Patent Grant
11666611
The technology described herein relates to the treatment or prevention of host pathology involving bacterial toxin production.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 28, 2018, is named 043214-090870WOPT_SL.txt and is 116,476 bytes in size.
The healthy human gastrointestinal tract is generally host to 300 to 1,000 or more different species of microorganisms. These commensal organisms serve a wide range of functions increasingly recognized as mutualistic and directly connected to the health of the host, including assisting digestion, metabolism, immune function, and colonization resistance against pathogens, among others (Guarner, F. “Enteric flora in health and disease.” Digestion 73 Suppl 1:5-12 (2006)).
Clostridium difficile (C. difficile) infection is the most common cause of nosocomial diarrhea, most often arising in individuals treated with antibiotics, accounting for 10-20% of antibiotic-associated diarrhea and most cases of colitis associated with antibiotic use. C. difficile can be present in the healthy gut microbiota, but the pathogen's biomass and toxin production is kept in check by the indigenous gut flora. Further, when stressed, C. difficile can generate spores that can tolerate extreme conditions many active bacteria cannot, and C. difficile spores are also resistant to antibiotics and many cleaners. Thus, as the normal intestinal flora is disturbed by antibiotic use, the C. difficile spores can remain, leading to their germination and proliferation a large population of C. difficile; perturbation of the healthy microbiota caused by antibiotic use is believed to provide an advantage to C. difficile, allowing it to proliferate and elaborate toxins. The mortality for C. difficile infection is estimated at 1-2.5%, contributing to 15,000-30,000 deaths annually in the U.S. (Ananthakrishnan, A. N. “Clostridium difficile infection: epidemiology, risk factors and management,” Nat Rev Gastroenterol Hapatol, 8:17-26 (2011).
While the importance of other microbial species in suppressing C. difficile populations is generally acknowledged, and some species, including Lactobacilli, Enterococci, and some Bifidobacteria and Bacteroides species, have shown varying degrees of inhibitory activity against C. difficile (Borriello, S. P., and Barclay, F. E. 1986. Ibid.; Naaber, P. et al. “Inhibition of Clostridium difficile strains by intestinal Lactobacillus species.” J Med Microbiol 53:551-4 (2004); Rolfe, R. D. et al. “Bacterial interference between Clostridium difficile and normal fecal flora.” J. Infect. Dis. 143:470-475 (1981); Lee, Y. J. et al. “Identification and screening for antimicrobial activity against Clostridium difficile of Bifidobacterium and Lactobacillus species isolated from healthy infant faeces.” Int J Antimicrob Agents 21:340-6 (2003)), it is not clear which species are essential for suppressing C. difficile in the healthy gut.
The technology described herein is related to the discovery of commensal bacteria that can suppress toxin production by Gram-positive toxigenic bacteria such as C. difficile and thereby treat or prevent the development of toxin-mediated pathology. Indeed, it was found that as few as a single species of bacterium can provide complete protection from otherwise fatal C. difficile infection in murine models described herein. Suppression of toxin production provides an alternative route to treatment of C. difficile-mediated pathology, in that it can be sufficient for treatment to just suppress production of the pathology-generating toxin without necessarily killing the microbe.
Described herein are defined therapeutic microbiota that treat and/or prevent C. difficile infection, as the term is used herein, including recurrent C. difficile infection. Highly effective therapeutic microbiota include species that are highly proteolytic and that promote Stickland fermentation by C. difficile. In one embodiment, the single, highly proteolytic, Stickland fermenting species Clostridium bifermentans can provide complete protection against fatal C. difficile infection. In another embodiment, a defined consortium of just two species, C. bifermentans and C. scindens, another proteolytic, Stickland fermenting Clostridial species, provides highly effective protection against pathology caused by C. difficile and its toxin production. It is anticipated that other proteolytic, Stickland fermenting species can also provide benefits, including, as non-limiting example, other proteolytic, Stickland fermenting Clostridia.
The examination of the mechanisms through which the identified bacterial species affect C. difficile toxin production provide insight into the ability to suppress the expression of toxins by other toxigenic species of Gram positive, spore-forming anaerobes, as well as insights into the properties of other species likely to perform the same toxin-suppression.
Also described herein is the identification of a biomarker profile, comprising two groups of Clostridial species, that is predictive of the likelihood of recurrent C. difficile infection and/or susceptibility to initial C. difficile infection. When the relative abundance of these groups in a microbiota sample are low relative to healthy gut microbiota, it is considerably more likely that C. difficile infection will arise and/or be recurrent.
Also described herein are diagnostic methods that exploit markers such as proteolytic activity or protease expression, markers of Stickland fermentation (at the protein, nucleic acid, and/or metabolite levels), including: i) methods for the determination of the efficacy of bacteriotherapy for infection with a toxigenic, Gram positive, spore-forming bacterium such as C. difficile; ii) methods for predicting the likelihood of recurrent infection with a toxigenic, Gram positive, spore-forming bacterium such as C. difficile; and iii) methods of determining the likelihood of infection with a toxigenic, Gram positive, spore-forming bacterium such as C. difficile.
In one aspect, described herein is a pharmaceutical composition comprising an oral formulation comprising C. scindens and C. bifermentans bacteria.
In another aspect, described herein is a composition comprising C. scindens and C. bifermentans bacteria, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form.
In another aspect, described herein is a pharmaceutical composition comprising a formulation comprising C. scindens and C. bifermentans bacteria, wherein the composition does not comprise Bacteroides species or Escherichia species.
In one embodiment of these aspects and all such aspects described herein, one or both of the C. scindens and C. bifermentans bacteria are in spore form.
In another embodiment of these aspects and all such aspects described herein, one or both of the C. scindens and C. bifermentans bacteria are not in spore form.
In another embodiment of these aspects and all such aspects described herein, the C. scindens and C. bifermentans bacteria are present as a mixture of metabolically active and spore forms.
In another embodiment of these aspects and all such aspects described herein, the composition comprises a capsule or microcapsule, or a composition formulated for enteric delivery.
In another embodiment of these aspects and all such aspects described herein, wherein one or both of the C. scindens and C. bifermentans bacteria are in dried viable form.
In another embodiment of these aspects and all such aspects described herein, the composition does not comprise C. sardiniensis bacteria.
In another embodiment of these aspects and all such aspects described herein, the composition does not comprise any other Clostridium species.
In another embodiment of these aspects and all such aspects described herein, the composition does not contain Bacteroides species or Escherichia coli.
In another embodiment of these aspects and all such aspects described herein, the formulation comprises no other bacteria.
In another aspect, described herein is a pharmaceutical composition comprising an oral formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium.
In another aspect described, herein is a composition comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form.
In another aspect, described herein is a pharmaceutical composition comprising a formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium wherein the composition does not comprise Bacteroides species or Escherichia species.
In one embodiment of these aspects and all such aspects described herein, one or both of the C. scindens and C. bifermentans bacteria are in spore form.
In another embodiment of these aspects and all such aspects described herein, one or both of the C. scindens and C. bifermentans bacteria are not in spore form.
In another embodiment of these aspects and all such aspects described herein, the C. scindens and C. bifermentans bacteria are present as a mixture of metabolically active and spore forms.
In another embodiment of these aspects and all such aspects described herein, the composition comprises a capsule or microcapsule, or a composition formulated for enteric delivery.
In another embodiment of these aspects and all such aspects described herein, one or both of the C. scindens and C. bifermentans bacteria are in dried viable form.
In another embodiment of these aspects and all such aspects described herein, the composition does not comprise C. sardiniensis bacteria.
In another embodiment of these aspects and all such aspects described herein, the composition does not comprise Bacteroides species or Escherichia coli.
In another embodiment of these aspects and all such aspects described herein, the composition does not comprise any other Clostridium species.
In another embodiment of these aspects and all such aspects described herein, the composition in which the formulation comprises no other bacteria.
In another embodiment of these aspects and all such aspects described herein, further comprises a prebiotic.
In another embodiment of these aspects and all such aspects described herein, the composition further comprises a microbe that supports C. scindens and/or C. bifermentans.
In another embodiment of these aspects and all such aspects described herein, the microbe that supports C. scindens is Ruminococcus obeum.
In another embodiment of these aspects and all such aspects described herein, the composition is for use in the treatment of C. difficile infection.
In one embodiment of this aspect and all such aspects described herein, the use comprises suppressing the expression of C. difficile toxin.
In another embodiment of this aspect and all such aspects described herein, the use comprises promoting a shift towards use of the proline reductase pathway of Stickland fermentation in C. difficile.
In another embodiment of this aspect and all such aspects described herein, the use comprises inducing CodY activity or expression in C. difficile.
In another embodiment of this aspect and all such aspects described herein, the use comprises promoting ethanolamine utilization by C. difficile.
In another aspect described herein is a method comprising administering a composition of any embodiment of the aspects above to a subject in need thereof.
In one embodiment of this aspect and all such aspects described herein is a method, the subject has or has been diagnosed with C. difficile infection.
In another embodiment of this aspect and all such aspects described herein, the C. difficile infection is recurrent.
In another embodiment of this aspect and all such aspects described herein, the subject is at risk of C. difficile infection or recurrent C. difficile infection.
In another embodiment of this aspect and all such aspects described herein, the administration is oral.
In another embodiment of this aspect and all aspects described herein the method comprises administering a composition directly to the colon of a subject in need thereof.
In another embodiment of this aspect and all such aspects described herein, administration is via colonoscopy or enema.
In another embodiment of this aspect and all such aspects described herein, the method the subject is receiving or has recently received antibiotic treatment.
In another aspect, described herein is a method of treating an infection, the method comprising administering an antibiotic and a composition of the aspects described herein.
In another aspect, as described herein is a method of treating an infection compromising administering an antibiotic and a composition as described herein, wherein the composition is administered before or concurrently with the antibiotic.
In another aspect, described herein is a method of treating an infection compromising administering an antibiotic and a composition as described herein, wherein the composition is administered after a course of an antibiotic.
In another aspect, described herein is a method of predicting recurrence of C. difficile infection in a subject, the method comprising:
In another embodiment of this aspect, the reference sequences for C. scindens and C. hylemonae are 16S rDNA sequences.
In another embodiment of this aspect, the determining steps are performed on samples taken before, during or after the subject has been treated with antibiotics for C. difficile infection.
In another embodiment of this aspect, the determining steps are performed on samples taken after the subject has been treated with antibiotics for C. difficile infection.
In another embodiment of this aspect, and all such aspects described herein, the method further comprises the step, when the sum of relative abundances is at or below 1%, of administering a composition as described herein above.
In another aspect, described herein is a method of suppressing expression of a bacterial toxin in a subject, the method comprising administering a defined bacterial microbiota comprising at least one bacterial organism that encodes and secretes a protease and/or performs Stickland fermentation.
In another aspect, described herein is a method of treating or preventing a pathology caused by expression of a bacterial toxin, the method comprising administering a defined bacterial microbiota comprising at least one bacterial organism that encodes and secretes a protease and/or performs Stickland fermentation.
In another aspect, described herein is a method of promoting CodY expression or activity in a C. difficile bacterium in a subject, the method comprising administering a defined bacterial microbiota comprising a bacterial organism that encodes and secretes a protease and/or performs Stickland fermentation.
In another aspect, described herein is a method of promoting ethanolamine utilization by a C. difficile bacterium in a subject, the method comprising administering a defined bacterial microbiota comprising a bacterial organism that encodes and secretes a protease and/or performs Stickland fermentation.
In one embodiment of these aspects and all such aspects described herein, at least one bacterial organism encodes and secretes at least one protease selected from the group consisting of: a protease of PATRIC ID fig|186802.30.peg.279; a protease of PATRIC ID fig|186802.30.peg.290; a protease of PATRIC ID fig|186802.30.peg.313; a protease of PATRIC ID fig|186802.30.peg.414; a protease of PATRIC ID fig|186802.30.peg.543; a protease of PATRIC ID fig|186802.30.peg.2205; a protease of PATRIC ID fig|186802.30.peg.2313; a protease of PATRIC ID fig|186802.30.peg.2680; a protease of PATRIC ID fig|186802.30.peg.2745; a protease of PATRIC ID fig|186802.30.peg.2746; a protease of PATRIC ID fig|186802.30.peg.830; a protease of PATRIC ID fig|186802.30.peg.921; a protease of PATRIC ID fig|186802.30.peg.936; a protease of PATRIC ID fig|186802.30.peg.3000; a protease of PATRIC ID fig|186802.30.peg.3018; a protease of PATRIC ID fig|186802.30.peg.3019; and a protease of PATRIC ID fig|186802.30.peg.3065.
In another embodiment of these aspects and all such aspects described herein, at least one protease performs the proteolysis reaction of enzymes of Enzyme Commission number (E.C. number) EC 3.4.21.-; EC 3.4.21.53; or EC 3.4.21.92.
