Patent Application
20230310518
The mammalian gastrointestinal (GI) tract harbors a diverse microbial community that is usually maintained in symbiotic balance. Interactions between microbes within the microbial populations, and between the microbes and the host, affect both the host and the internal microbial community. In some individuals, this symbiotic balance is disrupted. This state can lead to increased susceptibility to pathogens and the development of disease. One such disease, Clostridioides difficile infection (CDI) is the leading cause of health care associated diarrhea, with approximately a half million cases and 29,000 deaths in the United States. CDI is associated with antibiotic-induced dysbiosis, and treatment typically consists of terminating administration of the antibiotic followed by antimicrobial therapy.
Recurrent Clostridioides difficile infection (rCDI) describes a clinical condition where Clostridioides difficile bacterial infections recur in a single patient after treatment for the original infection. Fecal Microbiota Transplantation (FMT) has been widely used therapeutically for recurrent rCDI, since its superiority to vancomycin was demonstrated. (See, e.g., Ooijevaar, R. E., et al., Annu. Rev. Med. 70, 335-351 (2019)). With no FDA-approved drug, FMT is currently largely used under enforcement discretion in the USA. Although thousands of FMTs have been conducted over the last decade, many questions remain about the efficacy of different FMT formulations and the reasons for the long-term success or failures of different formulations. Open questions include, for example, identifying which FMT donor strains engraft in recipients, whether any FMT strains last beyond days or months, identifying the proportion of donor, recipient and environmental strains that ultimately survive, and how these different factors affect relapse, if at all.
A significant impediment to answering the above questions is the ability to and need for obtaining strain level resolution of the microbiome of the human gut. Previous microbiome analyses utilized a level of resolution that was incapable of delineating bacterial strains within a particular species. See, e.g., Knight, R. et al., Nat. Rev. Microbiol. 16, 410-422 (2018). Pure metagenomics approaches, meanwhile, require very deep sequencing to track strains via SNPs in marker genes, do not model the microbiota as a defined set of discrete strains, and primarily provide non-quantifiable inferences related to sharing of metagenome-assembled bacterial contigs or SNPs across FMT samples. See, e.g., Olm, M. R. et al., Nat. Biotechnol., 1-10 (2021). A higher level of resolution is required to determine the efficacy of any FMT formulation and its ultimate impact on the host.
In addition, recent FDA advisories have documented adverse events associated with FMT and have raised safety concerns about using FMT formulations that contain whole stool material. Moreover, FMT formulations are undefined, contain hundreds of strains, and can include both beneficial and potentially harmful microbes (including antibiotic resistant strains). A goal in the field is to generate a defined cocktail of microbes with demonstrated safety and efficacy that can be used instead of FMT to treat conditions such as rCDI. Another goal is to achieve consistent strain level monitoring methodologies that can be used to track disease and treatment efficacy.
The present disclosure provides for the first-time compositions for use in treating Clostridioides difficile infections, including for treating recurrent CDI, in the form of a Live Biotherapeutic Product (LBP).
The LBP of the present disclosure contains a live, cultured bacterial composition for engraftment into human patients suffering from gastrointestinal disorders, particularly Clostridioides difficile infections.
The LBP of the present disclosure contains FMT donor strains: that have been isolated and purified; that engraft consistently into recipient gut microbiotas.
The LBP of the present disclosure includes: live bacterial strains that have been isolated, purified and cultured; that engraft consistently into recipients; and that are susceptible to treatment with multiple antibiotic classes.
The LBP of the present disclosure includes: live bacterial strains that have been isolated, purified and cultured; that engraft consistently into recipients; that are susceptible to treatment with multiple antibiotic classes; and where none of the strains is resistant to any of the last line of antibiotics.
The present disclosure provides a composition comprising a formulation of bacterial strains for treating diseases, disorders, or maladies of the human gastrointestinal tract, wherein the formulation comprises a mixture of isolated, cultured bacteria selected from the group consisting of: Bacteroides ovatus; Bacteroides vulgatus; Bifidobacterium longum; Bacteroides uniformis; Bacteroides thetaiotaomicron; Ruminococcus obeum; Parabacteroides distasonis; Coprococcus comes; Bacteroides fragilis; Dorea longicatena; Parabacteroides merdae; Bacteroides cellulosilyticus’; Bifidobacterium pseudocatenulatum; Odoribacter splanchnicus; Ruminococcus torques; Bacteroides caccae; Alistipes putredinis; Alistipes onderdonkii; Eubacterium rectale; Collinsella aerofaciens; Blautia massiliensis; Bacteroides stercoris; Barnesiella intestinihominis; Alistipes senegalensis; Bifidobacterium adolescentis; Eggerthella lenta; Clostridium ramosum; Bifidobacterium bifidum; Clostridium leptum; Streptococcus parasanguinis; Eubacterium siraeum; Streptococcus salivarius; Roseburia faecis; Bacteroides intestinalis; Escherichia coli; Bacteroides clarus; Bacteroides xylanisolvens; Parabacteroides johnsonii; Anaerotruncus colihominis; Bacteroides massiliensis; and Alistipes shahii.
The present disclosure also provides for the first-time a high throughput hybrid approach for identifying bacterial strains in the microbial genome of a subject. The method involves collecting comprehensive cultures of bacterial strains from FMT donors or recipients and tracking the composition of the cultures across metagenomic samples using computational analysis and comparing the genomic results to reference sequences of the cultured strains.
