THE MICROBIOME-ASD CONNECTIONNew Discoveries in The Gut Microbiome’s Role in

ASD Risk and Potential Probiotic Supportive Therapies

Current NEW Understanding – The Sourceu The Human Microbiome Project (HMP) – NIH + 5 years

u Why the HMP project?

u Human flora has never been completely characterized. Limitations of microbiology with obligate anaerobes

u Metagenomics — This has allowed for the characterization and study of the total GI microbial population as it relates to health, wellness and disease

u Launched by the NIH 5 years ago — 200 researchers, 80 research institutes

u We are more bacteria than human! — 10 trillion human cells vs. 100 trillion bacteria cells

u There are over 1,000 different species of commensal organisms in the GIT out of 35,000 possible

u Bacterial genes outnumber human genes more than 150 to 1. (25,000 human genes vs. 3.3 million microbial genes)

u No two individuals have the exact same composition – not even twins. Phylum level conserved —Species level vast difference

u The intestinal microbiota is not homogeneous

Facts from the Human Microbiome Project (HMP)

Spatial and Temporal Aspects of Intestinal Microbiota Composition

Sekirov I et al. Physiol Rev 2010;90:859-904©2010 by American Physiological Society

MOM

SEEDING

THE ENVIRONMENT

CONDITIONING

Where do we get our Microbiota?

838 Days to adult-like microbiota

ASD and The Microbiomeu Initially the focus on ASD research was focused on the brain and genes.

u The most updated science is clear that ASD is a multi-factorial disorder – both intrinsic and extrinsic factors seem to play a role – ex:

u Environmental Chemicals

u Diet Alterations

u Metabolic Status

u Microbiota

COMMON CORRELATIONS WITH GIT DISRUPTORS AND ASD DEVELOPMENT

§ Both prenatal and perinatal acetaminophen exposure as well as acetaminophen exposure during childhood have been associated with the development of ASD. Coincidently acetaminophen has also been associated with GI problems, including acute upper GI emergencies.

§ Exposures to antibiotics, either early in life or during pregnancy, have been linked to the later developmental of ASD, and some have suggested that early antibiotic exposure could cause ASD

§ Proton-pump inhibitors, which are used to treat gastroesophageal reflux, a disorder not uncommonly associated with ASD, also effects GI pH levels and thus can cause dysbiosis.

§ Pesticides and heavy metals that have been linked to ASD also cause mitochondrial dysfunction and GI abnormalities.

GLYPHOSATE: DESTRUCTOR OF HUMAN HEALTH AND BIODIVERSITY by Rosemary Mason MB ChB FRCA

Number of children with autism plotted against glyphosate use on GE corn and soy. Autism data were obtained from the U.S. Department of Education, under the Individuals with Disabilities Education Act (IDEA).

This plot is shown using data from USDE for the number of autistic children receiving services. By kind permission of DrNancy Swanson.

ASD and The MicrobiomeØ Comorbidity with GI disturbances is often observed in ASD kids – some estimates indicate 70%-90%

Ø In Human Studies the presence of the following microbes have been positively associated with ASD:Ø Clostridium Tetani (1998 study) – not present in non-ASD kids and produces a toxin that impacts

neurotransmitter release.Ø Clostridium Bolteae was shown to be significantly elevated in kids with ASD in a 2004 study.Ø A follow up study with the same group showed 9 species of clostridium present in the stool of ASD kids.Ø Clostridium Histolyticum is another toxin producing bacteria found in ASD kids. – A further interesting

fact is that non-autistic siblings showed moderate levels of the same strain.Ø Desulfovibria, Bacteroides VulgatusØ Low Bifidobacterium levels and high Akkermansia levels are found in ASD kids.

Ø In most cases, the clinical severity of the GI issues were positively correlated with severity of ASD.

Ø A 2009 Study showed that vancomycin treatment improved symptoms in children with ASD but the effect was only transient and when the drug was stopped, the symptoms came back.

Ø ASD children tend to have higher levels of ammonia in stool.

Ø Microbiome of the mouth could play a role – mouth dysbiosis has been associated with severity of epileptic seizures.

Ø One study suggests that vaginal dysbiosis from maternal stress may effect off spring neurobehavioral development

HOW DOES DYSBIOSIS INFLUENCE ASD?

HOW DOES DYSBIOSIS INFLUENCE ASD?

Ø By using the enteric nervous system and vagus nerve, microbes and their components can get directly to the brain.

Ø Direct infection manipulates vagus nerve, ex: campylobacter jejuni infections creates sudden and rapid anxiety behavior by directly effecting the vagus nerve.

Ø Release of bacterial metabolites – these can directly effect inflammation and function in the brain, for example d-lactate and ammonia cause brain swelling.

