Download Biofilm and Chronic Infections

Autism ONE
Chicago
May 2009
Anju Usman,MD
True Health Medical Center
Many patients with autistic symptoms have persistent
dysbiosis. Treatment of GI issues often alleviates the
symptoms we call autism.
Hypothesis:
Patients with autism, who have toxic metal burdens and toxic
chemical burdens are likely to grow resistant organisms in their GI
tract. This resistance to treatment is perpetuated by the production
of biofilms. Treatment of biofilms will help to eradicate dysbiotic
flora and improve the symptoms we call autism.
BIOFILM
 What is Biofilm?
 What is the implication
of biofilm production in
ASD?
 How are they formed?
 Where do they grow?
 Possible treatments being
researched?
What is Biofilm?
 A biofilm is a collection of microbial
communities enclosed by a matrix of
extracellular polymeric substance (EPS) and
separated by a network of open water
channels.
 These communities adhere to manmade and
natural surfaces, such as metals and teeth,
typically at a liquid-solid interface . Their
architecture is an optimal environment for
cell-cell interactions, including the
intercellular exchange of genetic material,
communication signals, and metabolites,
which enables diffusion of necessary nutrients
to the biofilm community.
 The matrix is composed of a negatively
charged polysaccharide substance, held
together with positively charged metal ions
(calcium, magnesium, and iron).
Staphylococcus aureus biofilm
 The matrix in which microbes in a biofilm are
embedded protects them from UV exposure,
metal toxicity, acid exposure, dehydration
salinity, phagocytosis, antibiotics,
antimicrobial agents and the immune system.
How is Biofilm formed?
5 stages of biofilm development.
 Stage 1, initial attachment; stage 2, irreversible attachment;
stage 3, maturation I; stage 4, maturation II; stage 5, dispersion.
 Each stage of development in the diagram is paired with a
photomicrograph of a developing P. aeruginosa biofilm.
Nature: May 2002
Where do they grow?
•Biofilm formation appears common near polluted
and toxic areas and environments.
Account for the majority of all microbial infections
of the human body.
•
Device-related infections,
intravenous catheters,
joint prostheses
•
•Human body –
Mouth, teeth, pancreaticbiliary tracts, lungs, sinuses,
adenoids, tonsils and
the intestinal tract….
Why are they so difficult to treat?
• Remarkably difficult to treat with antimicrobials, resistant to
doses of antimicrobials 100- to 1000-fold over the minimum
lethal dose for microbes outside of biofilms.
• Antibiotics do not penetrate the polysaccharide matrix.
• Highly resistant to both immunological and non-specific
defense mechanisms of the body.
• Difficult to diagnose, difficult to culture.
• Microbes impart genetic material to one another to maintain
resistance.
• Colonies communicate with one another through the use of
quorum sensing molecules.
• Colonies fail to express OMP (outer membrane proteins).
“Testing the Susceptibility of Bacteria in Biofilms to Antibacterial Agents” Antimicrobial Agents
and Chemo. Nov 1990.
Quorum Sensing
bacteria communicate with each other via signal molecules or autoinducers
Autoinducers drive
bacterial gene expression
and regulate
 Bioluminescence
 Virulence
 Biofilm
 Conjugation
 Motility
 Presence of Normal Bacteria
QS signals can be blocked
 Gram negative bacteria produce
lactones
 E. coli produce epinephrine
 Brominated furanones (marine
algae) are quorum sensing
inhibitors
 Gram positive bacteria produce
cyclic peptides
 RIP, a synthetic peptide,
interferes with biofilm
infections in animal models
“Cell to Cell Signaling in Intestinal Pathogens” Curr Issues Intest Microbiol,Mar 2004
“Bacteria-Host Communication: The Language of Hormones”Microbiology. May 2003
Is it possible my child has Biofilm
producing bacteria and yeast
overgrowth?
 Consider if
 Persistent /Recurrent Dysbiosis
 Recurrent Sinusitis/Otitis
 Initially does well with Antibiotics and Antifungals
 History of frequent Antibiotics
 Frequent flaring of yeast/bacteria during DETOX
 Concomitant – gingivitis, tonsillitis, dental caries
 Stool and Urine Cultures negative, but patient does well
when placed on antifungals/antibiotics
What type of biofilm control strategies are
being researched?
