Patent Application
20210038663
Clostridium difficile infection (CDI) is a symptomatic infection due to the spore-forming bacterium, Clostridium difficile. C. difficile infection is spread by bacterial spores found within feces. Risk factors for infection include antibiotic or proton pump inhibitors use, hospitalization, other health problems, and older age. Its symptoms including watery diarrhea, fever, nausea, and abdominal pain, CDI makes up about 20% of cases of antibiotic-associated diarrhea. About 453,000 cases C. difficile infection occurred in the United States in 2011, resulting in 29,000 deaths. Each year, C. difficile infections accounts for health care cost of approximately $1.5 billion. Globally, rates of C. difficile infection have increased between 2001 and 2016, typically with more women than men affected by the infections.
Fecal microbiota transplantation (FMT) is highly effective for the treatment of CDI, especially among patients suffering from recurrent CDI. Also known as stool transplant, FMT involves a process of transplanting fecal matter containing microorganism from a healthy individual into the gastrointestinal tract of a recipient. The goal of FMT is restoration of the gut microflora disrupted due to CDI by introducing (or re-introducing) healthy bacterial flora via various means of infusion of a healthy individual's stool, e.g., by colonoscopy, enema, orogastric tube, or by mouth in the form of a capsule containing freeze-dried material obtained from a healthy donor. Aside from CDI, FMT is increasingly being used to treat other intestinal and extra-intestinal diseases, including other gastrointestinal diseases, such as inflammatory bowel disease (IBD), antibiotic-resistant bacterial infection, diarrhea, constipation, irritable bowel syndrome, autism, depression, obesity, diabetes, alopecia, and the like. In addition, FMT has been used for treating certain neurological conditions, such as multiple sclerosis and Parkinson's Disease. Considering the prevalence of CDI and other conditions treatable by FMT in the human population and their significant economic implications, there exists an urgent need for developing new and improved methods for treating CDI and other disorders by FMT with enhanced efficacy. The present invention fulfills this and other related needs.
The invention relates to novel methods and compositions useful for more effectively treating Clostridium difficile infection (CDI) and other diseases suitable by fecal microbiota transplantation (FMT) treatment. In particular, the present inventor discovered that, when elevated level of the yeast species Candida albicans is present in the gastrointestinal tract of an FMT recipient or in the stool of an FMT donor, therapeutic efficacy of FMT is negatively impacted. This finding allows the inventors to devise methods and compositions that can improve FMT effectiveness. Thus, in the first aspect, the present invention provides a novel method for assessing the likelihood of effective FMT. The method includes a step of determining C. albicans level in a stool sample obtained from a potential recipient prior to FMT is performed, i.e., before the recipient is to receive transplantation of a donor fecal material.
In some embodiments, the C. albicans level is a percentage relative abundance, or is expressed as a percentage over the total level of all fungal species in the sample. In some embodiments, when the C. albicans level is determined as greater than 10% of total fungi in the sample of the recipient, FMT is assessed as unlikely to be effective for the potential recipient. Under such a determination, the recipient in some cases will not receive FMT treatment but will receive another different therapy; in other cases, the recipient is administered an effective amount of an antifungal agent that suppresses C. albicans growth before FMT is performed. Optionally, after the administration of the antifungal agent, the C. albicans level in the recipient (e.g., in a recipient's stool sample) is again measured and determined to have been lowered before the recipient is transplanted with a composition containing donor fecal material. In some cases, C. albicans level is again determined in a stool sample obtained from the recipient after FMT.
In some embodiments, when the C. albicans level is no greater than 10% of total fungi in the sample, FMT is assessed as likely to be effective for the potential recipient. In some cases, the potential recipient is then immediately given FMT, without any further treatment or preparation such as administration of an antifungal agent in the effective amount. In some embodiments, the method further involves a step of determining total fungal load in the stool sample. A potential recipient whose total fungal load in his stool sample is found to be relatively lower than that of a second potential recipient is expected to have a higher likelihood of having a successful FMT than the second recipient. In some embodiments, multiple potential recipients of FMT are tested prior to FMT using this method for assessing their relative likelihood of success upon receiving FMT treatment. For instance, C. albicans level is determined in a first stool sample obtained from a first potential recipient prior to FMT, and in the meantime C. albicans level is determined in a second stool sample obtained from a second potential recipient prior to FMT. In some embodiments, when the first potential recipient has a lower C. albicans level than the second potential recipient and is therefore assessed to have a higher likelihood of effective FMT than the second potential recipient. In some embodiments, the second potential recipient is then administered an effective amount of an antifungal agent that suppresses C. albicans growth before FMT, whereas the first potential recipient may or may need antifungal agent treatment prior to FMT. In some embodiments, C. albicans level is determined by quantitative polymerase chain reaction (PCR). In some embodiments, the levels of all fungal species present in the sample is determined by the Internal transcribed spacer 2 (ITS2) sequencing. In some embodiments, the recipient or recipients suffer from inflammatory bowel disease (IBD) with concurrent Clostridium difficile infection (CDI). The C. albicans level in the stool may be determined before and after their FMT process. An elevated C. albicans level before or after FMT is indicative of a higher likelihood of poor outcome or ineffective FMT.
In a second aspect, the present invention provides a novel, improved method for identifying a suitable donor who would provide stool or fecal material for FMT. The method includes the step of determining C. albicans level in a stool sample obtained from a candidate who is being considered as a potential donor for FMT.