In another embodiment of these aspects and all such aspects described herein, at least one bacterial organism encodes and expresses one or more of D-proline reductase, Glycine reductase, Thioredoxin, or Choloylglycine hydrolase.
In another embodiment of these aspects and all such aspects described herein, the at least one bacterial organism falls within Clostridial cluster I, XI, or XIVa, and does not express a pathology-causing bacterial toxin.
In another embodiment of these aspects and all such aspects described herein, the bacterial organism in Clostridial cluster I is selected from C. sporogenes, and C. histolyticum.
In another embodiment of these aspects and all such aspects described herein, the bacterial organism in Clostridial cluster XI is selected from C. bifermentans, C. hiranonis, and P. anaerobius.
In another embodiment of these aspects and all such aspects described herein, the bacterial organism in Clostridial cluster XIVa is selected from C. scindens, C. clostriiforme, and C. nexile.
In another embodiment of these aspects and all such aspects described herein, the at least one bacterial organism inhibits sorbitol/mannitol fermentation by C. difficile.
In another embodiment of these aspects and all such aspects described herein, the at least one bacterial organism promotes Stickland fermentation through the acceptor amino acid proline, or activation of proline reductase.
In another embodiment of these aspects and all such aspects described herein, the at least one bacterial organism promotes 5-aminovalerate production.
In another embodiment of these aspects and all such aspects described herein, the bacterial toxin is a C. difficile toxin.
In another embodiment of these aspects and all such aspects described herein, the bacterial organism is C. bifermentans and/or C. scindens.
In another embodiment of these aspects and all such aspects described herein, suppressing expression of a bacterial toxin comprises inhibition of butyrate, codY, ccpA, tcdR, and/or tcdA production.
In another aspect, described herein is a method of suppressing expression of a bacterial toxin in the gut of a subject, the method comprising administering at least one amino acid that is metabolized by Stickland fermentation.
In another aspect, described herein is a method of treating or preventing a pathology caused by expression of a bacterial toxin, the method comprising administering at least one amino acid that is metabolized by Stickland fermentation.
In one embodiment of this aspect and all such aspects described herein, the at least one amino acid is a Stickland donor or Stickland acceptor.
In another embodiment of this aspect and all such aspects described herein, the Stickland donor is selected from the group consisting of: alanine, leucine, valine, isoleucine, tryptophan, tyrosine and phenylalanine.
In another embodiment of this aspect and all such aspects described herein, the Stickland acceptor is selected from the group consisting of: glycine and proline.
In another embodiment of this aspect and all such aspects described herein, the amino acid is a branched-chain amino acid, a branched-keto amino acid, or an aromatic amino acid.
In another embodiment of this aspect and all such aspects described herein, the at least one amino acid promotes 5-aminovalerate production.
In another embodiment of this aspect and all such aspects described herein, the bacterial toxin is a C. difficile toxin.
In another embodiment of this aspect and all such aspects described herein, suppression of the expression of a bacterial toxin comprises inhibition of butyrate, codY, ccpA, tcdR, and/or tcdA activity or production.
In another aspect, described herein is a method of determining the therapeutic efficacy of a bacterial organism for treatment of a pathology involving expression of a toxin, produced by a Gram-positive spore-forming bacterium, the method comprising measuring in a biological sample obtained from an individual administered the bacterial organism one or more of:
In one embodiment of this aspect and all such aspects described herein, the bacterial toxin is produced by a Gram-positive, spore-forming bacterium.
In another embodiment of this aspects and all such aspects described herein, the bacterial toxin is a C. difficile toxin.
In another embodiment of this aspect and all such aspects described herein, the pathology comprises expression of a toxin by C. difficile.
In another embodiment of this aspect and all such aspects described herein, Stickland donor amino acids are selected from leucine, isoleucine, valine, alanine, phenylalanine, tryptophan, tyrosine and Stickland acceptor amino acids are selected from glycine, proline, and hydroxyproline.
In another embodiment of this aspect and all such aspects described herein, the sample is a stool sample or a sample from within the colon of the individual.
In another aspect, described herein is a method to predict the risk of developing a disease involving a toxin produced by a Gram positive, spore-forming bacterium, the method comprising measuring in a biological sample obtained from an individual one or more of the following:
comparing the amount, concentration or activity measured in one or more of (a)-(e) to a reference, wherein an amount, concentration or activity in one or more of (a)-(e) below the reference indicates increased risk of developing a disease involving a toxin produced by a Gram positive, spore-forming bacterium.
In one embodiment of this aspect and all such aspects described herein, the disease involves expression of a toxin by C. difficile.
In another embodiment of this aspect and all such aspects described herein, the reference comprises a biological sample from a healthy individual.
In another embodiment of this aspect and all such aspects described herein, the biological sample is a stool sample or a sample from within the colon of the individual.
In another embodiment of this aspect and all such aspects described herein, two or more of (a)-(e) are measured.
In another embodiment of this aspect and all such aspects described, three or more of (a)-(e) are measured.
In one embodiment of this aspect and all such aspects described herein, four or more, of (a)-(e) are measured.
In another aspect, described herein is a method of identifying a candidate bacterial organism that is likely to suppress the expression of a toxin by a Gram-positive, spore-forming bacterial pathogen, the method comprising:
In one embodiment of this aspect and all such aspects described herein, the candidate bacterial organism is not an opportunistic gut pathogen in humans.
In one embodiment of this aspect and all such aspects described herein, the proline reductase enzyme is an enzyme of E.C. 1.21.4.1.
In another aspect, described herein is a method to predict the risk of developing a spore-forming, toxin-producing Gram-positive bacterial pathogen in the gut or other location, or its recurrence, comprising measuring in a biological sample
wherein an increase in the amount or activity of at least one of (a)-(d) relative to a biological sample obtained prior to administration identifies a risk of developing a spore-forming, toxin-producing Gram-positive bacterial pathogen in the gut or other location.
In one embodiment of this aspect and all such aspects described herein, the method further comprising, prior to measuring, administering the bacterial organism or amino acid to the subject.
In another embodiment of this aspect and all such aspects described herein, the biological sample is obtained from a subject.
In another embodiment of this aspect and all such aspects described herein, the biological sample is a stool sample.
In another embodiment of this aspect and all such aspects described herein, the biological sample is obtained from the gut.
In another embodiment of this aspect and all such aspects described herein, the gram-positive bacterial pathogen is C. difficile infection.
In another embodiment of a composition comprising a defined bacterial microbiota as described herein, the composition further comprises an amount of one or more free Stickland donor and/or Stickland acceptor amino acids effective to promote Stickland fermentation by a species in the composition or by C. difficile after the composition is administered to a subject.
In another embodiment of a composition comprising a defined bacterial microbiota as described herein, the composition further comprises an amount of a polypeptide substrate effective for proteolysis by proteolytic activity of a bacterial species in the composition to generate amino acids fermentable by Stickland fermentation.
In one embodiment, the polypeptide substrate comprises casein, collagen and/or gelatin. In another embodiment, the polypeptide substrate comprises a synthetic polymer or copolymer polypeptide hydrolysable by a proteolytic activity of a species in the composition to generate Stickland fermentable amino acids.
In one embodiment, the synthetic polymer comprises a poly[N] polymer, where N is a Stickland donor amino acid selected from leucine, isoleucine, valine, alanine, phenylalanine, tryptophan, and tyrosine or a Stickland acceptor amino acids selected from glycine and proline.
In another embodiment, the synthetic copolymer comprises a poly[N,X] copolymer, where N and X are Stickland donor amino acids selected from leucine, isoleucine, valine, alanine, phenylalanine, tryptophan, and tyrosine or Stickland acceptor amino acids selected from glycine and proline.
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
Definitions of common terms in microbiology, molecular biology and medicine can be found, for example, in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-O-911910-19-3); Robert S. Porter et al. (eds.), Black, Jacquelyn G. Microbiology: Principles and Explorations, 9th Edition: Wiley; 9th Edition, 2014, Moore, Veranus A. Principles of Microbiology: A Treatise on Bacteria, Fungi and Protozoa: Forgotten Books, 2012, The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strober (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
It should be understood, as discussed elsewhere herein and as known in the art, that healthy individuals can have detectable levels of C. difficile bacteria in their gut, but that the pathogen biomass and toxin production are kept in balance by components of the healthy gut microbiota and do not cause disease until that balance is disturbed, often, but not necessarily exclusively, by antibiotic treatment for a different infection. Thus, as used herein, the term “C. difficile infection” or “CDI” refers not to the mere presence of C. difficile bacteria, but to expression of C. difficile toxin by such bacteria at a level that causes symptoms, and/or to an overgrowth of C. difficile bacteria relative to levels in healthy individuals, with accompanying gastrointestinal pathology, including diarrhea, among other signs and symptoms. C. difficile overgrowth includes or results in the elaboration of C. difficile toxins (which include C. difficile toxins A and B, and cytolethal distending toxins A and B), which are detectable in stool samples of those with C. difficile infection using, for example, commercially available immunoassays (e.g., lateral flow “dipstick” assays). C. difficile overgrowth also increases (as that term is used herein) the relative biomass of C. difficile in the stool as compared to healthy stool.
“Recurrent” C. difficile infection refers to C. difficile infection that occurs after an initial treatment for C. difficile infection, generally antibiotic therapy. Recurrence can occur, for example, days, weeks or months after an initial infection has been treated, including up to 8 months or more after the initial infection.
As used herein, “bacteriotherapy” refers to the administration of live bacteria for the treatment or prevention of a disease or disorder, including, but not limited to C. difficile infection. As the term is used herein, the bacteria administered are viable and metabolically active, or become metabolically active after administration. Thus, bacteriotherapy can include administration of metabolically active bacteria and/or the administration of dried (e.g., lyophilized) viable bacteria or bacterial spores or a mixture or combination of these. It is preferred that bacteriotherapy as described herein result in the proliferation of the administered bacteria, and preferably establishment of a persistent presence of a population of such bacteria in the gut. A “persistent” presence is one that is detectable at least one week or more after administration, including, for example, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks or more, including essentially indefinite establishment of the administered species or strain in the gut of the subject. To the extent that administration does not result in a persistent presence, re-administration, including re-administration at regular intervals is specifically contemplated.
As used herein, the term “oral formulation” refers to a pharmaceutical formulation formulated to be suitable for oral administration. Oral formulations include, for example, elixirs and suspensions including an active agent or agents that render the agent or agents suitable, or more suitable than the agent alone, for oral administration, and can include, for example, cryoprotectants, reducing agents to protect against oxygen exposure, sweeteners or other agents to enhance palatability. Oral formulations also include, for example, formulations in capsules or microcapsules, which can be standard or designed to dissolve substantially only after passage through the acidic environment of the stomach—i.e., enteric encapsulation. Oral formulations can also include, for example, an antacid, which can modify the pH of the stomach and thereby promote survival of a bacteriotherapy composition. Oral formulations also include, for example, an active agent or agents in admixture with or as a component of a foodstuff.
As used herein, the term “viable” encompasses metabolically active, proliferative as well as dried forms of bacteria that can be reconstituted to provide metabolically active, proliferative bacteria, and spores that can germinate to provide metabolically active, proliferative bacteria. It is contemplated that non-viable, killed cells or an extract or preparation derived therefrom may have benefit for inhibiting C. difficile infection. It is also contemplated that while not proliferative, bacterial cells that have been rendered non-proliferative, e.g., by irradiation, yet retain metabolic activity, can also provide a benefit for inhibiting C. difficile infection. In such instances, regular administration of preparations of killed or proliferatively inactivated bacteria as described herein would be specifically contemplated.
As used herein, the term “dried, viable form” refers to a preparation of bacteria that have been dried, e.g., lyophilized, yet retain the capacity, upon re-hydration, to become metabolically and proliferatively active. Methods for the preparation of dried, viable bacteria are known to those of skill in the art. Indeed, C. scindens, C. bifermentans and C. hylemonae species available from American Type Culture Collection (ATCC) are shipped in dried, viable form.
As used herein, the term “does not comprise species X,” “does not contain species X,” means, at a minimum, that a composition does not comprise viable species X. In one embodiment, the composition does not contain species X at all—i.e., even dead or inactivated bacteria of species X are excluded. Similarly, the term “comprises no other bacteria,” means, at a minimum, that the subject composition does not comprise any viable bacteria other than those specified. In one embodiment, “comprises no other bacteria” means the formulation does not comprise any other bacteria at all, whether dead or alive.
As used herein, a “prebiotic” refers to an ingredient that allows or promotes specific changes, both in the composition and/or activity in the gastrointestinal microbiota that may (or may not) confer benefits upon the host. A prebiotic is not directly digestible by humans, but that is readily digestible by and promotes the growth or establishment of one or more probiotic and/or commensal microbes. Non-limiting examples of prebiotics include but are not limited to inulin, fructooligosaccharides, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides. In one embodiment, a prebiotic comprises a sugar or carbohydrate e.g., a starch or other carbohydrate polymer, or protein formulation that can be digested by C. bifermentans and C. scindens, but not readily by other commensals, and therefore promotes selective expansion of these species.
