claimed
on the Yakult bottle.
• Not just bacteria, fungi and viruses identified.
• phages change the proportion of reads for bacteria
Quality Control can be tested at different stages:
The starter culture, The inoculum, Pre-packaged product
Batches, Ready to shipout samples, Samples from outlets
using Nanopore sequencing technology for contamination.
42. • According to the World Health Organization, Probiotics are ‘live organisms
which when administered in adequate amounts confer a health benefit on the
host’.
• Probiotics promotes the growth or activity of the gut microbiome which
helps to maintain the health of the body.
• Maintaining the digestive health of an individual is crucial for healthy life as
the Gastrointestinal (GI) tract acts as an interface between the host and its
environment.
• Alteration in the gut microbiome composition may render the Probiotics as
alternatives for antibiotics in future.
• Probiotics acts as a broad-spectrum antibiotic capable of eradicating "bad"
bacteria and recolonizing the GI tract with "good" bacteria.
Antibiotics Probiotics in Digestive health
43. (1) Inhibiting the pathogenic bacterial adherence and decolonization
of the gas producing, bile salts deconjugating bacteria.
(2) Alteration of bacterial flora by acidification of the colon by
nutrient fermentation.
(3) Enhancement of epithelial barrier function.
(4) Reduction of stress response.
(5) GI tract immunity modulation by altering immune
regulatory mechanisms in a strain-specific manner.
Probiotic microbes delivered orally must survive varying
environments in the GI tract, including acidic gastric juices in the
stomach, and bile in the small intestines.
Potential Role of Probiotics in Human Health
44. Probiotics and Immune System
A) Probiotic microbes delivered orally must survive varying environments of the GI Tract.
B) At intestinal epithelia, probiotics adhere in high numbers, leading to competitive
exclusion of pathogens by producing bacteriocins and other antimicrobial agents which
may antagonize pathogens in the lumen.
C) Probiotics bound in the mucus and epithelial layers are proximal to dendritic cells of the
mucosal immune system, leading to immunomodulation.
Antonie van Leeuwenhoek (2014) 106:141–156.DOI 10.1007/s10482-014-0171-y
45. Roles and Benefits of Probiotic Bacteria in the GI Tract
Antonie van Leeuwenhoek (2014) 106:141–156.DOI 10.1007/s10482-014-0171-y
46. Roles and Benefits of Probiotic Bacteria in the GI Tract
Antonie van Leeuwenhoek (2014) 106:141–156.DOI 10.1007/s10482-014-0171-y
47. Microbial-Host Metabolism and The Effect On
Behavioral Function of Brain
GH- Glycoside Hydrolases; PL- Polysaccharide Lysases; SCFAs-Short-Chain Fatty Acids;
GPR- G-Protein Coupling Receptors; PYY- Peptide YY.
Gut-Microbiota and Mental Health: Current and Future Perspectives.
Thakur et al. 2014. J Pharmacol Clin Toxicol 2(1):1016.
48. The P4H Continuum Model
M Sagner,....Leroy Hood,... SK Brahmachari ... et al. Progress in Cardiovascular Disease (2016).
49. Ayurveda : Ancient Indian system of Predictive, Preventive
and Personal Medicine with Participatory Approach
Touching base with the people for understanding their biological variability, health
care needs and their knowledge of the same.
Tapping the wisdom -Observing the health status , trends and Life Style practices of
populations for predictive, preventive and curative healthcare .
Connecting baseline inter individual variability with their geo- climatic conditions to
understand needs and develop personalized health care solutions!
Ayurveda has been practicing it and has documented the methods since 1500 B.C
It advocates examination of the patient in his /her context viz, habitat, ethnicity, time/
season and Age along with his/ her constitution to arrive at customized solutions that are
acceptable, accessible and affordable to them!
Need for population participation for development and delivery of P4 medicine
www.trisutra.in
http://www.ayurvedicpoint.it/pdf/Ayurgenomics.pdf
http://www.scienceandcultureisna.org/jan2011/02%20Mitali%20Mukerji.pdf
50. A
B1 B2 C D1 D2
Modern Medicine
Ayurveda
Health
Sanchaya
(Initiation of Dosha
accumulation)
• Normal Individuals
Biological Expression
(Signs )
• Blood pressure, Body habitus,
Blood glucose, Biomarkers,Gut
Microbiome etc.
Prakopa
(Dosha aggravation at site)
•No Phenotypic Expression
•Biochemical changes
starts
Clinical Expression
(Symptoms)
Fatigue, Pain,
Shortness of breath etc.
Prasara
(Spread of Dosha to other
tissues/systems )
•Biochemical changes
spreads to tissues
Disease
Diseasome
Sthana
Samsharya
(Interaction & effect on
local target systems)
•Phenotypic Expression
•Major Biochemical
changes
Vyakti
(Disease manifestation
•Detectable
Phenotypic Expression
•Major Biochemical
changes
Bheda
(Further
differentiation)
•Chronic stage
•Disease
complication
Health to Disease Transition- Modern Medicine vs Ayurveda
A
B DC
•Apparently healthy
• Normal health
metrics
51. Big Data Analysis of Traditional
Knowledge Based Medicine
(Ayurveda)
53. (a): Distribution of patients in the order of age group.
(b): Distribution of patient in the order of diseases.
Pie Charts Representing Distribution of
Patients Based on the Age Groups and
Diseases
54. Chronicity Distribution of 287K
Telemedicine Patients
(a): Represents the Chronicity distribution in patients and it clearly appears that vast
majority of the patients have been chronic for at least a year or more.
(b): Represents distribution for patients with Chronicity greater than one year.
Out of 318K telemedicine data, 287K have reported Chronicity.
55. Alluvial Plots Representing Patient
Prevalence by Age
(a): Alluvial plot for total patient prevalence by age.
56. Three most prevalent disease elements of six
major disease systems
(1): Digestive System
61. Future of Medicine for Lifestyle Disorders:
Probiotics and Functional Food
In all stages, Stool test (metagenomic analysis) will be crucial before and
after treatment to detect and decide the amount of pathogenic load and its
reduction on treatment.
Normal
Healthy
• Probiotics
Pre-
Disease
• Probiotics +
Functional
Food
Onset of
Disease
• Antibiotic
Treatment
or Pancha
Karma +
Probiotics +
Functional
Food
Disease
Phenotype
62. The P4H Continuum Model for Policy Makers
M Sagner,....Leroy Hood,... SK Brahmachari ....et al. Progress in Cardiovascular Disease (2016).
http://dx.doi.org/10.1016/j.pcad.2016.08.002
63. The advantage you have yesterday, will be replaced by the
trends of tomorrow. You don’t have to do anything wrong, as
long as your competitors catch the wave and do it RIGHT, you can
lose out and fail.
To change and improve yourself is giving yourself a second
chance. To be forced by others to change, is like being discarded.
Those who refuse to learn & improve, will definitely one day
become redundant & not relevant to the industry. They will learn
the lesson in a hard & expensive way.
- Ziyad Jawabra
“We didn’t do anything wrong,
but somehow, we lost”
- Nokia CEO
