Emerging and Future Opportunities for Modifying Gut Microbial Populations

CSIRO HEALTH & BIOSECURITY

Dr Michael ConlonOctober 2018

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Focus Areas of the Presentation

• Lifestyle and Programs

• Foods with Enhanced Characteristics (e.g. high amylose grains-resistant starch)

• Dietary Fibre, Resistant Starch and Microbes Producing Butyrate

• Dietary Polyphenols as Modulators of Gut Microbes

• Inflammatory Bowel Disease – A Commonly Used Microbial Dysbiosis

• Some Microbes of the Future

• Delivery via Nano- and Micro- Encapsulation

• Faecal (or Substitute Stool) Microbial Transplantation

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Key Factors Contributing to Gut Health –Applicable to Gut Microbes

Gut Health

Diet

Genes

Gut Microbiota

Lifestyle

Environment Endocrine System

Immune SystemBrain

Multiple Factors Can Impact Health via Gut Microbes

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ProteinsLipids

CarbohydratesMineralsAlcohol

Supplements/drugs

StressTravel

Geography/EconomyExercise (or lack of)Sunlight - Vitamin D

SleepHygiene-Sanitation

SmokingEnvironment-Pollution

Birth & Nurture MethodsGut Microbiota:Numbers

TypesActivitiesProducts

HEALTH

DIET LIFESTYLE

CASE STUDY

ImpromyTM Health and weight management program

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CSIRO developed ImpromyTM health and weight management program in collaboration with CSIRO's partner Probiotec Ltd.

ImpromyTM is a health improvement program involving point of care testing to assess weight and risk factors such as cholesterol, blood pressure and blood glucose.

The program is delivered through pharmacies and includes meal replacements, high protein meals and provides ongoing support by trained pharmacy staff.

Can we modify the gut microbiota through innovative programs that include diet & lifestyle improvements?

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How about high profile books that promote the consumption of dietary fibre in the community?

Includes a description of how gut microbes are importantto health

Introduces concepts such asprobiotics, prebiotics, leaky gut,the gut-brain connection

Key goals are to promotedietary fibre diversity but to also increase consumption ofResistant Starches (RS) – whichpromote production of SCFA bygut microbes

High Amylose Barley – Successful Substantiation and Commercialisation of a Product Influencing Gut Health

CSIRO’s novel cereal ingredients

Cereal Composition Health function

BARLEYmax™ High in several types of fibre Digestive, metabolic

High amylose wheat High resistant starch Digestive, metabolic

High fructan barley High fructans Digestive, metabolic, bone

High β-glucan wheat High soluble fibre Cardiovascular, metabolic

Low-gluten barley Ultra low gluten Digestive

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Dietary Fibre - Components

Dietary Fibre Consists Largely ofNon-Digestible Carbohydrates ofPlant Origin, and Includes:

Non-Starch Polysaccharides (NSP)

Resistant Starches (RS)

Oligosaccharides

Synthetic and ChemicallyModified Carbohydrates

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Probiotics and Prebiotics

Probiotic: A live microorganism which when administered in adequate amounts confers a health benefit on the host

Prebiotic: A dietary component which leads to “the selective stimulation or growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host”.

The current suite of probiotics, and the bacteria quantified as part of prebiotic measures, are largely (but not exclusively) based on a few genera and species (Bifidobacterium and Lactobacillus) which often represent only a few percent of the adult gut microbiota

Other probiotics and prebiotic targets commonly include Enterococcus, Bacillus, E.coli. Streptococcus and the yeast Saccharomyces cerevisiae (boulardii)

Are Dietary Fibres Prebiotics?

• Fibres and prebiotics are both usually non-digestible carbohydrates which are then fermented by gut microflora

• However, some definitions have suggested that a prebiotic differs from fibre in that it must result in selective growth and use in the gut (and imply that fibre broadly does not)

• Fructooligosaccharides are presently the most widely accepted forms of prebiotics due to selective stimulation of Bifidobacteria and Lactobacilli (these bacteria have been regarded as the primary indicators of prebiosis)

• Some forms of fibre (according to current definitions) can promote the selective proliferation of these prebiotic markers (eg. resistant starch in the form of high amylose maize starch (Bird et al., 2009))

• If selective stimulation of bacteria other than Bifidobacteria and Lactobacilli are considered a stronger case can be argued for fibre as prebiotics