As used herein, the term “defined bacterial microbiota” refers to one or a combination of known bacteria in a composition to be administered for bacteriotherapy. In a defined bacterial microbiota, the component bacteria are all known and prepared from pure culture; a defined bacterial microbiota excludes stool or fecal material. In one embodiment, the relative proportions of members of a defined microbial microbiota are defined and deliberately set.
As used herein, the term “probiotic” refers to microorganisms that form at least a part of the transient or endogenous flora or microbial consortium and thereby exhibit a beneficial prophylactic and/or therapeutic effect on the host organism. Probiotics are generally known to be clinically safe (i.e., non-pathogenic) by those skilled in the art.
As used herein, a microbe that “supports” another produces one or more metabolites that promotes the growth or maintenance of another microbe. Alternatively, or in addition, a microbe that “supports” another microbe provides or contributes to an environment, e.g., a pH, redox status, nutritional status, or other environmental component that promotes growth of the other, or, for example, inhibits the growth of a third species or class of microbes that inhibits the desired species.
As used herein, the term “at risk” as applied to risk of C. difficile infection refers to an individual in a high risk category for C. difficile infection. These include, for example, those who are receiving or have recently completed a course of antibiotic therapy for a different infection (or for a first infection with C. difficile), those of advanced age, those who are hospitalized or under nursing home care, those who have had prior C. difficile infection, those taking medication for gastric acid suppression (e.g., with proton pump inhibitors or histamine-2 receptor antagonists), those undergoing gastrointestinal procedures, those undergoing chemotherapy, those with inflammatory bowel disease, and those who are immunosuppressed. Additional risk factors include low relative abundance of the biomarker or indicator species C. scindens and/or C. hylemonae, as described herein below.
As used herein, the term “recently received,” in reference to antibiotic treatment refers to having received the last dose of a course of antibiotics to treat C. difficile or another infection. In this context, one has “recently received” antibiotic treatment if the last dose of a course was given two days, three days, four days, five days, six days, one week, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, three weeks, twenty-two days, twenty-three days, twenty-four days, twenty-five days, twenty-six days, twenty-seven days or four weeks previously.
As used herein, the term “operational taxonomic unit (OTU, plural OTUs)” refers to a terminal leaf in a phylogenetic tree and is defined by a specific genetic sequence and all sequences that share a specified degree of sequence identity to this sequence. The specific genetic sequence may be the 16S rRNA sequence or a portion of the 16S rRNA sequence, or it may be a functionally conserved housekeeping gene found broadly across the eubacterial kingdom. OTUs generally share at least 97%, 98%, or 99% sequence identity in the reference sequence, although lower numbers can be applied—it follows that the lower the % identity to the reference sequence, the more species will be encompassed, and the more variation between members of the OTU grouping.
As used herein, the term “clade” refers to the set of OTUs or members of a phylogenetic tree downstream of a statistically valid node in a phylogenetic tree. The clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit.
In microbiology, “16S sequencing” or “16S rRNA” or “16S-rRNA” or “16S” refers to sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500 nucleotides in length and is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to a second isolate using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most bacteria, as well as fungi.
The “V1-V9 regions” of the 16S rRNA refers to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature. Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU. In another embodiment, the full length of a 16S rRNA is used to characterize an OTU. A person of ordinary skill in the art can identify the specific hypervariable regions of a candidate 16S rRNA by comparing the candidate sequence in question to the reference sequence and identifying the hypervariable regions based on similarity to the reference hypervariable regions.
As used herein, a “protease” is an enzyme which degrades proteins or peptides into smaller components or amino acids. A “proteolytic” species of bacteria or a “proteolytic” anaerobe is an anaerobic bacterial species that expresses and secretes a protease enzyme that generates amino acids fermentable by Stickland fermentation. A “highly” proteolytic species expresses and secretes proteolytic activity at least as great or greater than that of C. scindens as measured by methods known in the art or described herein. In one embodiment, a “highly” proteolytic species expresses and secretes a proteolytic activity at least as great or greater than that of C. bifermentans.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. Where applicable, a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.
As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate mammal such as a non-human primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, or canine species, e.g., dog, fox or wolf. Mammals other than humans can be advantageously used as subjects that represent models of human disease e.g., C. difficile infection. A subject can be male or female. The terms “individual,” “patient” and “subject” are used interchangeably herein.
A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. C. difficile infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors. Risk factors for C. difficile infection include, but are not limited to, receiving antibiotic therapy for a different infection (or for a first infection with C. difficile), advanced age, hospitalization or nursing home care, prior C. difficile infection, gastric acid suppression (e.g., with proton pump inhibitors or histamine-2 receptor antagonists), gastrointestinal procedures, chemotherapy, inflammatory bowel disease, and immunosuppression. In addition, in those infected with C. difficile, low levels of vitamin D have been shown to be an independent predictor of poor outcome and are associated with higher recurrence. Additional risk factors include low relative abundance of the biomarker or indicator species C. scindens and/or C. hylemonae as described herein below.
A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
As used herein, the term “suppressing expression of a bacterial toxin” refers to conditions or treatment(s) that inhibit or reduce, as the terms are defined herein, the expression of a toxin by a bacterium in a subject. In one embodiment, the inhibition or reduction is sufficient to reduce one or more symptoms or one or more markers of bacterial infection in a subject. In one embodiment, the inhibition or reduction is to a level that does not cause symptoms or detectable pathology in the subject. In another embodiment, the suppression acts upon toxin production without necessarily reducing biomass or viability of the bacterium.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. C. difficile infection, including recurrent C. difficile infection. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality. Treatment can include suppression of bacterial toxin production, e.g., to a degree such that pathology or symptoms caused by the toxin are reduced.
As used herein, the term “pharmaceutical composition” refers to an active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found to occur in nature. Where the active agent includes one or more bacterial species, whether in active metabolic, dried viable or spore form, the carrier will be compatible with the bacteria and will not interfere with viability or activity of the bacteria.
As used herein, the term “administering,” refers to the placement of a therapeutic or pharmaceutical composition as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising agents as disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. Common routes of administration for bacteriotherapies include oral administration, often, but not necessarily in the form of one or more capsules, and direct administration to the lower GI tract, e.g., via enema or colonoscope.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean±1%.
As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the technology.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
Other terms are defined within the description of the various aspects and embodiments of the technology of the following.
By its very name, C. difficile is notorious for being difficult to treat. Not only is the C. difficile organism resistant to multiple antibiotics commonly used to treat bacterial infections in clinical settings (e.g., aminoglycosides, lincomycin, tetracyclines, erythromycin, clindamycin, penicillins, cephalosporins, and fluoroquinolones), but the use of such antibiotics to treat other infections is one of the greatest risk factors for developing C. difficile pathology. Add to that the ability of C. difficile to sporulate and essentially wait until even an antibiotic that kills its metabolically active form is gone, and it truly deserves its name and reputation.
Toxigenic strains of C. difficile cause pseudomembranous colitis, a severe infectious disease of the colon. Infection often arises with disruptions to the gut microbiota, commonly after use of antibiotics that ablate protective commensals. C. difficile elaborates toxins when starved for carbon, nitrogen or energy (=high levels of NAD+). The toxins rapidly destroy the gut epithelial barrier enabling release of host-derived proteins and carbohydrate sources into the gut lumen which the pathogen then metabolizes. When co-existing with “beneficial” commensal species, C. difficile remains energetically stable and does not elaborate significant toxin. For patients failing multiple rounds of antibiotic treatment, who develop recurrent C. difficile infections, fecal microbiota transplant (FMT) is highly efficacious through its restoration of healthy gut microbial communities. However, the mechanisms of action of FMT, and specific microbes needed to halt toxin production and reduce pathogen biomass remain ill-defined. While microbial conversion of primary host bile acids to secondary acids have been hypothesized to play a key role, mechanisms of action have highlighted effects upon germination of C. difficile spores (primary bile acids stimulate; secondary bile acids inhibit) and less so on metabolic factors that modulate toxin production or overall pathogen biomass.
The technology described herein is related to the discovery of commensal bacteria that can suppress toxin production by Gram-positive toxigenic bacteria such as C. difficile and thereby treat or prevent the development of toxin-mediated pathology. Indeed, it was found that as few as a single species of bacteria can provide complete protection from otherwise fatal C. difficile infection in murine models described herein. Suppression of toxin production provides an alternative route to treatment of C. difficile-mediated pathology, in that it can be sufficient for treatment to just suppress production of the pathology-generating toxin without necessarily killing the microbe.
It is discovered that commensal bacteria that suppress toxin production by Gram-positive toxigenic bacteria such as C. difficile strongly express and secrete protease enzyme activity that generates free amino acids that can be fermented by C. difficile via Stickland fermentation, which metabolizes free amino acids to generate energy. Without wishing to be bound by theory, the picture that emerges is that in order to avoid toxin production by Gram-positive toxigenic bacteria such as C. difficile, the gut environment needs to provide energy and nutrients sufficient to keep such bacteria from becoming stressed and responding by toxin production. Thus, commensal bacteria that secrete proteolytic enzymes can provide therapeutic benefits in suppressing toxin production by Stickland-fermenting Gram-positive toxigenic bacteria such as C. difficile. It is noted that among the protective commensal species found to have protective effects were bacteria that are themselves capable of Stickland fermentation, including, for example, C. bifermentans and C. scindens.
Examination of the effects of protective commensals as described herein provides insights into the mechanisms of protection and further targets for manipulation of toxin production and biomarkers useful for evaluation of, for example, the likelihood of C. difficile recurrence, or the efficacy of treatment.
The following provides additional details regarding C. difficile and the discoveries of highly protective/therapeutic commensal species, and considerations to facilitate the performance of the methods and compositions for the prevention, treatment, prognosis and diagnosis that exploit these discoveries.
Clostridium difficile
Clostridium is a genus of Gram-positive bacteria including around 100 species, which includes several significant human pathogens, including the causative agent of botulism and an important cause of diarrhea, Clostridium difficile. Pathogenic Clostridium strains include but are not limited to C. botulinum that can produce botulinum toxin in food or wounds and can cause botulism. Clostridium difficile can flourish when other members of the gut microbiota are killed during antibiotic therapy, leading to superinfection and potentially fatal pseudomembranous colitis (a severe necrotizing disease of the large intestine). Clostridium perfringens causes a wide range of symptoms, from food poisoning to cellulitis, fasciitis, and gas gangrene. Clostridium tetanii causes tetanus. Clostridium sordellii can cause a fatal infection in rare cases after medical abortions.
C. difficile is a Gram-positive, anaerobic, spore-forming and toxin-producing bacillus, belonging to cluster XI of the Clostridium genus and commonly occurs in the hospital environment, and in the intestines of humans and domesticated animals. C. difficile can cause a spectrum of clinical conditions in humans, collectively known as C. difficile infections (CDI), which range from mild and possibly recurrent diarrhea to life-threatening complications such as pseudomembranous colitis (PMC), toxic megacolon and colonic perforation. As discussed elsewhere herein, C. difficile can occur in the gut of healthy individuals, but be maintained at a level that does not cause illness and/or have the expression of C. difficile toxin suppressed to a degree that the organism does not cause illness.
The clinical spectrum of C. difficile ranges from asymptomatic colonization, mild and self-limiting disease to a severe, life-threatening pseudomembranous colitis, toxic megacolon, sepsis and death. CDI is defined when there is the presence of symptomatic diarrhea defined by three or more unformed stools per 24 h and at least one of the following criteria: a positive laboratory assay for C. difficile toxin A and/or B or toxin-producing C. difficile organism in a stool sample or pseudomembranous colitis or colonic histopathology characteristic of CDI revealed by endoscopy. Toxin is detected, e.g., by immunoassay for the A and or B toxin proteins, and/or by RT-PCR for the toxin-encoding nucleic acids. CDI can be associated with an increased abundance of toxin-producing C. difficile strains, leading to high toxin concentrations within the colon resulting in inflammation and damage of the colonocytes.
The various strains of C. difficile may be classified by a number of methods. One of the most commonly used is polymerase chain reaction (PCR) ribotyping in which PCR is used to amplify the 16S-23S rRNA gene intergenic spacer region of C. difficile. Reaction products from this provide characteristic band patterns identifying the bacterial ribotype of isolates. Toxinotyping is another typing method in which the restriction patterns derived from DNA coding for the C. difficile toxins are used to identify strain toxinotype. The differences in restriction patterns observed between toxin genes of different strains are also indicative of sequence variation within the C. difficile toxin family. Toxin B shows sequence variation in some regions. For example, there's an approximate 13% sequence difference with the C-terminal 60 kDa region of toxinotype 0 Toxin B compared to the same region in toxinotype III.
