Q1A: What is a fecal transplant (also called a fecal microbiome transplant, or a poop transplant)? Fecal transplant is to obtain fecal, or poop, sample, mix it with saline, and place it in patients. This treatment is used to treat clostridium difficile infection which is caused invasion of an anaerobic, gram-negative, sporeforming bacteria. This invasion is due to the treatment of antibiotics. When a patient acquires antibiotics, all the good bacteria is suppressed, and the purpose of fecal transplant is to replace the bad bacteria with good bacteria (The Fecal Transplant Foundation).
Q1B: Find three recent scientific studies (published in 2013 or later) that provide evidence for or against fecal transplant as an effective therapy for C. difficile infection in humans. I strongly recommend using Pubmed to answer this question. For each publication, the required components of your answer are the following: (10 points per publication) • Full publication citation (Title, authors, date, journal) • Number of patients treated with fecal transplant • Source of fecal matter used for transplant
• A 4-5 sentence summary of the findings of the research and its significance. Focus on the big picture. Kelly C. R. , Khoruts A. , Staley C. , Sadowsky M. J. , Abd M. , Bakow B. , Curran P. , McKenney J. , Tisch A. , Reinert S. E. , Machan J. T. , Brandt L… “Effect of Fecal Microbiota Transplantation on Recurrence in Multiply Recurrent Clostridium difficile Infection: A Randomized Trial. ” Annals of Internal Medicine. 2016 Nov 1; 165(9): 609-616. For this study, there was 46 patients with 3 reoccurrence of C. difficile infection after they have been administered vancomycin for their previous illness. The sources of fecal matter used for fecal transplant were stool from healthy donor and the patients’ own tool. The goal of this study was to determine if the patients remain symptom-free without the need to do additional fecal transplantation. As a result, patients with donor stool as their treatment acquired a higher percentage at curing C. difficile than patients with their own stool as the treatment. Those patients who were treated with their own stool were cured after being treated with donor stool.
The analysis for this result came out to be that the microbiota from healthy donor colonized the unhealthy patients and atients and made them hea d it is more efficient in treating C. ifficile than it is for analogous stool as the treatment. Alexander Link, Tim Lachmund, Christian Schulz, Jochen Weigt, Peter Malfertheiner. “Endoscopic peroral jejunal fecal microbiota transplantation. ” Digestive and Liver Disease. 2016 November; 48(11): 1336-1339. In this study, there was 6 patients tested positive for C. difficile toxin. These patients were previously administered metronidazole, vancomycin, fidaxomycin. The source of the stool originated from 2 unrelated, healthy donors. These two donors were tested for antibiotic susceptibility.
At least 50g of stool dosage was administered to each patient varying in the amount of stool dosage within each patient. The fecal transplantation was administered via endoscope directly in jejunum. The result of this study was that all the patients were cured from C. difficile. However, there was FMT-reflux in two cases; the first one led to affecting the stomach due to deep endoscopic jejunum application; the second one led to a serious case of pneumonia. Broecker, Felix et al. “Long-Term Changes of Bacterial and Viral Compositions in the Intestine of a Recovered Clostridium Difficile Patient after Fecal Microbiota Transplantation.
Cold Spring Harbor Molecular Case Studies 2. 1 (2016): a000448. PMC. Web. 15 Nov. 2016. The patient for this study was a 51-years-old woman with 6 episodes of reoccurring CDI. The donor of this study was the patient’s sister who was tested in negative bacterial and viral pathogens. As a result, the patient remained symptom free after four and a half year of the treatment. This indicated that FMT is efficient in treating CDI in a long-term. Even though the patient remained C. difficile free, Caudovirales phages and Chlorella viruses were found in her gut.
Based on this finding, it was determined that fecal transplant colonize the patients with CDI with good bacteria; however, this colonization could lead to the emergence of viral and other bacterial pathogens. Q1C: What is the V6 region of bacterial 16S rRNA, and why is it useful for phylogenetic analysis of microbial communities? V6 is a 58 nucleotide region that could be used to distinguish the all of the CDC-defined select agents (Soumitesh, 1). V6 has a sequence that matches 90% to the classification of accuracy of microbial populations (Dea, 5).
This region has better resolution within species and it changes over time which means it does not interfere with the region that is viable for cell replication, transcription, and translation. The function of 16S rRNA is to bind to the Shine-Dalgarno sequence on mRNA and ensure translation is completed correctly. 16S rRNA is useful for phylogentic studies because every cell has RNA, it changes slowly over time, and it does not undergo horizontal gene transfer. Q1D: Briefly describe lon Torrent sequencing in 4-5 sentences. How does it work, and what is it used for? Genomic DNA is prepared by fragmentation, ligation, and amplification.
Bounded DNA polymerase and primers are inserted through loading port. Beads are placed into sensor wells when the chip is spinning in a centrifuge. Complementary nucleotides are inserted singularly during the spin building onto the growing DNA strand. Signal processing software detects each nucleotide that is incorporated into the DNA strand (Porterfield). It is used to sequence bases at a massive measure (Thermofisher). Q2A: Microbiome: microbial cells, their genes, and the sites they inhabit. Q2B: Dysbiosis: imbalance of gut flora caused by an overpopulation of bad bacteria and too little of beneficial bacteria.
Q2C: Vancomycin: antibiotic used to treat gram-negative bacteria like Clostridium difficile. 02D: Metronidazole: antibiotic used to treat bacteria that cause infection in vagina, reproductive system, gastrointestinal tract. Q2E: Kirby-Bauer test: this test is used to measure the sensitivity of bacteria to antibiotics. Q2F: Operational taxonomic unit: a group of species that closely related by a shared characteristic.
