The authors would like to acknowledge the Infectious Disease Department at Brigham and Women&#8217;s Hospital especially Michael S. Calderwood, MD, MPH for helping us construct this protocol. We would also like to thank the Institution&#8217;s endoscopy center and it&#8217;s leadership team for their support.

NOTE: At the time of this manuscript publication an Investigational New Drug (IND) application is not required to perform an FMT for recurrent CDI in clinical practice. However, if FMTs are being performed as part of a study or for another indication, an IND or equivalent application may be required18 . Regulatory agencies will likely continue to assess the safety and efficacy of FMT as applied to CDI and other medical conditions and so it is advised to check the regulatory requirements in your respective location prior to the initiation of an FMT program and every one to two months once actively performing FMT.
NOTE Candidates must be willing to consent to the fecal transplant as well as the colonoscopy.
1. Identification of Appropriate Candidates for FMT
<ol>
	<li>Patients who have relapsing CDI or patients with refractory disease that is unresponsive to traditional antibiotics are appropriate candidates for FMT. 
	NOTE: relapsing CDI is defined as a history of three or more episodes of CDI confirmed by toxin testing or PCR, or two episodes that required hospitalization.</li>
	<li>Ensure the patient is able to undergo a colonoscopy safely. This is at the discretion of the endoscopist.</li>
</ol>
2. Stool Donor Selection
NOTE: Stool Donor selection applies to the treatment of CDI and may not be applicable to the treatment of other disorders.
<ol>
	<li>Ensure the patient identifies a donor. Potential donors should be present with the recipient at the time of the initial gastroenterology clinic visit. Donors may be spouses, family members, or friends. Donors may live in the same residence as the recipient as long as other criteria are met.</li>
</ol>
3. Donor Eligibility
<ol>
	<li>Inclusion Criteria: Ensure that the donor is a healthy adult (&#8805;18 years old) and has a daily formed bowel movement.</li>
	<li>Exclusion Criteria:
	<ol>
		<li>Ensure that the donor is not taking antibiotics or has not taken antibiotics in the prior three months or any medication for an infection.</li>
		<li>Ensure that the donor does not have a history of constipation or use laxatives chronically. Additionally, ensure the donor does not have a history of Inflammatory Bowel Disease, chronic diarrhea, microscopic colitis, a personal history of intestinal/colonic malignancy, or a history of gastric bypass/other weight loss surgery.</li>
		<li>Ensure the donor has not recently traveled (within the last three months) to an area where infectious diarrhea is a concern. If they have traveled and experienced diarrhea they should be exluded.</li>
		<li>Ensure that the donor is not taking any immunosuppressive agents and is not on systemic chemotherapy.</li>
		<li>Ensure that the donor does not have metabolic syndrome, autoimmune conditions or atopic diseases.</li>
	</ol>
	</li>
	<li>Obtain written consent from the donor and recipient prior to screening given the sensitive nature of the screening tests. Reassure both parties that test results will not be shared with the other party.</li>
</ol>
4. Screening
NOTE: This screening protocol was adapted for the patients at Brigham and Women&#8217;s Hospital in Massachusetts, U.S.A. Consider consulting the infectious disease department and hospital infection control prior to initiating the FMT protocol to make sure all appropriate screening tests have been considered.
<ol>
	<li>Recipient:
	<ol>
		<li>At the time of the initial clinic visit, send the following laboratory tests for recipient screening (to establish baseline testing for these infectious diseases and to ensure there is no question of transmission in the future).</li>
		<li>Perform Hepatitis A (IgG and IgM), Hepatitis B (HbsAg/Ab and HbcAb), Hepatitis C Ab, HIV-1/2 (Ab and viral load) and Syphilis (TP-IgG) laboratory blood tests.</li>
		<li>Obtain a thorough history of food allergies at this initial assessment.</li>
	</ol>
	</li>
	<li>Donor:
	<ol>
		<li>Coordinate with the microbiology laboratory before screening to ensure formed stool is not discarded.</li>
		<li>Order the below laboratory tests for donor screening if the donor meets eligibility criteria as per section 3.</li>
		<li>Perform Hepatitis A (IgG and IgM), Hepatitis B (HBsAg/Ab and HBcAb), Hepatitis C Ab, HIV-1/2 (Ab and viral load) and Syphilis (TP-IgG) laboratory blood tests.</li>
		<li>Perform C. difficile (by culture), routine stool culture, Giardia antigen, Cryptosporidium antigen and ova and parasites laboratory stool tests.</li>
		<li>Screen the donor no more than 30 days but preferably 1 - 2 weeks prior to the scheduled procedure. 
		NOTE: If the donor develops diarrhea or liquid stool they are not longer eligible for donation.</li>
	</ol>
	</li>
	<li>Counsel the donor on foods to avoid based on the recipient&#8217;s food allergies. The donor should not ingest foods to which the recipient is allergic for the 5 days before the donation.</li>
</ol>
5. Pre-procedure Preparation
<ol>
	<li>Recipient:
	<ol>
		<li>At the initial clinic visit, if the recipient is not already on oral vancomycin, start vancomycin 125 mg every 6 hr for at least seven to ten days prior to the procedure.</li>
		<li>Provide the recipient with the endoscopy center&#8217;s instructions for pre-colonoscopy bowel preparation to be done the day before the scheduled procedure. Recipients should also follow the endoscopy center&#8217;s standard dietary instructions for the week prior to the procedure.</li>
		<li>Instruct the recipient to take their last dose of vancomycin the night before the procedure.</li>
