Mar 30, 2014
Mar 27, 2014
Paleo - sweet potato apple hash
Ingredients
2 sweet potatoes, diced small
2 green apples, diced small
1 large yellow onion, diced small
2 Tbsp lard (or coconut oil)
1 lb. breakfast sausage (optional)
salt and pepper to taste
Instructions
In a large cast iron skillet over medium heat, melt lard.
Sautee potatoes and onions until potatoes just begin to soften.
Add sausage and apples, continue to cook until sausage has browned, breaking up larger pieces.
Add salt and pepper and serve.
http://paleoparents.com/
Mar 25, 2014
Mar 22, 2014
REVIEW+WINACTIE: COCOS LOCO / HEALTHY EATS
REVIEW+WINACTIE: COCOS LOCO / HEALTHY EATS
Bijna alles van de kokosboom kan worden gebruikt door mensen. Vooral de kokosnoten worden gebruikt voor consumptie. De mensen waarvan u hebt horen zeggen dat kokos vol verzadigde vetten zitten, hebben volkomen gelijk. Maar als ze erbij zeggen dat het daarom ongezond is, moeten we ze verbeteren. De verzadigde vetten in kokos(olie) bestaan namelijk grotendeels uit middellange vetzuurketen. Dit heeft als gevolg dat de lever de kokos(olie) erg snel kan afbreken. Dit heeft vooral een positief effect op ons metabolisme, omdat dit hierdoor versnelt wordt.
Het eten en/of drinken van kokos heeft nog meer voordelen. Zo zit het vlees van de kokosnoot bomvol vezels. Deze zijn erg belangrijk voor een gezond darmstelsel en werken als een soort ‘smeermiddel’ voor de darmen. Ook de spijsvertering heeft baat bij de vezels in kokos. In 100 gram kokosvlees zit 12 a 14 gram aan vezels. Aanbevolen wordt om dagelijks zo’n dertig gram aan vezels te eten, waarbij kokos dus prima in kan voorzien.
Kokos zitten naast deze vezels ook bomvol vitamines en mineralen. Deze zijn essentieel voor het goed functioneren van ons lichaam. Er zit o.a. veel vitamine B11 in kokos.
Kokos bevat veel anti-oxidanten
In kokos zitten veel anti-oxidanten, die ook erg gezond zijn voor ons lichaam. Anti-oxidanten beschermen ons lichaam tegen vrije radicalen, die o.a. ziektes als kanker kunnen veroorzaken. Daarnaast verhogen deze vrije radicalen de kans op hart- en vaatziektes. Het consumeren van kokos verkleint deze kans.
Boost your Health had de eer om de kokosnoten van Cocos Locos te proeven en heerlijk uit te drinken!
Tevens kregen we heerlijke gezonde mixed raw coco cookies om uit te proberen.
Deze smaken heerlijk en zijn ideaal voor een gezonde snack tussendoor, bijvoorbeeld bij de thee.
Ze hebben een stevige bite en smaken heerlijk, het vult ook goed dus je blijft langer verzadigd dan bij andere ongezonde snacks.
Deze smaken heerlijk en zijn ideaal voor een gezonde snack tussendoor, bijvoorbeeld bij de thee.
Ze hebben een stevige bite en smaken heerlijk, het vult ook goed dus je blijft langer verzadigd dan bij andere ongezonde snacks.
WINACTIE!
Jullie kunnen ook kans maken op deze heerlijke raw coco cookies in de smaken choco, lemon en mixed cookies van Healthyeats.nl!
- LIKE & SHARE de facebookpagina van Cocos Locos
- Laat een berichtje achter onderaan deze blog
*De winactie loopt t/m 31 maart 2014.
Meer informatie? http://www.cocoslocos.nl/
- See more at: http://boostyourhealth.nl/2014/reviewwinactie-cocos-locos/#pinglist-container
De website van “Boost your health” bestaat uit geweldige deals op het gebied van health- en lifestyle producten.
Daarnaast biedt “Boost your health” nog meer nieuws, blogs, recepten, recensies van nieuwe producten en boeken, winacties en geweldige evenementen voor body en mind!
De gedachte achter “Boost your health” is om een website te creĆ«ren waarbij de bezoeker alles in 1 keer terug kan vinden en daarmee de bezoeker laat genieten van alles op het gebied van health, beauty, lifestyle en events.
Enjoy!
- See more at: http://boostyourhealth.nl/2014/reviewwinactie-cocos-locos/#pinglist-container
Mar 17, 2014
Coconut Candy
Coconut Candy Ingredients
- 1 cup of coconut cream concentrate (coconut butter)
- 1 cup coconut oil
- 1/3 cup peanut/almond butter
- 1 cup peanuts/almonds, chopped
- 1 cup shredded coconut
- 1/2 cup chocolate chips ( or nibs )
- 1/3 cup rapadura sugar (optional) or a bit of stevia or xylitol
Coconut Candy Steps
- Warm the coconut cream concentrate so that it can be stirred.
- In a mixer, combine coconut cream concentrate and coconut oil until well mixed. (This step can be done by hand but the coconut cream concentrate is much easier handled in a mixer.)
- Add nut butter. Blend well.
- Add sugar (optional). Blend well.
- On a cookie sheet greased with coconut oil, sprinkle shredded coconut, nuts, and chocolate chips.
- Drizzle the coconut cream mixture over the chocolate and coconut, taking care to stir in the sugar so that it is well blended.
- When the coconut cream mixture forms a thin layer on top of the chocolate chips, nuts, and coconut, move the cookie sheet to the freezer until mixture is frozen.
- After about 30 minutes in the freezer, remove the cookie sheet and break the mixture into chips using a spatula. A thin layer of the coconut cream mixture will be very easy to break apart.
- Move the pieces into a freezer container for storage.
Serve the candy immediately from the freezer — the coconut oil will melt in your hands if kept too long.
adapted from http://www.traditional-foods.com
Mar 14, 2014
St Patty's Day - Shamrock Shake
Ingredients
- ½ Frozen Banana
- 1 cup Ice (optional)
- 1 cup Coconut Milk (or other milk of your choice raw goat milk if you can get it)
- 1 large handful of Spinach
- ½ teaspoon Vanilla Extract or vanilla bean powder
- 1-5 drops Peppermint Essential Oil to taste or (a few fresh mint leaves are nice too)
- Garnish with Coconut Whip Cream
Instructions
- Combine banana, ice, spinach, vanilla, peppermint essential oil and/or mint leaves, coconut milk throw it into your blender and blend until smooth.
- Garnish with Coconut Whipped Cream, if you really have the time to make it.
vanilla extract-
get your vanilla beans , split them in 1/2 lengthwise to expose the seeds, and your vodka, now just combine them and let them sit for 8+ weeks
use 3-4 vanilla beans per cup of vodka, the whole half-pound bag of vanilla beans will mix will 1 gallon of vodka, so liter of vodka and about 15 vanilla beans.
use 3-4 vanilla beans per cup of vodka, the whole half-pound bag of vanilla beans will mix will 1 gallon of vodka, so liter of vodka and about 15 vanilla beans.
coconut whipped cream -
1. Place the can of coconut milk in the refrigerator for at least 6 hours (or overnight). This will cause the cream to separate from the milk.