C. difficile uses a variety of carbon and nitrogen sources for energy and metabolism. Within the gut environment, these sources can originate from the diet, from other commensals, or from the host, particularly when elaborating toxin to disrupt mucosal barriers. Known carbon sources include sugars such as glucose, fructose, mannose, mannitol or sorbitol, the latter two of which are poorly absorbed in the gut and reach the colon. Sources of carbon and nitrogen include amino groups and ethanolamine, a breakdown product of host phosphatidyl ethanolamine from eukaryotic cell membranes, other commensals, or the diet. As noted elsewhere herein, when preferred energy sources or nutrients for C. difficile run low or become limiting, C. difficile toxin production is induced.
C. difficile Toxins
As noted above, C. difficile encodes two toxin proteins, referred to as TcdA and TcdB, or simply herein as C. difficile toxin A and toxin B. TcdA and TcdB are broadly classified as AB toxins, wherein a B subunit is involved in the delivery of an enzymatic A subunit into the cytosol of a target cell. The enzymatic A subunit of TcdA is an N-terminal glucosyltransferase domain (GTD) that inactivates members of the Rho family of small GTPases by glucosylation. The B subunit is composed of three regions: a combined repetitive oligopeptides (CROPS) domain, a delivery/pore-forming domain and an autoprocessing domain (APD).
TcdA is encoded by the tcdA gene. Sequences for TcdA are known for a number of species, e.g., for C. difficile 630 (the TcdA NCBI Gene ID is 4914076) and polypeptide sequence (e.g., YP_001087137.1 (SEQ ID NO: 1). The sequence for the TcdA polypeptide is as follows (SEQ ID NO: 1):
The tcdA gene sequence for C. difficile 630 is as follows (SEQ ID NO:2):
TcdB is encoded by the tcdB gene. Sequences for TcdB are known for a number of species, e.g., for C. difficile 630 (the TcdB NCBI Gene ID is 4914074) and polypeptide sequence (e.g., YP_001087135.1 (SEQ ID NO: 3). The sequence for the TcdB gene product is as follows (SEQ ID NO: 3):
The tcdB gene sequence for C. difficile 630 is as follows (SEQ ID NO: 4):
Outbreaks of CDI have been reported with Toxin A-negative/Toxin B-positive strains, which indicates that Toxin B is also capable of playing a key role in the disease pathology. TcdA and TcdB are 308 and 270 kDa proteins, respectively. The toxins belong to the family of large clostridial toxins (LCTs), which are a group of homologous, high molecular weight proteins that further include the lethal and hemorrhagic toxins from C. sordellii (TcsL and TcsH, respectively), α-toxin from C. novyi (Tcnα) and a cytotoxin from C. perfringens (TpeL). The homologous proteins intoxicate host cells through a multistep mechanism that involves (i) receptor binding and endocytosis, (ii) pore formation and translocation of the GTD across the endosomal membrane, (iii) autoprocessing and release of GTD into the cytosol, and (iv) glucosylation of host Rho GTPase. Both Toxins A and B also contain a second enzyme activity in the form of a cysteine protease which appears to play a role in the release of the effector domain into the cytosol after translocation. The C. difficile binary toxin modifies cell actin by a mechanism which involves the transfer of an ADP-ribose moiety from NAD onto its target protein. Given the similarities in toxin structure and the genetic similarities of Clostridial species, it is likely that the expression of toxins of other spore-forming toxigenic Clostridium species are regulated in a similar manner to that of C. difficile, i.e., in a manner sensitive to environmental conditions that can be influenced by commensal bacteria as described herein.
Additional bacterial toxins, including additional Clostridial toxins, are described in Table 1.
Clostridium difficile
Bacillus anthracis
Bordetella pertussis
Clostridium botulinum
Clostridium perfringens
Clostridium tetani
Corynebacterium diphtheria
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Pseudomonas aeruginosa
Shigella dysenteriae
Staphylococcus aureus
Staphylococcus aureus
Streptococcus pneumonia
Streptococcus pyogenes
Streptococcus pyogenes
Vibrio cholera
Regulation of C. difficile Toxin Gene Expression
C. difficile's pathogenicity locus, PaLoc, contains the toxin operon with genes tcdA, tcdB and tcdE that respectively encode the A and B portions of the toxin and a putative holin involved in toxin export. In addition to the toxins TcdA and TcdB, the PaLoc in pathogenic strains encodes TcdR, a member of the extracytoplasmic function family of alternative sigma factors that plays a critical role in activating the expression of tcdA and tcdB; transcription of the tcdA and tcdB genes requires TcdR to enable RNA polymerase to have specificity to bind the toxin gene promoters. Within C. difficile, multiple nutritional regulators influence PaLoc gene expression at the level of tcdR and the tcdAEB operon genes. In particular, C. difficile primarily elaborates toxin under starvation conditions to extract nutrients from the host and to also enable shedding of spores. Exogenously added glucose, amino acids—proline and leucine in particular, as well as cysteine, inhibit toxin production through codY, ccpA, rex and/or prdR activation. Exogenously added butyrate induces toxin production, while butanol does not. The symptoms of CDI correlate with the expression of TcdR. The genes encoding TcdA (tcdA) and TcdB (tcdB) are located within a 19.6-kb chromosomal region that makes up the PaLoc. The activity of TcdR is modulated by TcdC, an anti-sigma factor that destabilizes the TcdR-core RNA polymerase complex. TcdC seems to be most active in rapidly growing cells.
Stickland Fermentation
Stickland reactions couple metabolism of pairs of amino acids in which one amino acid, acting as an electron donor, is oxidatively deaminated or decarboxylated and a second amino acid, acting as an electron acceptor, is reduced or reductively deaminated (Stickland, L. H., Biochem. J. 28: 1746-1759 (1934). The most efficient electron donors are leucine, isoleucine, and alanine, and the most efficient acceptors are glycine, proline. Hydroxyproline is also an efficient Stickland acceptor.
C. difficile, like other cluster XI species of clostridia, uses Stickland fermentations to extract energy from amino acids. Donor amino acids include the electron-rich branched chain amino acids leucine, isoleucine and valine (BCAA), aromatic amino acids phenylalanine, tyrosine and tryptophan, and acceptor amino acids glycine and proline. Cellular proline and glycine reductases transfer electrons from Stickland donor amino acids to recipients proline and glycine. The reaction consumes one NADH to one NAD+ in the reductive pathway, with release of ammonia and the branched short-chain fatty acids isocaproate, isobutyrate and isovalerate, and regenerates 2 NAD+ to NADH in the oxidative pathway. to regenerate NAD+ to NADH.
The reduction of the Stickland acceptors glycine and proline is performed by two selenium-dependent reductases, glycine reductase (GR) and D-Proline reductase (PR), respectively. GR catalyzes the reductive deamination of glycine to acetyl phosphate and ammonium, and PR reductively cleaves D-proline to 5-aminovalerate. The glycine reductase and proline reductase operons, respectively, mediate these reactions. Each reductase is comprised of multiple polypeptides and is dependent upon selenocysteine residues for activity.
Genes encoding the C. difficile GR and PR subunits are clustered in two distinct genetic loci (grd and prd, respectively) on the chromosome of C. difficile strain 630. The grd locus contains eight genes. Two of the genes, grdA and grdB, likely encode the selenocysteine-containing subunits of GR. The seven genes of the prd operon include prdF, that encodes a d-proline racemase, and prdB, that encodes the selenium-containing subunit of PR.
In one embodiment, prdR activates the proline reductase operon and represses the glycine reductase operon. prdR suppresses the expression of C. difficile toxins by inhibiting butyrate, coDY, ccpA, tcdR, and/or tcdA production. The Stickland fermentation pathway using proline as the Stickland acceptor generates NADH and the metabolite 5-aminovalerate from proline. The acetate-generating pathway generates NADH and the metabolite acetate from glycine. C. difficile consumes large amounts of NADH in the carbohydrate metabolism pathway from acetate. Therefore, acetate-generating pathways are more prone to inducing a stress response pathway, leading to the generation of butyrate, codY, ccpA, tcdR, and/or tcdA toxins.
In one embodiment, the method of treating or preventing a pathology caused by expression of a bacterial toxin compromises administering at least one bacterial organism that encodes and expresses one or more of D-Proline reductase (PR), Glycine reductase (GR), thioredoxin, or choloylglycine.
As noted above, the C. difficile pathogen can also utilize sugars, sugar alcohols, and ethanolamine for energy production (carbohydrate metabolism and other energy production approaches used by C. difficile are discussed further below). Toxin production in C. difficile responds to environmental conditions, including the availability of specific nutrients, temperature changes, and alteration of the redox potential. The presence of a rapidly metabolizable carbon source or certain amino acids inhibits toxin gene expression. When cells are grown in rich medium, the toxin genes are transcribed only when the cells reach stationary phase. While not wishing to be bound by theory, this is fully consistent with the discovery that protective commensal species are highly proteolytic and induce genes in C. difficile that, for example, promote the use of ethanolamine in the gut environment as an energy source.
Nutritional regulators within C. difficile, including codY, ccpA, and rex sense aspects of amino acid, sugar, Stickland reactions and NAD+/NADH pools respectively. Each of these regulons also exerts effects on PaLoc gene expression through direct and indirect mechanisms.
Upon binding of BCAA and GTP, codY strongly represses tcdR transcription, with subsequent reduction of tcdAEB gene expression. ccpA, active under carbon starvation conditions, binds the tcdAEB operon promoter, enhancing toxin expression if the tcdR sigma factor has been expressed. Thus, starvation where carbohydrates and Stickland amino acids are limiting induces expression of toxin. Under conditions of nutrient sufficiency, codY, ccpA and rex act coordinately through direct and indirect mechanisms to repress the expression of PaLoc genes. Notably, exogenous in vitro addition of Stickland amino acids or carbohydrate energy sources represses C. difficile toxin expression through the above mechanisms, while exogenous addition of butyrate, a key end product of anaerobic carbohydrate fermentation, can alone induce toxin expression. Together, these gene regulatory systems promote C. difficile toxin expression under conditions of starvation and energy limitation by sensing preferred sources for intracellular energy production.
NAD+/NADH
The balance of NAD+ to NADH influences the expression of C. difficile toxin. The Stickland reaction involves the coupled oxidation and reduction of pairs of amino acids to generate ATP and NAD+. The oxidative pathway generates ATP and NADH, while the reductive pathway regenerates NAD+ from NADH. Proline reductase (PR) and glycine reductase (GR) expression are specifically induced in the presence of proline and glycine, respectively, and carry out the respective reduction of these amino acids. Moreover, the addition of proline to the growth medium decreases the expression of GR-encoding genes, suggesting a preferential utilization of proline for NAD+ regeneration. PrdR, which is a regulator that responds to proline, mediates both the proline-dependent activation of PR and the proline-dependent repression of toxin genes and the GR operon. When proline is limiting in the medium or if PrdR or PR is inactive, the alternative reductive pathways are induced. In fact, both PrdR and a functional PR are indirectly required for the proline-dependent regulation of the alternative reductive pathways in response to the intracellular concentration of NADH and NAD+. This process involves the global redox-sensing regulator Rex. In a number of Gram-positive bacteria, Rex acts as a repressor of genes that are important for growth using fermentation. Rex directly senses changes in redox status and is only active as a DNA-binding protein when the intracellular NADH/NAD+ ratio is low. The protein Rex is stimulated by NAD+ but inhibited by NADH. Although Rex, like PrdR, controls the proline-responsive expression of these alternative reductive pathways, Rex also mediates the proline-dependent repression of toxin gene expression, probably through the regulation of butyrate production. As a result, when proline is not limiting the NADH/NAD+ ratio is low and Rex is active as a repressor of the alternative NAD+ regeneration pathways. In contrast, if proline becomes limiting, the NADH/NAD+ ratio increases and NADH prevents Rex-dependent repression of the alternative pathways. The regeneration of NAD+ using these alternative reductive pathways leads to an accumulation of butyrate, a compound that stimulates toxin synthesis as shown in
In one embodiment, the method of treating or preventing a pathology caused by expression of a bacterial toxin compromises administering at least one bacterial organism that promotes Stickland fermentation by C. difficile. In another embodiment, the bacterial organism itself performs Stickland fermentation—bacteria that have evolved to perform Stickland fermentation tend to express, among other things, extracellular proteolytic activities that can feed amino acids into the Stickland fermentation pathway for C. difficile.
The following discusses the C. difficile toxin regulatory factors involved in sensing environmental conditions.
TcdR
TcdR or “alternative RNA polymerase sigma factor TcdR” is encoded by the tcdR gene. Sequences for TcdR are known for a number of species, e.g., for C. difficile 630 (the TcdR NCBI Gene ID is 4914073) and polypeptide sequence (e.g., YP_001087134.1 (SEQ ID NO: 5). The sequence for the TcdR gene product is as follows (SEQ ID NO: 5):
The tcdR gene sequence for C. difficile 630 is as follows (SEQ ID NO: 6):
TcdR polypeptide expression can be detected or measured via immunoassay, e.g., via an ELISA using antibodies raised against TcdR. TcdR expression can also be detected or measured by RT-PCR using primers specific for the tcdR mRNA.