In some embodiments, the C. albicans level determined in this method is a percentage relative abundance. In some cases, when the C. albicans level is no greater than 0.1% of all fungi present in the sample, the candidate is identified as a suitable donor for FMT. Optionally, fecal matter such as stool is immediately collected from the candidate for use in FMT. In some cases, when the C. albicans level is greater than 0.1%, the candidate is deemed unsuitable as a donor for FMT. As a result, either no fecal matter is collected from the candidate at all; or fecal matter is collected for processing to be used in FMT after the candidate is given an effective amount of an antifungal agent that suppresses C. albicans growth and again tested to find a satisfactorily reduced C. albicans level in the stool sample (e.g., no greater than 0.1% of total fungi in the sample). In some embodiments, the stool sample of a candidate is tested for Saccharomyces level and Aspergillus level in addition to C. albicans level. In some embodiments, C. albicans level is determined by quantitative PCR. In some embodiments, the levels of all fungi present in the sample is determined by ITS2 sequencing. In some embodiments, the method includes the additional step of determining Escherichia level and Proteus level in the stool sample of a potential donor. Among multiple potential donors, one with a relatively high Escherichia level and a relatively low Proteus level is deemed a more suitable donor than one with a relatively low Escherichia level and/or a relatively high Proteus level. In some embodiments, the method further includes a step of determining the total fungal load in the stool sample taken from the potential donor. A potential donor whose total fungal load in his stool sample is found to be relatively lower than that of a second potential donor is expected to be a more desirable donor, i.e., provide a higher likelihood of a successful FMT, than the second donor.
In a third aspect, the present invention provides a method for improving FMT efficacy. The method includes the step of administering to an FMT recipient prior to FMT being performed an effective amount of an antifungal agent that suppresses C. albicans growth. In some embodiments of this method, C. albicans level is first determined in a stool sample from the FMT recipient prior to administration of the antifungal agent. In some embodiments, C. albicans level is determined in a stool sample from the FMT recipient after administration of the antifungal agent. In some embodiments, C. albicans level is determined by quantitative PCR. In some embodiments, the levels of all fungi present in the sample is determined by ITS2 sequencing. In some embodiments, the method further includes a step of administering to the recipient prior to FMT an effective amount of an agent (e.g., an anti-fungal agent, such as a broad-spectrum fungicide), which reduces total fungal load in a stool sample taken from the recipient prior to FMT. In some embodiments, the recipient is a patient suffering from inflammatory bowel disease (IBD) with concurrent Clostridium difficile infection (CDI).
In a fourth aspect, the present invention provides kits and compositions useful for enhanced FMT treatment with improved efficacy. In some embodiments, a kit for improving FMT efficacy includes a first composition comprising a donor stool material and a second composition comprising an effective amount of an antifungal agent capable of suppressing the growth of C. albicans. Typically, the first and second compositions are kept in two separate containers. In some embodiments, the first composition contains processed donor fetal matter and is formulated for FMT by direct transfer to the GI tract (e.g., via colonoscopy or via nasal intubation) or by oral ingestion. In some embodiments, the first composition comprises donor fecal matter further fortified with an additional and effective amount of one or more fungal species belonging to the genus Saccharomyces and/or the genus Aspergillus. In some embodiments, the second composition is formulated for administration of the antifungal agent (such as fluconazole) to the recipient by injection, oral ingestion, or rectal deposit. In some embodiments, the kit may further comprise, either in the second composition or in a third composition, an effective amount of an agent that reduces total fungal load. In the alternative, the kit may comprise a third composition, which comprises an effective amount of an agent that reduces total fungal load, but not the second composition. In some cases, the kit may further include printed user instructions.
Related compositions useful in FMT with improved efficacy may comprise (1) a donor stool material containing live fecal microorganisms and (2) an antifungal agent that specifically suppresses the growth or proliferation of C. albicans but exhibits no such suppressive or inhibitory effect against other fungal species. Instead of a broad-spectrum fungicide, such specific anti-C. albicans agent may be short polynucleotide in nature of (e.g., a small inhibitory RNA, microRNA, miniRNA, lncRNA, or an antisense oligonucleotide that is capable of disrupting the expression of a key gene in the life cycle of C. albicans) that is capable of specifically targeting the species only but not other closely related fungal species.
C. albicans compromises FMT efficacy in eradicating C. difficile infection in mice. A) Schematic diagram of C. albicans administration and stool infusion (FMT) in a murine C. difficile infection (CDI) model. Antibiotic treatment was ceased before gavage of C. albicans (CA) and C. difficile. B) Diarrhoea in mice on day 1 after stool infusion. C) Representative H&E-stained colonic sections on day 2 after stool infusion (shaded star denotes inflammatory cells infiltration, hallowed star denote ulceration, asterisk denotes oedema, arrow denotes goblet cell loss and triangle denotes herniated crypts). Scale bar, 150 μm. n=5 mice per group. D) Enumeration of C. difficile in feces of mice on day 0 before FMT and day 1 post FMT (n=9 mice per group). Statistical significance represents comparisons between FMT-treated mice with C. difficile infection versus other groups by unpaired Mann-Whitney test. *P<0.05, **P<0.01. E) Enumeration of C. albicans in feces of mice both on day 0 before FMT and day 1 post FMT (n=9 mice per group). Statistical significance represents comparison between C. albicans load on day 0 before FMT and day 1 post FMT, by paired Mann-Whitney test. *P<0.05. Dot graphs show means±s.e.m, performed at least two times independently.
Definitions
The term “fecal microbiota transplantation (FMT)” or “stool transplant” refers to a medical procedure during which fecal matter containing live fecal microorganisms (bacteria, fungi, and he like) obtained from a healthy individual is transferred into the gastrointestinal tract of a recipient to restore healthy gut microflora that has been disrupted or destroyed by a variety of medical conditions. Typically, the fecal matter from a healthy donor is first processed into an appropriate form for the transplantation, which can be made through direct deposit into the lower gastrointestinal tract such as by colonoscopy, or by nasal intubation, or through oral ingestion of an encapsulated material containing dried and frozen fecal matter. Clostridium difficile infection (CDI) is the condition most commonly treated by FMT, although a number of other diseases and disorders including in the digestive system and in the nervous system have been reported to be successfully treated by FMT.