Q3A: What problem were the authors trying to solve, and what was the overall goal of this study? The problem the authors were trying to solve is that fecal transplant can be used as a treatment for C. ifficile, but during the process of transmission of stool between donor and recipient, there could also a transmission of disease; therefore, the authors are trying to find an effective way to treat C. difficile without causing additional diseases to the patients. In this research, the authors use a stool substitute sample to cure C. difficile. Q3B: Summarize, in your own words, how the authors generated “RePOOPulate. ” In your answer, be sure to state where the bacteria were isolated from, and what steps the authors took to determine that the bacteria were probably ‘safe’ for therapeutic use.
The generation of RePOOPulate was formulated by culturing a stool sample of a healthy middle age female donor. After the culturing of the stool sample, 62 microbial isolates were obtained through many types of media. These isolates were tested to determine whether or not they were susceptible to antibiotics by measuring the zone of inhibition when using minimal inhibition concentration of antibiotics noting that any isolates that are resistant to antibiotics were not used. These purified cultures were sequenced by 16S rRNA sequencing to compare their ratio to the metagenomic database of a human stool sample.
Q3C: Summarize the state of health of the two patients before and after RePOOPulate administration. For Patient 1, before the treatment, she had 6 times reoccurrences of CDI over 18 months. Before CDI, she went into her knee surgery and was administered cefazolin. This caused her first CDI. After the fecal transplant treatment, her bowel cycle was normal and C. difficile was not detected in her stool sample 10 days after the treatment. Because of her urinary problems, she was administered to additional antibiotics; however, no sign of CDI occurred.
For Patient 2, because she had cellulitis, she was administered cefazolin and had 3 times of reoccurrences of CDI. After the treatment, she bowel movement normalized. She was prescribed to ceftriaxone for her cellulitis after the treatment and still remained symptom-free because her bowel movement and stool were normal. Both stool samples from the patients contained ribotype 078 preceding the treatment. Q3D: After describing the clinical outcomes for the two patients, the authors spend the rest of the paper describing analyses of the fecal microbiome of the two patients.
Specifically, they isolated DNA from fecal material from the patients, used PCR to amplify the V6 region of bacterial 16S rRNA, and then sequenced the PCR products using lon Torrent technology. They then compared the sequences back to public databases in an attempt to identify the bacterial species colonizing the gastrointestinal tracts of the two patients. What do you think the authors were trying to prove with these analyses?
The authors were trying to prove if the microbial formulated in RePOOPulate could stably colonize the gastrointestinal tracts of those who are administered which their analysis matched their hypothesis in hat the microbe in RePOOPulate stably colonized the gastrointestinal tracts after the treatment. They concluded that because the microbe in RePOOPulate was isolated from a multispecies sample they are predisposed to colonize in a communal environment. Q3E: This is an opinion question. Do you think that the authors provided sufficient evidence to show that RePOOPulate is a viable treatment strategy for recurrent C. difficile infection? If you are convinced by their evidence, state dence that convinced vou. If you are not convinced, state the weaknesses you identified, and what they could do to convince you.
I think that the authors provided sufficient evidence to show that RePOOPulate is a viable treatment because they included the results after administering the stool substitution to Patient 1 and 2 in that both patients remained symptom-free. Their explanation for this result is that after the treatment, the microflora of the two patients adapted to the characteristics of the stool substitution which then proved that these microbes from RePOOPulate stably colonized the gastrointestinal tracts of the diseased patients, indicating that the stool substitution treatment was successful in treating patients with CDI.
Q4A: The authors state under the “Competing interests” section that some of the authors have filed a provisional patent. What is a provisional patent, and why is it important for inventors? Provisional patent is when an inventor fills out an application consists of description and drawing of the invention; by doing this application, it allows the inventors to have their invention as “patent pending” status for 12 months. This is important because it allows for the inventors to claim their work as their and prevent other manufacture from stealing their invention. This is also important because it marks the date at which the invention is made.
Q4B: This is an opinion question. If the authors could patent RePOOPulate as a novel invention, do you think that the original feces from the human source of RePOOPulate is also patentable? Why or why not? The original feces from the human source of RePOOPulate is patentable because it was used as the main source of this novel invention. When the donor donated her feces, it was indicated that she allowed the researchers to use it as a part of their experiment. By using her feces, they studied that C. difficile could be treated and remained harmless. Therefore, the original feces from the donor is also patentable. Q5A: ClinicalTrials. ov is a registry and database for clinical studies using human subjects.
For this question, I want you to search Clinical Trials. gov and find a study where fecal transplantation is being evaluated for treatment of a condition other than C. difficile infection. In your answer, include the following: (4 points) 1. A link to the clinical trial entry on clinicaltrials. gov https://clinicaltrials. gov/ct2/show/NCT02516384? term=fecal +transplantation&rank=5 2. What disease or condition is being treated? Fecal transplant is used to treat inflammatory bowel disease which is a disease caused by dysfunctional gastrointestinal immune system.
Q5B: This is an opinion question. Would you be willing to try a fecal transplant as an alternative to antibiotic therapy, if you had a C. difficile infection? Why or why not? || would try fecal transplant because it is effective in treating C. difficile proven by the study with two patients. This treatment results in symptom-free and regulates bowel movement. Furthermore, fecal transplant lead to stably colonization of the good bacteria in human gastrointestinal tract which indicates that this treatment cures C. difficile infection.