Microbes Colonise the Gut at Birth – Effects on Microbes During Infancy May Have Long-term Consequences

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Interactions with your immune system begin

Mode of birth (Caesarean?) and diet during infancy (Breast or Bottle?) have profound effects on gut microbiota and long-term health outcomes

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Your Gut Microbiota Profile is Unique

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16S rRNA based-Eubacterial primer

week of study

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Faecal SCFA Differences of IndividualsMaintained over Months

Faecal Microbiota Population Profile Differencesof Individuals Maintained over Months

nMDS plot of DGGE bacterial profiles of individuals over time

mean butyrate (mmol/g) concentration

Subject in order of ascending butyrate.mmol/g, diet N

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Human Faecal Butyrate Variation: Responses to RS (Adults)

Bu

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Subjects in Order of Ascending Butyrate Concentration

46 healthy individuals consumed a diet high in NSP or high in NSP +resistant starch (RS) for 4 wks in a dietary cross-over(25 g NSP or 25 g NSP + 22 g RS)

-Faecal acetate, butyrate and total SCFA concentrations were increased by RS relative to NSP diet and entry

-Faecal weights and excretions of all SCFA were higher for RS and NSP diets relative to entry

(Mc Orist et al. (2011) J Nutr 141:883-889)

27% variation explained by BMI, 16% variation explained by dietary protein intake, 6% by dietary starch, 4% by fibre

Acetate Range: 22-110Propionate Range: 4-29

Despite Microbiota Variation – Prebiotics (RS) may be Effective in Most

Biological Effects Of Butyrate – Primary Energy Source of Cells Lining The Colon

Metastasis & Angiogenesis

Induction of Apoptosis

Inhibition of Cell Cycle& Proliferation

Induction of Cell Differentiation

Enhancement of the Immune Response

Butyrate

Modulation of Electrolyte Transport

Inhibition of Inflammatory Response

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Analyses Using Gut Bacteria Microarray

Entry controls vs RS

Enterococcus faecium

Ruminococcus bromii

Faecalibacterium prausnitzii

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Resistant starch

Several sulphate-reducers abundant(in low v high butyrate)

FEMS Microbiol Ecol (2008)

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Dietary Polyphenols

In addition to being fibre sources, many plant-based foods (e.g. berries, tea, coffee) are also rich sources of polyphenolic compounds with diverse biological functions (e.g. anti-oxidant capacity)

Polyphenols are plant secondary metabolites that contain an aromatic phenol ring

Most phenolic compounds can reach the colon where they are degraded by gut microbes and form metabolites with distinct metabolic functions

- If they have beneficial effects on health via selective stimulation of gut microbes and activities can they be considered prebiotics?

- What if the beneficial effects result from inhibition of the growth/activity of specific microbes?

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Resveratrol Protects Against Cardiovascular Disease by Altering Microbial Activity in the Gut

Moderate red wine consumption has been linked with a varietyof health benefits, and these effects have been attributed toresveratrol component.

Resveratrol, a phenolic compound found in the skin of many berries, is a potent anti-oxidant and widely consumed as a health supplement

Trimethyl-N-oxide (TMAO) is a known risk factor for artherosclerosis.

In a 2016 study, resveratrol increased levels of Lactobacillus and Bifidobacterium in the gut, and these microbes inhibited TMAO production by altering production of the precursor molecule.

Chen et al. mBio. 2016; 7(2): e02210-15. DOI:10.1128/mBio.02210-15

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Propolis:-a polyphenol-rich, resinous substance collected by honeybees from plants.-has a range of bioactivities (anti-inflammatory, inhibition of microbial pathogens).-consumed more regularly in some populations (e.g. Chinese) to promote health.-opportunity to engage propolis producers in other countries to characterise, develop and distinguish local product and to enhance uptake by consumers for health benefits.

Rodent study, Dr Kai Wang (Bee Research Institute, Beijing-CSIRO, Adelaide): - Addition of Chinese propolis to a western style diet reduced the severity of colitis

induced by DSS, reduced gut SCFA production (microbial analyses completed)- Follow-up studies show dietary Chinese and Brazilian propolis–induced attenuation

of DSS-colitis is linked to a reduction in numbers of gut Bacteroides spp.