The term “inhibiting” or “inhibition,” as used herein, refers to any detectable negative effect on a target biological process, such as RNA/protein expression of a target gene, the biological activity of a target protein, cellular signal transduction, cell proliferation, and the like. Typically, an inhibition is reflected in a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater in the target process (e.g., growth or proliferation of fungal cells), or any one of the downstream parameters mentioned above, when compared to a control. “Inhibition” further includes a 100% reduction, i.e., a complete elimination, prevention, or abolition of a target biological process or signal. The other relative terms such as “suppressing,” “suppression,” “reducing,” and “reduction” are used in a similar fashion in this disclosure to refer to decreases to different levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater decrease compared to a control level) up to complete elimination of a target biological process or signal. On the other hand, terms such as “activate,” “activating,” “activation,” “increase,” “increasing,” “promote,” “promoting,” “enhance,” “enhancing,” or “enhancement” are used in this disclosure to encompass positive changes at different levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or greater such as 3, 5, 8, 10, 20-fold increase compared to a control level) in a target process or signal.
As used herein, “C. albicans” refers to a fungal species belonging to the Candida genus, Saccharomycetaceae family, Saccharomycetales order, Saccharomycetes class, and Ascomycota division. A common member of the human gut flora, C. albicans is a potential yeast pathogen capable of causing opportunistic infection in humans, especially in those with compromised immune system.
The term “antifungal agent” refers to any substance that is capable of inhibiting, suppressing, or preventing the growth or proliferation of fungal species, especially those of the Ascomycota division, such as C. albicans. Known agents with fungicidal activity include amphotericin B, echinocandin, fluconazole, nystatin, and clotrimazole.
“Percentage relative abundance,” when used in the context of describing the presence of a particular fungal species (e.g., C. albicans) in relation to all fungal species present in the same environment, refers to the relative amount of the fungal species out of the amount of all fungal species as expressed in a percentage form. For instance, the percentage relative abundance of C. albicans can be determined by comparing the quantity of C. albicans-specific DNA (e.g., determined by quantitative polymerase chain reaction) in one given sample with the quantity of all fungal DNA (e.g., determined by quantitative PCR and sequencing based on the Internal transcribed spacer 2 or ITS2 sequence) in the same sample.
“Absolute abundance,” when used in the context of describing the presence of a particular fungal species (e.g., C. albicans) in the feces, refers to the amount of DNA derived from the fungal species out of the amount of all DNA in a fecal sample. For instance, the absolute abundance of C. albicans can be determined by comparing the quantity of C. albicans-specific DNA (e.g., determined by quantitative polymerase chain reaction) in one given sample with the quantity of all fecal DNA in the same sample.
“Total fungal load” of a fecal sample, as used herein, refers to the amount of all fungal DNA out of the amount of all DNA in the fecal sample. For instance, the absolute abundance of fungi can be determined by comparing the quantity of fungal specific DNA (e.g., 18S rDNA determined by quantitative polymerase chain reaction) in one given sample with the quantity of all fecal DNA in the same sample.
The term “effective amount,” as used herein, refers to an amount of a substance that produces a desired effect (e.g., an inhibitory or suppressive effect on C. albicans growth) for which the substance (e.g., an antifungal agent) is used or administered. The effects include the prevention, inhibition, or delaying of any pertinent biological process during C. albicans growth or development to any detectable extent. The exact amount will depend on the nature of the substance (the active agent), the manner of use/administration, and the purpose of the application, and will be ascertainable by one skilled in the art using known techniques as well as those described herein.
As used herein, the term “about” denotes a range of value that is +/−10% of a specified value. For instance, “about 10” denotes the value range of 10+/−10×10%, i.e., 9 to 11.
The invention provides a novel approach for assessing the likelihood of effective FMT prior to the procedure being performed as well as for improving the effectiveness of the FMT procedure. During their studies, the present inventors discovered that the presence and relative abundance of certain fungal species both in a recipient's gastrointestinal tract and in a donor's stool directly correlate with the outcome of FMT. In particular, the fungal species Candida albicans of the Saccharomycetaceae family is found to negatively impact the effectiveness of FMT. The detection of C. albicans in a potential donor's stool thus can be used to guide donor selection, whereas analysis of C. albicans level in an FMT recipient can determine whether the subject is immediately ready for FMT or should be treated with an antifungal agent that suppresses C. albicans growth prior to FMT in order to optimize the therapeutic outcome.
Patients suffering from CDI, especially recurring CDI, are often considered as recipients for FMT treatment. Aside from CDI, other diseases and conditions, including those of digestive system or nervous system such as colitis, irritable bowel syndrome, multiple sclerosis, Parkinson's Disease, diabetes mellitus, and obesity are also beginning to be considered for FMT treatment.
Fecal matter used in FMT is obtained from a healthy donor and then processed into appropriate forms for the intended means of delivery in the upcoming FMT procedure. Up until now, the general criterion for an FMT donor is simply that the donor is a healthy individual without any known diseases or disorders especially in the digestive tract, although some preference is often given to the members of the same household as the recipient.
The present inventors have discovered in their studies that elevated presence of one particular fungal species, C. albicans, in a recipient's gastrointestinal tract or in a donor stool (which is used in the transplantation after being processed) can significantly reduce efficacy of FMT treatment in a patient. In contrast, successful FMT has been observed as correlating with elevated presence of other fungal species, such as those belonging to the genus of Saccharomyces or Aspergillus, in a recipient or in a donor stool. This revelation enables the initial screening of individuals as appropriate FMT donors as well as the initial screening of patients as likely candidates for successful FMT treatment: if a candidate donor's stool contains an elevated level of C. albicans (e.g., greater than 0.1% of total fungi), the candidate is deemed as unsuitable as an FMT donor, and his stool should not be taken or used in FMT; if a candidate's stool sample shows no or only low level of C. albicans (e.g., no greater than 0.1% of total fungi), then the candidate is deemed an appropriate FMT donor and his fecal material can be immediately retrieved for processing and later used in FMT. On the other hand, if a patient who has been proposed to receive FMT treatment, and his stool sample shows an elevated level of C. albicans (e.g., greater than 10% of total fungi), then the patient is deemed to be unsuitable to receive FMT and is therefore not to be given FMT, as the therapy is likely to be unsuccessful; if a patient's stool sample shows no or only a low level of C. albicans (e.g., no greater than 10% of total fungi), the patient is deemed an appropriate recipient for FMT who is likely to enjoy therapeutic success from FMT, and thus can start FMT treatment immediately without other steps of preparation or pre-treatment.