2. Open the can of coconut milk and scrape out the cream into a medium sized bowl.You can use the saved milk for smoothies and other recipes.3. Add the vanilla, cinnamon, raw honey and cardamom (optional).
4. Whip the cream with a hand mixer until fluffy. Place the bowl of whipped cream in the fridge until ready to serve.
adapted from http://www.paleocupboard.com/
and http://www.healthstartsinthekitchen.com/
Mar 11, 2014
NO NUTS Raw lemon pie, this is for you.
IF you just keep this stuff in the kitchen all the time, life will be much easier, so go ahead, shop well, enjoy the moments of reading those labels and picking the healthy stuff, then you can easily make the real feel good mind blowing body balancing delicious treats at home in less time than it takes to read all the bla bla stories of how someone came up with this.
Instead of going to the mall for clothes and other things you don't need, buy this stuff.
- 1 cup shredded coconut
- 1/3 cup buckwheat oats, you know the stuff that looks like oatmeal (not groats)
- 1/3 cup flaxmeal you can also throw in some chia seeds (you know those little poppyseed looking things with loads of omega 3 if you have a coffee grinder pulverise them into powder)
- 7 Medjool dates, or whatever dates are on sale, if they are small use a few more.
- 1 tsp. lime zest, lemon zest will also do just fine, just scrape the thing, as much as you like.
- 1/8 tsp. Celtic sea salt or a pinch will do
Filling
- 2 avocados three if they are small
- 3/4 cup lime juice, or just squeeze that lemon you scraped, or two.
- 1/4 cup maple syrup or whatever sweetener you're turned on to, just NO refined sugar please
- 1/8 cup lemon juice -can don't have to
- 1 tsp. vanilla essence, or seeds
- 1/4 tsp. Celtic sea salt
- 1/4 cup coconut butter - make it yourself, take that shaved coconut about 3 cups will do it and run it in the food processor until its like butter - done.
Coconut Icing - tastes darn good and looks nice if you want to be fancy shmancy, not a must if you are in a hurry just throw some fruit on top, or vanilla seeds or whatever.
- 1 cup coconut meat or keep 2 cans of coconut milk in the fridge over night, and scrape out that hard stuff- use it instead
- 1/3 cup maple syrup - honey works too preferably raw
- 1/4 cup coconut water or just use that left over coconut milk from the can you left in the fridge.
- 1/3 cup coconut butter -that you just made in the food processor
- 1/2 tsp. vanilla essence
- 1/8 tsp. Celtic sea salt
Decorate with something like
- lime zest
- lime wedges
This is how to do it:
Combine all the ingredients for the base together and blend. The crust should be able to hold together. If you haven't pre-soaked the nuts you will need to add a bit of water then press the base mix lightly into the bottom of whatever pie plate you are using and leave it in the fridge for 30 minutes to harden
Combine the cake filling ingredients together
Spoon the filling on top of the base
Place in freezer to set for 1 hour
Combine all ingredients of the icing together and spread evenly over the filling
Sprinkle chopped up zest and decorate with lime wedges if you want, then put it in the fridge for as long as you can stand it, without sticking your finger in it to taste.
Mar 7, 2014
HEMP - THE CLEANEST COMPLETE PROTEIN SOURCE WITH OMEGA-3!
superfood + hemp powder just
GET HEMP
Hemp - THE CLEANEST COMPLETE PROTEIN SOURCE WITH OMEGA-3!
• Suitable for All Dietary Preferences •
- vegetarian
- vegan
- paleo
- omnivore
- gluten-free
- coeliacs
- raw foodists
- pescetarian
Hemp is a miracle food for humans! It is a complete protein source containing all 8 essential amino acids required for building protein in the body, plus also contains healthy essential fatty acids. These essential fatty acids such as Omega-3 help to boost mood, immunity and the assimilation of nutrients.
superfood + hemp powder or just Hemp powder or Hemp seeds are arguably the cleanest complete protein source on the planet, easy to digest and taste great.
The tiny shelled seed of the amazing hemp plant has a pleasant
nutty flavor,
similar to sunflower seeds. The seeds are packed with nutrients -
- they are an excellent source of the essential fatty acids (EFAs),
delivering these EFAs in a balanced 3.75:1 ratio.
Hemp seed is a source of complete protein, containing all the
essential amino
acids.
-Gabriel Cousens, M.D.
"There is a Cure for Diabetes"Coconut Flour Pie Crust
Coconut Flour Pie Crust
Ingredients
- 1/2 cup Grass Fed Butter, Melted
- 2 Eggs, Preferably Local Pastured
- 1/4 Teaspoon Sea Salt
- 3/4 Cup Coconut Flour
- 1-3 Tablespoons Raw Honey
Directions
1)Preheat oven to 400 degrees. In a medium bowl beat butter, eggs, honey and salt together with a fork. Then add coconut flour. Stir until dough holds together.
1)Preheat oven to 400 degrees. In a medium bowl beat butter, eggs, honey and salt together with a fork. Then add coconut flour. Stir until dough holds together.
2)Gather the dough into a ball, then pat into a 9″ greased pie pan. Prick the dough with a fork. Bake for 9 minutes. Let cool.
Mar 5, 2014
How to make any Raw Soup
A basic raw soup, consists of any vegetables – carrots, celery, tomatoes, cucumber broccoli, you name it….It could be one or more of these.
These will give your soup its heartiness. Squash is of course a popular option for Thanksgiving. Acorn squash and spaghetti squash seem most common in raw recipes.
Next you’ll want your liquid base. This would usually be water, but could also be vegetable juice (if you have enough vegetables on hand to make carrot juice or some combination of vegetables that would be yummy juiced). Your liquid base could also be coconut water, coconut milk, or apple juice.
You may want to add an avocado and/or coconut meat for creaminess.
Now you want to add your flavorings. I think lemon juice is almost essential for a good raw soup! One lemon should do for a blender-full of soup, but you could experiment with the juice of up to two lemons. A few tablespoons of olive oil adds a nice body to your soup.
Other strong flavorings to add might be garlic, ginger, onion, green onion, honey and of course sea salt.
Put all of it in the Vitamix or other blender and blend away until smooth and creamy or slightly chunky if you prefer. You might blend half creamy first and the other half chunky for a nice chunky soup.
Or you can add finely chopped vegetables to your creamy soup.
Some nice toppings are fresh herbs like chives, dill, parsley, thyme, marjoram, and/or rosemary.
Of course you stir in some spices like curry, turmeric, italian seasonings, paprika, or any seasoning you like!
CARROT GINGER SOUP
3 carrots
CARROT GINGER SOUP
3 carrots
1 tomato
½ avocado
4 small radishes
2 T coconut aminos (or bragg's aminos, tamari)
1 t turmeric
1 t cayenne
2 inches ginger, grated
1-2 cloves garlic
1 c water
makes ~3 cups
top with chopped cilantro and fresh pepper, optional
Blend everything together until smooth. The soup can be served cold or slightly warmed on the stove.You can always double the recipe if need be.
http://cleanrawliving.blogspot.nl
Make your own Whey
Equipment:
- Fine strainer
- cheesecloth
Ingredients:
raw milk or yogurt
Preparation:If you are using yoghurt, no advance preparation is required. You may use homemade yoghurt or good quality commercial plain yoghurt. If you are using raw milk, place the milk in a clean glass container and allow it to stand at room temperature 1-4 days until it separates. For the raw milk to separate into curds and whey properly the air must be around 73 degrees Fahrenheit.