• Probiotics and Prebiotics
• EDTA
• Iron chelating compounds
• Enzymes - mucous degrading
• Others
Probiotics to Prevent the Need For,
and Augment the Use Of, Antibiotics
Can J Infect Dis Med Microbiol. 2006 Sep
Gregor Reid, BSc, PhD MBA
 Reduce the risk of antibiotic-induced super infections
in the gut and the vagina;
 Secrete antibacterial substances that lower pathogenic
bacterial populations locally and at distant mucosal
sites, and disrupt biofilms, making it easier for
antibiotics to function;
 Enhance generalized mucosal immunity, which in turn
aids in the eradication of the organisms at the mucosal
site.
Microbial biofilms in the human
gastrointestinal tract.
J Appl Microbiol. 2007 May;102(5):1187-96.
Macfarlane S, Dillon JF.
There is mounting interest in mucosal biofilms in the
colon, especially with respect to their role in
inflammatory bowel disease. Because bacteria growing
in biofilms are more resistant to antibiotics than
unattached organisms, it is often difficult to modify the
structure and composition of these communities, or to
eradicate them from the body. However, recent work has
shown that there is considerable potential to alter the
species composition of mucosal biofilms in a beneficial
way using synbiotics.
The Efficacy of EDTA Against Biofilm Bacteria
(Kim, 2005)
 Biofilms = complex communities of micro-organisms
attached to surfaces held together by EPS (extracellular
polysaccharides, that are negatively charged and held
together by positively charged cations, specifically Fe2+,
Ca2+, and Mg2+.
 EDTA complexes with cations in the extracellular matrix.
 Neither Vancomycin or EDTA alone detached Staph biofilm.
 EDTA plus Vancomycin together caused biomass removal.
Chelator-Induced Dispersal and Killing of
Pseudomonas aeruginosa Cells in a Biofilm
(Banin, 2005)
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EDTA is a potent Pseudomonas biofilm disrupter.
1000x killing when EDTA combined with Gentamicin.
EDTA causes dispersal and killing of biofilm cells.
Ca, Fe, and Mg protect biofilm.
When Ca or Fe are added, killing and detachment are
completely blocked.
EDTA as an Adjuct Antifungal Agent for
Invasive Pulmonary Aspergillosis in a Rodent
Model
(Hachem, 2006)
 Immunosuppressed rats infected with Aspergillus who were
treated with an antifungal drug and EDTA in combination had
less severe disease and greater survival
 EDTA acts like an antifungal –enhancing agent
 Absence of direct toxic effect of EDTA, no tissue damage
associated with EDTA
Iron Chelating Compounds
 Outer membrane proteins(OMP) are expressed when
iron is restricted.
 If OMP are not expressed, the immune system is not
alerted appropriately, and can not illicit a normal
immune response.
 Transferrin and Lactoferrin
 Synthesized by host to inhibit bacterial growth by
sequestering free Iron.
 Pathogenic bacteria secrete iron chelators (siderophores)
to compete with transferrin and lactoferrin for Iron.
A Component of Innate Immunity Prevents
Bacterial Biofilm Development.
Nature. 2002 May 30;417(6888):552-5.
Singh PK, Parsek MR, Greenberg EP, Welsh MJ.
Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from
bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent
acute invasive infections. In many chronic infections, however, bacteria live in biofilms, which are
distinct, matrix-encased communities specialized for surface persistence. The transition from a freeliving, independent existence to a biofilm lifestyle can be devastating, because biofilms notoriously
resist killing by host defence mechanisms and antibiotics. We hypothesized that the innate immune
system possesses specific activity to protect against biofilm infections.
Here we show that
lactoferrin, a ubiquitous and abundant constituent of human
external secretions, blocks biofilm development by the opportunistic
pathogen Pseudomonas aeruginosa. This occurs at lactoferrin concentrations below those that kill or
prevent growth
. By chelating iron, lactoferrin stimulates twitching,
a specialized form of surface motility, causing the bacteria to
wander across the surface instead of forming cell clusters and
biofilms. These findings reveal a specific anti-biofilm defence mechanism acting at a critical
juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate into
durable communities.