Ø Suppression of Serotonin and dopamine function – 2015 study showed that 95% of circulating serotonin is made in the gut. Dysbiosis can disrupt this biosynthesis. Around 30 percent of patients with autism have abnormal blood levels of serotonin

Ø Micronutrient Deficiencies – especially B12. Methylated B12 is produced by a healthy gut microbiome and absorbed in the ilium. Dysbiosis reduces B12 production and absorption as well. Deficiencies impair formation of myelin sheets on nerves, production of neurotransmitters, activation of genes and repairing the gut lining. Vitamin D is another example.

Ø Leaky gut!! Intestinal permeability and the associated blood-brain barrier permeability may play the largest role yet in the increased risk and symptomology of ASD as it relates to the microbiome.

ASD and LEAKY GUTThere is mounting evidence that gut permeability, starting from birth can dramatically influence the risk of developing ASD.

Ø One line of evidence is a high-fat diet, which has been shown to cause a microbiome shift and increase intestinal permeability which leads to higher concentrations of endotoxins in the blood and more neuroinflammation. This pathology is associated with increased ASD risk and symptomology.

Ø Going on a gluten free and casein free diet has shown promise in improving symptoms of ASD as both gluten and casein have been shown to cause inflammation and gut permeability in a significant portion of the population.

Ø In a Harvard study, altered intestinal permeability was found in 43% of autistic patients, but not found in any of the control subjects.

Ø The presence of toxin producing harmful bacteria like clostridium tetani seem to increase inflammation on the lining and increase permeability – may be the mechanism by which these species are closely associated with ASD.

Ø Researchers studying Maternal Immune Activation using deliberate viral infections during pregnancy showed that mice that were infected had “leaky gut” and gave birth to mice that demonstrated autism behavior. When they extracted the endotoxins from these mothers and injected it into normal mice, they developed autism symptoms.

Ø Patricia D’Eufemia and colleagues published a seminal paper on gut hyperpermeability—leaky gut—in relation to autism. The researchers reported that altered intestinal permeability in nearly half of her ASD participants, compared with none of the controls examined.

“Autism is primarily a disorder of the brain, but research suggests that as many as nine out of 10 individuals with the condition also suffer from gastrointestinal problems such as inflammatory bowel disease and “leaky gut.” – Scientific American 05/2016

u Post-prandial Endotoxemia

Probiotic function of sporesLEAKY GUT – THIS JUST MAY BE THE REAL CULPRIT

ASD AND PROBIOTIC THERAPY

Because of the strong gut connect with ASD, it has been proposed that probiotic therapy may play a supportive role in the management of the condition.

In the following 2015 Review paper titled; Microbiome Disturbances and Autism Spectrum Disorders, the researchers outline the following important features of a probiotic that could make it useful as part of ASD therapy:

• Ability to alter host immunity – increase sIgA, lower inflammation markers, etc.• Restoration of normal commensal flora• Suppression of microbial pathogens• Ability to fix/improve intestinal barrier • Promotes the synthesis of antioxidants (Lutgendorff, et al 2009)

The Current Solutions in Dietary Supplements

The Food Standards Agency (FSA) study w/ Reading University (UK)Dr. G.R. Gibson, Dr. G. Rouzaud, Dr. J. Brostoff and Dr. N. RaymentPurpose:

1) Evaluate the probiotic effect of commercial products in the human gut. Any affect on gut flora.

2) 35 strains from commercial products were studied. Primarily lactobacillus sp. and bifidobacterium sp.

3) Evaluate the survivability of common probiotics through the GIT.

STEP 1: Survival through gastric juices in the stomach. 20 minutes at pH 1-3

pH 1 = None survived with any viability. Unable to recovery

pH 2-3 = 18 of the 35 showed around 50% survivalSTEP 2: Survivors through the stomach

were tested for survival in the upper small intestines via bile acid tolerance

Of the 18 tested, only 6 showed viability in the presence of bile acid salts and would have a chance to make it to the large intestines.

STEP 3: The 6 strains were then chosen for survival testing in lower intestines

Of the 6 strains, 4 strains showed viability in lower intestinal conditions

Only 4 of 35 strains would survive to enter the large intestine and the survivors would have less than 50% survival

Survival of Probiotics in Simulated Gastric FluidSilliker, Inc.- Food Microbiology Department

u Study Designu 4 Products tested: 1 leading retail brand supplement; 1 leading yogurt and

1 leading Greek yogurt vs. leading spore-form probiotic supplements

u USP Simulated Gastric Solution — pH 1.3 for 2 hour then bile salts

u Data is based on percent survival of bacterial population

u Test products were sourced directly by the lab from store shelves

Survival of Probiotics in Simulated Gastric FluidSilliker, Inc.- Food Microbiology Department, cont.