Line a large strainer set over a bowl with cheesecloth or a clean dish towel. Pour in the yogurt or separated milk, cover and let stand at room temperature for several hours (longer for yogurt). The whey will run into the bowl and the milk solids will stay in the strainer. Tie up the towel with the milk solids inside, being careful not to squeeze. Tie this little sack to a wooden spoon placed across the top of a container so that more whey can drip out. When the bag stops dripping, the cheese is ready. Store whey in a mason jar and cream cheese in a covered glass container. Refrigerated, the cream cheese keeps for about 1 month and the whey for up to 6 months.
more info:
It's
true: In an ideal world, everyone would obtain their nutrients from
traditional whole foods like tart, golden, homemade whey. However,
realizing that a significant piece of modern wellness is making informed
choices that best fit your lifestyle, the convenience of a powdered
form of whey is completely understandable. Over the past few years have
been unwaveringly committed to finding a powdered whey from happy,
healthy grass-fed cows, in its complete, non-denatured form or goat whey, this prepared whole food supplement is
incredibly rich with alkalizing minerals and electrolytes. This mineral
whey can be very helpful for the improvement digestive distress, weak
and painful joints and to replenish the mineral reserves depleted by
stress and devitalized foods. While these powders cannot by used in
place of fresh whey in cooking and baking, you can try blending them
into a superfoods smoothie for an effortless way to incorporate the
health benefits of whey into your diet.
Resources
more info:
When made properly in
small batches from cultured dairy, whey has incredibly unique healing
properties. Rich with biologically active proteins and protein
fractions, it has a high concentration of essential amino acids that are
readily used to support vital biological functions in the body. Among
these beneficial factors is:
- Lactoferrin, a multifunctional protein with iron-binding properties that acts as a powerful antimicrobial and anti-inflammatory
- Bovine serum albumin (BSA), a substance high in amino acids that has been shown to support infection-fighting white blood cells, increase antioxidant activity and maintain healthy cholesterol levels in the body
- Immunoglobulins to support disease control by bolstering immunity
- Probiotic organisms to promote optimal digestion and full nutrient absorption by balancing the gut flora
- Essential amino acids in a highly bioavailable form to act as building blocks for proteins
- Glutathione precursors, to boost production of the body's most powerful antioxidant
- Minerals such as potassium, iron and zinc in balanced amounts
- Vitamins notably vitamin B2 or riboflavin which helps the body to convert carbohydrates into fuel
2 cups yoghurt or kefir*
1 large glass bowl
1 strainer
1 thin, clean dishtowel or unbleached cheesecloth
1 wooden spoon
1 pitcher
1. Line a large strainer with an
unbleached cheesecloth or thin, clean dish towel and set strainer in
large bowl. Pour yogurt or kefir into the cloth, cover and allow to sit
out at room temperature for several hours. The liquid whey will begin to
drip into the bowl, while the milk solids will stay collected in the
cloth.
2. When the dripping slows, tie up the
cloth into a sac with the milk solids inside. Although tempting, do not
squeeze the curds to get the remaining liquid out. Instead, tie the
cloth to a wooden spoon, placing it over a pitcher so that the sac
containing the milk solids is suspended inside. Allow this to hang for
an additional several hours (I generally leave mine overnight) or until
the dripping completely stops. At that point, pour the liquid whey from
the large bowl or pitcher into a small glass jar for storage- it will
last 6 months refrigerated. The curds left in the strainer are a tasty
cream cheese rich with healthy fats, that can be used as a nutrient
dense spread or dip.
This second step ensures that you are getting all of the whey to separate from
the curds. There is no one way to do this either; get creative with
straining methods! For example, you can tie the cloth to the kitchen
faucet and place a bowl in the sink to collect the whey, or onto a knob
on your cabinets so that the whey collects into a container on the
counter. Find out what works best with your kitchen set-up.
*You can also use raw milk in this process, if first left
out to separate for about 1-4 days. I prefer to use cultured yogurt and
kefir because of their superior probiotic profile. If you use a high
quality store bought yogurt, make sure that it is free of gelatin and
pectin as these will bind the solids to the liquids, and prevent
separation of curds and whey. Although different dairy produces
different results- a rule of thumb is that 1 cup of dairy yields about
1/2 cup of whey.
Finding a Non-Denatured Whey Powder
It's
true: In an ideal world, everyone would obtain their nutrients from
traditional whole foods like tart, golden, homemade whey. However,
realizing that a significant piece of modern wellness is making informed
choices that best fit your lifestyle, the convenience of a powdered
form of whey is completely understandable. Over the past few years have
been unwaveringly committed to finding a powdered whey from happy,
healthy grass-fed cows, in its complete, non-denatured form or goat whey, this prepared whole food supplement is
incredibly rich with alkalizing minerals and electrolytes. This mineral
whey can be very helpful for the improvement digestive distress, weak
and painful joints and to replenish the mineral reserves depleted by
stress and devitalized foods. While these powders cannot by used in
place of fresh whey in cooking and baking, you can try blending them
into a superfoods smoothie for an effortless way to incorporate the
health benefits of whey into your diet.Resources
radiantlifecatalog.com/
Mar 3, 2014
Raw Walnut Fudge
Raw Walnut Fudge
Description
This recipe by Betty Rawker It can be made 2 ways: with walnut pulp flour leftover from making nut milk, or with whole, raw walnut halves. The flour version will turn out lighter, the whole walnut version will be denser and more chewy. Both are delicious! EnjoyIngredients
Choose either or:1/2 to 1 cup (approx.) Walnut Pulp (left over from making 1 batch walnut milk)*
OR 1 cup raw Walnut halves
The following ingredients stay the same:
1/2 cup raw cacao powder
1/2 cup softened/melted Expeller Pressed coconut oil
1/2 cup maple syrup
2 teaspoons vanilla
2 pinches sea salt
Optional: 1 Tablespoon cold pressed walnut oil, pumpkin seed oil, or virgin oil of your choosing (depending on what extra flavor you want to impart while adding a little extra richness)
GARNISH: 3/4 cup rough chopped raw walnuts
Methods/steps
1) If you use left over walnut pulp, you can follow the full directions here for making a fresh quart of walnut milk. (Basically soak the walnuts for 2 hours, rinse well, blend with 4 cups water, strain through a nut milk bag, and save left over walnut “pulp” fiber for making this fudge!)
2) Blend walnut pulp OR 1 cup raw walnut halves, cacao powder, softened coconut oil, maple syrup, vanilla, sea salt and walnut oil together in a food processor (basically everything except 3/4 cup walnuts used for the garnish).
3) Line a small square dish. I used a tupperwear container that was 4”x5” LINED with a parchment paper or saran wrap.
4) Pour & scoop fudge batter into square dish.
5) Top with chopped walnuts, pushing some in as you go, and add a finishing layer.