	</ol>
	</li>
	<li>Donor:
	<ol>
		<li>Provide the donor with several stool collection cups. Label toilet inserts (hats) and cups with the recipient&#8217;s label as well as 3 empty specimen containers. Ask the donor to begin stool collection no more than 6 hr prior to processing (time of procedure). 
		NOTE: If the donor provides multiple stool samples, they should be labeled with dates and times. Use the freshest sample (one filled specimen cup will suffice).</li>
		<li>Ensure that the recipient to brings the donor&#8217;s stool sample with them to the procedure.</li>
		<li>Store the stool samples at room temperature. Exclude any unformed stool. Discard any unused stool in a sanitary sewer system (unless saving for research purposes).</li>
	</ol>
	</li>
</ol>
6. Stool Preparation
NOTE: Ensure this procedure is discussed and approved by the hospital&#8217;s infection control department.
<ol>
	<li>Wear personal protective equipment when preparing the stool.</li>
	<li>Prepare stool in the endoscopy room in a designated work area in which there is an eighteen inch splash zone that prevents contamination of other materials.</li>
	<li>Prior to stool preparation, clean the designated area with hospital-approved disinfectant.</li>
	<li>Cover the area with a disposable pad to avoid contamination.</li>
	<li>Transfer the formed stool into a fourteen ounce single speed blender and add 500 cc of normal saline.</li>
	<li>Blend the contents for at least one minute or until it reaches a liquid slurry consistency. Be careful to avoid spillage when removing the lid of the blender.</li>
	<li>Pour the solution through the strainer into a plastic eight quart basin to eliminate any solid waste. Discard the strainer.</li>
	<li>Draw up the strained solution into 60 cc luer lock syringes (approximately eight to nine for a standard FMT by colonoscopy). Draw up an extra 60 cc of normal saline for flushes.</li>
</ol>
7. Fecal Transplant via Colonoscopy
<ol>
	<li>Wear personal protective equipment in accordance with the endoscopy unit policies while performing or assisting with the infusion.</li>
	<li>Consider light sedation, if possible, so that the patient may be able to cooperate with retaining the stool after the procedure.</li>
	<li>Perform a colonoscopy to the terminal ileum (or as far as technically feasible). On insertion of the colonscope, lavage and suction any residual liquid stool.</li>
	<li>Inspect the mucosal walls for evidence of inflammation but mucosal biopsies are not routinely performed during the FMT procedure. 
	NOTE: Determine whether or not a full colonoscopy is safe due to the degree of inflammation. If not,consider a more limited flexible sigmoidoscopy with infusion (use half of the stool slurry).</li>
	<li>Infuse 60 cc (one syringe) once in the terminal ileum via the biopsy channel of the colonoscope using the biopsy channel cap with extension tubing.</li>
	<li>Between applications of stool syringes, flush the channel with normal saline to avoid clogging.</li>
	<li>Infuse another 60 cc of stool into the cecum and then repeat in the right colon.</li>
	<li>Continue infusing 30 cc every five to ten cm while evenly distributing the slurry on all four colon wall quadrants until the mid-transverse colon is reached. Remember to flush with saline in between stool syringes. 
	NOTE: If the slurry is very viscous it may be necessary to push with considerable force on the syringe. Be careful that the biopsy channel cap does not separate from the colonoscope and cause spillage of stool contents. Frequent flushing will allow for easier passage of the slurry. If there is continued difficulty in pushing the slurry, consider re-straining the remaining donor stool for a second time or diluting further with normal saline.</li>
	<li>Aspirate excess air upon withdrawal when changing syringe barrels while being careful not to suction any liquid stool. 
	NOTE: If the patient has known fecal incontinence, the last syringe should be infused no lower than the hepatic flexure to avoid leakage during and post procedure.</li>
	<li>Once the colonoscopy with FMT is complete, advise the patient to retain the stool as long as they can. They may resume their regular diet and medications two hours after the colonoscopy is complete.</li>
</ol>
8. Colonoscopy with FMT Clean Up
<ol>
	<li>Perform post procedure equipment reprocessing, environment cleaning/disinfection and waste disposal as per hospital protocol.</li>
	<li>Dispose of all fecal material in the appropriate biohazard container.</li>
	<li>Bring the colonoscope to the decontamination area for routine processing per standard protocol.</li>
	<li>Dispose of the blender and strainer with any other soiled waste. 
	NOTE: If single use blenders and strainers are not feasible at an individual institution alternate mixing methods can be considered. One example: passing the stool through a stainless steel potato ricer that can be cleaned in an autoclave. We recommend discussing any potential methods with your facility&#8217;s infection control department.</li>
	<li>Decontaminate all surfaces that were used for the procedure using the facility-approved products.</li>
</ol>
9. Follow Up
<ol>
	<li>Arrange a follow up appointment in gastroenterology clinic for the patient approximately 30 days after the FMT.</li>
	<li>Counsel the patient about the risk of C. difficile recurrence with future courses of antibiotics.</li>
</ol>
10. Reporting of Adverse Events
<ol>
	<li>Although FMT is considered to be a safe procedure, it is strongly advised to keep track of all adverse events felt to be associated with the procedure and to report them to the appropriate regulatory boards.</li>
	<li>In order to document the long-term safety of FMT, keep a reliable database that can be periodically queried.</li>
</ol>