The effects of lactoferrin on P. aeruginosa biofilms after
three days.
Left image: Without lactoferrin, the cells congregate.
Right image: With lactoferrin, the cells wander freely.
©Nature
Enzymatic Degradation
Dispersing biofilms with engineered enzymatic bacteriophage.
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11197-202. Epub 2007 Jun 25.
Lu TK, Collins JJ.
Synthetic biology involves the engineering of biological organisms by using modular and generalizable
designs with the ultimate goal of developing useful solutions to real-world problems. One such problem
involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections
and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by
we engineered bacteriophage
to express a biofilm-degrading enzyme during
infection to simultaneously attack the bacterial cells
in the biofilm and the biofilm matrix, which is
composed of extracellular polymeric substances. We
show that the efficacy of biofilm removal by this twopronged enzymatic bacteriophage strategy is
significantly greater than that of nonenzymatic
bacteriophage treatment. Our engineered enzymatic phage substantially
host immune systems. To address this issue,
reduced bacterial biofilm cell counts by approximately 4.5 orders of magnitude ( approximately 99.997%
removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work
demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial
biofilms and the applicability of synthetic biology to an important medical and industrial problem.
Biofilm formation of Staphylococcus aureus strains isolated from
impetigo and furuncle: role of fibrinogen and fibrin.
J Dermatol Sci. 1997 Nov
Akiyama H, Ueda M, Kanzaki H, Tada J, Arata J.
The formation of membranous structure (thickness from the plastic tissue-culture coverslip (hematoxylineosin) > 1 mm; periodic acid-Schiff-positive) was more prominent with Staphylococcus aureus (S.
aureus) strains isolated from impetigo (coagulase types I.V origin) than with S. aureus strains isolated
from furuncle (coagulase type IV origin) (P < 0.05) in the plastic tissue-culture coverslip in human
plasma after 72 h. Attachment of S. aureus cells to a plastic tissue-culture coverslip was more marked in
0-3% fibrinogen/tryptic soy broth (TSB) than in plasma (P < 0.05). The formation of the membranous
structure was observed on the plastic tissue-culture coverslip with 0.3% fibrinogen/human serum but
not with 0.3% fibrinogen + 5% glucose/TSB. Electron
microscopy revealed abundant
fibrin around S. aureus cells at 4 h and Ruthenium red-positive materials increased at 24
and 72 h in plasma. Staphylococcus aureus cell attachment to the plastic tissue-culture coverslip in
plasma decreased by addition of levofloxacin (LVFX) at 1/2 minimum inhibitory concentration (MIC)
and clarithromycin (CAM) at 1/4 MIC. Polysaccharide production of S. aureus cells on the plastic tissueculture coverslip in plasma decreased with the addition of CAM at 1/4 MIC. Fibrinogen
is
closely related to initiation of infection but biofilm formation requires
the conversion of fibrinogen to fibrin. Thus, attachment of S. aureus cells to the plastic
tissue-culture coverslip, conversion of fibrinogen to fibrin by coagulase-prothrombin complex, and
production of abundant glycocalyx by S. aureus cells are at least required for the production of biofilm in
staphylococcal skin infection.
Proteolytic enzymes: a new treatment strategy
for prosthetic infections?
Antimicrob Agents Chemother. 1993 Dec
Selan L, Berlutti F, Passariello C, Comodi-Ballanti MR, Thaller MC.
Among the different mechanisms of bacterial resistance to
antimicrobial agents that have been studied, biofilm formation is one
of the most widespread. This mechanism is frequently the cause of
failure in the treatment of prosthetic device infections, and several
attempts have been made to develop molecules and protocols that are
able to inhibit biofilm-embedded bacteria. We present data suggesting
the possibility that proteolytic enzymes could significantly enhance the
activities of antibiotics against biofilms. Antibiotic susceptibility tests
on both planktonic and sessile cultures, studies on the dynamics of
colonization of 10 biofilm-forming isolates, and then bioluminescence
and scanning electron microscopy under seven different experimental
conditions showed that serratiopeptidase greatly enhances the activity
of ofloxacin on sessile cultures and can inhibit biofilm formation.