6) Freeze for an hour or more.
7) Chop into squares, ENJOY! Best stored in the freezer.
2) Blend walnut pulp OR 1 cup raw walnut halves, cacao powder, softened coconut oil, maple syrup, vanilla, sea salt and walnut oil together in a food processor (basically everything except 3/4 cup walnuts used for the garnish).
3) Line a small square dish. I used a tupperwear container that was 4”x5” LINED with a parchment paper or saran wrap.
4) Pour & scoop fudge batter into square dish.
5) Top with chopped walnuts, pushing some in as you go, and add a finishing layer.
6) Freeze for an hour or more.
7) Chop into squares, ENJOY! Best stored in the freezer.
RePOOPulate ‘super-probiotic’
Though childish songs make crude jokes, there’s nothing funny about diarrhea. Aside from the painful, twisting feeling in your guts, there’s just something psychologically upsetting about losing control of your bowels. It’s embarrassing. It’s disgusting. And we’ve all been there.
But for many, diarrhea is more than a shameful stain to be washed away in an impromptu laundry load; in the US alone, more than 500,000 suffer and 15,000 die every year from uncontrollable diarrhea caused by infection with Clostridium difficile. These rod-shaped bacteria are commonly found in the environment and even in our bodies, but have lately become a major concern in hospitals where antibiotics leave patients without the natural flora that protect their bodies. When C. difficilepopulations grow unchecked, toxins produced by the bacteria cause inflammation and cell death, leading to the explosive symptoms that, if not controlled, can lead to severe dehydration, kidney failure, holes in the intestines and death. Patients already weakened by other illnesses are particularly at risk of succumbing. Epidemics of C. difficile have become such a serious problem that the infection now rivals the superbug MRSA as one of the top emerging disease threats.
A diagram of how a stool transplant is performed.
With more and more strains of C. difficile becoming resistant to antibiotics, doctors have had to find creative ways to treat the infection. One of the most promising (if not revolting) treatments that has been tested in recent years is called fecal bacteriotherapy or ‘stool transplant’, which involves taking donor poop from a healthy patient and inserting it into the gut of an infected one as a form of probiotics, seeking to replace the protective flora. I wish I was kidding. The procedure involves collecting crap from a close relative that has been screened for other microbial pathogens, mixing it with saline or milk until it reaches the right consistency, and then placing it directly in the patient’s digestive system through a tube inserted either up the anus or down from their mouths. Though it might sound gross, the dose of healthy, helpful bacteria has shown to be very effective.
Of course, most patients aren’t exactly thrilled with the suggestion of shoving someone else’s bowel movements up their rectums or down their throats. Now, University of Guelph researchers have developed a more sanitary way of achieving the same results: synthetic poop.
The researchers created fake feces, aptly named RePOOPulate, after careful examination of bacterial colonies grown from the stool of healthy volunteers. Once the right ratio of species was determined, 33 different bacteria were grown in a robotic intestine simulator affectionately called Robo-gut to create a ‘super-probiotic’ stool substitute. According to the scientists, the bacterial mixture is much more palatable than what it mimics, and smells significantly better. Two patients treated with RePOOPulate showed marked improvement after three days, remaining C. difficile-free months after treatment. Tests of their intestinal flora showed that the fake crap successfully introduced beneficial bacteria to the patients’ guts.
It’s hard to say exactly how effective the new treatment is off of such a small test, but the results are very promising. Because scientists can control the bacterial mixture in RePOOPulate, there is less risk of introducing potentially harmful bacteria than with regular stool transplants, and the treatment can be tweaked to meet the needs of different patients. This proof-of-concept paper opens the doors for future testing. In time, RePOOPulate may prove a safe and effective treatment for C. difficile infection, as well as other gut diseases caused by the imbalance of beneficial bacteria like inflammatory bowel disease.
Abstract
Background
Fecal bacteriotherapy (‘stool transplant’) can be effective in treating recurrentClostridium difficile infection, but concerns of donor infection transmission and patient acceptance limit its use. Here we describe the use of a stool substitute preparation, made from purified intestinal bacterial cultures derived from a single healthy donor, to treat recurrent C. difficile infection that had failed repeated standard antibiotics. Thirty-three isolates were recovered from a healthy donor stool sample. Two patients who had failed at least three courses of metronidazole or vancomycin underwent colonoscopy and the mixture was infused throughout the right and mid colon. Pre-treatment and post-treatment stool samples were analyzed by 16 S rRNA gene sequencing using the Ion Torrent platform.
Results
Both patients were infected with the hyper virulent C. difficile strain, ribotype 078. Following stool substitute treatment, each patient reverted to their normal bowel pattern within 2 to 3 days and remained symptom-free at 6 months. The analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples but constituted over 25% of the sequences up to 6 months after treatment.
Conclusion
This proof-of-principle study demonstrates that a stool substitute mixture comprising a multi-species community of bacteria is capable of curing antibiotic-resistant C. difficile colitis. This benefit correlates with major changes in stool microbial profile and these changes reflect isolates from the synthetic mixture.
Trial registration
Clinical trial registration number: CinicalTrials.gov NCT01372943
Background
Clostridium difficile infection (CDI) is a bacterial disease of the gastrointestinal tract caused by C. difficile, a toxin-producing, Gram-positive, anaerobic, spore-forming bacillus. CDI accounts for 15 to 25% of antibiotic-associated diarrhea[1]. The infection occurs most commonly when patients receive antibiotics that alter their normal enteric gut bacteria, allowing overgrowth of C. difficile. Recommended therapy for CDI consists of either metronidazole or oral vancomycin [2].
Recurrent CDI is defined as complete resolution of CDI while on appropriate therapy followed by recurrence of CDI after treatment has been stopped [3]. An association has been made between recurrent disease and intestinal dysbiosis[4], and an inability of certain individuals to re-establish their normal intestinal bacteria is thought to play a leading role in recurrence. Unfortunately, few effective treatments exist for those patients with multiple recurrences of CDI, a debilitating disease. Fecal bacteriotherapy, or ‘stool transplant’ – infusing donor stool into the intestine of the recipient to re-establish normal bacterial microbiota – has shown promising results in preliminary studies [3,5] but concerns about pathogen transmission, patient acceptance and inability to standardize the treatment regimen remain. The aim of this study was to determine whether the same disease resolution could be accomplished with a stool substitute. Here we report the successful outcome of two patients with recurrent CDI unresponsive to conventional therapy who received a stool substitute, a preparation of 33 different intestinal bacteria isolated in pure culture, from a single healthy donor.
Methods
Study design
The study protocol, in accordance with Good Clinical Practice Guidelines, was approved by the Human Research Ethics Boards at Queen’s University and the University of Guelph, and meets the provisions of the Helsinki Declaration (1964, amended in 2008) of the World Medical Association.
Inclusion criteria for the study included a history of previous CDI, confirmed byC. difficile fecal toxin immunoassay, new onset of symptoms after completing a full course of medication for CDI, positive C. difficile toxin assay confirming recurrent CDI, and age 18 years or older. Patients were assessed by specialists in Infectious Disease and Gastroenterology, and other possible causes of diarrhea were ruled out. Two patients who fulfilled the inclusion criteria were enrolled in the study and written informed consent was obtained.