The present disclosure fulfills the abovementioned needs by identifying for the first time a Live Biotherapeutic Product (LBP), which includes a defined sample of bacterial strains that are effective in treating gut disorders and in generating a durable, long-term change to the recipient’s microbiome following a single administration. The present disclosure also provides methods for treating rCDI patients by quantifying the efficacy and long-term stability of FMT and LBP strains engrafted into patients with rCDI and modifying patient treatment accordingly.
In the present description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the present subject matter. Aspects of the present disclosure, including the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are contemplated herein.
References in the specification to “one embodiment”, “an embodiment”, “an example embodiment” or “some embodiments,” etc. indicate that the embodiments described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, such feature, structure, or characteristic may be effected in connection with other embodiments whether or not explicitly described.
The term “Live Biotherapeutic Product” or “LBP” as used herein refers to a composition containing a defined population of isolated, purified, and cultured bacterial strains that are effective for treating disorders of the gastrointestinal tract, particularly Clostridioides difficile infections, including rCDI. The population of bacteria in the LBP are susceptible to at least two different classes of antibiotics and can be sensitively and precisely detected in the recipient.
The term “Clostridioides difficile infection” or “rCDI” as used herein, refers to a clinical situation where a patient is diagnosed with a Clostridioides difficile infection, which has been clinically identified by symptoms, usually diarrhea, and a positive assay result for C. difficile toxin or detection of a toxin-producing C. difficile strain. The term “recurrent Clostridioides difficile infection” or “rCDI” is defined by resolution of CDI symptoms while on appropriate CDI therapy, followed by reappearance of symptoms within two to eight weeks after treatment has been stopped.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, the term “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.
The below examples provide specific embodiments. The specific embodiments show exemplary compositions that can be made according to the teachings contained herein. The specific embodiments also show methods for staging, treating, and tracking the progression of treatment for rCDI that can be accomplished using the teachings herein. The use of these specific examples, however, is not intended to be limiting
The inventors isolated and sequenced the largest collection of 2,987 bacterial isolates representing 1,008 unique strains (207 species) from 9 FMT healthy donors and 13 rCDI FMT recipients (Table 1). Similar to previous analyses performed by the inventors, bacterial isolates with <96% whole genome similarity were defined as unique strains, otherwise they were considered as multiple isolates for the representative strain.
Seven donors provided their fecal material for FMT to 13 patients with either rCDI or both rCDI and IBD. Fecal metagenomics was performed on all stool samples. Donor strains from all the donors were isolated and tracked in matching recipient metagenomes over time. Strains were also isolated from a few recipients both pre- and post-FMT. M and C indicate that metagenomics or culturing respectively were performed at an indicated time point. The underline highlight denotes that a sample was collected after repeat FMT (due to initial failure of FMT). Success indicates that no relapse was noted for that patient.
The inventors sequenced 85 metagenomes from donor fecal samples used for the transplant, and recipient samples taken prior to and for up to 5 years after FMT. The cultured strains represented the majority of the metagenome with 70% (sd =16%) of bacterial metagenomic reads mapping to the cultured strain genomes (
The present disclosure overcomes one of the central challenges behind strain tracking from metagenomics data, i.e., the identification of a set of informative sequence features or k-mers from the bacterial genome that can uniquely identify a given strain. Bacterial species often contain numerous closely related distinct strains that share a majority of their genomic content (
The inventors first confirmed the ability of Strainer to accurately detect the bacterial strains in gnotobiotic mice sequentially gavaged with defined culture collections of bacteria isolated from 3 different human fecal samples and a subset of 10 unique strains of the common human gut commensal bacterium Bacteroides ovatus (
In the present disclosure, the dataset of strains isolated from matched and metagenomically sequenced FMT samples provides for the first time an in vivo experimental benchmark for rigorous comparison of SNP based inference approaches for tracking SNP strain proxies in metagenomics. The inventors tested the previously published Strain Finder, ConStrains and inStrain algorithms on the present gnotobiotic mice dataset. These SNP proxy algorithms, that were developed on synthetic and in vitro datasets, are inferior to the present disclosure because these proxy algorithms must first infer the strains from the metagenomes themselves. Any strain not inferred leads to false negatives across the dataset, and any strain incorrectly inferred propagates false positives in any sample where it is falsely detected. An illustration of the difference between the SNP proxy algorithms and the present disclosure can be seen by comparing the relative abilities of the different algorithms to estimate the correct number of B. ovatus strains in each mouse. Each of the SNP proxy algorithms struggled to do so; however, the ability of the Strainer algorithm of the present disclosure to detect the correct number of strains generated results that were in line with the actual number of B. ovatus strains gavaged into the mice (
Confirmation of the difference in performance between the proxy algorithms and Strainer was obtained by examining whether the inferred strains match those gavaged to the germ-free mice. To make the comparison, the inventors provided the raw unassembled sequencing reads (~2.1 M) for every strain (from its pure culture) as a distinct metagenomic “truth” sample and examined whether any of the algorithms could match the unassembled strain reads with the correct, corresponding metagenomics sample. None of the SNP proxy algorithms was able to do so (
To evaluate Strainer further, the inventors tested it in the context of several complex human gut microbiota communities with high species overlap but little to no strain overlap. This is a representative model for the use-case application for FMT where a potentially transmitted bacterial strain must be precisely detected across multiple individuals, while differentiating it from other related commensal strains from the same species. The inventors sequenced the fecal metagenome of 10 unrelated individuals as well as the genome of 261 bacterial strains isolated from the same fecal samples and then evaluated the ability of Strainer to detect these strains in the correct individual’s metagenome, while not falsely detecting it in the other nine other samples.