Various methods have been reported in the literature for determining the levels of all fungal species in a sample, for example, amplification (e.g., by PCR) and sequencing of fungal polynucleotide sequence by using the Internal transcribed spacer 2 (ITS2) sequence. On the other hand, the level of any given fungal species may be determined by amplification and sequencing of its signature 18S rRNA sequence. A percentage abundance is often used as a parameter to indicate the relative level of a fungal species in a given environment.
The discovery by the present inventors revealing the direct correlation between an elevated level of C. albicans in FMT donor or recipient and reduced efficacy of FMT treatment not only allows one to devise an initial screening process to identify appropriate donors and recipients for the FMT procedure, it also enables different methods for improving FMT efficacy by reducing the level of C. albicans in a donor and in a recipient prior to the FMT treatment.
As discussed in the above section, when a candidate donor's stool is tested and found to contain an elevated level of C. albicans (e.g., greater than 0.1% of total fungi), the candidate is deemed as unsuitable as an FMT donor, and his stool should not be taken for use in FMT as it is unlikely to result in a successful FMT treatment if used. Similarly, when a patient or proposed FMT recipient whose stool is tested and found to contain an elevated level of C. albicans (e.g., greater than 10% of total fungi), the patient is deemed as an unsuitable recipient for FMT, and he should not immediately undergo FMT due to the high probability of an ineffective outcome. Yet these cases of expected unsuccessful treatment outcome can be readily improved in view of the inventors' discovery.
First, for a patient who has been considered for receiving FMT but who has also been deemed an unsuitable recipient of FMT due to an elevated level of C. albicans (e.g., above 10% of total fungi) found in his/her stool sample, which indicates a diminished chance of a successful FMT, measures can be taken to lower his/her level of C. albicans before FMT is commenced so that a much greater efficacy can be achieved for the FMT procedure. For instance, an antifungal agent capable of suppressing the growth or proliferation of C. albicans can be administered to the patient in an effective amount such that the level of C. albicans in the patient's digestive track and in the feces is significantly reduced (e.g., no more than 10% of total fungi) prior to the start of the FMT procedure. In this case, the patient's C. albicans level is to be determined twice: once at the initial screening stage, a second time after the initial level is deemed too high for an effective FMT and after an antifungal agent has been given to the patient. Once the C. albicans level is confirmed as lowered to a percentage that would allow satisfactory FMT outcome, the patient is then ready to undergo FMT treatment.
Second, for a candidate who has been deemed improper to serve as an FMT donor due to a higher level of C. albicans in his stool, the expected undesirable FMT outcome can be remedied by treating the candidate donor with an effective amount of an antifungal agent capable of suppressing the growth or proliferation of C. albicans can be administered. Since the donor's body, especially the gastrointestinal track, contains a vast collection of microorganisms many of which are important for the health of gut microflora and for the success of FMT, a useful antifungal agent for this purpose cannot be a broad-spectrum fungicide. Rather, it should be an agent that narrowly and precisely targets the species of C. albicans without significantly affecting other fungal species, including those closely related to C. albicans. Although the agent may be of any chemical compound in nature, small polynucleotides (e.g., siRNAs, miRNAs, miniRNAs, lncRNAs, or antisense DNAs/RNAs) may be the most effective in achieving the specific task of disrupting the expression of one or more key genes in the life cycle of C. albicans so as to specifically inhibit the proliferation of the target species only without significant impact on other closely related fungal species.
Immediately upon completion of FMT procedure, the recipient may be further monitored by continuous testing of the level of C. albicans in the stool samples on a daily basis for up to 5 days post-FMT while the clinical symptoms of the condition being treated are also being monitored in order to assess FMT outcome and the corresponding C. albicans level in the recipient.
The present invention also provides novel kits and compositions that can be used for improving FMT efficacy. For example, in a kit for treating a patient in need of FMT, a first composition intended for transplantation into a patient or FMT recipient and a second composition intended to be administered to the recipient for reducing the level of C. albicans in the recipient. The first composition comprises a fecal material from a donor, which has been processed, formulated, and packaged to be in an appropriate form in accordance with the delivery means in the FMT procedure, which may be by direct deposit in the recipient's lower gastrointestinal track (e.g., wet or semi-wet form) or by oral ingestion (e.g., frozen dried encapsulated). The second composition comprises an antifungal agent capable of suppressing the growth/proliferation of C. albicans, which may be a broad-spectrum fungicide or a specific inhibitor of the C. albicans species, and one or more pharmaceutically acceptable excipient. The composition is formulated for the intended delivery method of the antifungal agent, for example, by injection (intravenous, intraperitoneal, intramuscular, or subcutaneous injection) or by oral ingestion or by local deposit (e.g., suppositories). The first and second compositions are often kept separately in two different containers in the kit. Typically, the kit will further include printed material providing detailed instructions for users of the kit, such as providing information of the schedule and dosing arrangement for administering the first and second compositions to a recipient.
In another aspect of this invention, alternative compositions useful in FMT with improved efficacy may be devised to contain at least these two components: (1) a donor stool material containing live fecal microorganisms, and (2) an antifungal agent that specifically suppresses the growth or proliferation of C. albicans but exhibits no such suppressive or inhibitory effect against other fungal species. Component (2) preferably is not a broad-spectrum fungicide; rather, it should be a specific anti-C. albicans agent. For example, it may be short polynucleotide in nature of, e.g., a small inhibitory RNA, microRNA, miniRNA, lncRNA, or an antisense oligonucleotide, that is capable of disrupting the expression of at least one key gene in the life cycle of C. albicans, such that the agent is capable of specifically targeting the species only without significantly affecting other closely related fungal species. Component (2) is particularly useful in the case of a donor's stool containing a level of C. albicans too high to permit a satisfactory FMT outcome, as it is capable of locally and specifically suppressing the proliferation of C. albincans so as to ensure the success of FMT despite the less than desirable quality of the donor fecal material.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.