Microbiology
Development and preparation of RePOOPulate
The RePOOPulate human probiotic or synthetic stool mixture was developed by extensively culturing the microbial diversity from the stool of a healthy, 41-year-old female donor. Sixty-two different bacterial isolates were recovered on various media types (including Brain Heart Infusion agar, Wilkins–Chalgren agar, Reinforced Clostridial Agar, and deMan, Rogosa & Sharpe agar) using strict anaerobic conditions (to recover both strict and facultative anaerobes). Purified isolates were identified by 16S rRNA gene sequencing and were subjected to antibiotic susceptibility profiling. Susceptibility to antimicrobials was determined either by directly measuring susceptibility or through inference based on other cultivated representatives. For instance, in cases where minimum inhibitory concentration breakpoints are not documented, susceptibility was determined using Kirby–Bauer discs for select antibiotics known to have anaerobic activity; if the bacterial lawn grew up to the edge of the disc, then it was considered resistant and that isolate was not used. For isolates where there was a zone of inhibition of questionable significance, an acceptable level of inhibition was inferred based on other cultivated representatives. If there was any doubt, and the organism was at all suspected to be resistant, then it was not used in the mixture.
Thirty-three isolates, representing commensal species that were generally sensitive to a range of antimicrobials and were relatively straightforward to culture, were selected for the final stool substitute formulation. Full-length 16 S rRNA sequences were aligned with the NAST server [6], and were then classified using the GreenGenes classification server [7]. The most specific name in the GreenGenes classification was used and we report the DNA maximum likelihood score for each classification (Table 1).
Table 1. Composition of stool substitute (RePOOPulate)
To infer a relative ratio of the selected isolates for inclusion in the formulated RePOOPulate product, a comparison of our list of cultured bacterial species was made with the MetaREP metagenomic database collection of stool sample datasets from healthy donors [8]. Using the taxonomy browser, the dataset that most closely matched our profile of cultured isolates (SRS058723) was selected and used as a guide for inference of relative abundance of each species – with the exception that Bifidobacterium spp. were added to higher abundances, reflecting the widely observed underestimated abundances of Actinobacteria, and specifically this genus, in metagenomic analyses of human stool [9,10]. An approximate ratio based on cultured cell biomass, measured using standard 10 Ī¼l microbiological loops, was generated (Table 1).
Each of the 33 isolates was individually cultured on Fastidious Anaerobe Agar (Lab M Ltd. Heywood, Lancashire, UK) under anaerobic conditions, and then cultures were formulated into the predetermined ratio, as described above, in 100 ml pre-reduced sterile 0.9% normal saline to an estimated concentration of 3.5 × 109 colony-forming units/ml. The bacterial suspension was placed in a reduced atmosphere in a double-sealed container at 4°C, and used within 24 hours of preparation.
Isolation and ribotyping of C. difficile from patient stool samples
C. difficile was isolated from stool samples according to methods described previously [11], using selective media of moxalactam norfloxacin broth (CDMN; Oxoid, Nepean, Ontario, Canada) enriched with 0.1% sodium taurocholate. Isolates were typed using the PCR ribotyping method described by Bidet and colleagues [12].
Administration of stool substitute
Antibiotic therapy was withheld for 2 days and the patients underwent standard colon cleansing the evening prior to colonoscopy. The following morning during colonoscopy, one-half (50 ml) of the solution was deposited in the region of the cecum/proximal ascending colon and the other half was drizzled throughout the transverse colon as the colonoscope was withdrawn. Both patients were noted to have significant diverticular disease. Immediately post procedure, patients were maintained in the Trendelenburg position for 60 minutes before being discharged home. Patients were instructed to eat a fiber-rich diet and not to consume products containing probiotics. Patients were followed by a study nurse to obtain stool samples and closely monitor their clinical response.
Sequence analysis
gDNA extraction from stool samples
gDNA was extracted using a protocol involving a combination of bead beating, the E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek, Norcross, Georgia, USA) and the Maxwell® 16 DNA Purification Kit (Promega, Madison, Wisconsin, USA). Briefly, 200 Ī¼l stool sample, 300 Ī¼l E.Z.N.A. Kit SLX buffer, 10 Ī¼l of 20 mg/ml proteinase K (in 0.1 mM CaCl2) and 200 mg glass beads were added to a screw-capped Eppendorf tube and disrupted in a bead beater for 3 minutes. Following subsequent incubation at 70°C for 10 minutes and at 95°C for 2 minutes, 100 Ī¼l E.Z.N.A. Kit Buffer P2 was added to each sample and incubated on ice for 5 minutes. Samples were then centrifuged at 14,000 × g for 5 minutes, and the supernatant transferred into new tubes, each containing 200 Ī¼l E.Z.N.A. Kit HTR reagent. Following thorough mixing, samples were incubated at room temperature for 2 minutes and centrifuged at 14,000 × g, and the supernatant was transferred into Maxwell® 16 DNA Purification Kit cartridges. The remainder of the DNA extraction protocol was carried out in the Maxwell® 16 Instrument according to the manufacturer’s instructions (Promega).
V6 rRNA amplification
PCR amplification of the bacterial V6 rRNA region was carried out with the left-side primer CWACGCGARGAACCTTACC and the right-side primer ACRACACGAGCTGACGAC. These primer sequences were chosen because they are exact matches to >95% of the rRNA sequences from organisms identified in the human microbiome project (GBG, unpublished observations). In addition the left-side primers contained the standard Ion Torrent (Ion Torrent Systems Inc., Guilford, Connecticut, USA) adapter and key sequence at their 5′ end (CCATCTCATCCCTGCGTGTCTCCGACTCAG). One of the following 5-mer barcodes was located between the 3′ end of the key sequence and the 5′ end of the primer: TATCG, TAGAC, TGCAT, ATGAG, ACAGT, AGATG, CTCAC, CTGTA, CGTGA, CGACT, AACTC, or CCTAT. Duplicate samples did not use the same barcodes. The right-side primer had the other standard Ion Torrent adapter sequence (CCTCTCTATGGGCAGTCGGTGAT) attached to its 5′ end. Amplification was performed for 25 cycles in 40 Ī¼l using the colorless GO-Taq hot start master mix (Promega) according to the manufacturer's instructions with the following three-step temperature profile: 95°C, 55°C and 72°C for 1 minute each step, then 5 Ī¼l of the resulting amplification were quantified using the QuBit broad-range double-stranded DNA fluorometric quantitation reagent (InVitroGen, Life technologies Inc., Burlington, Ontario, Canada). Samples were pooled at approximately equal concentrations and purified using a Wizard PCR Clean-Up Kit (Promega).
Sequencing
Sequence reactions were carried out on the Ion Torrent 314 and 316 chip platform. Up to 12 samples were multiplexed on each chip through use of individual sequence tags. Data from all runs were pooled when samples were run on more than one chip.
