Multidrug Antimicrobial Resistance Testing and Fecal Transplant Therapy Risks

Introduction

Following the death of one patient and another falling seriously ill (reported in June 2019), the U.S. Food and Drug Administration (FDA) has called for greater controls over fecal transplants and the introduction of microbial pathogen screening. The patients became infected with a multi-drug resistant bacterial infection.¹

Fecal transplants are novel therapies and ones that are not fully regulated, as with more proven pharmaceutical therapies. As it stands, the safety profiles of these types of medicinal intervention have yet to be fully evaluated in controlled clinical trials.² Despite several years of study, most of which demonstrate the effectiveness of fecal transplants, the longer-term process remains difficult to monitor (and to indicate what exactly are the numbers and concentration of beneficial bacteria; there is also evidence that the intestinal ecosystem of each individual patient reacts very differently to fecal transplantation. Plus, there are no standardized methods for administering the bacteriotherapy. Furthermore, those clinical trials underway do not need to conform to the agency’s investigational new drug (IND) regulations (21 CFR Part 312) (and, as the U.S. National Library of Medicine indicates, at the time of writing, there are several clinical trials in progress).³ Other challenges relate to a lack of suitable of donors.

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A further concern relates to the scenario where the transferred material contains additional pathogens to those that the therapy is designed to treat. This appears to have been the situation with the recent case. The FDA warning issued in 2019 relates to the risk of serious or lifethreatening infections occurring due to the use of fecal microbiota for transplantation, linked to bacterial infections caused by multidrug resistant organisms. With the recent cases that sparked the FDA warning, two immunocompromised adults received investigational fecal microbiota. The two patients developed invasive infections that were triggered by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. The therapeutic agent administered to the two people was prepared from stool obtained from the same donor.

Fecal Bacteriotherapy

With fecal transplants (or ‘fecal bacteriotherapy’) the aim is to restore the balance between good bacteria and bad bacteria in the colon (a condition known as dysbiosis), by replacing or replenishing the microbial communities that thrive in the intestinal tract. Essentially, it is theorized that the transfer of microbe-laden stool from a healthy donor can restore the equilibrium and beneficial functions of the microbiome of the recipient patients. This could be via a complete repopulation of the gut microbiota or by boosting microbial diversity. The exact mechanisms are not fully known. There are also some concerns with side effects. In one case, a woman who was successfully treated for a recurrent C. difficile infection with stool from an overweight donor rapidly gained weight herself ).⁴ The most common forms of the procedure involve either single to multiple infusions (such as by enema) of bacterial fecal flora originating from a healthy donor, or a fecal transplant in pill form.

While the process may come across as relatively imprecise, the extent of the number of transfers being conducted and the extending range of conditions that they are intended to address means that the therapy is crossing the boundary between medical microbiology and medicinal (pharmaceutical) products. This is particularly so with some forms of fecal bacteriotherapy that exist in pill form (and a more palatable alternative to the use of other routes of administration like colonoscopies, nasal tubes, and enemas). In practice, there are a range of different forms of fecal bacteriotherapy: hand-mixed fresh stool, freeze-dried portions, microbial-enriched excrement, formulated pills, and synthetic slurries, and no common framework to assess their overall suitability.

Much of the research, advanced by a more detailed understanding of the human microbiome,⁵ has been orientated to the treatment of infections causes by Clostridium difficile, which can infect the bowel and cause diarrhea. One study, published in The New England Journal of Medicine, indicated that fecal transplants were almost twice as effective as antibiotics in treating patients with recurring C. difficile.⁶ Treatment of C. difficile remains the most common application and it is this area for which the FDA has given special dispensation for (as detailed below). Another study has expanded the idea of reducing the population of pathogens by targeting two pathogens residing in the same gut at the same time. The Memorial Sloan-Kettering Cancer Center study looked at applying fecal material to combat the effects of vancomycin-resistant Enterococcus faecium and multi-drug resistant Klebsiella pneumonia.⁷ There are other investigational lines that are assessing different areas of medical inquiry. For example, research sponsored by Ohio State University into eighteen children with autism and moderate to severe gastrointestinal problems suggests that, following the fecal therapy, positive changes (assessed via the Childhood Autism Rating Scale) have been observed following eight weeks of fecal bacteriotherapy treatment.⁸ Other areas of clinical trial investigation extend to Crohn’s disease and ulcerative colitis (chronic inflammatory bowel diseases).