Fecal microbiota transplantation (FMT) is effective in treating recurrent Clostridium difficile infection (CDI) and is increasingly being utilized in other human diseases. Whilst bacteria colonization in recipients after FMT has been established, little is known of the role of the gut mycobiota. In this study the present inventors show gut fungal dysbiosis in CDI and identify that donor-derived fungi colonization in recipient is associated with FMT response. Mycobiota profiling in CDI reveals over-presentation of C. albicans and decreased fungal diversity, richness and evenness compared with healthy controls. Cure after FMT was observed when donor-derived fungal taxa predominated in recipients' mycobiota. FMT responders display a high prevalence of Saccharomyces and Aspergillus whilst non-responders and individuals treated with antibiotics display a dominant presence of Candida. High abundance of C. albicans in recipient before FMT and in donor stool nullifies FMT efficacy in eradicating CDI. Furthermore, C. albicans compromises FMT efficacy in a mouse model of CDI, while anti-fungal treatment reestablishes its efficacy. This study furthers the knowledge of human gut mycobiota dynamics and their contribution to FMT, and it enables an understanding of personalized donor-recipient selection in future FMT studies for various human diseases.
The past decade has witnessed an increasing use of fecal microbiota transplantation (FMT) as a promising treatment option for several diseases 1-3, yet success rates are variable with a cure rate of 85-90% in recurrent Clostridium difficile infections (CDI)3-5 and a response rate of 30-40% in inflammatory bowel disease6-8. Such variations may be related to disease traits, recipient factors or donor characteristics. The mechanisms underlying a successful FMT and its relationship with gut microbial profiles in donor-recipient pairs remain elusive. To date, the efficacy of FMT has been mostly ascribed to the restoration of the bacterial microbiota and a sustained co-existence of donor and recipient bacterial strains9-12. Recently, bacteriophages have been shown to be altered in CDI after FMT and these changes were associated with treatment outcome 13-15. The human gastrointestinal tract is also colonized by a large population of fungi, collectively referred to as the mycobiota, which play an important role in human health16,17. Gut mycobiota contribute to normal human physiology and in some cases can recapitulate the benefit of intestinal bacteria via regulating host immunity and maintaining intestinal homeostasis16,17,18. Whether donor-derived mycobiota can colonize a recipient host, the fate of donor and recipient mycobiota after FMT and their relationship with treatment outcomes are unknown. The inventors performed internal transcribed spacer 2 (ITS2) and 16S rDNA sequencing in FMT-treated subjects with CDI to explore the effects of FMT on the gut mycobiome in association with treatment outcome. A proof-of-causality study was conducted in C. difficile-infected mice to confirm the role of gut mycobiota in FMT response.
The fecal mycobiomes were compared between 31 CDI subjects and 23 healthy controls. There was a significant decrease in fungal diversity, evenness and richness in CDI compared with controls (Mann-Whitney test, p=0.0120, 0.0309, and 0.0043, respectively,
Over-presentation of C. albicans in absolute abundance in CDI was confirmed through quantitative PCR (
Donor fungi colonization in recipient is associated with FMT response
It was then explored whether FMT leads to colonization of donor-derived fungi and its association with treatment efficacy. Changes in the gut mycobiomes of recipients after FMT were monitored at multiple time points in 16 CDI subjects, using pre-FMT samples of each donor-recipient pair as a baseline for FMT (
FMT Alters the Gut Mycobiota Distinct from Antibiotic Treatment
CDI subjects who responded to FMT showed a significant increase in fungal richness and diversity (Wilcoxon matched-pairs singed rank test, p=0.0273 and p=0.0474 respectively,
C. albicans markedly decreased after FMT (Wilcoxon matched-pairs singed rank test, p=0.0458,
The effect of antibiotics on the gut mycobiota was also assessed across longitudinal time-points in 8 CDI subjects treated with vancomycin (STD treatment,
FMT and vancomycin led to an increase in the gut fungal diversity in 81.3% (13 out of 16) and 37.5% (3 out of 8) of CDI subjects, respectively (Chi-square test p=0.032,
Taxonomical analysis was performed to further elaborate the effect of antibiotics on the fungal community and to discern differences between FMT and antibiotics in modulating the gut mycobiota. After vancomycin treatment, fungal compositions exhibited similar configurations during follow-up across STD subjects, with a marked expansion of the genus Candida (
Trans-kingdom Interactions Between Gut Mycobiota and Bacterial Microbiota are Associated with Treatment Outcome
To characterize the ecological network of the gut mycobiota and bacterial microbiota, the correlation of the a-diversity (diversity, evenness and richness) of the fungal community with that of the bacterial community was evaluated. Among the post-treatment samples of FMT responders, significant positive correlations were found between fungal diversity and bacterial diversity, and between fungal richness and bacterial diversity, evenness, and richness (Spearman's correlation, permutation test, P<0.05,
C. albicans Compromises FMT Efficacy in a Murine Model of CDI
C. albicans was the most prominent species associated with treatment failure of FMT in CDI, suggesting a possible causal relationship. This assumption is further supported by reports whereby CDI recurrence was observed after antibiotics treatment3,19, as antibiotics contribute to the expansion of Candida. To determine the causal relationship between C. albicans and response to FMT, the efficacy of FMT in eliminating C. difficile was assessed using a C. difficile induced-diarrhea murine model in three groups of mice: (i) mice infused with human stool preparation, (ii) mice colonized with C. albicans then infused with human stool preparation, and (iii) mice infused with human stool preparation supplemented with C. albicans during fecal transplantation (
The total fecal fungal load was significantly higher in CDI than in controls [Mann-Whitney test, p=0.0004, Loglog transformed effect size 1.32 (95% CI: 0.62-1.97), FIG.17].
Total Fungal Load and C. albicans in Inflammatory Bowel Disease (IBD)
The total fecal fungal load was significantly higher in patients with IBD, including patients with Crohn's disease (CD) and Ulcerative colitis (UC)—two subtypes of IBD, than in controls (Mann-Whitney test, p=0.0003, p=0.0225, respectively,
The total fecal fungal load was significantly higher in IBS than in controls [Mann-Whitney test, p=0.0237, FIG.19].