Sequence data processing
Five sequencing reactions were carried out on the Ion Torrent platform: three reactions on a 314 chip and two reactions on the 316 chip. The chips differ only in the density of the spots, and hence in the amount of sequence that can be obtained (the 316 chip is about five to six times as dense as the 314 chip). The sequence was provided in fastq format. All sequences were then filtered according to the following criteria: exact matches to the barcodes used, exact match to the left-side primer including redundant positions in the primer, an exact match to the first six nucleotides of the right-side primer, and a length between the left-side and right-side primer of between 71 and 83 nucleotides. This length was chosen because it encompasses the predicted amplicon product size from all human-associated bacterial organisms that have been cultured and sequenced as part of the human microbiome project.
Approximately 40 to 50% of the reads passed these filters in the most recent Ion Torrent runs; reads not passing the filters were not examined further. Reads were processed as described by Gloor and colleagues [13] except that clustering with USEARCH was performed at 97% identity. Chimera detection was performed with UCHIME (version v5.2.32) using the de novo method [14]. Only four chimeric sequences were observed out of 30,419 unique sequences in the merged dataset, and all were rare. This frequency is similar to that reported previously for amplification and sequencing of the V6 rRNA region using the Illumina platform [13]. Chimeric sequences were not considered an issue in this dataset.
A table of counts for sequences grouped at the 97% operational taxonomic unit (OTU) and 100% identical sequence unit identity level were generated for each sample as before [13], keeping all identical sequence unit or OTU sequences that were represented in any sample at a frequency >0.5%. Reads that were never abundant in any sample (<0.5%) were grouped into the remainder and discarded. Between 12.6 and 51.9% (median 31%) of the identical sequence unit reads and between 1.4 and 17.2% (median 5.8%) of the OTU reads were in the remainder group. These values are approximately five times greater than those observed for identical sequence units sequenced on the Illumina platform but are about equivalent to the Illumina platform observations when reads were clustered.
Taxonomic classification
Classification of the sequences by either the GreenGenes or RDP classifiers proved to be unreliable because of the short length of the V6 region. Classification of the sequences present in the count table was therefore performed using the RDP closest match option on the full-length, high-quality, isolated subset. The maximum number of best hits was identified, and the taxonomic classification of the best match and ties was collected. The classification of those hits was adopted for all levels where the classification was identical across all best matches, otherwise the classification was marked as undefined. The V6 region is not able to resolve the genus or species level of a number of clades, so all analyses were carried out at the family level. This strategy worked for all abundant families – with the exception of theBifidobacterium, which were annotated as such from BLAST searches of the NCBI microbial 16 S rRNA database. The taxonomic classification was added to the sequence count table and the data were presented in formats that could be accepted by QIIME 1.5.0 [15] as follows. Sequence alignments were built using Muscle [16] and a neighbor-joining tree was generated by ClustalW2 [17]. Beta-diversity was calculated by the UniFrac algorithm [18]. Tables were imported into MacQIIME, which is an OS X bundled version of QIIME 1.5.0, and were analyzed using the default parameters.
Results
Clinical outcomes
Patient 1
Patient 1 was a 74-year-old Caucasian woman who presented with six episodes of recurrent CDI (confirmed by C. difficile toxin assay) over an 18-month period, all of which required hospitalization. The first episode of CDI developed after elective orthopedic surgery (knee arthroplasty), when the patient received preoperative cefazolin (Figure 1A). After treatment with the stool substitute, she reverted to her normal bowel pattern of a formed stool every 1 or 2 days. No C. difficile was detectable by C. difficile toxin assay at 10 days post procedure. Patient 1 did receive several courses of antibiotics for recurrent urinary tract infections in the subsequent weeks following her stool substitute treatment, but her diarrhea did not recur. She remained symptom-free at the last evaluation, 24 weeks after treatment. Two different strains of C. difficilewere isolated from the pre-treatment sample. One strain was identified as ribotype 078; the other was a less common toxinotype 0 ribotype (data not shown).
Figure 1. Clinical timeline of events for Patients 1 and 2. Sequence of events for the first two patients enrolled in the study. (A) Patient 1 had Clostridium difficile initially occurring after a pre-operative course of cefazolin for elective total knee arthroplasty. (B) Patient 2 had C. difficile initially occurring after a course of cefazolin for cellulitis. Both patients had multiple courses of antibiotic treatment for theC. difficile infection with both vancomycin and metronidazole prior to enrollment, as indicated. In addition, Patient 1 received the probioticSaccharomyces boulardii. Prior to treatment with the stool substitute preparation RePOOPulate (RP), stool collection on each patient was carried out at 2 days pre treatment (PT), day 2 post treatment (D2), week 2 post treatment (W2), week 4 post treatment (W4), and 6 months post treatment (6 M). Toxin assays for C. difficile were also performed (purple boxes), with results as shown. Incidental antibiotic use post treatment is indicated. AMX, amoxicillin; CFZ, cefazolin; CIP, ciprofloxacin; CLI, clindamycin; CRO, ceftriaxone; LEX, cephalexin; MTZ, metronidazole; NIT, nitrofurantoin; SXT, trimethoprim-sulfamethoxazole; VAN, vancomycinPatient 2
Patient 2 was a 70-year-old Caucasian woman with a history of peripheral neuropathy, which predisposed her to recurrent skin and soft tissue infections. She developed her initial CDI after receiving cefazolin for cellulitis and presented to the clinic with a history of three episodes of recurrent CDI, the last of which had failed standard medical therapy (Figure 1B). After receiving the study treatment, she reported formed bowel movements within 72 hours. She remained symptom-free for 3 weeks, then developed recurrent cellulitis and was placed on ceftriaxone by her physician. She was monitored closely while on ceftriaxone but did not develop loose stool or diarrhea. She suffered from several skin and soft tissue infections in the subsequent weeks and received several additional courses of broad-spectrum antibiotics for these infections. Nevertheless, she remained symptom-free with no diarrhea at last evaluation, which was 26 weeks post procedure. Ribotype 078 was also isolated from this patient’s pre-treatment sample.
Sequence analysis
Reproducibility of the data
The Ion Torrent instrument has not previously been used for community microbial composition analysis with amplified rRNA variable regions. We therefore first examined the reproducibility of the reads obtained on the instrument by performing three separate PCR amplifications of the V6 rRNA region and sequencing these amplifications on four separate Ion Torrent runs. The PCR reactions were amplified by two separate individuals on separate days. A separate library was prepared from each amplification. Each library was run on either a 314 or a 316 Ion Torrent chip, with one library run on two separate chips. In this way the technical replication both of the amplification and of the sequencing reaction could be assessed.
The number of reads obtained for these sequencing reactions was often small – especially for the initial run on the 314 chip, which has limited capacity – and is summarized in Table 2. Reads were processed by the standard pipeline outlined in Methods, and an unweighted pair group method with arithmetic mean distance tree was generated from the beta-diversity output by QIIME. The result (shown in Figure 2) demonstrates that all the amplifications from each of the four replicates clustered together by sample – with the exception of one of the Patient 1 day 2 and week 2 samples, which showed clustering together by the amplification. Note, however, the very short branch lengths in this clade, indicating that these samples are probably indistinguishable. All further analyses used pooled reads across all replicates for each sample.
