probiotic symposium chennai 3 dec 2016

P4 Medicine: Genomics of Microbiota and Probiotics

Prof. Samir K. BrahmachariJ.C. Bose National Fellow

Academy Professor, AcSIRFounder Director CSIR-IGIB

Former Director General, CSIR and Secretary DGIR, GOI.

3rd December, 2016

8th India Probiotic Symposium, Chennai

2014-PresentCSIR-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-).

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 inFrance, 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

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

39 Trillion bacteria

30 Trillion human cells

Human Genome in Equilibrium

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

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

Y axis is on log scale

Genotype to Phenotype Gaps

• 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

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

“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

Global prevalence of diseases may vary due to:

Cultural practices : Spread of lactose tolerance with dairy farming

or parasite load for e.g. malaria

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.

CONSUMER DRIVEN

HEALTHCARE

SYSTEMS BIOLOGY AND

MEDICINE

P4 MEDICINE

Normal

Pre-disease

Disease onset

ProgressComplications & side effectsPrevention

Diagnosis & screening

Customization of therapydrug, diet,probiotic & life style

Maintain Health

Maintain Quality of life

Aim of Personalised Medicine in genomics era

Prognosis

Monitoring

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

Wearable Technology…The New Frontier of Healthcare

Smart Contact LensMeasure Glucose levels in tears to manage diabetes

Electronic Sensor TattoosMonitor skin hydration, temperature & electric signals from brain activity

Fitness BandsMeasure and monitor physical activity and hours of sleep

Smart SocksMonitor heart rate and coach on running techniques in real time to prevent injuries

Smart WatchesMonitor heartbeat, read pulse, all-day calories burnt

Pain Relief Patches Track pain for pain management

Reactive Medicine Proactive P4 MedicineReactiveSymptoms based response

Proactive and preventivePre-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-BiologyDisease-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

NCD’s and Gene-Environment Interactions

Bousquet J…..Brahmachari Samir, et.al Genome Med. 2011; 3(7): 43.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221551/

Iterative mathematical modeling to increase knowledge on NCDs.

Bousquet J…..Brahmachari Samir, et.al Genome Med. 2011; 3(7): 43.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221551/

•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 Consortium2008, 115 million USD

Genus- and phylum-level classification of bacteria colonizing a composite subject

Host – Microbiome distribution ???

Interpersonal variations????

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 EcosystemThe Gut ecosystem predominantly includes the following Enterotypes: Bacteroides, Prevotella, Ruminococcus sp.

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

• 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

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

Predominant Bacterial Species in Different Disease Conditions

Disease Name of prevalent bacteria

Type 2 Diabetes

Akkermansia muciniphilaBacteroides intestinalisBacteroides sp. 20_3Clostridium bolteaeClostridium ramosumClostridium sp. HGF2Clostridium symbiosumColstridium hathewayiDesulfovibrio sp. 3_1_syn3Eggerthella lentaEscherichia coli

Disease Name of prevalent bacteria

Obesity/IBD/CD

Acidimicrobidae ellin 7143Actinobacterium GWS-BW-H99Actinomyces oxydansBacillus licheniformisDrinking water bacterium Y7Gamma proteobacterium DD103Nocardioides sp. NS/27Novosphingobium sp. K39Pseudomonas stramineaSphingomonas sp. AO1

IBD-Irritable Bowel Disease; CD- Crohn’s disease

Mandal RS et al. Genomics Proteomics Bioinformatics 13 (2015) 148–158

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.

Probiotic and Dairy products: Metagenomes unveiled using

3rd Generation sequencing

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

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.

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 .

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.