Bacterial Alterations in CDI after FMT in Association with FMT Outcome
The present inventors explored the composition of the bacterial microbiota after FMT in relation to FMT outcomes, at various taxonomic levels (
This is the first study to characterize the gut mycobiota in CDI and to elucidate mycobiota alterations after FMT in relation to treatment outcome. Patients with CDI showed enteric fungal dysbiosis. Importantly, disease recurrence after FMT was associated with several important findings including persistent fungal dysbiosis, low levels of donor-derived fungal colonization, high abundance of C. albicans in the recipient stool before FMT and the presence of C. albicans in the donor stool. The observations that disease cure requires both fungal and bacterial colonization from the donor provides new and important insights into the potential therapeutic importance of the gut mycobiota in treatment outcome in FMT, beyond the bacterial microbiota. These data also highlight a new concept in FMT that the abundance of fecal C. albicans both in recipient before treatment and in donor are critical components when considering implementation of FMT. Integration of more in-depth mycobiota analysis in donor-recipient pair may lead to personalized and targeted gut microbial therapy in the future.
Although studies of the gut mycobiota have lagged behind that of the gut bacterial microbiota, fungi are increasingly being considered as important players of the gut and interactions between pathogenic and commensal fungal and bacterial communities are crucial in the maintenance of human health and disease pathogenesis16. Furthermore, disruption of the gut mycobiota has deleterious effect on host immunity17. Despite high interpersonal variability of the gut mycobiota in patients with CDI, there was a significant expansion of the genus Candida and the species C. albicans. Interestingly, FMT induced an increase in the genus Saccharomyces along with a marked contraction of Candida and C. albicans after FMT in treatment responders, while recipients who demonstrated high abundance of C. albicans in the stool after FMT (10%) or those whose donor had a high abundance of (0.1%) of C. albicans were more likely to have disease recurrence after FMT. Over-presentation of C. albicans in the recipient stool after FMT may largely contribute to treatment failure.
The role of fungal commensals in educating the human immune system has gained new appreciation in intestinal disease. In the steady state, bacterial communities keep fungi in check in the gut. Fungi are major causes of infections among immunocompromised or hospitalized patients with serious underlying diseases and comorbidities. Candida species remain the most important cause of opportunistic infections worldwide, affecting predominantly elderly patients20. Candidalysin was recently unveiled as a fungal toxin from C. albicans critical for mucosal infection21. Commensal bacteria inhibit C. albicans colonization through activation of HIF-1α and LL-3722. Antibiotic treatment selectively and effectively eradicates the bacterial community but consequently leads to fungal outgrowth, particularly the Candida species23,24. Antibiotics or immunosuppressants are effective in the short term but they likely compromise the immune system in the longer term. A compromised immune system creates a more favourable environment to expansion of Candida and overgrowth of Candida can alter the recovery of the gut bacterial microbiota after cessation of antibiotic treatment 25,26. In this study, the over-presence of Candida species in recipients might account for the high failure rate of FMT in CDI. In DSS-induced colitis mouse model as well as patients with inflammatory bowel disease (IBD), C. albicans and Candida were significantly enriched27-29. Antifungal treatment decreased Candida prevalence and ameliorated inflammatory responses in DSS colitis mice29. However, disruption of fungal communities by long-term use of antifungals aggravated severity of DSS colitis and allergic airway30. Collectively, these data implicate the importance of the gut fungal-bacterial homeostasis in host health. These data suggest that the establishment of a balanced gut fungal and bacterial community via FMT is important to eradicate CDI, as FMT non-responders showed abrogated fungi-bacteria correlations in a-diversity and taxa when compared with responders.
In conclusion, gut mycobiota alterations may determine treatment outcome in FMT. The persistence of fungal dysbiosis, particularly the presence of C. albicans, can incur CDI recurrence. The findings disclosed herein highlight the importance of both “optimal” donor selection and pre-FMT eradication of C. albicans in recipient during FMT practice, where future FMT therapy should incorporate detailed characterization and stratification of both donor and recipient fecal mycobiomes. These results provide a framework for future investigations into the contribution of donor/recipient mycobiota profiles and gut fungi-bacteria interactions in FMT treatment for various human diseases.
Study subjects and treatment outcome
The current study was a sub-study from a randomised controlled trial (RCT) of FMT versus vancomycin (standard therapy, STD) for patients with CDI. Consecutive CDI subjects enrolled into this randomised controlled trial were invited to participate in a substudy of assessment of fecal microbiota. Patients were included if they had three or more loose or watery stools per day for at least two consecutive days or eight or more soft or loose stools in 48 hours and a positive stool test for C. difficile based on a two-step testing algorithm in our hospital, a positive GDH (Glutamate dehydrogenase) screening test followed by a positive polymerase chain reaction (PCR) test of C. difficile. A total of 31 subjects with CDI and 24 healthy household controls were recruited and stool samples at baseline were obtained for analyses of fungal and bacterial microbiomes. Among them, 24 CDI subjects consented to have stool samples collected serially after treatment for microbiome analysis. 16 CDI subjects were treated with FMT and 8 were treated with vancomycin, and they were followed up at baseline and at weeks 2, 4, 10 and 16 after treatment (
Treatment response was defined as an absence of diarrhea or persistent diarrhea that could be explained by other causes with a negative stool test for C. difficile toxin, while relapse was defined as diarrhea with a positive stool test for C. difficile toxin. Treatment cure is defined as symptom resolution and a negative Clostridium difficile toxin in stool until the last follow-up (last follow-up is referred to as the last stool collection time point, as shown in
Study design
Patient inclusion criteria:
1. C. difficile infection was defined as diarrhea (≥3 soft, loose or watery stools per day for at least 2 consecutive days or ≥8 soft or loose stools in 48 hours) and a positive stool test for C. difficile toxin; and
2. Age≥18; and
3. Written informed consent obtained
Patient exclusion criteria:
1. The presence of human immunodeficiency virus (HIV) infection with a CD4 count of less than 240
2. Pregnancy
3. GI Bleeding
4. Acute coronary syndrome
Donor screening:
Donors included individuals who are spouses or partners, first-degree relatives, other relatives, friends, and individuals unknown to the patient. They were screened with a questionnaire and excluded if they had taken antibiotics within the preceding 3 months; were on major immunosuppressive agents, including chemotherapeutic agents; had known or recent exposure to HIV, hepatitis B or C; had a current communicable disease; participated in high-risk sexual behaviors; used illicit drugs; traveled within 6 months to areas with endemic diarrheal illnesses; or had history of inflammatory bowel disease, irritable bowel syndrome or chronic diarrhea, gastrointestinal malignancy or polyposis. In addition, donor was screened for HBsurface Ag, Anti-HBc, Anti-HCV, Anti-HIV, Syphilis EIA, stool microscopy, culture and sensitivity, stool cyst, ova, parasite, norovirus and C. difficile (cytotoxin and PCR assay). All subjects and collected stool samples are listed in Table 1.