<ol>
	<li>Eiseman, B., Silen, W., Bascom, G. S., Kauvar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+enema+as+an+adjunct+in+the+treatment+of+pseudomembranous+enterocolitis.">Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis.</a> Surgery. 44 (5), 854-859 (1958).</li><li>Zhang, F., Luo, W., Shi, Y., Fan, Z., Ji, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+we+standardize+the+1,700-year-old+fecal+microbiota+transplantation.">Should we standardize the 1,700-year-old fecal microbiota transplantation.</a> Am J Gastroenterol. 107 (11), 1755-1710 (2012).</li><li>Borody, T. J., Paramsothy, S., Agrawal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+microbiota+transplantation:+indications,+methods,+evidence,+and+future+directions.">Fecal microbiota transplantation: indications, methods, evidence, and future directions.</a> Curr Gastroenterol Rep. 15 (8), 337 (2013).</li><li>Mattila, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+transplantation,+through+colonoscopy,+is+effective+therapy+for+recurrent+Clostridium+difficile+infection.">Fecal transplantation, through colonoscopy, is effective therapy for recurrent Clostridium difficile infection.</a> Gastroenterology. 142 (3), 490-496 (2012).</li><li>Brandt, L. J., Aroniadis, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+fecal+microbiota+transplantation:+techniques,+indications,+and+outcomes.">An overview of fecal microbiota transplantation: techniques, indications, and outcomes.</a> Gastrointest Endosc. 78 (2), 240-249 (2013).</li><li>Aas, J., Gessert, C. E., Bakken, J. 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R., de Leon, L., Jasutkar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+microbiota+transplantation+for+relapsing+Clostridium+difficile+infection+in+26+patients:+methodology+and+results.">Fecal microbiota transplantation for relapsing Clostridium difficile infection in 26 patients: methodology and results.</a> J Clin Gastroenterol. 46 (2), 145-149 (2012).</li><li>Lund-Tonnesen, S., Berstad, A., Schreiner, A., Midtvedt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile-associated+diarrhea+treated+with+homologous+feces.">Clostridium difficile-associated diarrhea treated with homologous feces.</a> Tidsskr Nor Laegeforen. 118 (7), 1027-1030 (1998).</li><li>MacConnachie, A. A., Fox, R., Kennedy, D. R., Seaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faecal+transplant+for+recurrent+Clostridium+difficile-associated+diarrhoea:+a+UK+case+series.">Faecal transplant for recurrent Clostridium difficile-associated diarrhoea: a UK case series.</a> QJM. 102 (11), 781-784 (2009).</li><li>Mellow, M. H., Kanatzar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonoscopic+fecal+bacteriotherapy+in+the+treatment+of+recurrent+Clostridium+difficile+infection--results+and+follow-up.">Colonoscopic fecal bacteriotherapy in the treatment of recurrent Clostridium difficile infection--results and follow-up.</a> J Okla State Med Assoc. 104 (3), 89-91 (2011).</li><li>Polak, P., Freibergerova, M., Jurankova, J., Kocourkova, H., Mikesova, L., Svacina, R., Husa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+experiences+with+faecal+bacteriotherapy+in+the+treatment+of+relapsing+pseudomembranous+colitis+due+to+Clostridium+difficile.">First experiences with faecal bacteriotherapy in the treatment of relapsing pseudomembranous colitis due to Clostridium difficile.</a> Klin Mikrobiol Infekc Lek. 17 (6), 214-217 (2011).</li><li>Rohlke, F., Surawicz, C. 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The authors have nothing to disclose relevant to the material presented in this manuscript.