With 10 M metagenomic reads per sample, the inventors reached a precision of 93.9% at a recall of 72.4% with an AUC of 0.86 (
(A) Strainer can accurately detect the correct Bacteriodes ovatus strain(s) in gnotobiotic mice, from other closely related strains. Each column represents an independent germ-free mouse gavaged with the specific B. ovatus strain(s) with or without a diverse human gut bacterial culture library of strains. Strains F and G were contained in human culture library 1 and 2 respectively. Human culture library 3 contained no B. ovatus, while the remaining B. ovatus isolates were isolated from other human fecal samples. Green box indicates the strain was introduced in the mice and detected in metagenomics (true positive), Grey indicates the strain was not detected and (true negative), Orange indicates the strain was detected but was not introduced (false positive) and Yellow indicates the strain was not detected but was gavaged in the mice (unknown as gavaging a strain does not always lead to stable colonization).
(B) Performance of SNP-inference based strain detection algorithms, ConStrains, Strain Finder, inStrain and our Strainer approach on detecting the number of Bacteriodes ovatus strain(s) in gnotobiotic mice.
(C) Precision-Recall curves to assess the performance of SNP-inference based strain tracking approaches and Strainer on real datasets ranging from sequential gavaging of a defined set of strains in gnotobiotic mice, FMT donor recipient pairs, and tracking the strain stability in a healthy individual over time.
(D) Performance assessment of Strainer’s ability to match strains to the metagenome of the sample from which they were isolated. Solid lines denote the results at different sequencing depth after application of our algorithm on 261 strains isolated from healthy controls (HC). The color blue indicates the sequencing depth of 2.5 M reads, while the dashed line indicates the result after application of Strainer on 56 strains isolated from patients with rCDI and the dotted curve is for 54 strains from patients with IBD. AUC of the Precision-Recall curves is in the legend box.
(A) Proportion of bacterial reads in the metagenomics sample that are explained by the genome sequences of the cultured strain library for that sample. Each point in the boxplot corresponds to a separate sample.
(B) Proportion of bacterial reads explained by the cultured strain library for a donor after gavaging (n = 3) germ-free mice with stool from (n = 3) corresponding human donors and performing metagenomics on the mouse fecal samples. Each point corresponds to a separate sample.
(C) Percentage similarity between 96 different isolates of species Bacteriodes ovatus and the reference strain AAXF00000000.2. Similarity is found by comparing sequence k-mers of length 31 between genomes.
(D) Proportion of bacterial reads in the metagenomics sample that are explained by the genome sequences of the cultured strain library for that sample. Each point in the boxplot corresponds to a separate sample.
(E) Overview of our algorithm Strainer.
The algorithm has 3 modules, where Module-1 involves finding the unique and likely informative sequence k-mers for each strain by removing those shared extensively with unrelated sequenced strains in NCBI, unrelated metagenomics samples, and those cultured and sequenced in this study. Next, the inventors decompose each sequencing read in the metagenomics sample of interest into its k-mers, and find reads that have k-mers belonging to multiple strains, or have <95% of informative k-mers for a single strain. The inventors further remove these non-informative k-mers from the previous set. In Module-2, the inventors assign sequencing reads from the metagenomics sample of interest, with a majority of informative k-mers (>95%) to each strain. Next, the inventors map these reads to the genome of the corresponding strain, and consider the non-overlapping ones only. This step normalizes for sequencing depth across samples and checks for evenness of read distribution across the bacterial genome. Finally, in Module-3 the inventors compare the read enrichment in a sample to unrelated samples or negative controls and present summary statistics for presence or absence of a strain in a sample.
Engraftment in FMT recipients: In the clinical cohort, seven FMT donors each provided their sample to a single recipient (which was sampled at multiple timepoints post-FMT), while one donor provided the sample to seven different patients (Table 1,
Previous FMT approaches demonstrated sharing of microbiota between the donor and the recipient post-FMT, but none has demonstrated precise quantification of engraftment. The inventors used Strainer to measure the engraftment of donor strains in the recipients and defined the Proportional Engraftment of Donor strains (PED) metric as the number of donor strains detected in a recipient post-FMT divided by total number of strains isolated from the donor. The inventors tracked 10 non-relapsing recipients for up to five years after FMT and found consistently high engraftment of donor strains at all time points (
The inventors found that 50 out of 51 strains belonging to order Bacteriodales, which engrafted at 8-weeks, remained stably engrafted at a longer time-scale of 6-months or more (
The isolation and sequencing of the transmitted strains from both the donor and the recipient represents a gold standard validation and verification of the commensal Koch’s postulates. To date, there is no large study demonstrating transmission of donor bacterial strains from multiple species and across different FMT interventions by culture. The inventors cultured strains from 6 recipients both pre- and post-FMT (
Bacteroides ovatus
Bacteroides vulgatus
Bifidobacterium longum
Alistipes finegoldii
Bacteroides uniformis
Bifidobacterium bifidum
Parabacteroides distasonis
Parabacteroides merdae
Bacteroides caccae
Bacteroides thetaiotaomicron
Bifidobacterium adolescentis
Bifidobacterium pseudocatenulatum
Collinsella aerofaciens
Odoribacter splanchnicus
Bacteroides cellulosilyticus
Bacteroides fragilis
Butyricimonas faecalis
The vast majority of these (46/48) strains were also detected independently in metagenomics samples from the same timepoint when they were cultured, and the other 2 were detected at an earlier timepoint, highlighting the Strainer algorithm’s capability to track and study engraftment of strains post-FMT.