Enhancement of the Fibrinolytic Activity in
Plasma by Oral Administration of Nattokinase.
Acta Haematol. 1990;84(3):139-43
Sumi H, Hamada H, Nakanishi K, Hiratani H.
The existence of a potent fibrinolytic enzyme (nattokinase, NK) in the
traditional fermented food called 'natto', was reported by us previously.
It was confirmed that oral administration of NK (or natto)
produced a mild and frequent enhancement of the
fibrinolytic activity in the plasma, as indicated by the
fibrinolytic parameters, and the production of tissue
plasminogen activator. NK capsules were also administered orally
to dogs with experimentally induced thrombosis, and lysis of the
thrombi was observed by angiography. The results obtained suggest
that NK represents a possible drug for use not only in the treatment of
embolism but also in the prevention of the disease, since NK has a
proven safety and can be mass produced.
Dentists
understand
biofilm!
“Normal mouthwashes can only
clean the surface, which is why
bad breath returns quickly and
gum disease is a constant
problem. With the new patented
technology in Biotene PBF
mouthwash, you can dissolve the
biofilm, expose hidden bacteria
colonies and kill germs.
In addition, Biotene PBF contains
the proven LP3 salivary enzyme
system to strengthen the body’s
antibacterial action, dissolving
biofilm and inhibiting excessive
bacteria – maintaining a healthy
oral balance.”
Chitosans and Biofilm
Effects of chitosans with different molecular
weights on Streptococcus sanguis biofilm
Ma R, Zhu M, Liu Z.
Streptococcus sanguis biofilm was formed on saliva-coated glass (SCG) in a
flow culture system, then exposed to 2% chitosans with different molecular
weights (5 cps, 80 cps, 600 cps) for 3, 10, 30 minutes. Confocal laser scanning
microscope and Vital/Dead fluorescent staining technique (vital stained
green, dead stained red) were combined to observe the biofilm thickness,
bacterial density. Analysis of variance was used for PMID:
the biofilm thickness and bacterial density
reduced significantly after treatment with 2% chitosan.
Low molecular weight chitosan seems most effective at
detaching biofilms.
Antimicrobial effects of chitosans and
chitooligosaccharides, upon Staphylococcus aureus
and Escherichia coli, in food model systems.
RESULTS:
Food Microbiol. 2008 Oct .Fernandes JC
True Health Medical Center
Gut Biofilm Approach

Step 1 Lysis/Detachment
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Step 2 Microbial Killing
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Step 3 Clean up
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Step 4 Rebuilding/Nourishing the Gut Lining
Step 1: Lysis/Detachment
Enzyme (polysaccharidase, disaccharidase)
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Staph use fibrinolytic like lumbrokinase,
serratiopeptidase, or nattokinase
Pseudomonas or thick mucus production use
serratiopeptidase
May use pineapple or papaya for sensitive patient
Disodium EDTA (oral only) or Apple Cider Vinegar
Lactoferrin (especially for Pseudomonas)
NAG/Chitosan (especially for Strep Biofilm, do not give if
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shellfish allergy)
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Works best on an empty stomach
Start one supplement at a time, go slow
Do not give Enzymes on an empty stomach to patients
with severe GI issues
Do not give Lactoferrin to patients with dairy allergy
Avoid giving Iron, Calcium or Magnesium with
supplements above
Careful with sensitive patients or weak patients
Step 2: Killing
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Consider Natural Antimicrobials first
Do not start with Pharmaceuticals
Vary agents depending on microbiology and
mycology testing
Bacterial Cultures tend to show Gram positive
overgrowth or Gram negative
Dysbiosis may seem to worsen initially
Watch for die off, treat accordingly
Consider the use of antibiotics or antifungal meds if
organisms remain persistent
Start low and go slow
Step 3: Clean up

Fiber, insoluble/soluble

Activated Charcoal if needed
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Alginates, Brown Algae
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Modified Citrus Pectin
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Very important step
Helps prevent symptoms of die off