Fecal transplants have been shown to be an effective and safe treatment for recurrent CDI. We have outlined a standard protocol for FMT via colonoscopy and included our results for 22 patients who underwent the procedure for recurrent CDI. Our results thus far are comparable to published data for recurrent disease with only one patient recurring one month post FMT. A critical step of the procedure is the prescreening visit to ensure that the donor is suitable and that the patient has no contraindications to unde...

Fecal transplants for the treatment of pseumembranous colitis were first described in 19581 . However, reports of ingesting feces for the treatment of food poisoning and severe diarrhea date back as early as fourth century China2 . Fecal microbiota transplantation (FMT) is also termed in the literature &#8220;fecal bacteriotherapy&#8221;, &#8220;human probiotic infusion&#8221;, &#8220;stool transplant&#8221;, &#8220;intestinal microbiome restoration&#8221;, and &#8220;fecal transfer&#8221;. It involves collecting stool from a healthy, pre-screened donor and delivering a prepared slurry into the gastrointestinal tract of the recipient via nasogastric tube, esophagogastricduodenoscopy, colonoscopy, or enema 3 . Many case series have reported the efficacy of administration via NGT and enema, however more recent studies have shown that administration of donor stool via a colonoscopy is safe and highly effective4 . While other methods such as nasogastric tube administration have been reported, delivery of donor stool via colonoscopy has less immediate side effects (e.g. abdominal bloating and nausea)4 and we have found that patients are more accepting of the concept of FMT when it is performed by colonoscopy. One of the therapeutic advantages of FMT via colonoscopy is the reduction in colonic biomass due to the bowel lavage that is performed prior to the procedure4.
Multiple studies have investigated the role of FMT for the treatment of colitis and diarrhea caused by the opportunistic pathogen C. difficile (CDI). The incidence of CDI continues to rise and is a major cause of morbidity and mortality with a large economic burden throughout the world. First line treatment for CDI consists of antibiotic therapy, however recurrence rates have been reported between 15-35%5 . Several case series and reports have documented the safety and efficacy of FMT for CDI refractory to standard medical treatment with antibiotics4,6-16 . A study looking at long term follow up of patients after FMT via colonoscopy for CDI reported a 91% primary cure rate in 77 patients17.
At our Center (Brigham and Women&#39;s Hospital Division of Gastroenterology, Hepatology and Endoscopy), we initiated a fecal transplant program for patients with recurrent or refractory CDI. Appropriate candidates are defined as patients who have recurrent CDI (a history of 3 or more episodes, or 2 episodes that required hospitalization), or patients with refractory disease that is unresponsive to traditional antibiotics. We believe a systematic approach to all phases of this procedure maximizes efficacy. In this manuscript and accompanying video, we detail the protocol we have employed at our Center, which includes patient and donor screening, stool preparation, and the delivery of stool at the time of colonoscopy. This method has yielded positive results comparable to the published literature.
CASE PRESENTATION:
This patient represents a typical patient referred for fecal transplantation.
The patient is a 69 year-old woman with a history of chronic lymphocytic leukemia who had relapse of disease requiring further treatment with chemotherapy. She suffered from three episodes of CDI in the past year, and was therefore referred to our clinic for consideration of FMT prior to re-initiating chemotherapy. Her first episode of CDI occurred in October 2012. She had not had any preceding antibiotics. She was very ill in the ICU after presenting with septic shock and underwent diverting loop ileostomy with antegrade vancomycin enemas for a 6 week course in combination with oral vancomycin given the severity of her illness. She did very well with resolution of her diarrhea and she was able to come off antibiotics. She developed a hernia around her stoma so it was reversed. Shortly after the ostomy reversal she again developed diarrhea and was found again to be C. difficile toxin positive. She completed another 6 week course of oral vancomycin and was able to taper off of it successfully. However several months later, she again developed diarrhea that was C. difficile toxin positive and was started on her third course of oral vancomycin. She was doing well on antibiotics when it was noted that her white count increased to &#62;100 and she has found to have a recurrence of her CLL on bone marrow biopsy. Her oncologists were concerned about initiating chemotherapy without complete resolution of her CDI and so we were asked to perform FMT. &#8206;