The inventors quantified tracking performance on these gold standard strains (which were isolated either in one person across multiple timepoints, or between the donor and the recipient using different algorithms) and found that the present disclosures method for FMT tracking had overall sensitivity of 92.9 (with 1 false positive) while inStrain had 25.3, Strain Finder had 0 and ConStrains had 1.4 (
Studies have shown that resident microbiota strains create ecological niches, which in turn can influence the engraftment of donor microbes post-FMT. Thus, it is important to identify the bacterial strains present pre-FMT and resolve their persistence dynamics after transplantation. Here, the inventors isolated and sequenced the pre-FMT resident strains in 7 recipients and tracked them for up to 5 years in each recipient’s metagenome. Similar to the PED metric, the inventors defined Proportional Persistence of Recipient Strains (PPR) as the ratio between the strains of the recipient observed post-FMT to total recipient strains cultured pre-FMT. Unlike the rapid high engraftment of donor strains, the inventors found a more graduated decline in the PPR (
(A) Overview of FMT study design indicating the dates of metagenomic sequencing and bacterial strain culturing. The genome sequences of the cultured bacterial strains are used to track each strain across metagenomic samples using Strainer.
(B) Strains from the donor remain stably engrafted in successful post-FMT patients for at least 5 years after transplant.
(C) Strains isolated from a recipient prior to FMT are rapidly lost with a small proportion persisting at longer timescales.
(D) Proportion of donor, recipient, and environment strains detected in patients post-FMT. Environmental strains are non-donor and non- recipient (prior to FMT) in origin, which are both cultured and metagenomically detected post-FMT.
(E) Count of strains detected in patients post-FMT subclassified by major phylogenetic taxa (at order level) and colored based on their origin.
The inventors investigated whether donor and pre-FMT recipient strains lead to complete niche occupancy of the host, or whether there is further engraftment of gut microbes from other individuals and environmental sources. The inventors isolated and tracked strains from 5 subjects post-FMT and found 24 strains that were non-donor and non-recipient in origin that were metagenomically detected and cultured in recipients post-FMT. On average in a patient post-FMT, 8.9% strains persisted from the recipient pre-FMT, 79.6% strains engrafted from the donor, and 11.5% strains were non-donor or non-recipient in origin (
(A) Trajectory of proportional strain engraftment of donor strains in each recipient at all available timepoints (in days). The donor recipient pair ids are at the top of each plot.
(B) Number of strains that transmit and engraft for at least 8-weeks in patients post-FMT (single FMT donor to recipient setting) grouped by taxonomic order.
(C) The number of strains colonized at 8 weeks (short term) that engraft for at least 6-months or more (long-term) in patients post-FMT (both single FMT donor to single and multiple recipients setting) grouped by taxonomic order.
(D) Trajectory of proportional persistence of recipient’s strains post-FMT at all available timepoints (in days). The donor recipient pair ids are at the top of each plot.
(E) The number of the recipient’s original strains that persist for at least 8-weeks post-FMT, grouped by taxonomic order.
(F) The number of environment strains (i.e. non-donor and non-recipient in origin) that engraft in patients stably over multiple timepoints (>1 week) post-FMT, grouped by taxonomic order.
Eight weeks is the typical timepoint for evaluating the efficacy of FMT interventions, which can be accomplished by comparing the number of patients that achieved the clinical endpoint with those that failed to do so. PED provides a potential quantitative surrogate marker to understand FMT clinical success or relapse. In the two patients in this cohort who experienced an early relapse within 8-weeks of FMT, the inventors found significantly reduced PED (
Individuals that undergo repeat FMT often respond to treatment the second time. Therefore, the inventors evaluated if the present PED metric can elucidate the outcome of repeat-FMT in such patients. The 2 recipients (R095 and R311) that had an early failure, received a repeat dose of FMT and reported clinical success (i.e., no relapse with rCDI recurrence) at future timepoints (including at 5 year for R095). The inventors found that PED was significantly higher after the repeat dosage (
Since PED was able to explain both relapse and outcome of repeat-FMT in patients, the inventors evaluated the overall predictive power of the present disclosure on all available FMT samples where clinical evaluation was independently noted (
(A) Proportional Engraftment of donor’s (PED) strains at 8-weeks can predict early relapse of FMT in patients with rCDI.
(B) PED metric can elucidate the successful outcome of repeat-FMT in patients that relapsed with rCDI after the initial-FMT.
(C) Predictive power of our approach on all available FMT samples where clinical evaluation was independently noted. Whenever we report clinical success we find engraftment to be above the threshold of 17% (n = 19 true positives) with 1 false negative. Clinical relapse was always independently associated with low engraftment (n = 2 true negatives) with no false negatives
(D) Bacterial strain engraftment and identification of highly transmissible strains that stably engraft in multiple recipients. The first 4 columns are weekly metagenomic samples from the donor, while the 5th column is the donor sample from 5 years later. The next 6 columns are from the FMT recipients that did not have an early relapse. The last column is from one of the recipient 5 years later. Strainer was used to find the presence (green) or absence (yellow) of each bacterial strain from the corresponding metagenomics sample.