Probiotic and Dairy Products Sample Types for Metagenome Analysis

Metagenomics using Nanopore: Workflow (4-6 hrs)

DNA Extraction

Barcoding

Library Preparation

Nanopore Sequencing Run

Analysis of Reads for Microbial Diversity

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

Nanopore Sequencing Run

Real Time Data obtained Time taken: 1.5 hours

Analysis of Reads for Microbial Diversity

Sample Barcode Reads Species_1 Species_2 Species_3

Fermented drink BC01 254 Lactobacillus casei Lactobaccilus Casei froupi Lactobaccilus paracasei

Curd #1 BC02 160Streptococcus thermophilus

Streptococcus thermophilus LMD-9

Streptococcus thermophilus MN-ZLW-002

Lowfat Curd BC03 71Streptococcus thermophilus

Lactococcus lactis subsp. lactis

Lactococcus lactis subsp. cremoris UC509.9

Fermented Milk BC04 271Streptococcus thermophilus saccharomyceta

Candida dubliniensis CD36

Sweet Curd #2 BC05 142 saccharomycetaCandida dubliniensis

CD36 Saccharomycetales

Curd #3 BC06 373Streptococcus thermophilus

Streptococcus phage Alq132

Streptococcus phage TP-778L

Homemade Curd #1 BC07 112Streptococcus thermophilus

Streptococcus thermophilus JIM 8232 Streptococcus phage DT1

Sweet Curd BC08 68Streptococcus thermophilus

Streptococcus thermophilus LMD-9 saccharomyceta

Probiotic Capsule #1 BC10 39 Enterococcus faecium Bacillus coagulans 36D1Enterococcus faecium

NRRL B-2354

Probiotic Capsule #2 BC11 107Lactobacillus rhamnosus

GGLactobacillus rhamnosus

LOCK900Lactobacillus reuteri

SD2112

E.coli,M.smeg,Lambda Phage Control 43 Escherichia coli

Mycobacterium smegmatis str. MC2 155 Lambdalikevirus

Microbiome Table made in 6 hours by Nanopore ( It takes 24 to 48 hours by microbiological methods manually)Green – expected Red- unexpected Black -allowed

Taxonomic tree

Bar Chart

Donut Chart

Microbial Diversity in Fermented drink

Unexpected

Species Reads Classification_ScoreLactobacillus

rhamnosus GG 30 0.0519Lactobacillus

rhamnosus LOCK900 11 0.0252Lactobacillus reuteri

SD2112 10 0.0825Lactobacillus casei ATCC

334 9 0.0089Lactobacillus plantarum 5 0.2624

Bifidobacterium animalis subsp. lactis 5 0.0684

Lactobacillus plantarum JDM1 4 0.0355

Lactobacillus casei 4 0.039Enterococcus 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 0.01Lactobacillus 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

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

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.

• 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

(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

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.


Roles and Benefits of Probiotic Bacteria in the GI Tract


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.

The P4H Continuum Model

M Sagner,....Leroy Hood,... SK Brahmachari ... et al. Progress in Cardiovascular Disease (2016). http://dx.doi.org/10.1016/j.pcad.2016.08.002

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.CIt 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

http://www.trisutra.in/

http://www.ayurvedicpoint.it/pdf/Ayurgenomics.pdf

http://www.ayurvedicpoint.it/pdf/Ayurgenomics.pdf

AB1 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

DiseaseDiseasome

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

AB DC

•Apparently healthy• Normal health metrics

Big Data Analysis of Traditional Knowledge Based Medicine (Ayurveda)

Prescriptions Per day

fdfd

2500

45

350K

5.2MILLION

Clinics

Curated Patient DatabaseLast 3 Years Data

( Data under consideration )

Contact Database

Number of Physicians

300

1-800- Jiva Telemedicine

Data Background

(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

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.

Alluvial Plots Representing Patient Prevalence by Age

(a): Alluvial plot for total patient prevalence by age.

Three most prevalent disease elements of six major disease systems

(1): Digestive System

Skeleton


Endocrine System


Network Based Alluvial Plot for Disease Association Across Age Groups

Overall Distribution of Follow-up Relief

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.

NormalHealthy

• Probiotics

Pre-Disease

• Probiotics + Functional Food

Onset of Disease

• Antibiotic Treatment or Pancha Karma + Probiotics + Functional Food

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

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