Propolis: Impact on Colitis

0.3% propolisreduced diseaseactivity

Wang et al 2017, Nutrients 9:875; Wang et al 2018, Mol Nutr Food Res 62

The Role of Gut Microbes in Inflammatory Bowel Disease

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• Bacteria are essential for IBD to occur1

• Debated whether dysbiosis in IBD is primary or secondary to gut inflammation

• Injection of Bacteroides and clostridia into the rat intestinal wall induced inflammatory reaction, not seen with bifidobacteria and lactobacilli2,3

• Active IBD (ulcerative colitis and Crohn’s disease)

• Firmicutes/Bacteroidetes ratios altered4

• Depletion in the C.leptum group

– Faecalibacterium praunitzii4

Sulphate reducing bacteria (SRB) are also thought to

play an important role in IBD5

-Desulfovibrio piger

• Mucus of ulcerative colitis and Crohn’s disease

patients have increased numbers of mucolytic bacteria6

1) Cummings, J. H. et al. 2003. 2) Garcia-Lafuente, A. et al. 1997. 3) Mourelle, M. et al. 1998.

4) Sokol, H. et al. 2009. 5) Steed, H. et al. 2008. 6) Png, C.W. et al. 2010

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F.prausnitzii abundance in CD mucosa decreased

Identified reductions in mucosa associated F.prausnitzii were a key feature of CD and suggest the potential of using treatment with F.prausnitzii as a probiotic to counterbalance the dysbiosis in this condition

Butyrate-producing bacteria have promise as probiotics and as targets for prebiotics (resistant starch)

- Other key candidate butyrate-producers include R. intestinalis, E. rectale, E. hallii

- But how easy are they to cultivate and are there pre-conditions for some of their activities/benefits?

Faecalibacterium prausnitzii – key butyrate-producer – probiotic of the future?

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Akkermansia muciniphila - promoter of gut barrier function –another probiotic of the future?

-A.muciniphila probiotic prevented Western diet–induced inflammation in the

circulation and local atherosclerotic lesions.

-Gut permeability was decreased, tight junction protein expression was upregulated.

Autism, GI Disturbance and Microbiota

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Faecal Bacteria

-Stool collected from children with ASD, their siblings and community controls-Reported incidence of GI disturbance higher in ASD children-Higher faecal propionate than controls-Some urinary bacterial metabolites elevated in ASD. Indolyl-3-acryloylglycine (IAG) associated with leaky gut elevated, especially in children with reported GI disturbance.

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Nano- and Micro- Encapsulation of Probiotics and Supplements

Technology which incorporates an ingredient/probiotic within or surrounded by a protective matrix. The matrices can include:- Alginate, Chitosan, Xanthum Gum, Starch, Gelatin, Pectin, Casein, Whey

Protein, BSA, Soy Protein, Lipids

Key Advantages can include:- Enabling gastric survival of probiotic organisms or prebiotics/supplements and

hence an ability to impact lower reaches of the gut - The potential to control release of encapsulated components through

encapsulant properties- Reduceed loss of probiotic/supplement viability during processing or storage - Masking of unwanted tastes to facilitate human consumption (supplements

such as polyphenols often have a bitter taste)

CSIRO’s MicroMAX Technology Platform“High oil loading – Long shelf life”

• An innovative “technology platform” for protection and

delivery of bioactives into functional food

• Using natural food grade materials

• Using simple and clever chemistry

• Utilising standard food processing equipment

Emulsion based delivery system – suitable to deliver single or a combination of

bioactive cores with different solubilities

Standard microencapsulation process for probiotics using MicroMAX® technology

Protein

Carbohydrates

Other components

Emulsion

Probiotic cells

± Reaction Mixing Drying

Film formed

around bacteriaExcess bulk

encapsulant

-Spray dry

-Freeze dry

10 μm

Laser confocal micrograph

Lactobacillus sp.

pH ~ 4.5

Colour labelled

green = proteinaceous material

yellow/orange = oil droplets

rods (2-3 mm) = Lactobacillus bacteria

MicroMAX® Microcapsule at pH 4.5

10 μm

CSIRO©

Unencapsulated

Encapsulated

mmol

Results confirmed that co-administration or supplementation with microencapsulated probiotics for 3wks enhanced metabolism of flavanones in orange juice