The donors for the FMT group were healthy household controls and the donor stool samples analyzed were the same samples used for FMT. All subjects provided written informed consent.
Family members provided donor stool for subjects randomised to FMT arm. Cure after FMT or vancomycin therapy was defined as symptom resolution and negative Clostridium difficile toxin in stool at last follow-up by PCR assay. Relapse was defined as diarrhea with a positive stool test for C. difficile toxin.
This was a randomised but not blinded study. However for mycobiome and bacterial microbiome analyses on stool samples, assessments were initially performed by analysts who were blinded to the clinical outcome of the studied subjects. When the profiled mycobiome and bacterial microbiome data were available for each individual subject, correlation was then made to associate microbiome profiles with treatment outcomes of subjects.
In subjects who received FMT, a nasoduodenal tube was inserted with radiology guidance. Donor feces was diluted with 500 ml of sterile saline (0.9%), blended and the supernatant was strained with filter paper and poured in a sterile bottle. Within 6 hours after collection of feces by the donor, the solution was infused through a nasoduodenal tube (2 to 3 minutes per 50 ml). The tube was removed 30 minutes after the infusion, and patients were monitored for 2 hours. In subjects with received FMT, a minimum of 50g of donor stool was collected on the same day of infusion and used within 6 hours of collection.
Fecal DNA was isolated as described below. 100 mg fecal sample was pre-washed with 1 ml ddH2O and pelleted by centrifugation at 10,000×g for 1 minute. The fecal pellet was re-suspended in 800 μl TE buffer (pH 7.5), supplemented with 1.6 μl 2-Mercaptoethanol and 500 U lyticase (Sigma), and incubated at 37° C. for 60 min. The sample was then centrifuged at 10,000×g for 2 minutes and fecal DNA was subsequently extracted from the pellet using ZR Fecal DNA miniPrep kit (Zymo Research, Orange, Calif.) according to the protocol. Briefly, fecal pellet was added to the BashingBeadLysis Tube with 750 μl Lysis solution, and then processed at maximum speed for 10 minutes. The lysates were centrifugeed at ≥10,000×g for 1 minute. The supernatant was transferred to a Zymo-Spin™ IV Spin Filter in a collection tube and centrifuged at 7,000×g for 1 minute. About 1,200 μl of fecal DNA binding buffer was added to the filtrate in the collection tube, followed by concentration and purification in a new filter tube. Finally, a total of 50 μl eluted DNA with a concentration at 20-100 ng/μl was prepared for each sample.
The final fecal DNA for fungal sequencing was amplified based upon ITS2 (Internal transcribed spacer 2) region using primers as below and PrimeSTAR HS DNA Polymerase kit (TaKaRa, Japan). The primer pairs are ITS2-F: 5′-GCATCGATGAAGAACGCAGC-3′, ITS2-R: 5′-TCCTCCGCTTATTGATATGC-3′. ITS2 amplicons were generated with 38 cycles of 3-step PCR: 98° C. 10 s, 59° C. 10 s, and 72° C. 30 s. PCR samples were then sequenced on the Illumina MiSeq PE300 platform (2×300 bp, BGI, China), 151,524±97,694 (number±SD) clean sequences obtained on average (sequence statistics in Table 4).
Raw reads were filtered by SOAPnuke (v 1.5.3) (web site: soap.genomics.org.cn/) developed by BGI as follows: (i) adaptors removed, (ii) read removed if N base is more than 3% of the read, (iii) read removed if bases with quality low than 20 were more than 40% of read, (iv) all duplicates removed. Quality control and data analysis were further implemented in PIPITS (v 1.4.5)31. Briefly, PIPITS_PREP prepares raw reads from Illumina MiSeq sequencers for ITS extraction; PIPITS_FUNITS extracts ITS2 from the reads; and PIPITS_PROCESS analyses the reads to produce operational taxonomic unit (OTU) abundance tables and the RDP taxonomic assignment table for downstream analysis. The quality trimmed and ITS2 extracted reads were aligned to fungi UNITE database exploiting RDP classifier 2.10 for taxonomic assignment to produce operational taxonomic unit (OTU) abundance table (based on sequence identity≥97% identity) and phylotype abundance tables at different taxonomic levels, for downstream analysis.
The fungal OTU and phylptype abundance data were imported into R 3.2.3. Richness, diversity, and evenness calculation were performed using the estimated richness function of the phyloseq package. Spearman correlation and their significance were calculated using the cor and cor.test functions in R, respectively. For the fungal-bacterial taxa comparisons, Spearman correlations were calculated for the relative abundance of the differentially presented fungal taxa and the bacterial taxa determined to be significantly associated with disease by Lefse analysis. Correlation plots were generated using the corrplot package in R. Heat maps were generated using the pheatmap package in R.
Quantitative PCR for Detection of C. albicans in Human Fecal DNA Samples
C. albicans loads in human stools were quantified by qPCR analysis (SsoAdvanced SYBR Green Supermix, Bio-Rad) of extracted human fecal DNA using C. albicans specific primers: C. albicans-F 5′-CCTGTTTGAGCGTCGTTTCTC-3′; C. albicans-R 5′-TTTGGTTAGACCTAAGCCATTGTCA-3′. C. albicans abundance was determined using standard curves constructed with reference genomic DNA (gDNA) of C. albicans.
