Therapeutic poop eating

There are quite a few companies that sell human feces for fecal microbial transplantation. My thoughts are that stomach and small intestine protection of these microbes could allow for oral delivery. My other thought is that eating a yogurt cultured with a bacterium isolated from feces is one thing, eating poop from another person is only something one does when easy stuff has failed. Here are some companies selling “poop”

• The GutClub sells breast fed baby excrement for Do It Yourselfers. The Gut Club also sells tributyrin. .
• Human Microbes sells well screened human feces to doctors and clinics.
• Growing Youthful has a DIY page for using a trusted donor’s feces. Two methods include (1) inserting feces into an enteric coated capsule (2) the rectal route with a Higginson syringe.
• Open Biome has has a processing protocol: Stool from rigorously screened donors is processed into FMT treatments in a specialized, good manufacturing practice (GMP)-certified facility. Within our laboratories, HEPA filters and positively pressured airflow maintain a sterile environment. Our technicians follow a carefully validated set of standard operating procedures to ensure consistent quality production. First, stool is handled and examined for any signs of pathology under a Class II Biology Safety Cabinet that is UV-sterilized and cleaned with a sporicidal agent. Stool is then mixed with a glycerol buffer, filtered for particulates, and stored in bottles with tamper evident bands. To preserve the therapeutic potential of stool, FMT units are stored at -80 degrees Celsius and transported to clinical partners on dry ice with temperature verification. “We also offer FMT capsules for $2050 per dose.”
• FMTcapsules is ridiculous. This super donor is not even allowed to use cellphones or be around wifi.

Staley and coauthors compared the microbial diversity of freeze dried fecal samples delivered orally or through endoscopy. [1] Their basic protocol for making them came from a previous publication. [2] The basic design was to insert a size 0 capsule inside a size 00 capsule. The protocol seemed to indicate that both capsules were Hypromellose.