CodY
CodY or “transcriptional repressor CodY” refers to a protein encoded by the codY gene. CodY is a sensor of carbon and nitrogen, and binds GTP and leucine to become active. When active, it represses transcription through a number of genes. C. difficile actively undergoing Stickland fermentations (abundant leucine and energy) represses toxin production in part through CodY's effects on tcdR and tcd operon gene expression. Transcription of the tcdR gene is repressed during the rapid exponential growth phase by CodY. CodY is active in cells with an excess of branched-chain amino acids (isoleucine, leucine, and valine) and GTP. When the cells reach stationary phase, the intracellular concentrations of these ligands decrease and CodY is less able to bind as a repressor, leading to derepression of tcdR transcription.
Sequences for CodY are known for a number of species, e.g., for C. difficile 630 (the codY NCBI Gene ID 4915868) and polypeptide sequence (e.g., YP_001087769.1 (SEQ ID NO: 7). The sequence for the codY gene product is as follows (SEQ ID NO: 7):
The coDY gene sequence for C. difficile 630 is as follows (SEQ ID NO: 8):
CodY polypeptide expression can be detected or measured via immunoassay, e.g., via an ELISA using antibodies raised against CodY. CodY expression can also be detected or measured by RT-PCR using primers specific for the codY mRNA.
CcpA
Catabolite control protein A, CcpA, senses carbon state within the cell and is a global regulator of carbon metabolism in Gram-positive bacteria. In B. subtilis, fructose derivatives of glucose bind and activate CcpA. Regulation of C. difficile toxin production by carbon source is mediated at least in part by CcpA, which is a direct repressor of the tcdA and tcdB genes.
CcpA protein is encoded by the ccpA gene. Sequences for CcpA are known for a number of species, e.g., for C. difficile 630 (the CcpA NCBI Gene ID 4915199) and polypeptide sequence (e.g., YP_001087548.1 (SEQ ID NO: 9). The sequence for the CcpA gene product is as follows (SEQ ID NO: 9):
The CcpA gene sequence for C. difficile 630 is as follows (SEQ ID NO:10):
CcpA polypeptide expression can be detected or measured via immunoassay, e.g., via an ELISA using antibodies raised against CcpA. CcpA expression can also be detected or measured by RT-PCR using primers specific for the ccpA mRNA.
Rex
Rex or “redox-sensing transcriptional repressor” senses energy state of the cell, particularly the concentration of NADH/NAD+. When activated by low energy (high concentrations of NAD+), Rex is known to indirectly lead to toxin expression, though the mechanisms of action are not well described. High NAD+ and butyrate levels, the latter possibly reflective of NADH consumption, are believed to promote C. difficile toxin production through this pathway.
Sequences for Rex and the gene sequence encoding it are known for a number of species, e.g., for C. difficile 630 (the rex NCBI Gene ID 4914836) and polypeptide sequence (e.g., YP_001086640.1 (SEQ ID NO:11). The sequence for the Rex gene product is as follows (SEQ ID NO: 11):
The rex gene sequence for C. difficile 630 is as follows (SEQ ID NO:12):
Rex polypeptide expression can be detected or measured via immunoassay, e.g., via an ELISA using antibodies raised against Rex. Rex expression can also be detected or measured by RT-PCR using primers specific for the rex mRNA.
Energy status of C. difficile is important for determining whether the pathogen expresses toxin. The following considers various pathways in addition to Stickland fermentation that C. difficile uses to extract energy needed for metabolism from its environment.
C. difficile Carbohydrate Utilization
C. difficile metabolizes sugars such as glucose and fructose, and sugal alcohols such as sorbitol and mannitol for energy production. End metabolites and points of energy production are shown in
C. difficile Ethanolamine Utilization
Ethanolamine occurs abundantly in gut secretions, primarily from the breakdown of phosphatidyl ethanolamine from sloughed epithelial cells, as well as from dietary and other commensal sources. The C. difficile eut operons include a two-component ethanolamine sensing system and transporter, and downstream genes that encode the carboxysome, a polyhedral protein complex in which ethanolamine is metabolized to ammonia and acetaldehyde, resulting in the generation of NADH from NAD+. A schematic of the pathway is shown in
C. difficile Reductive Leucine Pathway
C. difficile also uses the reductive leucine pathway for energy. Mediated by the had gene operon, the pathway generates 2 ATP from 3 leucine molecules. No net NADH/NAD+ is consumed, and the end product of this pathway is isocaproate.
Cysteine and Threonine Use
C. difficile can convert these amino acids to pyruvate with branch points for conversion of pyruvate to acetate, propionate or butyrate, that follow the glycolytic pathways.
SCFA, bSCFA and 5-Aminovalerate
Exogenous acetate and succinate are hypothesized to be taken up and converted to butyrate by C. difficile if it needs energy and does not have sugars or amino acids readily available. The capacity of C. difficile to use exogenous branched short-chain fatty acids and 5-aminovalerate is not known. Other organisms can use these compounds for further energy derivation. Without wishing to be bound by theory, another potential mechanism of protection provided by Stickland fermenting species could be cross-feeding these compounds to C. difficile for energy, or through other mechanisms of C. difficile sensing its extracellular environment.
PrdR
PrdR regulates that proline and glycine reductase enzymes, and also has indirect effects on toxin expression. With abundant proline, PrdR activates the proline reductase operon and inhibits the glycine reductase operon. PrdR activates the proline reductase operon and represses the glycine reductase operon. prdR suppresses the expression of C. difficile toxins by inhibiting butyrate, coDY, ccpA, tcdR, and/or tcdA production. The Stickland fermentation pathway using proline as the Stickland acceptor generates NADH and the metabolite 5-aminovalerate from proline. The acetate-generating pathway generates NADH and the metabolite acetate from glycine. C. difficile consumes large amounts of NADH in the carbohydrate metabolism pathway from acetate
Therapeutic Microbiota
Described herein are therapeutic microbiota that suppress toxin production by Gram positive, spore-forming bacteria such as C. difficile. As described herein above, commensal species that provide C. difficile with amino acids and other energy sources that promote Stickland fermentation can provide therapeutic benefits by suppressing C. difficile toxin production. Such species include, for example, species that express and secrete one or more protease enzymes that generate a supply of amino acids for C. difficile to derive energy from via Stickland fermentation, as well as species that themselves perform Stickland fermentation.
Proteases Expressed by Therapeutic Species
In one embodiment, a method of suppressing bacterial toxin production or treating or preventing a pathology caused by expression of a bacterial toxin compromises administering at least one bacterial organism that encodes and secretes a protease enzyme. Protease enzymes catalyze protein catabolism by hydrolysis of peptide bonds. Proteases are produced by all living organisms, in which they display many physiological functions ranging from generalized protein degradation to more specific and regulated processes such as blood coagulation, hormone activation, or transport of secretory proteins across membranes. Proteases can be either exopeptidases, whose actions are directed by the amino- or carboxyl terminus of proteins, or endopeptidases, which cleave internal peptide bonds. Extracellular proteases are made as precursors, containing an amino-terminal signal peptide, which is removed during the export to generate a mature, extracellular protease.
Proteolytic Clostridia elaborate a number of extracellular and membrane-bound metalloprotein, serine and other classes of proteases. The proteases generate free amino acids from available extracellular protein. An estimated 10-15 grams/day of undigested dietary protein enters the large intestine and remains available for microbial digestion and growth. The proteolytic species commonly use Stickland fermentations as core metabolic processes for energy generation under anaerobic conditions. Protease activity can be measured biochemically with lysis assays of casein, gelatin and other proteins, and with, e.g., FITC-conjugated substrates that release the fluorophore for detection. Microbiologically, protease activity can be measured on solid agar (casein hydrolysis agar), or with casein gelatin or meat granule hydrolysis in liquid media.
Among Stickland-fermenting Cluster XI Clostridia, Clostridium bifermentans is among the most highly proteolytic species, preferring Stickland fermentations over glycolytic pathways for energy extraction. In contrast, C. sardiniense, a non-proteolytic and strongly glycolytic Cluster I Clostridial species, produces abundant butyrate through anaerobic fermentation of available carbon sources.
In one embodiment, a bacterial organism administered as described herein, e.g., to suppress C. difficile toxin production, encodes and secretes at least one protease selected from the group shown in Table 2 or a homologue thereof. In another embodiment, the species administered encodes and secretes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or all of the protease enzymes listed in Table 2. In another embodiment, the species administered expresses one or more (up to and including all) enzymes with the same Enzyme Commission (E.C.) number as an enzyme in Table 2. That is, the administered species can express and secrete one or more enzymes that catalyze the same reaction as an enzyme listed in Table 2. Additional bacteria that express and secrete such enzymes can be identified, for example, by analysis of bacterial genomic sequences for sequence encoding such secreted enzymes. For example, the sequence encoding a representative listed C. bifermentans protease can be used to query the genomic sequences of other non-pathogenic bacteria, including but not limited to other commensal bacteria, including but not limited to other Clostridial bacteria, for sequence encoding a homologous enzyme.
Clostridium bifermentans MHMC14 and the corresponding
In one embodiment, a bacterial organism administered as described herein, e.g., to suppress C. difficile toxin production, encodes an enzyme that participates in Stickland fermentation. Such enzymes include, for example, D-proline reductase and glycine reductase. Species that encode one or both of such enzymes can be identified, for example, by analysis of genomic sequences for sequence that encodes the enzymes.
D-Proline Reductase (PR)
“prdA” or “D-Proline reductase (PR)” is encoded by the prdA gene. Sequences for prdA are known for a number of species, e.g., for C. difficile 630 (the prdA NCBI Gene ID is 4916399 and polypeptide sequence e.g., YP_001089760.1), as well as for C. scindens ATCC 35704 (the prdA NCBI polypeptide sequence e.g., EDS06621.1) and for C. bifermentans ATCC 638 (the prdA NCBI polypeptide sequence e.g., EQK41327.1). Proline reductase levels can be measured, for example, via immunoassay or by measurement of RNA encoding the enzyme, e.g., via RT-PCR. The sequence for the prdA gene product for C. difficile 630 is as follows (SEQ ID NO: 13):
The prdA gene sequence for C. difficile 630 is as follows (SEQ ID NO:14):
Each reductase is comprised of multiple polypeptides of which the sequences are listed below. In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. bifermentans 638 is as follows (SEQ ID NO:15):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. bifermentans 638 is as follows (SEQ ID NO:16):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. bifermentans 638 is as follows (SEQ ID NO:17):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. bifermentans 638 is as follows (SEQ ID NO:18):
In one embodiment, the sequence for the prdA gene product for C. scindens 35704 is as follows (SEQ ID NO: 19):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:20):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:21):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:22):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:23):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:24):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:25):
In one embodiment, the prdA gene (D-proline reductase (EC 1.21.4.1)) sequence for C. scindens 35704 is as follows (SEQ ID NO:26):
The sequence for the prdA gene product for C. bifermentans 638 is as follows (SEQ ID NO: 27):
In some embodiments, it is useful to measure proline reductase levels or activity, e.g., as an indicator of the activity of the pathway in a sample, or, e.g., to identify a bacterial species as one that likely performs Stickland fermentation and/or can aid in suppressing C. difficile toxin expression. Measurement of proline reductase levels can be performed, for example, by immunoassay or by RT-PCR for the mRNA encoding the enzyme. Measurement of proline reductase activity can be performed, for example, with the fluorometric assay described by Jackson et al., J. Bacteriol. 188: 8487-8495 (2006), which is incorporated herein by reference. The assay follows the DTT- and d-proline-dependent production of δ-aminovaleric acid, which reacts with o-phthalaldehyde to generate a fluorescent product.
The nucleic acid and polypeptide sequences for the proline reductase expressed by C. difficile 630 are NCBI Gene ID 4916399, YP_001089760.1, respectively. The nucleic acid and polypeptide sequences for the proline reductase expressed by C. scindens 35704 NCBI Gene ID 167662491 and EDS06621.1, respectively. The nucleic acid and polypeptide sequences for the proline reductase expressed by C. bifermentans 638 are NCBI Gene ID 531765064 and EQK41327.1, respectively.
Glycine Reductase (GR)
The activity or expression of glycine reductase is important for Stickland fermentation via the glycine reductase pathway. In some embodiments, it is useful to measure glycine reductase levels or activity, e.g., as an indicator of the activity of the pathway in a sample, or, for example, to identify a bacterial species as one that likely performs Stickland fermentation. Measurement of glycine reductase levels in a sample can be performed, for example, by immunoassay and/or via RT-PCR for the mRNA encoding the enzyme. A biochemical assay for glycine reductase activity is described, for example, by Stadtman & Davis, J Biol Chem. 266(33):22147-53 (1991).
The nucleic acid and polypeptide sequences for the glycine reductase expressed by C. difficile 630 are NCBI Gene ID is 4915147 and YP_001088866.2, respectively.