Total fungal loads in human stools were quantified by TaqMan qPCR analysis (Premix Ex Taq™, TaKaRa) of extracted human fecal DNA using primers36: Fungi-quant-F 5′-GGRAAACTCACCAGGTCCAG-3′; Fungi-quant-R 5′-GSWCTATCCCCAKCACGA-3′, and probe: 5′-TGGTGCATGGCCGTT-3′.
To compare differences in the configurations of fungal and bacterial microbiomes between CDI patients and healthy controls, between FMT responders and non-responders, between FMT treatment and vancomycin (STD) treatment, Lefse analyses were performed on the Huttenhower lab Galaxy server (web site: huttenhower.sph.harvard.edu/galaxy/) by importing the viral and bacterial relative abundance values and associated sample metadata, with FDR adjusted p value<0.05 considered significant and effect size calculated.
In samples after FMT, if a fungal or bacterial OTU was not present in the recipient baseline sample but present both in the corresponding donor baseline sample and in the recipient post-FMT sample, the OTU was defined as “donor derived”; if an OTU was not present in the corresponding donor baseline sample but detected both in the recipient baseline sample and in the recipient post-FMT sample, the OTU was defined as “recipient exclusive”; if an OTU was present across the recipient baseline sample, the recipient post-FMT sample and the corresponding donor baseline sample, the OTU was defined as “donor-recipient co-existed.”
Bacterial 16S rRNA Sequencing and Data Analysis
The final fecal DNA samples were subject to bacterial 16S rRNA V4 region amplification and sequenced on the Illumina MiSeq PE250 platform (2×250 bp, BGI, China), 132,081±65,429 (number±SD) sequences obtained on average (sequence statistics in Table 5). Quality control and data analysis were implemented in mothur (v 1.38.0) as previously described32. Any sequences with ambiguous bases and anything longer than 275 bp were removed, and aligned against the non-redundant Greengenes database (v 13.8)33 using the NAST algorithm. Any sequences that failed to align with the V3-4 region were discarded. The remaining sequences were trimmed to the same alignment coordinates over which they fully overlapped, followed by removal of homopolymers and detection for the presence of chimeras by UChime.
The resulting sequences were classified against the Greengenes database and annotated with deepest level taxa represented by pseudo-bootstrap confidence scores of at least 80% averaged over 1,000 iterations of the naive Bayesian classifier. Any sequences that were classified as either being originated from archaea, eukarya, chloroplasts, mitochondria, or unknown kingdoms, were removed. The annotated sequences were assigned to phylotypes according to their consensus taxonomy with which at least 80% of the sequences agreed. Closed reference operational taxonomic units (OTUs) sharing 97% identity were clustered as well and assigned taxonomy according to the Greengenes database. Lefse analysis was performed to define bacterial taxa associated with CDI and healthy controls. The relative abundance of these abundance-differential taxa identified by LefSe in pre-FMT baseline samples and post-FMT last follow-up samples were plotted using pheatmap R package.
Mouse Husbandry and Model of C. difficile Infection
Studies were conducted on 4- to 6-week old demale C57BL/6 that were reared in groups of 9. Individual mice were randomized after arrival. Mice were subjected to a previously described model of CDI34. Briefly, mice were given an antibiotic cocktail of kanamycin (0.4 mg/mL), gentamicin (0.035 mg/mL), colistin (850 U/mL), metronidazole (0.215 mg/mL), and vancomycin (0.045 mg/mL) (all antibiotics were purchased from Sigma-Aldrich, St. Louis, Mo.) in their drinking water for 3 days. Mice were then given 2 days of recovery before administration of 107 spores of C. difficile in PBS via oral gavage. Animal grouping and research scheme were designed as shown in
For antifungal experiment, animal grouping and research scheme were designed as shown in
C. albicans Administration and Donor Stool Infusion in Mice
C. albicans (10231, purchased from ATCC, USA) was administered to mice (2×108 cfu per mouse) via gavage after 3-day antibiotic treatment or supplemented in donor stool slurry at the time of donor stool infusion. Human stool from a healthy volunteer (Chinese, male, age 28 years), without presence of C. albicans as measured by qPCR, was obtained with informed consent. For stool microbiota infusions, approximately 500 mg of stool samples were cut in an anaerobic chamber and suspended in 5 ml of phosphate-buffered saline. Mice were colonized by oral gavage of 150 μl of fecal slurry with or without supplementation of C. albicans on day 2 after C. difficile challenge.
Quantification of C. difficile and C. albicans Burdens in Mouse Feces
Mouse stool were collected both before and after stool infusion. Fecal C. difficile and C. albicans burdens on day 0 before and day 1 after stool infusion were measured by cultivation. Samples were diluted in PBS and respectively plated on taurocholate cycloserine cefoxitin fructose agar (TCCFA) for quantification of C. difficile burden, on Sabouraud dextrose agar (SDA) for quantification of C. albicans load. Stool samples prior to C. albicans colonization from antibiotic-treated mice were plated on SDA to ensure that mice were C. albicans culture negative.
Sequence data and accompanying metadata have been deposited to the NCBI Sequence Read Archive under BioProject accession numbers PRJNA419097 and PRJNA419104.
The fecal C. albicans level was investigated in three IBD patients with concurrent CDI, and subsequently followed them up after FMT (
All patents, patent applications, and other publications, including GenBank Accession Numbers, cited in this application are incorporated by reference in the entirety for all purposes.
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This application claims priority to U.S. Provisional Patent Application No. 62/625,705, filed Feb. 2, 2018, and U.S. Provisional Patent Application No. 62/679,417, filed Jun. 1, 2018, the contents of both are hereby incorporated by reference in the entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/074353 | 2/1/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62625705 | Feb 2018 | US | |
| 62679417 | Jun 2018 | US |