u Both yogurts had starting counts —regular yogurt had higher counts —but both yogurts saw over 99.99% of “probiotic” bacteria die off in the stomach

u The leading supplement brand had nearly a 4-fold overage but over 99.9% died off in the stomach.

u Spore form probiotics showed 100% survival with no loss at all

Issues: The Reseeding Plan

Quality:u Mislabeling of probiotic strains has been found to be an issue

u Several documented cases of mislabeled probiotics – a panel of EU experts in microbial characterization found the following:

1. Products were incorrectly labeled with the wrong strains

2. Products contained markedly reduced numbers than claimed

3. Found presence of strains not labeled – in some cases they were pathogenic.

u “Quality control of probiotics is lacking, study suggests:” (Nutraingredients-USA, 20-Nov-2015)

A new study by scientists at the University of California has found that contents of many bifidobacterialprobiotic products differ from the ingredients listed.

"After testing 16 probiotic products available in local Californian stores and also online, they found only one of the products exactly matched the bifidobacterial species claims on the label. Some products had pill to pill and lot to lot variation."

What are strategies for Probiotic Supplementation?

GOAL

CURRENT STRATEGY —RESEEDING THE GUT

REVISED STRATEGY —BACK TO BASICS

RECONDITIONING

The Importance of Environmental Bacteria

Ø Some of the last hunter-gatherer people on earth

Ø They live an ancient, ancestral life.

Ø Their environment hasn’t changed for 1000s of years.

Ø Massive exposure to ancestral microbial community

Ø Vastly different microbiota compared to westernized populations

Ø Virtually no common digestive diseases such as Crohn’s, UC, Colon Cancer, Reflux, etc.

STUDIES ON THE HAZDA TRIBE OF TANZANIA

What kind of features are required to be a probiotic?

Required Features of a Probioticu Naturally survive the harsh gastric environment

u Be of a strain that is found in the microbiota – It has to have a binding site and belong in the gut

u Must have evolutionary significance

u Must be a facultative anaerobe

u Must have a bi-phasic life cycle

u Must have clinical demonstration of safety and efficacy

So, What is nature’s design for supplemental probiotics?

“The Chuck Norris of probiotics: total tough guys.”

Dr. Michael Roizen

http://www.cleveland.com/healthfit/index.ssf/2011/10/spore_form_probiotics_keep_you.html

Bacterial Spores

u In particular Bacillus spores as they are the most widely studied and most widely used probiotics outside of the supplement market.

u Bacillus spores were the first commercial probiotics. Were also the first prescription probiotics starting in 1958:

u Enterogermina® (Sanofi-Aventis, Italy)

u Bacti-Subtil® (Aventis Pharma, France)

u Used extensively in agriculture and aquaculture

u AlCare®, BioGrow®, BioPlus ®2B, NeoFerm BS10, LiquaLife®, etc.

u Most widely used and well studied strains in humans are:

Ø Bacillus Subtilis, HU58

Ø Bacillus Coagulans

Ø Bacillus Clausii

Ø Introducing — Patented Bacillus Indicus HU36™

Key Features of Bacterial Spores

u They form robust endospore and can withstand harsh temps, desiccation, low pH, gastric barriers, antibiotics, UV radiation, solvents, enzymes and even high pressures

u They are found all over the environment (soil, vegetation, dust, rocks, aqua-environments, digestive systems of insects, marine life, mammals, etc.

u Spores Remain dormant over 50 million years

u They colonize very effectively in the Human GIT and have been found to colonize very effectively in the GIT of several different animals

u Are found as part of the normal human commensal flora

u LONG history of use in industries where efficacy is closely measured (pharma, agricultural)

u Extremely safe

u Its use as a probiotic is evolutionarily supported – true commensal organism

The Amazing Endospore

Function of Spores

u Colonizationu Numerous studies and a long history of use have confirmed

survivability of spores through the GIT

u Bacillus spores are found in the GI of insects, animals and humans -indicates a universal function as a commensal probiotic

u Studies now indicate that the environment is simply a vector to transfer the bacteria from host to host, the spores are better suited for life in the GI, but designed to be passed via the environment.

Function of Spores

u Microbiota Shift and BalanceSpores have the ability to shift the microbiota and favor the growth of good bacteria. They do this by:

u Producing a significant number of antibiotics that control bacterial growth (Example: Coagulin, Subtilisin, Amicoumacin, Surfactin, Iturins A, and Bacilysin)

u Competitive exclusion (CE) of pathogenic organisms. (they compete for space and nutrients)

u Increasing the growth of important GIT commensals, such as lactobacillus. (they produce compounds that feed the commensal)

u Gut Model study demonstrated a near 30% shift in microbiota population with continuous administration of our bacillus spores.