The “grdA” or “glycine/sarcosine/betaine reductase complex protein A” glycine reductase is encoded by the grdA gene. Sequences for grdA are known for a number of species, e.g., for C. difficile 630 (the grdA NCBI Gene ID is 4915147) and polypeptide sequence (e.g., YP_001088866.2 (SEQ ID NO: 28). The sequence for the grdA gene product is as follows (SEQ ID NO: 28):
The grdA gene sequence for C. difficile 630 is as follows (SEQ ID NO:29):
Defined Therapeutic Microbiota
Described herein are defined therapeutic microbiota that can be administered to suppress toxin expression by Gram positive, spore forming toxigenic bacteria such as C. difficile. In one embodiment, a defined therapeutic microbiota is or consists essentially of the single species, C. bifermentans. In another embodiment, the defined therapeutic microbiota comprises, consists essentially of or consists of C. scindens. In another embodiment, the defined therapeutic microbiota consists of, consists essentially of or comprises C. bifermentans and C. scindens. Strains of these species and others that express and secrete proteolytic enzymes into their surroundings and/or themselves perform Stickland fermentation are expected to promote suppression of C. difficile toxin expression. The following describes these species in further detail.
Clostridium Taxonomy: Clustering. As a starting point, species of the Genus Clostridium encompass a large number of anaerobic, spore-forming bacteria. In 1994, Collins et al. (Int. J. Systematic Biol. 44: 812-826 (1994), incorporated herein by reference) described a classification system that placed the species then known, and for which there was 16S rRNA sequence data available, into 19 “clusters,” termed Clostridium Clusters I-XIX, based upon similarities and differences in 16S rRNA sequences. This taxonomy and nomenclature has been retained to date, with some refinement, e.g., classifications of some of the larger clusters into smaller sub-clusters given alphabetic identifiers, e.g., Cluster XIVa. C. difficile is in Cluster XI.
Clostridium bifermentans
C. bifermentans are anaerobic, motile, Gram positive bacteria that colonize the healthy human gut, and are not commonly found to be pathogenic. C. bifermentans is a member of Clostridium Cluster XI, the same cluster as C. difficile. C. bifermentans induces the C. difficile ethanolamine pathway genes and maintains proline reductase activity, glucose and sorbitol metabolism in vivo. Through the greater than 10× repression of tcdR expression and greater than 30× suppression of toxin production, strong codY activation and repression of the PaLoc genes is indicated. The C. difficile metabolic pathways influenced by C. bifermentans are also indicated to activate ccpA, rex and prdR to also inhibit toxin production.
Both C. bifermentans and C. scindens possess the enzymatic machinery for Stickland fermentations. Of the two, C. bifermentans preferentially uses Stickland fermentations over glycolytic pathways, including in the presence of glucose or other sugars (see
A genome of C. bifermentans was sequenced as part of the Human Microbiome Project, and can be found at, e.g., accession #PRJNA212658.
In one embodiment, the C. bifermentans strain is 76 (ATCC #638). The 16S rRNA gene sequence for C. bifermentans strain 76 (SEQ ID NO: 30) is as follows:
In another embodiment, the C. bifermentans strain is a bacterial strain comprising a 16S rRNA sequence that is at least 97%, at least 98%, at least 99%, or more identical to the sequence of SEQ ID NO: 30. A C. bifermentans strain useful in the methods and compositions described herein should express at least 75% of the extracellular proteolytic activity of C. bifermentans strain 76 (ATCC #638) when assayed with a meat granule digestion microbiological assay as described herein. In one embodiment, the C. bifermentans strain expresses at least 80%, at least 85%, at least 90%, at least 95%, at least 100% or more of the proteolytic activity of C. bifermentans strain 76 (ATCC #638).
C. bifermentans is an anaerobic bacterium that can be cultured under anaerobic and microaerophilic conditions, and thus should be cultured accordingly, e.g., in a manner that limits or inhibits the bacteria's exposure to oxygen. (C. bifermentans is described as being able to tolerate up to 8% O2— see, e.g., Leja, K., Advances in Microbiology, 2014, however, growth under substantially anaerobic conditions is readily performed). Media and conditions for C. bifermentans growth in culture are known to those of ordinary skill in the art. As examples, C. bifermentans can be cultured under anaerobic conditions in, e.g., ATCC medium 2107 modified reinforced Clostridial agar/broth or ATCC medium 260 Trypticase soy agar/broth with defibrinated sheep blood (ATCC; Manassas, Va.), or grown on, e.g., Brucella Blood agar plates.
Clostridium scindens
C. scindens is an anaerobic, motile bacterium often found in the human gut, as well as in the soil. C. scindens is a member of Clostridium Cluster XIVa. C. scindens produces a 7-alpha bile acid dehydratase which converts primary bile acids to secondary bile acids that are strong inhibitors of C. difficile germination. It is noted that C. bifermentans has a choloylglycine deconjugating enzyme (which removes glycine from glycine-conjugated bile acids), but genomically does not possess comparable bile salt dehydroxylating enzymes.
While not wishing to be bound by theory, it is likely that at least part of the mechanism by which C. scindens promotes suppression of C. difficile infection is through bile acid metabolism to produce secondary bile acids (e.g., deoxycholic acid and lithocholic acid) that maintain an environment that is hostile to or not compatible with C. difficile and/or C. difficile infection. In this regard, C. scindens bacteria possess a complete secondary bile acid synthesis pathway, producing at least two enzymes active on the side-chain of the bile acid steroid nucleus and at least two enzymes active on the hydroxyl groups of the 7-position of bile acids. C. scindens expresses bile salt hydrolase/Choloylglycine hydrolase activity (E.C. #3.5.1.24), which catalyzes hydrolysis of the amide bond in conjugated bile salts, resulting in the release of free amino acids. This activity adds another source of free amino acid generation in the gut that can at least potentially contribute further substrate amino acids that promote Stickland fermentation.
A genome of C. scindens was sequenced as part of the Human Microbiome Project, and can be found at, e.g., accession #PRJNA18175.
C. scindens is an anaerobic bacterium, and thus should be cultured accordingly, e.g., in a manner the limits or inhibits the bacteria's exposure to oxygen. Media and conditions for C. scindens growth in culture are known to those of ordinary skill in the art. As examples, C. scindens can be cultured under anaerobic conditions in, e.g., ATCC medium 2107 modified reinforced Clostridial agar/broth or ATCC medium 260 Trypticase soy agar/broth with defibrinated sheep blood (ATCC; Manassas, Va.), or grown on, e.g., Brucella Blood agar plates.
In one embodiment, the C. scindens strain is VPI 13733 (ATCC #35704). The 16S rRNA gene sequence for C. scindens strain VPI 13733 (SEQ ID NO: 31) is as follows:
In another embodiment, the C. scindens strain is a bacterial strain comprising a 16S rRNA sequence that is at least 97%, at least 98%, at least 99%, or more identical to the sequence of SEQ ID NO: 1. In one embodiment, the C. scindens strain expresses at least the level of bile salt hydrolase/Choloylglycine hydrolase expressed by C. scindens strain VPI 13733 (ATCC #35704). C. scindens is also proteolytic, albeit to a lesser degree than C. bifermentans. In one embodiment, the C. scindens strain used in a defined microbiota consortium as described herein has at least the proteolytic activity of C. scindens strain VPI 13733 (ATCC #35704).
Clostridium hylemonae
C. hylemonae is a naturally-occurring anaerobic commensal bacterium of the human gut. As described herein below, the relative abundance of C. hylemonae has been found, in combination with that of C. scindens, to be reliably predictive of the recurrence of C. difficile infection in human patients. C. hylemonae is a member of Clostridium Cluster XIVa. The 16S rRNA gene sequence for the C. hylemonae strain (SEQ ID NO: 32) is as follows:
In one embodiment, the C. hylemonae bacterium useful in the reliable prediction of C. difficile infection recurrence or in the reliable prediction of initial C. difficile infection has a 16S rRNA gene sequence of SEQ ID NO: 32. In another embodiment, a C. hylemonae bacterium useful in the reliable prediction of C. difficile infection recurrence or in the reliable prediction of initial C. difficile infection has a 16S rRNA gene sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 32. It is also contemplated that C. hylemonae can provide therapeutic benefit, alone, or together with one or both of C. bifermentans and C. scindens.
Other potentially therapeutic Clostridial species that perform Stickland fermentation include, for example, C. cadaveris (Cluster I), and C. hiranonis, C. sticklandii, Peptostreptococcus anaerobius and C. sporogenes (Cluster XI).
The outcome of the studies described herein indicates that any or all of the following can have therapeutic benefit in combatting toxin production by Gram positive, spore-forming bacteria such as C. difficile:
A) Maintain metabolic and energy state in C. difficile through commensal proteolytic activity to release amino acids for Stickland fermentations (preferentially via the proline reductase pathway). Metabolites of Stickland fermentations can also be used or sensed by C. difficile through mechanisms that also reduce toxin production.
B) Induce or maintain expression of the C. difficile ethanolamine utilization pathway in C. difficile to help maintain energy state.
C) Prevent or limit production of butyrate by C. difficile and other commensals, to limit a stimulus of C. difficile toxin production.
D) Prevent C. difficile spore germination through production of secondary bile acids by C. scindens or other species that promote or produce such products.
E) Reduce biomass of C. difficile through, e.g., direct competition with C. difficile and by permitting recovery of other gut commensal species that directly compete and/or prevent spore germination through innate bile salt hydrolase activities.
F) Glycine Reductase activity or expression.
Defined Therapeutic Microbiota, and Compositions Comprising Them
It is demonstrated herein that C. bifermentans can completely protect GF and conventional mice from otherwise fatal infection with C. difficile.
In one aspect, then, described herein are compositions, including but not limited to pharmaceutical compositions, comprising an oral formulation comprising C. bifermentans bacteria. In one embodiment the C. bifermentans species is the only bacterial species in the formulation. In another embodiment, the C. bifermentans is the only Clostridial XI species in the formulation. In another embodiment, the C. bifermentans is the only Clostridial species in the formulation. In another embodiment, the C. bifermentans is the only bacterial species in the formulation. Such compositions can be used to treat or prevent C. difficile infection, including recurrent C. difficile infection, and/or toxin production.
Methods for the growth or preparation of C. bifermentans bacteria are known in the art. As noted herein above, this species is an obligate anaerobe, so culture under anaerobic conditions is required. Media for culture of Clostridium species are also known in the art, and are commercially available, as also noted elsewhere herein.
In another aspect, described herein is a composition, including a pharmaceutical composition, comprising C. bifermentans bacteria, wherein the C. bifermentans bacteria are in dried, viable form. Methods of drying Clostridium species in a manner that maintains viability upon re-hydration are known in the art.
In another aspect, described herein is a pharmaceutical composition comprising a formulation comprising C. bifermentans bacteria, wherein the composition does not comprise Bacteroides species or Escherichia species.
It is contemplated that killed or proliferatively inactive, but metabolically active C. bifermentans bacteria could have beneficial effect, e.g., if administered repeatedly. However, in each of the aspects noted above, it is preferred that the bacteria are viable as the term is used herein. In this context, the bacterial species for any of the compositions described herein can be present in vegetative, metabolically and proliferatively active forms, dried viable form, spore form, or a combination of these forms. Thus, in one embodiment, the C. bifermentans bacteria are in spore form. In another embodiment, the C. bifermentans bacteria are in metabolically active form, including, but not limited to vegetative and/or actively proliferative forms. In another embodiment, the C. bifermentans bacteria are not in spore form. In another embodiment, the C. bifermentans bacteria are present as a mixture of vegetative, metabolically active and spore forms. Methods for inducing sporulation of Clostridium species are known to those of ordinary skill in the art.
Another aspect described herein provides a pharmaceutical composition comprising an oral formulation comprising a bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium. In various embodiments, the bacterial population has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
Another aspect described herein provides a pharmaceutical composition comprising a bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium, wherein the C. bifermentans bacteria is in dried, viable form. In various embodiments, either or both of the bacterial populations has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
Another aspect described herein provides a pharmaceutical composition comprising a formulation comprising a bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium, wherein the composition does not comprise Bacteroides species or Escherichia species. In various embodiments, the bacterial population has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
In one embodiment of this or any other aspect described herein, the compositions described herein do not comprise viable C. sardiniense bacteria. As described herein, it was found that the presence of C. sardiniense in otherwise germ-free mice comprising pathogenic C. difficile bacteria increased the severity of the resulting C. difficile infection. Thus, in one embodiment, the technology described herein excludes C. sardiniense from a therapeutic composition as described herein.
