Restoration of the Fecal Microbiome After Antimicrobial Exposure With Autologous Fecal Flora Restoration Therapy
Phase: PHASE2
Conditions: Infectious Disease of Digestive Tract
Sponsor: Washington University School of Medicine

 
 
Study Protocol 
 
Restoration of the Fecal Microbiome after Antimicrobial Exposure with Autologous Fecal Flora 
Restoration Therapy 
 
Date: December 11, 2014 
 
ClinicalTrials.gov ID: 201312022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Restoration of the Fecal Microbiome after Antimicrobial Exposure with Autologous Fecal Flora Restoration Therapy 
 
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Methods 
Hypothesis: Autologous fecal microbiota therapy (auto FMT) will be able to rapidly, and safely, restore a 
patient’s fecal microbiome after antimicrobial exposure. 
 
Specific Aims: 
1. Determine the effects five days of amoxicillin/clavulanic acid on the fecal and urinary microbiomes of 
healthy volunteers. 
2. Determine whether an infusion of fecal filtrate collected prior to the antimicrobial exposure is capable of 
rapid restoration of a healthy fecal microbiome when infused after a course of antimicrobials. 
 
Patient Population: Ten healthy volunteers will be recruited to participate through the Washington University 
Research Participant Registry (see Resources). Inclusion criteria will be healthy adults 21 to 70 years of age. 
Exclusion criteria will include a history of allergic reaction to beta-lactam antimicrobials; any non-topical 
antimicrobial exposure or tube feeds as a primary source of nutrition in the past six months; pregnant or risk of 
becoming pregnant during the study period; gastroenteritis in the last 3 months; any non-elective 
hospitalization in the previous 12 months incontinent of stool; prior resection or alteration of the stomach; small 
bowel, or colon; unwillingness to receive an enema/FMT; known colonization with an MDRO; anticipated 
change in diet or medications, or elective surgery, during the study period; positive HIV test; evidence of 
chronic medical conditions seen during physical exam;  or a history of an intestinal disorder.  
 
Study Procedure After written informed consent is obtained, a full physical exam including vital signs, βHCG 
pregnancy test in women of childbearing age, and an HIV 1/2 antibody test will be performed.  The subject will 
be provided with a sealable stool collection device. The subject will be instructed to bring back stool and urine 
specimens at their convenience within the next week, preferably within two hours after the bowel 
movement/urination. If the bowel movement has been at room temperature for greater than 6 hours, the 
subject will be asked to bring back another stool specimen. In case the subject has a bowel movement after 
hours or is unable to deliver the specimen within two hours, the subject will be instructed to contact a pre-
identified courier to pick up the urine and stool specimens. Please see below on how stool and urine will be 
processed for microbiome interrogation (stool and urine specimens) and FMT (autologous FMT). After the first 
stool specimen is collected, the subject will be provided with a five day supply of amoxicillin/clavulanic acid. 
Explicit directions on how to take amoxicillin/clavulanic acid will be provided, as well as instructions to bring the 
pill bottle back to confirm all doses were taken. A return visit will be scheduled one or two days after 
amoxicillin/clavulanic acid is to be completed. The subject will be instructed to provide stool specimens after 
Figure  Study procedure overview. 10 patients will be enrolled. Half will be randomized to FMT and the other half 
will receive saline enemas. Stool will be collected at enrollment, after the course of antimicrobials, the first bowel 
movement after FMT/saline, and days 7, 30, and 90 after FMT. The stool specimens and FMT product will be 
interrogated with DNA-based microbiome methods and semi-quantitative culture 
 
 

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amoxicillin/clavulanic acid has been completed but before FMT/saline. The subject will be able to bring the 
specimens with them to the FMT/saline visit if they are less than two hours old, or drop off/courier specimens 
prior to the appointment. The FMT/saline will be administered by enema in the outpatient setting by trained 
research personnel. The subject will be instructed to collect the first bowel movement after FMT/saline 
(immediate release of enema material will be excluded), as well as urine at that time, and also bowel 
movements at day 7 ± 2, day 30 ± 2, and day 90 ± 2. The subject will be supplied with all necessary materials 
for stool collection and drop-off at each stool collection time point. 
 
Data Management: Demographic data, including age, gender, race/ethnicity, height, weight, comorbidities, 
and tobacco and alcohol history, will be obtained on enrollment. All medications the subject received in the 6 
months prior to first stool/urine collection will be recorded.  The subject will be provided a questionnaire on 
bowel movement consistency (per Bristol stool chart) and frequency, diet in past week, and medications taken 
to be completed at time of enrollment, FMT/saline, and all subsequent stool/urine collection time points. 
 