FDA Investigation

An investigation, for which the outcome was revealed in June 2019, found that the donor stool and resulting fecal microbiota for transplantation had not been pre-tested for ESBL-producing Gramnegative bacteria prior to use. Extended spectrum β-lactamases (ESBLs) are enzymes produced by several species of Gram-negative bacteria⁹ (typically members for the Enterobacteriaceae family such as Klebsiella pneumoniae and E. coli; and nonfermentative bacteria like Acinetobacter baumannii and Pseudomonas aeruginosa), which confer an increased resistance to commonly used antibiotics (such as penicillins and cephalosporins).¹⁰ The enzyme generally cleaves a ring common to all the chemical structures of these types of antibiotics. The enzymes can be carried on bacterial chromosomes (inherent to the bacterium); or resistance may be acquired through a plasmidmediated response, where the mechanism for resistance can move between bacterial populations. Subsequent examination, following notification of the adverse events, of the stool donor found the donation material to be positive for ESBL-producing E. coli. Metagenomic analysis further showed the contaminant to be identical to the organisms isolated from the two patients.

Current Regulatory Oversight

The use of fecal transplantation does not fall under the same level of strict guidances as with other pharmaceutical preparations. In 2013, the FDA permitted the use of discretion on the part of the treating medical doctor11 (which means the FDA practices “exercise enforcement discretion” and will not take any action against physicians performing fecal transplants in relation to the treatment of C. difficile infections). Under the agency guidance, the primary activity that the medical establishment is required to undertake is for the lead physician to seek informed consent from the individual set to receive the treatment and ensure that the patient is aware that fecal therapy products remain investigational and that an informed discussion is held with the patient as to the potential risks.

Need for a New Microbial Testing Regime

The recent case highlights the lack of microbial testing for some types of fecal transplant and the lack of a standardized approach. This leads to questions as to how does the producer of fecal bacteriotherapy control, quantify, and standardize the material? This becomes more challenging given that clinical trials have yet to establish which elements of the feces are therapeutic. Are specific microbial strains important, or is it a combinations of certain species? Does the concentration or population matter? This is an important question for quality control and specificity, as would be required for the laboratory analysis of a more conventional pharmaceutical treatment. Similarly, how is the material to be assessed for undesired organisms and those that could cause patient harm (the ‘objectionable organisms’ in the classic pharmaceutical microbiology sense)? What about microbial byproducts and toxins?

Following the incident, the FDA is calling for increased protection of the patient. The protections include the use of donor screening questionnaires, designed to address the types of risk factors that might indicate colonization with multi-drug resistant organisms with the aim of excluding individuals at higher risk of colonization with such antimicrobial resistant microorganisms. This should be followed by microbial testing of donor stool for multi-drug resistant organisms and the subsequent exclusion of any stool that tests positive for such organisms.

To assess samples of fecal material there are several classic culturebased laboratory methods and rapid alternative microbiological tests. With culture-based tests, a common test is for a small amount of a fresh fecal sample to be applied to a variety of selective culture media.

The physical characteristics of the colonies - their shape, color, and some of their chemical properties - allow them to be differentiated and for confirmatory identifications to be performed. While there will be variations between laboratories, example culture media include MacConkey agar; a selective/differential medium designed for the recovery of Salmonella and Shigella; a medium designed for the recovery of Campylobacter (such as charcoal-cefoperazonedeoxycholate agar); and a medium designed for the recovery of E. coli O157 (such as sorbitol-MacConkey agar) and/or enrichment broth for testing for the presence of Shiga toxins. Many laboratories also include a blood agar plate, for the recovery of Aeromonas spp., Plesiomonas spp., and Vibrio spp. Other selective media appropriate for fecal cultures include xylose-lysine-deoxycholate; salmonella-shigella; Hektoen enteric; and brilliant green or bismuth sulfite medium or a chromogenic medium designed for the recovery and detection of specific enteropathogens.¹²