Table 2. Read numbers for sequencing reactions obtained on the Ion Torrent platform
Figure 2. Distance tree of weighted UniFrac distances between samples for Patient 1 amplified and sequenced independently. Distance tree calculated by the unweighted pair group method with arithmetic mean. Branch tips are colored by sample: red, pre-treatment; blue, RePOOPulate formulation. Post-treatment samples are colored green (D2), cyan (W2), and purple (W4). Tip label fields are separated by an underscore character and the fields are: Ion Torrent run ID, person and time of amplification, sample identifier, barcode sequenceExamination of alpha-diversity
In total, there were between 3,758 and 76,752 V6 rRNA reads per sample for Patient 1 and between 19,751 and 64,200 reads per sample for Patient 2 using the Ion Torrent instrument as outlined in Methods. These reads were processed by a combination of custom scripts and the QIIME pipeline as described in Methods. Reads were clustered at 97% sequence identity for the analysis that follows, unless stated otherwise. Read counts were normalized using rarefaction to the minimum number of reads per sample in each patient, and Shannon's diversity index was plotted for each intermediate rarefaction level and for the nonrarefied data. We observed that the mean Shannon's diversity index of 10 rarefaction samples approximated the diversity index of the total dataset when the number of rarefied samples exceeded 1,000 (data not shown). This observation indicates that we obtained sufficient reads in all samples to accurately estimate the diversity. Shannon's diversity on the total dataset for all samples is given in Table 3, from which we see that the two patients had dramatically different Shannon's diversity scores before and after treatment. Patient 1 had a highly diverse microbiota that became less diverse after treatment, and over time tended to become more diverse. At 6 months post treatment, this patient had a diversity score that was almost the same as that at pre treatment. Patient 2 initially had a low diversity microbiota, which became more diverse following treatment and stabilized over the long term at a level that was more diverse than that at pre treatment.
Table 3. Shannon diversity values calculated for nonrarefied count values
Examination of beta-diversity
Taxonomic assignments of the seed sequences for each OTU were derived from best-hit analysis of the sequences in the RDP database as explained in Methods. Briefly, the full taxonomic lineage of the 20 best hits and ties was captured using a custom Perl script and added to the QIIME input tables. Any lineage where the best hits and ties were not in full agreement was annotated as undefined. Taxonomic assignment was carried out to the family level since the rRNA V6 region has poor resolution below this taxonomic level for several groups found in our dataset, such as the Gammaproteobacteria and Lachnospiraceae families. Beta-diversity taxonomic bi-plots at the family level were generated using the QIIME package with default values for the read counts of the samples derived from each individual patient including the initial RePOOPulate sample (Figure 3). In both patients, the first three principle components captured over 85% of the variation between the samples.
Figure 3. Principle component coordinates of patient time points and most abundant sequences clustered at family level. Weighted UniFrac principle coordinates were generated by QIIME for each patient independently. These time points are denoted PT for pre treatment, RP for the RePOOPulate formula, and as the day (D), week (W) or month (M) time point post treatment. The weighted mean abundance of family-level taxonomic groups is indicated by the size and position of the open circles. For example, for Patient 1 Bacteriodaceae are abundant in the day 2 and week 2 post-treatment samples, less abundant in the week 4 post-treatment sample, and are rare in all other samples. Only the 10 most abundant groupings of organisms are shown, and these differ between the two patients, although the Lachnospiraceae family is abundant in both
The taxonomic distribution of reads in the two patients was noticeably different, however, as shown in the barplots of Figure 4 – as was the trajectory of the microbiome composition after treatment. The microbiota of Patient 1 initially had a number of distinct families from the Firmicutes phylum. Samples collected at day 2 and at week 2 were largely composed of families from the Bacteriodetes. However, samples collected after 4 weeks were composed of similar fractions of families in these two phyla. After 6 months this patient had a microbiota that was largely composed of Firmicutes. In contrast, the microbiota of Patient 2 was largely composed of Proteobacteria before treatment, and was noticeably lacking in Actinobacteria and Bacteriodetes phyla. The fraction of Proteobacteria declined rapidly after treatment, initially displaced by families from the Bacteriodetes and Actinobacteria. At later time points (2 to 4 weeks post treatment) there was a reduction of Actinobacteria and an increase in Bacteriodetes and Firmicutes. The Proteobacteria were displaced completely by 2 weeks. After 6 months this patient’s microbiota was composed largely of families drawn from the Firmicutes and of Verrucomicrobia phyla.
Figure 4. Barplot of abundance at the family level.Operational taxonomic units (OTUs) that comprised more than 0.5% of the OTUs in any sample were grouped into the appropriate family and plotted. These plots show how the actual composition of each sample changes over time. Note that the two patients had very different initial microbiota compositions. The compositional differences were maintained at all time points, suggesting that environmental or genetic factors were important in shaping community structureLong-term colonization
We were interested in determining the ability of the organisms composing the RePOOPulate formulation to stably colonize the distal colon of the patients. We noted that the weighted UniFrac distances between the samples at pre treatment and 6 months post treatment in Patient 1 were less than those between either sample and any other. In contrast, the earliest time point for Patient 2 was most similar to the pre-treatment sample. These relationships can be seen in Figures 3 and 4, but are clearer in Figure S1 in Additional file 1. However, there was one common feature between the microbiota trajectories of the two patients. Figure 5 shows plots of the weighted fraction of sequences that were identical to the sequences found in the RePOOPulate sample clustered at 97% and 100% identity. Interestingly, even though the initial taxonomic distribution of the patients was very different (Figures 3 and 4), the initial fraction of reads identical to the RePOOPulate reads was <7% when clustered at 100% identity, and was between 7 and 9.5% when clustered at 97% identity. Not surprisingly, the fraction of reads identical to those derived from the RePOOPulate sample increased rapidly after treatment such that reads identical to the RePOOPulate reads 2 days to 2 weeks after treatment composed >70% of the total reads in Patient 1 (day 2 post treatment) and 50% in Patient 2 (week 2 post treatment). The fraction of the patient microbiota that was composed of reads identical to those found in RePOOPulate declined continuously from the sample 2 weeks post treatment onwards, and at 6 months these reads were found to compose between 25 and 36% of the total reads obtained from each patient. This emergent pattern of slow loss of reads identical to reads in the RePOOPulate sample was common to both patients.
Additional file 1. Figure S1. Unweighted pair group method with arithmetic mean distance tree of the weighted UniFrac distances between samples for Patients 1 and 2. The branch tips are labeled with the sample names for each patient. The scale bar is shown for each patient.
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Figure 5. Weighted abundance overlap at the identical sequence unit and 97%-clustered operational taxonomic unit levels. Proportion of sequence counts that correspond exactly to those in the RePOOPulate (RP) formulation and found in each patient sample as a function of time post treatment. Red, RP formulation; dark blue, samples from Patient 1; cyan, samples from Patient 2. There is an initial increase in reads identical to the RP reads immediately after treatment, and a steady decline in proportion for each patient with time since treatment. Both patients had similar RP-identical reads at 6 months post treatment, even though their microbiota profiles were differentDiscussion
This pilot study shows that a synthetic stool (stool substitute) may be an effective and feasible alternative to the use of defecated donor fecal matter (stool transplant) in the treatment of recurrent CDI. Given the marked differences between the two patients in the study, it is difficult to draw conclusions that might be broadly applicable to a larger patient population at this time and clearly more patients are needed. Nevertheless, the clinical cure achieved at 6 months of follow-up demonstrates feasibility of this approach as an alternative to conventional stool transplant. A synthetic stool substitute approach has multiple advantages: the exact composition of bacteria administered is known and can be controlled; the bacterial species composition can be reproduced, should a future treatment be necessary; preparations of pure culture are more stable than stool, which some groups recommend should be collected fresh and instilled into the recipient within 6 hours of collection[19]; an absence of viruses and other pathogens in the administered mixture can be ensured, thereby improving patient safety; and the administered organisms can be selected based on their sensitivity to antimicrobials, allowing an enhanced safety profile.