Stool specimens: Stool specimens will be collected as cited above. Fresh stool specimens will be cultured on 
to Brucella blood, Laked blood kanamycin vancomycin (LKV), PEA, Bacteroides Bile Esculin (BBE), 
MacConkey, Hectoen Enteric, Campylobacter blood, blood, Colistin-Naladixic, and cycloserine cefoxitin 
fructose (CCFA) agars semiquantitatively to assess viability of aerobic and anaerobic gram positive and gram 
negative organisms. DNA will be extracted prior to freezing, or processing of the pre-antimicrobial specimen, of 
residual stool using the MOBIO PowerSoil kit, as performed for the NIH Human Microbiome Project at 
Washington University (60;62). Parallel sequence datasets will be generated for each of the samples at the 
Genome Institute at Washington University as previously described for the HMP (58-60). This will involve 1) 
PCR amplification of bacterial rRNA genes using degenerate primers to capture rRNA genes from a broad 
diversity of bacteria, followed by sequencing of two separate segments of the 16S rRNA gene encoding either 
the V1 through V3 (V1–V3) or V3 through V5 (V3–V5) hypervariable regions on the 454 sequencing platform. 
Of note, the 454 sequencing primers used in this current study will be identical to the primers employed for 
characterizing the microbial communities in healthy individuals at different body sites, including the 
gastrointestinal tract by the Human Microbiome Project. All sequences will be processed as in the HMP to 
remove artifactual chimeric sequences and regions of low sequence quality, and then only sequences greater 
than a certain length (usually 200 bases) will be used for further analysis. Analyses will be performed by the 
methods used for previous studies at the Genome Institute (58;60;61) and will include quantitative statistical 
techniques to compare microbial community composition (taxa that are present) and structure (abundance of 
taxa). Where differences are found, it is usually possible to specify which species are most at variance 
between samples, and thus pinpoint potentially technical problems to be addressed in the preparation and re-
establishment of fecal microbiomes. We also note that this methodology will allow us to initially classify the 
subjects as to which enterotype (63;64) their microbiome belongs to, in case this is a variable that would 
require stratification in application of the method.  
 
We will also perform shotgun sequencing on a subset of the samples (the pre-antimicrobial sample, the 
enfusate, and the final 90 day sample). Shotgun sequencing allows for detection of non-bacterial entities such 
as viruses (50) and eukaryotic microbes, as well as detection of bacterial virulence factors, antibiotic resistance 
genes, and other relevant elements. Analyses will be as performed in the references cited above for the HMP 
in order to determine if these factors pose problems for the process.  
 
 
Autologous FMT: The pre-antimicrobial stool specimen will be processed for FMT within two hours of 
collection. Approximately 150 gm of stool will be homogenized with 250ml of non-bacteriostatic saline. The 
slurry will be passed through stainless steel strainers to remove particulate matter. Sterile pharmaceutical 
grade glycerol will be added to a final concentration of 10%, and the specimen will be frozen at -80˚C (43). 
After the antimicrobials have been discontinued and the patient has provided a post-antimicrobial bowel 
movement, the enema material will be thawed over 2 to 4 hours in an ice bath for the patient undergoing 
autologous fecal flora restoration therapy. A 10 ml aliquot of the material will be saved for viability count 

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cultures and microbiome interrogation per above. The remaining material will be administered to the patient by 
enema, and the patient will be instructed to retain the material for as long as possible. A 10 ml aliquot of the 
non-bacteriostatic saline enema given to patients undergoing the sham procedure will also be collected for 
viability count cultures and microbiome interrogation as well. 
 
Analysis: As mentioned above, analyses will be performed by the methods used for previous studies at the 
Genome Institute (58;60) and will include quantitative statistical techniques to compare microbial community 
composition (taxa that are present) and structure (abundance of taxa). Most methods are performed using 
functions in the R package, and include calculation of proximity measures such as Bray-Curtis index, 
comparison of communities using hierarchical clustering, multidimensional scaling, and/or Principal 
Component/Coordinate Analysis. The Genome Institute has recently developed additional metrics for 
community comparison using a tree-based approach (65). It is usually possible to specify which species are 
most at variance between samples, and thus pinpoint potentially technical problems to be addressed in the 
preparation and re-establishment of fecal microbiomes. Enterotype (63;64) analysis will also be performed on 
the initial subjects to determine if there is any correlation between this parameter and outcome.