Probiotic+juice Juice only Probiotic supplement daily (4 wks) | Juice only

collection 1baseline

Collection 1aAfter consumption

collection 2baseline

Collection 2a24h after OJ

consumption

Collection 324h before

Last supplement

Collection 3a24h after

last supplement

24h 24h wash out peroid 24h 24h week1 week4

Acute Study Four Weeks Supplementation Study

Microencapsulated probiotics added to

any food or beverage for 4 weeks

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

Before OJ After OJ Before OJ After OJ Before OJ After OJ

control (OJ only) OJ + Acute Probiotics OJ + 3 weeks Probiotics

3-Hydroxyphenyl acetic acid

3-Hyroxyphenylacrylic acid

3-Methoxy-4-hydroxyphenylacetic A

3-Methoxy4hydroxyphenylhydroacr

4-Hydroxybenzoic acid

4-Hydroxyphenylacetic acid

*

*

* *

Metabolites (at 24 h) in urine after ingestion of 196 mmol of orange juice flavanones.

Phenolic acids (at 0 & 24 h) in urine after ingestion of 196 mmol of orange juice flavanones.

Pereira-Caro et al, 2015, Free Radical Biology & Medicine, 84:206-214

B.longum

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Animal Kingdom• Coprophagia

Veterinary Medicine • Transfaunation

• Ruminal acidosis in cattle

FMT in Human Beings• Traditional Chinese Medicine

• Dong-jin dynasty 4th Century China

• Ming dynasty 16th Century China

• Bedouin of northern Africa

• 20th Century: Pseudomembranous colitis

• Eiseman et al 1958.

When All Else Fails: Faecal Microbial Transplantation (FMT)

Now used as the most effective therapy to treat recurrent Clostridium difficile infection (~95% success)

Short duration, low intensity pooled fecal microbiota transplantation induces remission in patients with mild-moderately active ulcerative colitis: A randomised controlled trial.(FIRST-UC study)

Samuel P Costello1, Oliver Waters5, Robert V Bryant1, Rosa Katsikeros2, Jesica Makanyanga5, Mark N Schoeman2, RemeMountifield3, Derrick Tee4, Stuart Howell2, Patrick Hughes2, Michael A Conlon6, Ian C Roberts-Thomson1, Jane M Andrews2

1. The Queen Elizabeth Hospital, 2.Royal Adelaide Hospital, 3. Flinders Medical Centre, 4. Lyell McEwin Hospital; South Australia.5. Fiona Stanley Hospital; Western Australia. 6. CSIRO Health & Biosecurity, Adelaide, South Australia

DDW, 9th May 2017

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• Double blind RCT

• Mild to moderate active ulcerative colitis (Mayo score 3 to 10)

• Endoscopic subscore of 2 or greater (to ensure symptoms are due to UC (not post-inflammatory IBS))

• Patients aged 18 to 75 years

• Colonoscopy FMT followed by 2 enemas in subsequent week

• Subjects receive Own or Donor stool

• Repeat colonoscopy at week 8 and 52

• Open label FMT access post blinded therapy available to placebo arm at week 8

• Frozen pooled donor stool (4 donors)

Study Design

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Outcomes

Microbial population diversity was greater in donor stool than UC stool and improved 4 wk after FMT

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14 cohort studies, 4 RCTs- Clinical remission in about 28% of RCT patients receiving donor stool- Clinical responses in about 49% of patients receiving donor stool- In cohort studies, 24% of patients achieved clinical remission

With a growing understanding of the roles of gut microbes will come an opportunity to tailor the donor stool or microbe mix and matrix to improve patient outcomes for IBD and other conditions

FMT: Restoring Gut Microbial Diversity and Resistance to Pathogens

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Transplantations using defined cocktails of microbes may become more popular as stool substitutes in the future.

The challenge is:- Defining the right microbes for the target individuals- Growing the microbes

Petrof et al (2013) created a mixture of 33 gut microbes from commensal species isolated from a healthy donor and used it to treat and restore bowel health in 2 patients with recurrent C.difficile infection

- They called it RePOOPulating the gut

- Microbes delivered shifted microbial profiles and 25% of the donated microbes could be detected 6 mo after colonic delivery

Transplantation of Defined Microbial Cocktails

Health & BiosecurityMichael ConlonSenior Research Scientist

t +61 8 8303 8909e michael.conlon@csiro.auw www.csiro.au

HEALTH & BIOSECURITY

Thank you