The inventors did find one case of very low engraftment in an otherwise successful FMT with no relapse occurred in patient R285 (
The inventors have developed a consortium of culturable, discrete strains for use in LBPs as a safer, scalable alternative to FMT. The inventors have demonstrated for the first time a consortium of a transferable, culturable engrafting fraction of human-tested donor fecal microbiotas, where strains that do not transfer are eliminated, and multi-drug resistant organisms (MDROs) are removed. Donor D283 was used for multiple (n = 5 non-relapsing) recipients, thus providing more power to detect engraftment consistency of single strains (
(A) Engraftment of donor D283 strains in recipient R285, which did not relapse but rather had a temporary loss in detectability of the donor strains during antibiotic treatment for severe diarrhea.
(B) Identification of a set of bacterial species for LBP, based on their culturing and engrafting efficacy across recipients. “Number of donors” correspond to the donors where strains from this species have been cultured or detected metagenomically. “Number of strains cultured” represents the unique strains cultured and metagenomically detected for this species. “Number of recipients transferred to” corresponds to number of FMT recipients (counted separately for each strain cultured from this species) which received a strain from this species. “Number of strains engrafted in recipients” represents the strains that engrafted for at least 8-weeks (a common clinical endpoint) in a recipient. “Engraftment efficacy” is calculated as the ratio of “Strain engraftment/Column 5” and “Recipients transferred to/Column 4”.
To be suitable for human trials, the strains in the bacterial consortium must be cultivatable in growth media that is free of animal products. The inventors discovered that all 16 bacterial strains can be cultured in a specific animal free media LYH_VIB (Table 7). All strains reach sufficient optical density (OD600) and potency (CFU/mL) cultured in LYH_VIB to be manufactured for human trials (Table 5). For safety considerations, the inventors focused on bacterial consortium strains that would be susceptible to multiple antibiotics. The inventors tested susceptibility to a range of antibiotics for all strains included in MTC01 and the minimal inhibitory concentration (MIC) was determined according to guidelines of the Clinical and Laboratory Standards Institute (CLSI). All strains were susceptible to multiple antibiotics (Table 6). A further consideration for the manufacture of these strains is the need to identify potential contaminant bacteria within the drug, most notably facultative anaerobic pathogens. USP<61> is an established assay for testing if a product is contaminated or does not have a high number of aerobic bacteria, yeast, and fungi in it. To apply this test in the context of a drug composed of bacteria, it is important that the bacteria are not aerobic or facultative aerobic organisms and that the drug strains do not inhibit the growth of other aerobic of facultative organisms used in the USP<61> assay. The inventors confirmed that all 16 strains were strict anaerobes with no bacterial growth documented for any of the strains under aerobic conditions as confirmed by total aerobic microbial count (TAMC). The inventors also confirmed that none of the 16 strains inhibited the growth of the USP<61> control organisms, S. aureus (ATCC6538); P. auruginosa (ATCC9027), B. subtilis (ATCC6633), C. albicans (ATCC10231) and A. brasiliensis (ATCC16404), as >50% recovery was demonstrated for these control organisms when incubated aerobically with each of the 16 therapeutic strains.
Table 6. Strain composition and antibiotic susceptibility of MTC01.
Each strain is susceptible to multiple antibiotics, and all strains are susceptible to three antibiotics (SAM, AMC, MEM). Minimum inhibitory concentrations (MIC) were determined by a CRO [Micromyx, LLC] according to CLSI standards and in-house by etest, keeping the highest value between the two methods: vancomycin (VAN), metronidazole (MTZ), tigecycline (TGC), ampicillin/sulbactam (SAM), amoxicillin/clavulanic acid (AMC), meropenem (MEM), piperacillin/tazobactam (TZP), clindamycin (CLI), ceftriaxone (CRO), moxifloxacin (MOX). All strains failed to grow aerobically in a USP<61> assay but did not inhibit the growth of positive control organisms validating USP<61> as a release assay for the master cell banks and drug product. Finally, multiple test fermentations were used to determine that the volumes for each manufacturing run are well within the 8L capacity of our current manufacturing set-up.