In one embodiment of this or any aspect described herein, a therapeutic microbiota composition does not comprise a Bacteroides species or Escherichia species. Bacteroides is a genus of Gam-negative, obligate anaerobic bacteria. Bacteroides species can be identified by sphingolipids in their membranes, as well as by 16S rDNA sequence. They make up a substantial proportion of the gut microbiota, and are commonly mutualistic. Escherichia species are Gram-negative, non-spore forming, facultative anaerobic bacteria from the family Enterobacteriaceae. The majority of Escherichia species are commensal gut flora, although certain Escherichia strains are human pathogens. A skilled person can identify Bacteroides and Escherichia species, e.g., using PCR-based methods to quantify 16S rDNA genetic markers, among others.
In one embodiment of this or any aspect described herein, the compositions described herein do not comprise any additional viable Clostridium species. In another embodiment of any aspect described herein, the compositions described herein do not comprise any additional viable bacteria of any kind. A skilled person can identify the presence of other viable bacteria (e.g., additional Clostridium species, among others) in the composition, e.g., using anaerobic culture, and identify organisms in such culture using, for example, PCR-based methods to quantify 16S rDNA genetic markers.
It is also demonstrated herein that a defined consortium of Clostridium species comprising C. scindens and C. bifermentans, as those species are defined herein, is sufficient to treat or prevent C. difficile infection, including, but not limited to recurrent C. difficile infection, and to suppress toxin production by C. difficile.
In one aspect, then, described herein are compositions, including but not limited to pharmaceutical compositions, comprising an oral formulation comprising C. scindens and C. bifermentans bacteria. In another embodiment of this and all other aspects described herein that involve the C. scindens and C. bifermentans species, the only bacterial species in the formulation are C. scindens and C. bifermentans. Methods for the growth or preparation of C. scindens and C. bifermentans bacteria are known in the art. As noted herein above, these species are obligate anaerobes, so culture under anaerobic conditions is required. Media for culture of Clostridium species are also known in the art, and are commercially available, as also noted herein above.
In another aspect, described herein is a composition, including a pharmaceutical composition, comprising C. scindens and C. bifermentans bacteria, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form. Methods of drying Clostridium species in a manner that maintains viability upon re-hydration are known in the art.
In another aspect, described herein is a pharmaceutical composition comprising a formulation comprising C. scindens and C. bifermentans bacteria, wherein the composition does not comprise Bacteroides species or Escherichia species.
It is contemplated that killed or proliferatively inactive, but metabolically active bacteria of these species could have beneficial effect if administered repeatedly. However, in each of the aspects noted above, it is preferred that the bacteria are viable as the term is used herein. In this context, the bacterial species for any of the compositions described herein can be present in vegetative, metabolically and proliferatively active forms, dried viable form, spore form, or a combination of these forms. Thus, in one embodiment, both of the C. scindens and C. bifermentans bacteria are in spore form. In another embodiment, both of the C. scindens and C. bifermentans bacteria are in metabolically active form, including, but not limited to vegetative and/or actively proliferative forms. In another embodiment, one or both of the C. scindens and C. bifermentans bacteria are not in spore form. In another embodiment, the C. scindens and C. bifermentans bacteria are present as a mixture of vegetative, metabolically active and spore forms. Methods for inducing sporulation of Clostridium species are known to those of ordinary skill in the art.
Another aspect described herein provides a pharmaceutical composition comprising an oral formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium. In various embodiments, either or both of the bacterial populations has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
Another aspect described herein provides a pharmaceutical composition comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form. In various embodiments, either or both of the bacterial populations has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
Another aspect described herein provides a pharmaceutical composition comprising a formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium wherein the composition does not comprise Bacteroides species or Escherichia species. In various embodiments, either or both of the bacterial populations has 16S rDNA with a sequence at least 97%, 98%, 99% or more identical to the 16S rDNA of the reference bacterium.
The above bacterial compositions can include, for example but are not limited to metabolically active bacteria, wet bacteria, dry viable bacteria (e.g., preparations including viable spray-dried cells, freeze-dried cells, vacuum-dried cells, drum-dried cells, vitrified etc.), and the like. Preparations of Clostridium species described herein can include, for example, suspensions of Clostridium bacteria, cultured cells of Clostridium bacteria (including bacterial cells, and optionally, supernatant and medium ingredients), and, for example, Clostridium culture biomass, removed from suspension culture, e.g., by centrifugation, filtration, or the like. While viable Clostridium bacteria are used in most applications considered herein, it is contemplated that in some embodiments, processed cells of Clostridium bacteria can include, for example, ground cells, crushed cells, liquefied cells (extracts etc.) and concentrates and preparations thereof, and the like.
Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term bacterial composition storage stability at temperatures elevated above cryogenic. If the bacterial composition comprises spore forming species and results in the production of spores, the final composition can be purified by additional means such as density gradient centrifugation.
Species effective for treatments as described herein are readily available. However, to maintain strain integrity over time, bacterial composition banking can be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of cryopreservation, a bacterial composition culture can be harvested by centrifugation to pellet the cells from the culture medium, the supernate decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C. for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.
C. bifermentans and/or C. scindens can be in spore form or not in spore form in the compositions described herein. Bacterial spores are dormant, non-reproductive structures produced by certain bacteria from the Firmicute phylum, for example in an environment lacking nutrients. Spores can be preserved as described above, and can be reactivated, e.g., by heating the endospore. In addition, C. bifermentans and/or C. scindens can be present in a mixture of metabolically active bacteria and spores. Metabolically active bacteria can actively metabolize nutrients, and will have little lag time from administration to active participation in treating or preventing C. difficile infection. They will also likely have little lag time in beginning to proliferate in the gut if conditions are appropriate.
In one embodiment, any of the defined therapeutic microbiota compositions described herein further comprises a prebiotic. Prebiotics promote the growth, survival, and activity of beneficial microorganisms, or probiotics. Prebiotics have been shown to alter the compositions of microorganisms (microflora) in the gut microbiota, alone or in combination with probiotic organisms. In addition, prebiotics have been shown to increase calcium and magnesium absorption in the gut, increase bone density, enhance the immune system, reduce blood triglyceride levels, and control hormone levels. Prebiotics include any of a number of compositions that are generally not directly digestible by humans, but that are readily digestible by and promote the growth or establishment of probiotic microbes. In one embodiment, a preferred prebiotic comprises a sugar or carbohydrate, e.g., a starch or other carbohydrate-comprising polymer, that can be digested by C. bifermentans and/or C. scindens, but not readily so by other commensals, to thereby favor an increase in the relative proportion or abundance of the administered species. Non-limiting examples of prebiotics include but are not limited to inulin, fructooligosaccharides, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carregenaan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides, and dietary proteins able to be digested by C. bifermentans and/or C. scindens.
In one embodiment, any of the defined therapeutic microbiota compositions described herein further comprise an effective amount of one or more free Stickland fermentable amino acids, e.g. a preparation of free proline alone or any combination of free amino acids selected from the group consisting of: alanine, leucine, valine, isoleucine, tryptophan, tyrosine, phenylalanine, proline or glycine.
In one embodiment, any of the defined therapeutic microbiota compositions described herein further comprise an effective amount of a polypeptide that can be proteolyzed by the administered species to generate free amino acids fermentable by Stickland fermentation. Inclusion of such a polypeptide provides a ready source of protein for an administered Stickland-fermenting bacterium to digest to amino acids useful for Stickland fermentation by C. difficile. Non-limiting examples of such polypeptides include, but are not limited to casein, gelatin, collagen, and an artificial polymer comprising Stickland acceptor amino acids and/or Stickland donor amino acids. A proline-rich or proline+ leucine-rich protein can also be used. Where an artificial polymer is used, the polymer can comprise Stickland donor amino acids selected from the group consisting of: alanine, leucine, valine, isoleucine, tryptophan, tyrosine and phenylalanine, and/or Stickland acceptor amino acids including proline and/or glycine. The polymer can comprise e.g., a poly[N] amino acid polymer, e.g. a poly[alanine], poly[leucine], etc., or e.g. a copolymer of one or more of the Stickland fermentable amino acids. As a non-limiting example, copolymers can include, e.g. poly[alanine, leucine], poly[alanine, isoleucine],[poly[alanine, tryptophan], etc. Polymers rich in proline would be expected to preferentially promote fermentation via the Stickland proline reductase pathway and repression of C. difficile toxin production.
In one embodiment, any of the compositions described herein, except those which expressly exclude any species other than C. scindens and/or C. bifermentans, further comprise a microbe that supports (e.g., the growth, or viability) C. bifermentans and/or C. scindens. Ruminococcus obeum is an exemplary microbe that has been shown to support C. scindens. Ruminococcus obeum is a genus of bacteria in the class Clostridia found in an abundance in the human gut. A skilled person will be able to determine if a microbe supports C. bifermentans and/or C. scindens, e.g., using complementation assays known in the art.
Establishment of administered C. scindens and/or C. bifermentans as described herein can be evaluated by monitoring the proportion of these species in gut microbiota samples taken over time. An administered species can be considered to be established if that species remains at a level increased relative to its level pre-administration for at least two weeks, preferably at least three weeks, one month, five weeks, six weeks, seven weeks, two months, nine weeks, ten weeks, eleven weeks, three months or more following administration. The presence of the administered species at a relative level of abundance of at least 0.3%, at least 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more, maintained over time as noted is preferred. Tracking of the administered species can be facilitated by modification of the species to carry a genetic difference or barcode, but this is optional.
Diagnostic Methods
One aspect of the present technology is a method of predicting or determining the likelihood of C. difficile infection or recurrence of C. difficile infection in a subject. Such a prediction can guide prophylactic and subsequent therapeutic treatment decisions. Markers of likely infection or recurrence include, but are not limited to markers in the following classes:
Proteolytic activity of the subject's commensal microbiota, measured, for example, by biochemical assay for proteolysis of a substrate, e.g., one including but not limited to casein or gelatin. Relatively high proteolytic activity is a predictor of reduced likelihood of infection or recurrence.
DNA- or RNA-based studies for commensal proteases and Stickland reductase genes as described herein. The presence and/or expression of commensal protease genes is a predictor of reduced likelihood of infection or recurrence, with higher levels providing benefit.
Microbiologic, biochemical or molecular assays for proteolytic Clostridia, Stickland fermenters, or other highly proteolytic commensal species. The presence of such species is a predictor of reduced likelihood of infection or recurrence, with higher levels providing benefit.
Detection of energy-producing substrates for C. difficile, e.g., substrates selected from proline, Stickland donor amino acids, ethanolamine, glucose, fructose, mannitol, sorbitol, cysteine and threonine. Greater energy-producing substrate levels, and particularly greater levels of substrates preferred by C. difficile are predictors of reduced likelihood of infection or recurrence.
Detection of metabolites of Stickland fermentation including, for example, 5-amino valerate, branched SCFA amino acid metabolites and other Stickland amino acid metabolites. Greater levels of such metabolites are predictive of reduced risk of infection or recurrence. Greater levels of metabolites associated with the proline reductive pathway are particularly predictive.
Detection of metabolites of anaerobic carbohydrate metabolism including, for example the volatile SCFA acetate, propionate or butyrate, and non-volatile SCFA succinate, lactate or pyruvate, where increased levels of volatile SCFA, and of succinate, are predictive of increased likelihood of infection or recurrence.
Detection of microbial energy transporters including NADH/NAD+, NADPH/NADP+, ATP/ADP and GDP/GTP as indicative of the energy state in tested materials from microbial metabolism.
In one embodiment, any or all of the markers noted above can be used with detection of toxigenic C. difficile by microbiologic, toxin ELISA or molecular methods to predict likelihood of infection or recurrence. The presence and/or levels of the various markers can be compared, for example, to a reference to determine likelihood of infection or recurrence. The reference can be, for example, a sample from a healthy individual, or as the case may be a sample from an individual with active C. difficile infection.
In one embodiment, a method is provided for determining the efficacy of therapy for C. difficile. In one embodiment, the therapy is a bacteriotherapy, for example, as described herein. In another embodiment, the therapy is or comprises administration of a pre-biotic, and/or administration of an amino acid or amino acid derivative. In one embodiment, the method comprises measuring in a sample from an individual being treated for C. difficile infection, one or more markers from one or more, two or more, three or more, four or more, or from each of the classes of the markers listed above. In one embodiment, the reference is a sample from an individual with active C. difficile infection. The reference can, but does not necessarily have to be, a sample from the subject being treated, taken before treatment began. A level of activity or expression of one or more markers or classes of markers that is increased relative to the reference indicates effective therapy. In one embodiment, the sample is a stool sample.
In another embodiment, a method is provided for predicting the likelihood of recurrence in a subject being treated or who has been treated for C. difficile infection. In one embodiment, the method comprises measuring in a sample from an individual who has been treated for C. difficile infection, one or more markers from one or more, two or more, three or more, four or more, or from each of the classes of the markers listed above. In one embodiment, the reference is a sample from a healthy individual. A healthy individual is one without active C. difficile infection and who has not received antibiotic treatment within the past three months. A level of one or more biomarkers from one or more of the classes listed above that is below that of the reference indicates an increased risk of recurring C. difficile infection. In one embodiment, the sample is a stool sample. The method can further comprise administering a bacteriotherapy as described herein to an individual for whom the level of such marker(s) is below the reference.