We are not aware of any other probiotic that has demonstrated the ability to fix dysbiosis.

Function of Spores

u Immunomodulationu Bacillus spores help develop the GALT. (It does this with the cooperation of

Bacteriodes Fragilis sp.)

u Bacillus spores increase circulating T and B Lymphocytes. (by becoming active in the small intestines and supports the activity in the Peyer’s patch.)

u Bacillus spores shifts the body from Th2 inflammatory to Th1 adaptive.

u Bacillus spores improve pattern recognition. (via Toll-like receptors(TLR) stimulation in Peyers Patch) – Allergies and Autoimmunity

u Bacillus spores shift from innate immune response to adaptive immune response (via dendritic activation.)

Function of Spores

uDigestive Aidu Bacillus probiotics has been shown to produce key enzymes that help the

digestion of food products and supports a healthy digestive system.

u Direct digestion of resistant starches, proteins, fats, plant matter and non-starch polysaccharides

u Detox – Bacillus has been shown to neutralize genotoxic compounds encountered in the GIT.

uExamples: Vomitoxin, also known as deoxynivalenol (DON), found in wheat, corn and other grains is neutralized by bacillus in the GIT.

uExample: Zearalenone (ZEA) is naturally produced by the fungus found on cattle, milk and even plants – impacts fertility and a number of other conditions. Bacillus has been shown to neutralize this toxin in the GIT.

Function of Spores u Key Nutrient Production

u Production of vitamins – Menaquinones, full array of b-vitamins, digestive enzymes, quinols, etc.

u NEW Bacillus Indicus HU36 – produces high levels of carotenoids that are of the highest bioavailability.

ASD AND PROBIOTIC THERAPY - REVIEWIn the following 2015 Review paper titled; Microbiome Disturbances and Autism Spectrum Disorders, the researchers outline the following important features of a probiotic that could make it useful as part of ASD therapy:

• Ability to alter host immunity – increase sIgA, lower inflammation markers, etc. –Bacterial spores do just that! The improve mucosal immunity and reduce inflammatory markers.

• Restoration of normal commensal flora – Bacterial spores shift the microbiome by eliminating the bad bacteria and helping the good bacteria to flourish.

• Suppression of microbial pathogens – Bacterial spores have the capability to competitively exclude pathogens.

• Promotes the synthesis of antioxidants (Lutgendorff, et al 2009) – Bacillus Indicus is the on antioxidant producing probiotic on the market.

• Ability to fix/improve intestinal barrier – Lets look at the data

“Chronic non-communicable diseases (NCDs) are the leading causes of work absence, disability, and mortality worldwide. Most of these diseases are associated with low-grade inflammation.”

Stress induces endotoxemia and increasing barrier permeabilityDe Punder, et al (2015).

“In combination with modern life-style factors, the increase in bacteria/bacterial toxin translocation arising from a more permeable intestinal wall causes a low-grade inflammatory state. We support this hypothesis with numerous studies finding associations with NCDs and markers of endotoxemia, suggesting that this process plays a pivotal and perhaps even a causal role in the development of low-grade inflammation and its related diseases.”

LEAKY GUT– THIS JUST MAY BE THE SOLUTION

GROUND ZERO OF MOST HEALTH DISORDERS

u Post-prandial Endotoxemia

Probiotic function of sporesLEAKY GUT – THIS JUST MAY BE THE SOLUTION

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Principal Investigator: Brian K. McFarlin, PhD, FACSM, FTOS University of North Texas

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The effect of 30-days of probiotic supplementation on post-prandial responses to a high-fat meal: An Expanded Pilot Study

Principal Investigator: Brian K. McFarlin, PhD, FACSM, FTOS University of North Texas

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Which strains are highly important?Bacillus Subtilis, HU58

•Widely used, safe and highly effective•Produces over 12 affective antibiotics•Makes nattokinase and vitamin K2•Supports immune development GALT

Bacillus Indicus, HU36•Supports immune health•Produces high level of carotenoids - lycopene, axtaxanthin, beta-carotene, lutein•Produces quinols and vitamins•The most effective antioxidants on the market

Bacillus Clausii•Most widely used probiotic drug in the world.•Supports immune health•Antibiotic resistant for use during antibiotic treatment

Bacillus Coagulans•Very well studied and long history of use.•Produces L+ optical form of lactic acid•Supports immune health

Introducing MegaSporeBiotic

• 100% spore-based probiotic

• Reduces serum LPS levels by 60% in just 30 days

• Increases microbial diversity by 40%

• Safe and effective for children

• Never requires refrigeration

• Shelf stable for 5 years