Alistipes onderdonkii A
Alistipes onderdonkii B
Alistipes onderdonkii C
Alistipes onderdonkii D
Alistipes onderdonkii E
Alistipes onderdonkii F
Alistipes putredinis A
Alistipes putredinis B
Alistipes senegalensis A
Alistipes senegalensis B
Alistipes shahii A
Alistipes shahii B
Alistipes shahii C
Alistipes shahii D
Alistipes shahii E
Anaerotruncus colihominis A
Anaerotruncus colihominis B
Anaerotruncus colihominis C
Anaerotruncus colihominis D
Anaerotruncus colihominis E
Bacteroides caccae A
Bacteroides caccae B
Bacteroides caccae C
Bacteroides caccae D
Bacteroides caccae E
Bacteroides caccae F
Bacteroides caccae G
Bacteroides caccae H
Bacteroides caccae I
Bacteroides cellulosilyticus A
Bacteroides cellulosilyticus B
Bacteroides cellulosilyticus C
Bacteroides cellulosilyticus D
Bacteroides cellulosilyticus E
Bacteroides cellulosilyticus F
Bacteroides cellulosilyticus G
Bacteroides cellulosilyticus H
Bacteroides cellulosilyticus I
Bacteroides cellulosilyticus J
Bacteroides cellulosilyticus K
Bacteroides cellulosilyticus L
Bacteroides cellulosilyticus M
Bacteroides clarus A
Bacteroides clarus B
Bacteroides clarus C
Bacteroides clarus D
Bacteroides fragilis A
Bacteroides fragilis B
Bacteroides fragilis C
Bacteroides fragilis D
Bacteroides fragilis E
Bacteroides fragilis F
Bacteroides fragilis G
Bacteroides fragilis H
Bacteroides fragilis I
Bacteroides fragilis J
Bacteroides fragilis K
Bacteroides fragilis L
Bacteroides fragilis M
Bacteroides fragilis N
Bacteroides intestinalis A
Bacteroides intestinalis B
Bacteroides intestinalis C
Bacteroides intestinalis D
Bacteroides intestinalis E
Bacteroides massiliensis A
Bacteroides massiliensis B
Bacteroides massiliensis C
Bacteroides massiliensis D
Bacteroides massiliensis E
Bacteroides massiliensis F
Bacteroides massiliensis G
Bacteroides massiliensis H
Bacteroides massiliensis I
Bacteroides ovatus a
Bacteroides ovatus A
Bacteroides ovatus b
Bacteroides ovatus B
Bacteroides ovatus C
Bacteroides ovatus c
Bacteroides ovatus D
Bacteroides ovatus d
Bacteroides ovatus E
Bacteroides ovatus e
Bacteroides ovatus F
Bacteroides ovatus f
Bacteroides ovatus G
Bacteroides ovatus g
Bacteroides ovatus H
Bacteroides ovatus h
Bacteroides ovatus i
Bacteroides ovatus I
Bacteroides ovatus J
Bacteroides ovatus K
Bacteroides ovatus L
Bacteroides ovatus M
Bacteroides ovatus N
Bacteroides ovatus O
Bacteroides ovatus P
Bacteroides ovatus Q
Bacteroides ovatus R
Bacteroides ovatus S
Bacteroides ovatus T
Bacteroides ovatus U
Bacteroides ovatus V
Bacteroides ovatus W
Bacteroides ovatus X
Bacteroides ovatus Y
Bacteroides ovatus Z
Bacteroides stercoris A
Bacteroides stercoris B
Bacteroides stercoris C
Bacteroides stercoris D
Bacteroides stercoris E
Bacteroides thetaiotaomicron A
Bacteroides thetaiotaomicron B
Bacteroides thetaiotaomicron C
Bacteroides thetaiotaomicron D
Bacteroides thetaiotaomicron E
Bacteroides thetaiotaomicron F
Bacteroides thetaiotaomicron G
Bacteroides thetaiotaomicron H
Bacteroides thetaiotaomicron I
Bacteroides thetaiotaomicron J
Bacteroides thetaiotaomicron K
Bacteroides thetaiotaomicron L
Bacteroides thetaiotaomicron M
Bacteroides thetaiotaomicron N
Bacteroides thetaiotaomicron O
Bacteroides thetaiotaomicron P
Bacteroides thetaiotaomicron Q
Bacteroides thetaiotaomicron R
Bacteroides thetaiotaomicron S
Bacteroides thetaiotaomicron T
Bacteroides thetaiotaomicron U
Bacteroides thetaiotaomicron V
Bacteroides thetaiotaomicron W
Bacteroides uniformis a
Bacteroides uniformis A
Bacteroides uniformis b
Bacteroides uniformis B
Bacteroides uniformis c
Bacteroides uniformis C
Bacteroides uniformis d
Bacteroides uniformis D
Bacteroides uniformis e
Bacteroides uniformis E
Bacteroides uniformis f
Bacteroides uniformis F
Bacteroides uniformis G
Bacteroides uniformis g
Bacteroides uniformis h
Bacteroides uniformis H
Bacteroides uniformis I
Bacteroides uniformis J
Bacteroides uniformis K
Bacteroides uniformis L
Bacteroides uniformis M
Bacteroides uniformis N
Bacteroides uniformis O
Bacteroides uniformis P
Bacteroides uniformis Q
Bacteroides uniformis R
Bacteroides uniformis S
Bacteroides uniformis T
Bacteroides uniformis U
Bacteroides uniformis V
Bacteroides uniformis W
Bacteroides uniformis X
Bacteroides uniformis Y
Bacteroides uniformis Z
Bacteroides vulgatus A
Bacteroides vulgatus a
Bacteroides vulgatus b
Bacteroides vulgatus B
Bacteroides vulgatus C
Bacteroides vulgatus c
Bacteroides vulgatus D
Bacteroides vulgatus d
Bacteroides vulgatus E
Bacteroides vulgatus e
Bacteroides vulgatus F
Bacteroides vulgatus f
Bacteroides vulgatus g
Bacteroides vulgatus G
Bacteroides vulgatus h