Step 4: Rebuilding/
Nourishing the Gut Lining
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Probiotics
Prebiotics
Fermented Foods
Healing, nutritious, non-toxic foods
Supportive Nutrients
Microbial Flora
 Bacteria are a major component of colonic material
 Hundreds of species/strains exist in the intestines
 Metabolic activity affects the host
 Digestion, Energy Production, Metabolism
 Modulation of the immune system
 Destruction of toxins and mutagens
 Repression of pathogenic microbial growth
 Preventing allergy
 Preventing inflammatory bowel disease and inflammation
 The Colon has an obligate need for bacterial fermentation products
(SCFA, short chain fatty acids)
 “the species composition and biochemical activities of the microbial
flora are determined primarily by diet and are strongly influenced by
carbohydrate availability”
“Composition and Metabolic Activities of Bacterial Biofilms Colonizing Food Residues in the Human Gut”
(Macfarlane Sept 2006)
Probiotics, Prebiotics, and Synbiotics: Approaches for
Modulating the Microbial Ecology of the Gut
M David Collins and Glenn R Gibson
American Journal of Clinical Nutrition, May 1999
 Probiotics
 A Lactobacillus acidophilus supplement given to formula-fed infants was thought to
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improve weight gain.
Oral rehydration that included a strain of L. casei promoted recovery from acute
diarrhea in children.
Oral administration of L. acidophilus has also been shown to be effective against
bacterially induced gastroenteritis .
Applications of bifidobacterial probiotics in infants have been directed toward
reducing the growth of Candida albicans and the incidence of enterocolitis.
Other organisms such as E. coli and the yeast. Saccharomyces boulardii have been
reported to have some beneficial effects in maintaining remission in IBD (Kruis,
Rembacken, Guslandi ).
 Prebiotics
are nondigestible food ingredients that beneficially affects the host
by selectively stimulating the growth of beneficial bacteria(inulin, oligosaccharides)
 Synbiotics are a combination of both prebiotics and probiotics
Probiotics – beneficial bacteria
 Bifidobacterium (breve, longum, infantis)
 Lactobacillus (acidophilus, casei, paracasei,
johnsonii, lactis, plantarum, reuteri, rhamanosus,
salivarius )
 Escherichia coli
 Enterococcus
 Streptococcus thermophilus
 D- Lactic Acid Free
Anti-oxidant rich foods
help reduce inflammation
 Inadequate antioxidant status is a major pathway for inflammation.
 Various free rdicals (ROS), including superoxide, peroxide, hydroxyl and
peroxynitrite, are generated through the inflammatory
prostaglandin/leukotriene pathways.
 These free radicals can damage or destroy virtually every cellular
biomolecule: proteins, fatty acids, phospholipids, glycoproteins, even
DNA, leading to cell injury or death.
 vitamins C and E are the two most important nutritional antioxidants.
 Vitamin C, E, alpha-lipoic acid, Co Q10 and NADH act as a team.
 One of the many ways pro-oxidants damage neurons
is to prevent the intracellular formation of glutathione.
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Biofilm Reducing Diet Strategies
Organic is best, Keep toxin exposure low
Hormone-free, antibiotic-free, pesticide free, grass-fed meat
Consider Specific Carbohydrate Diet (SCD)
Use Digestive Enzymes that break down Carbohydrates
Choose foods with high ORAC values
 ORAC corresponds to the amount of antioxidants in foods
 Fresh Vegetables and Fruits are rich in antioxidants (juicing)
 Choose foods with low AGEs(advanced glycation endproducts)
 Exogenous glycations and AGEs are typically formed when sugars are
cooked with proteins or fats at high temperatures
 High exogenous AGEs includes: donuts, barbecued meats, cake, dark
colored soda pop, and french fries
 Start fermenting foods
 Add kombucha , kefir and cultured foods
Supportive Healing Nutrients
 Xylitol, Aloe, Ribose, Omega 3 Essential Fatty Acids
 Okra, Slippery Elm, Marshmellow Root, Ginger
 N-Acetyl Glucosamine
Supports gastrointestinal function by enhancing mucosal integrity
 Provides a key building block molecule for connective tissue formation
 Better tolerated than glucosamine sulfate for those sensitive to sulfur

 Ecklonia cava
 rich in minerals, trace elements, iodine and especially mucopolysaccharides.