Disease	Preparation of inocula	Capsule materials	Dose and # of patients	Storage	Reference
rCDI	Single-donor fecal slurry was concentrated by centrifugation and resuspended (1:10) in saline with 10% glycerol added as a bacterial cryoprotectant.	Encapsulated using commercially available acid-resistant hypromellose capsules (DRcaps, Capsugel–acid resistant). Inocula added to size 0 capsules (650 µl) were closed and then secondarily sealed in size 00 capsules.	15 capsules administered on two consecutive days.	Capsules were frozen and stored at −80°C.	(Youngster et al., 2014a)
rCDI	Single-donor sample mixed in 200 cc of 0.9% normal saline and filtered using a stomacher bag to produce 180 cc of fecal slurry. The slurry was mixed with 20 cc of 100% glycerol (10% final conc.).	Encapsulated using No. 1 gelatin capsules (1889-02; Medisca) then secondarily sealed with No. 0 (2009-02; Medisca) and No. 00 (1109-02; Medisca) capsules. Gelatin capsules used were not acid resistant.	Single dose of 40 capsules (360 ml of fecal microbiota total).	Capsules were flash frozen at −55°C on dry ice and stored at −70°C.	(Kao et al., 2017)
rCDI	100 grams of stool/donor donation were processed within 4 h of passage by mixing a 1:5 dilution in 500 ml of sterile 0.85% NaCl containing glycerol followed by filtering twice through double-layered woven gauze prior to lyophilization.	Encapsulated using 00-size acid-resistant capsules.	2 doses of 100 g of fecal microbiota each. 24 hours apart.	Capsules were stored at 4°C.	(Jiang et al., 2018)
IBS	12 g of fresh donor stool was frozen with 30% glycerol prior to encapsulation.	Double encapsulated using Capsugel DRcaps size 0 and 00.	25 capsules every morning for 12 days. Each daily dose contained 12 g frozen fecal material.	Capsules were frozen at −20°C and stored at 5°C.	(Halkjær et al., 2018)
SIBO	250 ml of sterile normal saline was added to 100–150 g of fecal matter for homogenization. After the slurry was filtered, trehalose, a cryopreservant, was added for lyophilization. The final bacterial concentration was 60 mg/ml.	Encapsulated using enteric-soluble capsules of 0.9 g/grain.	16 capsules once a week for 4 weeks.	Capsules were frozen and stored at -80°C.	(Xu et al., 2021)
Obese adults with mild to moderate insulin resistance	Fecal samples were suspended in saline and sieved. The slurry was then resuspended in saline at one-tenth the volume of the initial sample with 10% glycerol for freezing.	Encapsulated using size 0 capsules (650 µl), which were closed and then secondarily sealed in size 00 capsules.	15 capsules (for two consecutive days, then 15 capsules once a week for the following 5 weeks. Each capsule contained approximately 1.6 g; frozen fecal material.	Capsules were frozen and stored at -80°C.	(Yu et al., 2020)
Obese adults without diabetes, metabolic syndrome, or steatohepatitis	FMT preparation was performed using OpenBiome’s microbiota services. Donor stool was frozen with glycerol and glycerol before being encapsulated.	Double encapsulated in size 00 capsules with a gelatin interior capsule and an acid-resistant exterior capsule.	30 capsules were given as induction dose with maintenance dose of 12 capsules at weeks 4 and 8.	Capsules were stored at -20°C.	(Allegretti et al., 2020)
Obese and/or dyslipidemic adults	Fecal samples were suspended in saline and sieved. The slurry was then resuspended in saline at one-tenth the volume of the initial sample and frozen with 10% glycerol.	Encapsulated using size 0 capsules (650 µl), which were closed and then secondarily sealed in size 00 capsules.	10 capsules containing 1 g fecal microbiota material each were administered on ten occasions over a 6-month period (100 capsules total).	Capsules were frozen and stored at -80°C.	(Rinott et al., 2021)
Obese adolescents	Each capsule contained 0.25 g of fresh fecal matter pooled from four donors. The donor stool was then mixed with 0.5 ml of a cryoprotective saline solution (0.9% NaCl, 15% glycerol) and frozen.	Double encapsulated acid-resistant DRcaps™.	28 capsules over two consecutive days. Each capsule contained 0.25 g of microbiota.	Capsules were frozen and stored at -80°C.	(Wilson et al., 2021)
HIV	FMT preparation was performed using OpenBiome’s microbiota services. Donor stool was frozen with glycerol and glycerol before encapsulated.	Double encapsulated in size 00 capsules with a gelatin interior capsule and an acid-resistant exterior capsule.	10 capsules were given in the first dose followed by 5 capsules weekly for seven weeks. (45 capsules containing 30 g of stool total was given over eight weeks.).	Capsules were stored at -20°C.	(Serrano-Villar et al., 2021)

Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment [4]

Asimple yet highly efficient method to coat gut microbes via biointerfacial supramolecular self-assembly. Coating can be performed within 15 min by simply vortexing with biocompatible lipids. Bacteria coated with an extra self-assembled lipid membrane exhibit significantly improved survival against environmental assaults and almost unchanged viability and bioactivity. This post totally skips the IBS treatment component and instead just presents some very interesting finds

Preparation of Lipid Coated Bacteria LCB.

1. .5 ml of bacterial sub-culture were washed and resuspended in 1 ml of ice cold calcium phosphate solution containing 12.5 mM of CaCl₂, DOPA (Avanti Polar Lipids, Alabaster, AL, USA) and cholesterol from a Chinese source. 
2. The DOPA and cholesterol thin film was mixed with the bacterial solution and vortexed to mix.
3. In this study the E coli seems to have been a strain of E coli resistant to two antibiotics so that the growth of other bacteria could be controlled.  These bacteria were also transfected with Green Fluorescent Protein, GFP.

These are some PubChem structures o DOPA and a lecithin. Lecithin has an amino group on the phosphate group.

Resistance assay.