Bacteroides vulgatus H
Bacteroides vulgatus I
Bacteroides vulgatus J
Bacteroides vulgatus K
Bacteroides vulgatus L
Bacteroides vulgatus M
Bacteroides vulgatus N
Bacteroides vulgatus O
Bacteroides vulgatus P
Bacteroides vulgatus Q
Bacteroides vulgatus R
Bacteroides vulgatus S
Bacteroides vulgatus T
Bacteroides vulgatus U
Bacteroides vulgatus V
Bacteroides vulgatus W
Bacteroides vulgatus X
Bacteroides vulgatus Y
Bacteroides vulgatus Z
Bacteroides xylanisolvens A
Bacteroides xylanisolvens B
Bacteroides xylanisolvens C
Bacteroides xylanisolvens D
Bacteroides xylanisolvens E
Bacteroides xylanisolvens F
Barnesiella intestinihominis A
Barnesiella intestinihominis B
Barnesiella intestinihominis C
Barnesiella intestinihominis D
Barnesiella intestinihominis E
Barnesiella intestinihominis F
Bifidobacterium adolescentis A
Bifidobacterium adolescentis B
Bifidobacterium adolescentis C
Bifidobacterium adolescentis D
Bifidobacterium adolescentis E
Bifidobacterium adolescentis F
Bifidobacterium adolescentis G
Bifidobacterium adolescentis H
Bifidobacterium adolescentis I
Bifidobacterium adolescentis J
Bifidobacterium adolescentis K
Bifidobacterium adolescentis L
Bifidobacterium adolescentis M
Bifidobacterium adolescentis N
Bifidobacterium adolescentis O
Bifidobacterium adolescentis P
Bifidobacterium adolescentis Q
Bifidobacterium adolescentis R
Bifidobacterium adolescentis S
Bifidobacterium adolescentis T
Bifidobacterium adolescentis U
Bifidobacterium bifidum A
Bifidobacterium bifidum B
Bifidobacterium bifidum C
Bifidobacterium bifidum D
Bifidobacterium bifidum E
Bifidobacterium bifidum F
Bifidobacterium bifidum G
Bifidobacterium bifidum H
Bifidobacterium longum a
Bifidobacterium longum A
Bifidobacterium longum b
Bifidobacterium longum B
Bifidobacterium longum C
Bifidobacterium longum c
Bifidobacterium longum D
Bifidobacterium longum d
Bifidobacterium longum E
Bifidobacterium longum e
Bifidobacterium longum f
Bifidobacterium longum F
Bifidobacterium longum g
Bifidobacterium longum G
Bifidobacterium longum H
Bifidobacterium longum h
Bifidobacterium longum I
Bifidobacterium longum i
Bifidobacterium longum j
Bifidobacterium longum J
Bifidobacterium longum k
Bifidobacterium longum K
Bifidobacterium longum L
Bifidobacterium longum M
Bifidobacterium longum N
Bifidobacterium longum O
Bifidobacterium longum P
Bifidobacterium longum Q
Bifidobacterium longum R
Bifidobacterium longum S
Bifidobacterium longum T
Bifidobacterium longum U
Bifidobacterium longum V
Bifidobacterium longum W
Bifidobacterium longum X
Bifidobacterium longum Y
Bifidobacterium longum Z
Bifidobacterium pseudocatenulatum A
Bifidobacterium pseudocatenulatum B
Bifidobacterium pseudocatenulatum C
Bifidobacterium pseudocatenulatum D
Bifidobacterium pseudocatenulatum E
Bifidobacterium pseudocatenulatum F
Bifidobacterium pseudocatenulatum G
Bifidobacterium pseudocatenulatum H
Bifidobacterium pseudocatenulatum I
Bifidobacterium pseudocatenulatum J
Bifidobacterium pseudocatenulatum K
Blautia massiliensis A
Blautia massiliensis B
Blautia massiliensis C
Blautia massiliensis D
Blautia wexlerae A
Blautia wexlerae B
Blautia wexlerae C
Blautia wexlerae D
Blautia wexlerae E
Blautia wexlerae F
Blautia wexlerae G
Blautia wexlerae H
Blautia wexlerae I
Blautia wexlerae J
Blautia wexlerae K
Blautia wexlerae L
Blautia wexlerae M
Blautia wexlerae N
Blautia wexlerae O
Clostridium A
Clostridium B
Clostridium C
Clostridium D
Clostridium E
Clostridium F
Clostridium G
Clostridium H
Clostridium I
Clostridium J
Clostridium K
Clostridium L
Clostridium M
Clostridium N
Clostridium O
Clostridium P
Clostridium Q
Clostridium R
Clostridium S
Collinsella aerofaciens A
Collinsella aerofaciens B
Collinsella aerofaciens C
Collinsella aerofaciens D
Collinsella aerofaciens E
Collinsella aerofaciens F
Collinsella aerofaciens G
Collinsella aerofaciens H
Collinsella aerofaciens I
Collinsella aerofaciens J
Collinsella aerofaciens K
Collinsella aerofaciens L
Collinsella aerofaciens M
Collinsella aerofaciens N
Collinsella aerofaciens O
Collinsella aerofaciens P
Collinsella aerofaciens Q
Collinsella aerofaciens R
Collinsella aerofaciens S
Collinsella aerofaciens T
Collinsella aerofaciens U
Collinsella aerofaciens V
Coprococcus comes A
Coprococcus comes B
Coprococcus comes C
Coprococcus comes D
Coprococcus comes E
Coprococcus comes F
Coprococcus comes G
Coprococcus comes H
Coprococcus comes I
Coprococcus comes J
Coprococcus comes K
Coprococcus comes L
Coprococcus comes M
Coprococcus comes N
Coprococcus comes O
Coprococcus comes P
Dorea longicatena A
Dorea longicatena B
Dorea longicatena C