 Fucoidans, component of brown seaweeds, a group of sulfated polysaccharides
 powerful anti-oxidant properties protect cellular DNA from free radical damage
 Laminaran, a complex sugar with laxative and diuretic properties.
 Colostrum / Whey
 Insulin-like growth factors (IgF) which stimulates cellular growth and repair,
 Transforming Growth Factors (TgF) which promote the synthesis and repair of RNA and
DNA and the repair of muscle tissue (particularly helpful for athletes),
 Epithelial Growth Factor EfG (which stimulates normal skin growth).
Potential Reactions
 Symptoms
 Irritability, aggression, behavioral issues
 Increased stimming, hyperactivity, sleeplessness
 Skin rash, diaper rash, fever
 Possible Causes
 Side effect of supplement or allergy to med
 Yeast or Bacterial Flare-up (Balancing act)
 Detox Reaction = Too rapid of an exodus of heavy metals leading to
vitamin or mineral deficiency, oxidative stress, liver or kidney stress
 Die off = Rapid death of gut bugs, leading to excess release of toxins
such as ammonia and subsequent liver or kidney stress
 Immune/Inflammatory??? = reaction to gut bugs
Our Statistics
and Case Discussions
 Biofilm Questionnaire n=20
 Parent Comments and Observations
 Labs
Biofilm Questionnaire
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20 Random Patients
Average duration of protocol 4.6 months (1 – 12 mo)
Parents rated benefit
Overall benefit (0 – 10)
16/20 reported positive changes
4/20 reported no or little change
 2 – quit early due to negative behaviors, which resolved
after stopping protocol
 2 – no negatives, no positives
 1 – no changes in ASD symptoms, positive effect on Hg
detox
Patient Reported Benefits of
Gut-Biofilm Protocol
n = 20
10
9
8
7
6
# of
Patients
5
4
3
2
1
0
Not helpful
Mildly helpful
Moderately
helpful
Very helpful
Parent Comments and Observations
 “the biofilm protocol was able to accomplish in one year what 5
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6 yrs of GI meds were not”
“ASO titers improved after years of unsuccessful treatments”
“couldn’t sit still, stimming worsened, poor attention, OCD
increased while on protocol”
“improved spontaneous conversation, open to new foods”
“better appetite, healthier, gained weight for first time”
“initial regression followed by improved cognitive, reading,
language, processing, and conversation”
“language doubled, more aware, more defiant”
“decreased OCD and motor tics dramatically”
“developed a strep infection during the protocol”
“improved verbal scripting, less tantrums, improved social play,
better school work”
Take home pearls
 Drink clean, non-toxic water
 Eat healthy, antioxidant rich, non-toxic food
 Let go of negative toxic thoughts
 Get plenty of pure sunshine
 Remember to laugh
 Remember to play
 Take care of yourself and your gut as well
“If you heal the gut, you heal the brain”
Jane Casey
Author and mother of twins
My Kids Won’t Eat it!!
 Neither would mine
 You can do this, I did with twins!!!
 A thoughtful approach….
What Diet should I implement?
 Gluten-Free/Casein-Free??
 Gradually start taking things out of the diet. And try not
freak out.
 After 3-6 month trial consider re-introduction
 Unless your child has a severe allergy, start adding
healthy, organic, fermented dairy or whey products.
 Start with dairy fat not protein, like ghee, butter, cream,
sour cream
 Consider sprouted organic grains
What is working for me?
 Alkalizing Drink upon first waking
 i.e. lemon water, etc.
 Probiotics
 Kombucha
 Coconut Oil
 Raw Butter
 Mixed with a little Manuka honey

¾ tsp raw butter with ½ tsp honey
Thank You!!
PHONE: (630) 995-4242
FAX:
(630) 995-4243