<table><tbody><tr><td>Blender</td><td>Hamilton Beach</td><td>51101</td><td>&lt;a href=&quot;http://www.amazon.com/Hamilton-Beach-51101-Personal-Blender/dp/B00065L6CU/ref=pd_sim_k_2?ie=UTF8&amp;amp;refRID=1Q06MXAE9XKTB4QG0AEN&quot; target=&quot;_blank&quot;&gt;Link to Amazon.com&lt;/a&gt;</td></tr><tr><td>Strainer</td><td>Winco</td><td>MS3A-8S</td><td>&lt;a href=&quot;http://www.amazon.com/Winco-MS3A-8S-Strainer-Single-Wooden/dp/B001VZ5E40/ref=sr_1_3?s=home-garden&amp;amp;ie=UTF8&amp;amp;qid=1395001295&amp;amp;sr=1-3&amp;amp;keywords=kitchen+strainer+wooden+handle&quot; target=&quot;_blank&quot;&gt;Link to Amazon.com&lt;/a&gt;</td></tr><tr><td>Disposable pads (chux), 17 x 24 in.</td><td>hospital standard</td><td></td><td>one package</td></tr><tr><td>Pink rectangular hospital standard wash basin, 8 quart</td><td>hospital standard</td><td></td><td>1 or 2</td></tr><tr><td>500 cc bottle of sterile normal saline</td><td>hospital standard</td><td></td><td>2</td></tr><tr><td>60 cc luer lock syringes</td><td>hospital standard</td><td></td><td>8 or 9</td></tr><tr><td>Biopsy channel cap with extension</td><td>hospital standard</td><td></td><td></td></tr></tbody></table>

fecal microbiota transplantation via colonoscopy for recurrent c. difficile infection

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods of FMT administration have been reported in the literature including nasogastric tube, upper endoscopy, enema and colonoscopy. FMT via colonoscopy yields excellent cure rates and is also well tolerated. We have found that patients find this an acceptable and tolerable mode of delivery. At our Center, we have initiated a fecal transplant program for patients with recurrent or refractory CDI. We have developed a protocol using an iterative process of revision and have performed 24 fecal transplants on 22 patients with success rates comparable to the current published literature. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool maximizes therapeutic success. Here we detail each step of the FMT protocol that can be carried out at any endoscopy center with a high degree of safety and success.

We have performed 24 FMTs on 22 patients at our Center (Table 1) using the above protocol. Nine patients had concurrent IBD (six with Crohn&#8217;s disease and three with ulcerative colitis). All nine had resolution of CDI. One of the patients with Crohn&#8217;s disease was status post a total colectomy for Crohn&#8217;s colitis. He initially underwent FMT via colonoscopy however after experiencing a recurrence two weeks post FMT he underwent a second FMT via upper endoscopy which resulted in CDI resolut...

Watch this Scientific Journal Video about Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection at JoVE.com

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation via colonoscopy is a safe and effective treatment for recurrent and refractory C.difficile infection. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool during the colonoscopy will maximize therapeutic success.

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods ...

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27.0K Views.  Brigham and Women‘s Hospital. Fecal Microbiota Transplantation via colonoscopy is a safe and effective treatment for recurrent and refractory C.difficile infection. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool during the colonoscopy will maximize therapeutic success.

Video: Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Cancer Research

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Immunology and Infection

Murine Ileocolic Bowel Resection with Primary Anastomosis

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn&#8217;s disease and colon cancer being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease.

Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease.

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn&#8217;s disease and colon cancer ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

Biology

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in ...

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Source: Daharsh, L. et al., <a href="https://app.jove.com/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice ...

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Summary

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Chapters in this video

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Murine Ileocolic Bowel Resection with Primary Anastomosis

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

Murine Fecal Isolation and Microbiota Transplantation

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model