Dorea longicatena D
Dorea longicatena E
Dorea longicatena F
Dorea longicatena G
Dorea longicatena H
Eggerthella lenta A
Eggerthella lenta B
Eggerthella lenta C
Eggerthella lenta D
Eggerthella lenta E
Eggerthella lenta F
Eggerthella lenta G
Eggerthella lenta H
Eggerthella lenta I
Eggerthella lenta J
Eggerthella lenta K
Eggerthella lenta L
Eggerthella lenta M
Eggerthella lenta N
Eggerthella lenta O
Eggerthella lenta P
Eggerthella lenta Q
Escherichia coli A
Escherichia coli a
Escherichia coli B
Escherichia coli b
Escherichia coli c
Escherichia coli C
Escherichia coli D
Escherichia coli d
Escherichia coli e
Escherichia coli E
Escherichia coli f
Escherichia coli F
Escherichia coli G
Escherichia coli g
Escherichia coli h
Escherichia coli H
Escherichia coli i
Escherichia coli I
Escherichia coli J
Escherichia coli K
Escherichia coli L
Escherichia coli M
Escherichia coli N
Escherichia coli O
Escherichia coli P
Escherichia coli Q
Escherichia coli R
Escherichia coli S
Escherichia coli T
Escherichia coli U
Escherichia coli V
Escherichia coli W
Escherichia coli X
Escherichia coli Y
Escherichia coli Z
Eubacterium rectale A
Eubacterium rectale B
Eubacterium rectale C
Eubacterium rectale D
Eubacterium rectale E
Eubacterium rectale F
Eubacterium rectale G
Eubacterium rectale H
Eubacterium siraeum A
Eubacterium siraeum B
Eubacterium siraeum C
Eubacterium siraeum D
Eubacterium siraeum E
Eubacterium tenue A
Odoribacter splanchnicus A
Odoribacter splanchnicus B
Odoribacter splanchnicus C
Odoribacter splanchnicus D
Odoribacter splanchnicus E
Odoribacter splanchnicus F
Odoribacter splanchnicus G
Odoribacter splanchnicus H
Odoribacter splanchnicus I
Parabacteroides distasonis A
Parabacteroides distasonis B
Parabacteroides distasonis C
Parabacteroides distasonis D
Parabacteroides distasonis E
Parabacteroides distasonis F
Parabacteroides distasonis G
Parabacteroides distasonis H
Parabacteroides distasonis I
Parabacteroides distasonis J
Parabacteroides distasonis K
Parabacteroides distasonis L
Parabacteroides distasonis M
Parabacteroides distasonis N
Parabacteroides distasonis O
Parabacteroides distasonis P
Parabacteroides distasonis Q
Parabacteroides distasonis R
Parabacteroides distasonis S
Parabacteroides distasonis T
Parabacteroides johnsonii A
Parabacteroides johnsonii B
Parabacteroides johnsonii C
Parabacteroides merdae A
Parabacteroides merdae B
Parabacteroides merdae C
Parabacteroides merdae D
Parabacteroides merdae E
Parabacteroides merdae F
Parabacteroides merdae G
Parabacteroides merdae H
Parabacteroides merdae I
Parabacteroides merdae J
Parabacteroides merdae K
Parabacteroides merdae L
Parabacteroides merdae M
Roseburia faecis A
Roseburia faecis B
Roseburia faecis C
Roseburia faecis D
Roseburia faecis E
Roseburia faecis F
Roseburia faecis G
Ruminococcus A
Ruminococcus B
Ruminococcus C
Ruminococcus D
Ruminococcus E
Ruminococcus F
Ruminococcus G
Ruminococcus H
Ruminococcus I
Ruminococcus J
Ruminococcus K
Ruminococcus L
Ruminococcus M
Ruminococcus N
Ruminococcus O
Ruminococcus P
Ruminococcus Q
Ruminococcus R
Ruminococcus torques A
Ruminococcus torques B
Ruminococcus torques C
Streptococcus parasanguinis A
Streptococcus parasanguinis B
Streptococcus parasanguinis C
Streptococcus parasanguinis D
Streptococcus parasanguinis E
Streptococcus parasanguinis F
Streptococcus parasanguinis G
Streptococcus parasanguinis H
Streptococcus parasanguinis I
Streptococcus parasanguinis J
Streptococcus parasanguinis K
Streptococcus pasteurianus A
Streptococcus salivarius A
Streptococcus salivarius B
Streptococcus salivarius C
Streptococcus salivarius D
Streptococcus salivarius E
Streptococcus salivarius F
Streptococcus salivarius G
Streptococcus salivarius H
Streptococcus salivarius I
Streptococcus salivarius J
Streptococcus salivarius K
Streptococcus salivarius L
Streptococcus salivarius M
Streptococcus salivarius N
Streptococcus salivarius O
Streptococcus salivarius P
Streptococcus salivarius Q
Streptococcus salivarius R
Streptococcus sobrinus A
This application is a § 371 national stage of PCT International Application No. PCT/US21/71018, entitled “Compositions and Methods for Treating Infections of the Gastrointestinal Tract” filed on Jul. 27, 2021, which claims the benefit of U.S. Provisional Pat. Application Serial No. 63/057,492, filed Jul. 28, 2020, and of U.S. Provisional Pat. Application Serial No. 63/069,931, filed Aug. 25, 2020, which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/071018 | 7/27/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63069931 | Aug 2020 | US | |
| 63057492 | Jul 2020 | US |