• strong acid solution (0.85% NaCl, pH 2.0), Almost total killing after just 3 hours. The liquid coated bacteria grew well .  They survived 5 hours well too.
• strong alkali solution (0.85% NaCl, pH 11.0),  This treatment was much better tolerated for 1 and 4 hours.   LCB CFU were twice the untreated control at both time points
• 30% ethanol (v/v),  Both EtOH treatments resulted in killing of untreated compared to the LCB.
• 50% ethanol (v/v),
• bile salts (0.3 mg ml^-1),
• SGF supplemented with pepsin (pH 1.2), or
• SIF containing trypsin (pH 6.8) and trypsin

incubated at 37 °C with gently shaking. At predetermined time points, 50 μl of each sample was taken, washed with fresh LB medium, and spread on LB agar plates containing

These data are interesting in that they say that untreated survive for short periods of time in the simulated stomach fluid… if there is a way to accelerate gastric emptying.  The Ecoli in this study was transfected to produce green fluorescent protein.  GFP.  This fluorescence was used to track the migration of the oral gavage administered bacteria through the mice.  EcN is the special strain of E coli.  LCB is the same bacteria after lipid coating.

The actual survival in mice was also measured.

The time courses of this study reinforce the notion of taking probiotics.  Even though the vast majority of the probiotic bacteria die, some live on.

Over the course of four days, more of this lipid coated special strain of E coli survived and flourished than the unprotected GFP strain of E coli.  Note that panel 4g is log scale.  The stomach seems to be the biggest site of demise of orally delivered bacteria

Encapsulated Fecal Microbiota Transplantation: Development, Efficacy, and Clinical Application [3]

The review was key word searched for “stomach” and “gastric”  These terms were not mentioned.

Lower endoscopic delivery of freeze‑dried intestinal microbiota results in more rapid and efficient engraftment than oral administration [2]

Fecal samples were homogenized by blending under N₂ gas, sieved to remove particles > 0.25 mm, trehalose was added (5% w/w), and freeze-dried. The freeze-dried microbiota was double encapsulated in hypromellose DRcaps from Capsugel (Morristown, NJ, USA) to obtain a final concentration of approximately 1 × 10¹¹ cells per capsule.

The fraction of bacteria with intact cell membranes was used as an indicator of viability and was determined using a Live/Dead BacLight Bacterial Viability assay kit (Invitrogen/ThermoFisher Scientific, Carlsbad, CA); this fraction did not decrease post-lyophilization relative to fresh microbiota and remained > 50%.  Each course of capsules represented only one fecal donation from one of two donors (67 and 71) Capsules were stored at − 80 °C

greater engraftment was observed within the first two weeks following eFMT relative to that seen with capsule  cFMT (78.0 ±5.0% vs. 55.0± 4.3%; Tukey’s post-hoc P= 0.002; Fig. 1A), although engraftment levels were similar between both routes of administration after the first two weeks.

The stool collection protocols have not been rigorously analyzed in this post. The bottom line seems to be that endoscope delivery results in a more donor authentic microbiota.

The encapsulation protocol really did not seem to be that good compared to Liu [5] It seems hard to conclude that endoscope delivery is better when the stomach protection protocol was really not that robust in the Hamilton study. [2]

References

1. Staley C, Halaweish H, Graiziger C, Hamilton MJ, Kabage AJ, Galdys AL, Vaughn BP, Vantanasiri K, Suryanarayanan R, Sadowsky MJ, Khoruts A. Lower endoscopic delivery of freeze-dried intestinal microbiota results in more rapid and efficient engraftment than oral administration. Sci Rep. 2021 Feb 25;11(1):4519 PMC free article
2. Staley C, Hamilton MJ, Vaughn BP, Graiziger CT, Newman KM, Kabage AJ, Sadowsky MJ, Khoruts A. Successful Resolution of Recurrent Clostridium difficile Infection using Freeze-Dried, Encapsulated Fecal Microbiota; Pragmatic Cohort Study. Am J Gastroenterol. 2017 Jun;112(6):940-947. PMC free article
3. Halaweish HF, Boatman S, Staley C. (2022) Encapsulated Fecal Microbiota Transplantation: Development, Efficacy, and Clinical Application. Front Cell Infect Microbiol. 2022 Mar 17;12:826114. PMC free article
4. Cao Z, Wang X, Pang Y, Cheng S, Liu J. Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment. Nat Commun. 2019 Dec 19;10(1):5783. PMC free article
5. Lin S, Mukherjee S, Li J, Hou W, Pan C, Liu J. Mucosal immunity-mediated modulation of the gut microbiome by oral delivery of probiotics into Peyer’s patches. Sci Adv. 2021 May 12;7(20):eabf0677. PMC free article