Advances with rapid microbiological screening permits a faster and more accurate assessment of pathogens. Detection of bacterial 16S rRNAs using PCR amplification or dot blot hybridization with specific probes has been shown to be successful.¹³ For example, a molecular platform like the BD MAX™, can screen for the presence of Salmonella spp, Campylobacter spp, Shigella spp, Enteroinvasive E. coli and Shiga toxin producing E. coli. This example system performs nucleic acid extraction and real-time PCR.¹⁴ An alternate method for the identification of single cells within complex ecosystems is fluorescent in situ hybridization (FISH) with specific 16S rRNA-based oligonucleotide probes, and this technique has also been used to quantify bacteria in the human gut.¹⁵

Whichever methods are adopted, the importance is that they are optimized and standardized, under controlled protocols, and that methods for collection, homogenization, microbial disruption, extraction, testing and results interpretation are detailed. These issues are especially important as what was a novel clinical therapy transitions over to a more controlled, pharmaceutical medicinal therapy.

Synthetic Material

Although not obviating the necessity for testing, an improved pharmaceutical preparation for bacteriotherapy may lie in a synthetic variant in pill form. This would involve preparing defined concentrations of multi-species community of bacteria. This would help to address concerns about pathogen transmission and concerns about standardizing the treatment regimen. Such an approach may also assist with patient acceptance, drawing closer parallels with the use of probiotics.

Summary

Fecal transplants (or ‘fecal bacteriotherapy’) appear to have a high success rate. The technique aims to restore the balance between good bacteria and bad bacteria in the colon, and there is clinical evidence that this process reduces the incidence of specific pathogens (particularly C. difficile) and such therapies are showing signs of success against certain metabolic diseases. However, the June 2019 warning issued by the FDA highlights a lack of control around screening for other pathogens and it appears that it is possible to transfer high levels of a different pathogen into a patient in addition to the pathogen that is intended to be treated. Hence, if the goal for fecal bacteriotherapy is to transition to the status of a consistently produced and manufactured pharmaceutical preparation then standardized microbiological quality control testing is required, both to assess the numbers and concentrations of beneficial bacteria that are required and in order to screen for the presence or absence of pathogens that might cause patient harm.

References

1. U.S. FDA Important Safety Alert Regarding Use of Fecal Microbiota for Transplantation and Risk of Serious Adverse Reactions Due to Transmission of Multi-Drug Resistant Organisms, at: https://www.fda.gov/vaccines-blood-biologics/safety-availabilitybiologics/important-safety-alert-regarding-use-fecal-microbiota-transplantation-andrisk-serious-adverse
2. Federal Register of February 25, 2013 (78 FR 12763) 
3. 3. U.S. National Library of Medicine, Clinical Trials at: https://www.clinicaltrials.gov/ct2/results?cond=Fecal+microbiota+transplant&Search=Apply&recrs=a&recrs=f&recrs=d&age_v=&gndr=&type=&rslt= (accessed 16th June 2019)
4. Alang , N. and Kelly, C. R. (2015) Weight Gain After Fecal Microbiota Transplantation, Open Forum Infectious Diseases, 2 (1): https://doi.org/10.1093/ofid/ofv004
5. Wilder, C., Sandle, T. and Sutton, S. (2013). Implications of the Human Microbiome on Pharmaceutical Microbiology, American Pharmaceutical Review, 16 (5): pp17-21 
6. van Nood, E.., Vrieze, A.., Nieuwdorp. M. (2013) Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile, N Engl J Med; 368:407-415
7. Caballero, S., Carter, R., Ke, X. et al (2015) Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae, PLOS Pathogens: https://doi.org/10.1371/journal.ppat.1005132
8. Kang, D-W., Adams, J.B., Gregory, A. C. (2017) Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study, Microbiome, 5 (10): https://doi.org/10.1186/s40168-016-022
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11. U.S. FDA Guidance for Industry Enforcement Policy Regarding Investigational New Drug Requirements for Use of Fecal Microbiota for Transplantation to Treat Clostridium difficile Infection Not Responsive to Standard Therapies, at: https://www.fda.gov/media/86440/download
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