Recurrent CDI is thought to be largely due to the inability of the normal intestinal microflora to recover and re-establish itself [4,20,21]. We used the Ion Torrent platform to analyze the 16 S rRNA gene profiles of stool samples collected from each patient during the study, and carried out exhaustive quality control of our data. We concluded that this sequencing platform, together with the PCR amplification protocol and bioinformatic analysis pipeline, was adequate to reproducibly separate both technical replicate samples (Figure 2).
Our study showed that the microbiota of both patients adapted characteristics of the stool substitute mixture yet still retained some of their original microbiota, similar to that described for stool transplants [20]. However, our data suggest that decreased diversity as a risk factor for recurrent CDI may be less important than the actual organisms present in the mixture per se, since Patient 1 actually had a very diverse microbiome at the outset but still suffered from severe recurrent CDI. Sequences identical to those of the stool substitute bacteria were initially rare in the pre-treatment samples for both patients (<7%), but became transiently abundant and constituted over 25% of the sequences up to 6 months after stool substitute treatment was given. Hence, we conclude that some of the administered bacteria are stably colonizing the colon, an important observation since most commercially available probiotics only transiently colonize the intestine. These data along with the very different set of RePOOPulate organisms that were initially abundant suggest that other factors, such as diet, may play an active role in influencing the microbial communities over time. These data suggest that a multi-species derivative community such as that used here will be more generally useful than a single organism probiotic or a mixed culture of such probiotic species, because the microbes in RePOOPulate are derived from a community and probably retain some community structure that enables them to colonize the appropriate environment. In addition, the data also suggest that the relative proportions of different bacterial strains in the formulation are of only minor importance. Experiments to determine the extent of the influence of therapeutic community composition on the resulting community profile in the host are currently underway. Longer term experiments colonizing animal models with the stool substitute formulations will clarify the extent to which organisms introduced as part of a gut microbial ecosystem are perturbed by antibiotic use, and the extent to which they persist over longer time frames.
The question of whether age-matching of donors and recipients of stool would be of relevance, since the isolates used to formulate our stool substitute were derived from a younger individual, is largely a matter of debate. Many stool transplants have been performed successfully in older patients using young donors that are often related to the patient [22,23]. The treatment seems equally effective when the young donors are not related [19,23]. We therefore suspect that age-matching will not turn out to be a critical factor in treatment success, although this is a question that should be addressed by careful meta-analysis of clinical trial data. Similarly, the relevance of patient enterotype [24], which may turn out to be more important than age-matching, has not been fully explored. Experiments are currently underway in our laboratories to develop further therapeutic synthetic stool mixtures to represent the different enterotypes known to exist in humans.
Both patients were infected with ribotype 078, an emerging hypervirulent strain that has been associated with community-associated CDI, as well as with livestock (pigs, cattle, poultry), and food products [25]. Like NAP1/027, its prevalence seems to be increasing in many countries [25]. Whether ribotype 078 will be more commonly found in patients with recurrent CDI, whether it is an emerging pathogen in this region or whether its presence in these two non-epidemiologically related patients was a chance finding remains to be seen. Regardless, the stool substitute preparation used here was effective at eradicating disease that had failed all other treatment regimens. In addition, this study also suggests that a defined microbial community, isolated from a single healthy donor, may be robust enough to withstand further perturbations by antibiotics as illustrated by the patients in our study. In the case of Patient 1, who suffered from occasional urinary tract infections, the antibiotics used post procedure (ciprofloxacin, nitrofurantoin and amoxicillin) were for short courses only, up to a maximum of 7 days. For Patient 2, her recurrent skin and soft tissue infections occasionally necessitated a broad-spectrum antibiotic combination (for example, cephalexin and metronidazole) of much longer duration (4 weeks in one case). Despite post-procedure administration of these incidental antibiotics for infections unrelated to C. difficile colitis, neither patient developed further recurrent CDI. However, at this time it remains unclear whether antibiotic administration affected the long-term colonization by the microbial community used as treatment, or to what extent the differences in microbial profile in the 6-month samples between patients is driven by the different antibiotics administered. More controlled studies in animal models may help to address some of these questions.
Conclusion
Further research is needed to define the mechanisms and precise role for this stool substitute in treating CDI. However, once a patient fails oral vancomycin therapy, treatment options for recurrent CDI are very limited. Ecosystem therapeutics or repopulating the bowel with defined communities of normal intestinal bacteria offers another effective therapy for treating recurrent CDI.
Abbreviations
CDI: Clostridium difficile infection; gDNA: genomic DNA; OTU: Operational taxonomic unit; PCR: Polymerase chain reaction; QIIME: Quantitative Insights Into Microbial Ecology; V6 rRNA: Variable region 6 of the 16S ribosomal RNA gene.
Competing interests
EOP, EA-V and SJV have filed a provisional patent together. The remaining authors declare that they have no competing interests.
Authors' contributions
EOP and GBG contributed equally to this work. EOP and EA-V conceived of the study, developed its design and coordination and drafted the manuscript. DC carried out the sequencing work. GBG carried out the bioinformatic analyses and helped to draft the manuscript. SJV participated in the clinical design and helped to draft the manuscript. JSW cultured C. difficile isolates from patients and determined their ribotypes. MCD, EMB and KS isolated, characterized and cultured the stool substitute components, and prepared gDNA for sequencing from patient samples. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the Southeastern Ontario Academic Medical Association and the Physicians’ Services Incorporated Foundation (to EOP, EA-V, SJV), as well as a University of Western Ontario Academic Development Fund grant (to GBG). Part of this work was presented at a Canadian Institutes of Health Research-sponsored meeting in October 2011 in Toronto, Ontario, and at the Canadian Digestive Diseases Week in February 2012 in Montreal, Quebec.
The authors would like to acknowledge the following people who contributed to the study:
clinical investigators (collected data, provided and cared for study patients) – Lawrence Hookey, Cathy Lowe, Mark Ropeleski, Gerald Evans, Christopher A Smith, Robert Bechara; laboratory and technical assistance (processed samples and collected data) – Curtis Noordhof, Adriana Breen, Karen Solomon, Christian Ambrose; and bioinformatics assistance (helped with QIIME analysis and barplot production) – Jean Macklaim.
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Additional Tips
**NOTE: You can use raw virgin coconut oil, OR if you are sharing this raw super fudge with someone who does not care for coconut flavors, you can use expeller pressed coconut oil which is virtually tasteless. Just make sure to use a brand like this one that does not use solvents in the extraction process.