In another embodiment, a method is provided for predicting the risk of an individual for developing a first infection with C. difficile. This is applicable, for example, to those who are elderly, immunocompromised, hospitalized, in a nursing home, receiving antibiotics, or receiving proton pump inhibitors. In one embodiment, the method comprises measuring in a sample from a subject in one or more of these categories one or more markers in one or more of the classes listed above, and the subject is at increased risk of contracting active C. difficile infection if the level of such marker(s) is below a reference. In one embodiment, the reference can be a sample from a healthy individual as above. In another embodiment, the sample from the subject is assayed to determine the presence and/or amount of Stickland fermenting and/or proteolytic bacterial species, including, for example, non-pathogenic Clostridial species that are proteolytic and/or Stickland fermenting. A lack of such species indicates an increased risk for developing active C. difficile infection. A reduced number relative to a healthy reference also indicates increased risk. In one embodiment the method further comprises administering a bacteriotherapy as described herein to an individual for whom such species are lacking or for whom the level of such marker(s) is below the reference.
Further, the markers noted above can be used to define and monitor the efficacious activity of a therapeutic regimen, including, but not limited to a therapeutic regimen comprising administration of bacteriotherapeutic products, e.g., a defined therapeutic microbiota composition or product as described herein.
Also described herein are predictive methods that examine the presence of certain commensal species as a marker of likelihood of C. difficile infection and/or C. difficile toxin production. In one embodiment, such a method comprises: (a) determining the relative abundance of all operational taxonomic units (OTUs) in a sample of the subject's stool that are >90% identical to a reference sequence of C. scindens; (b) determining the relative abundance of all operational taxonomic units (OTUs) in a sample of the subject's stool that are >90% identical to a reference sequence of C. hylemonae; and (c) summing the relative abundances determined in steps (a) and (b), wherein a sum of relative abundances less than or equal to 1% indicates an increased risk of C. difficile recurrence relative to a subject in which the sum of relative abundances is greater than 1%. In one embodiment, relative abundance of the marker species indicative of increased risk is between 1% and 0.01%. As used herein, “relative abundance” refers to comparison with all species of microbes identified in the subject's sample, and is not limited to Clostridium species. The relative abundance of species in a sample can be measured, for example using Roche/454 pyrosequencing or Illumina sequencing for 16S rRNA gene sequencing. This approach, combined with multiplexing produces thousands of 16S rRNA sequences per sample. Microbiome sequencing techniques are further reviewed in, e.g., Grice, E A, and Segre J A. Annu Rev Genomics Hum Genet. 2012; 13:151-170. Relative abundance can additionally be measured using, e.g., using amplicons for microbiologic or microbial products and/or other gene-level targets (e.g., qPCR for genes (e.g., the bai gene, or the gene encoding a Stickland enzyme, a bile acid hydrolase, etc.)).
The predictive method can also be applied to prediction of susceptibility to a first C. difficile infection. Thus, in one embodiment, a sample can be taken from a patient who is at risk of having, has, or has previously had at least one C. difficile infection. A sample can be taken from a subject who has never had a C. difficile infection, but who is in a risk category as noted herein. A stool sample can be collected using standard techniques, e.g., passing stool directly into a clean, dry container.
At least one sample is taken from the subject for the predictive method. However, repeated sampling can also be performed. For example, a sample can be taken from a subject once a day, once a week, twice a month, once a month, or every 3 months following a C. difficile infection to assess the risk of a recurrent C. difficile infection, or a sample can be taken from a subject once a year following a C. difficile infection to assess the risk of a recurrent C. difficile infection. It has even been found that measurement of the relative abundance of the noted species (C. scindens and C. hylemonae) during treatment for an initial C. difficile infection can be predictive of likelihood of a recurrence. Thus, at least one sample can be taken from a subject during a C. difficile infection, e.g., a sample can be taken once a day for the entirety of the infection.
A sample can be taken from a subject who has not previously been treated with antibiotics to treat a C. difficile infection. Alternatively, a sample can be taken from a subject who has been treated with antibiotics to treat a C. difficile infection. The sample can be taken from the subject before, during, or after administration of antibiotics to treat a C. difficile infection. A sample can be taken from a subject before, during, and after administration of an antibiotic (e.g., a sample is taken from a subject during and after administration of an antibiotic).
The method can further comprise the step of administering a further therapeutic or prophylactic treatment, including, for example, an FMT or any of the bacterial compositions described herein to a subject when the sum of relative abundances relative level is at or below 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.
In this context, the reference sequence of C. scindens can be a sequence comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 31. Similarly, in this context, the reference sequence of C. hylemonae can be a sequence comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 32.
In another aspect of any of the embodiments, described herein is a method of treating a pathology involving expression of a bacterial toxin from a Gram positive, spore-forming species in a subject in need thereof, the method comprising: a) determining that the subject has a reduced amount and/or activity of secreted proteolytic enzymes in a gut or stool sample relative to healthy individual as described herein; and b) administering a therapeutic bacterial species as described herein to the subject. In some embodiments of any of the aspects, the step of determining that the subject has a reduced amount and/or activity of a secreted proteolytic enzyme can comprise i) obtaining or having obtained a biological sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of the proteolytic enzyme in the subject. Methods to measure amount of secreted proteolytic enzymes and/or activity are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody. For example, the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques. Methods to measure the activity of secreted proteolytic enzymes are known to a skilled artisan. For example, a protease activity assay that uses casein or gelatin as a substrate can be used to measure the activity of a protease in a biological sample (Cat. No. Ab111750; Abcam, Cambridge Mass.).
In another aspect, described herein is a method of treating a pathology involving expression of a bacterial toxin brom a Gram positive spore-forming bacterium in a subject in need thereof, the method comprising: a) determining that a sample from the subject has a decreased amount and/or activity of proline reductase relative to a sample from a healthy individual; and b) administering a therapeutic bacterial species as described herein to the subject. In one embodiment, the step of determining that a sample from the subject has reduced level or activity of proline reductase can comprise i) obtaining or having obtained a biological sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of the proline reductase in the subject. Methods to measure amount or activity of proline reductase are known to a skilled artisan and/or described herein. Such methods can include measurement of gene expression products, e.g., protein level, and include for example, ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.
In another aspect of any of the embodiments, described herein is a method of treating a pathology involving expression of a bacterial toxin brom a Gram positive spore-forming bacterium in a subject in need thereof, the method comprising: a) determining that a sample from the subject has a decreased amount and/or activity of glycine reductase relative to a sample from a healthy individual; and b) administering a therapeutic bacterial species as described herein to the subject. In one embodiment, the step of determining that a sample from the subject has reduced level or activity of glycine reductase can comprise i) obtaining or having obtained a biological sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of the glycine reductase in the subject. Methods to measure amount or activity of glycine reductase are known to a skilled artisan and/or described herein. Such methods can include measurement of gene expression products, e.g., protein level, and include for example, ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.
Dosage, Administration and Formulations
In one aspect, any of the compositions described herein is administered to a subject that has, or has been diagnosed with C. difficile infection. In one embodiment, the C. difficile infection is a recurrent C. difficile infection. Recurrent C. difficile infections can be caused by the same or a different C. difficile strain that caused a previous C. difficile infection. In one embodiment, the subject is at risk for a C. difficile infection or a recurrent C. difficile infection.
A clinician can diagnose a subject as having a C. difficile infection using standard methods to detect toxins that are produced by C. difficile bacteria. For example, a stool sample from subject suspected of having a C. difficile infection can be analyzed via an enzyme immunoassay or even dipstick/lateral flow immunoassay for the C. difficile toxin(s), PCR-based assays, GDH/EIA tests, or cell cytotoxicity assays are also commonly used to definitively determine C. difficile infection. Imaging, e.g., colonoscopy or abdominal x-ray or CT scan can also be used to assist the diagnosis of C. difficile infection.
A clinician can determine is a subject is at risk of having a C. difficile infection by assessing a subject's risk factors, including but not limited to the subject's proximity to an individual who has or has recently had a C. difficile infection, current medications that promote C. difficile growth in the intestine, age, antibiotic use (length of antibiotic regimen, use of broad-spectrum antibiotics, or use of multiple antibiotics, use of gastric acid inhibitors such as proton pump-inhibitors and or histamine-2 receptor antagonists. At present, a subject who has had previous C. difficile infection is at risk of a recurrent C. difficile infection; approximately 20% of subjects who have had a C. difficile infection will have a recurrent C. difficile infection. In a further embodiment, risk of recurrence can be evaluated with the method described herein above that measures the relative abundance of C. scindens and C. hylemonae. Risk of recurrence can also be predicted via one or more of the diagnostic methods described herein.
Dosage
The dosage ranges for the bacterial species described herein in a defined therapeutic microbiota composition depends upon the potency (including viability), and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of a C. difficile infection in a treated subject. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage can vary with the type of illness, e.g., first infection vs. recurrent infection, and with the age, condition, and sex of the patient. The dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication.
For use in the various aspects described herein, an effective amount of cells in a composition as described herein comprises at least 1×105 bacterial cells, at least 1×106 bacterial cells, at least 1×107 bacterial cells, at least 1×108 bacterial cells, at least 1×109 bacterial cells, at least 1×1010 bacterial cells, at least 1×1011 bacterial cells, at least 1×1012 bacterial cells or more. In one embodiment, the microbial consortium or the individual bacterial components thereof can be obtained from a microbe bank. Members of a therapeutic or preventive/prophylactic consortium are generally administered together, e.g., in a single admixture. However, it is specifically contemplated herein that members of a given consortium can be administered as separate dosage forms or sub-mixtures or sub-combinations of the consortium members (e.g., C. scindens and C. bifermentans can be comprised in two separate compositions that are administered as separate doses). Thus, a consortium of e.g., C. scindens and C. bifermentans, can be administered, for example, as a single preparation including all members (in one or more dosage units, e.g., one or more capsules) or as two separate preparations that, in sum, include all members of the given consortium. While administration as a single admixture is preferred, a potential advantage of the use of e.g., individual units for each member of a consortium, is that the species administered to any given subject can be tailored, if necessary, by selecting the appropriate combination of, for example, single species dosage units that together comprise the desired consortium. With respect to the administration of two separate preparations, the route of administration can be the same for each preparation (e.g., the first and second preparation can be administered orally), or different for each preparation (e.g., the first preparation can be administered orally and the second preparation is administered directly to the colon via colonoscope, to the small intestine via endoscope, or to the colon via suppository or enema).
From the conventional mouse model (administer microbes after onset of symptomatic Cdiff infection) it was found that orally administered C. bifermentans rapidly changes the cecal environment 6-7 hr after administration (transit time through the gut). This does not occur with C. sardiniense. It is noted that C. bifermentans persistence in most conventional mice is only a few days but is sufficient to correct the gut environment and cause Cdiff to rapidly halt toxin production. The administered bacteriotherapy can, for example, provide long-term engraftment, e.g., weeks, months or longer, or, it can provide shorter term persistence or presence of the administered species. Where long-term engraftment does not occur, repeat dosing is warranted, for example to provide continuing protection for as long as needed, including days, weeks, months, years or longer, e.g., indefinitely if needed. The need for such continued dosing can be evaluated, e.g., using methods of evaluating risk of infection or recurrent infection as described herein.
One can also easily adjust the ratio of one species to another if separate dosage forms are administered. It is contemplated that the ratios of C. scindens to C. bifermentans, for example, can be varied, e.g. over a range of 1:10 to 10:1 with benefit to the patient under certain circumstances, especially where, for example, the persistence of one species is found to be less than that of the other. Such a finding would warrant, for example, increasing the ratio in favor of the species with the lower degree of persistence. In one embodiment, the ratio of C. scindens to C. bifermentans in a composition described herein is 1:1. The ratio of C. scindens to C. bifermentans in a composition described herein can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The ratio of C. bifermentans to C. scindens in a composition described herein can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The ratio should be such that the amount of each bacteria in the composition is sufficient to promote engraftment (i.e., in vivo replication) of each species in a subject's GI tract following administration of the composition.
A pharmaceutical composition comprising a microbial consortium can be administered by any method suitable for depositing in the gastrointestinal tract, preferably the colon, of a subject (e.g., human, mammal, animal, etc.). Examples of routes of administration include rectal administration by colonoscopy, suppository, enema, upper endoscopy, or upper push enteroscopy. Additionally, intubation through the nose or the mouth by nasogastric tube, nasoenteric tube, or nasal jejunal tube can be utilized. Oral administration by a solid such as a pill, tablet, a suspension, a gel, a geltab, a semisolid, a tablet, a sachet, a lozenge or a capsule or microcapsule, or as an enteral formulation, or re-formulated for final delivery as a liquid, a suspension, a gel, a geltab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or as an enteral formulation can be utilized as well. Also contemplated herein are food items that are inoculated with a microbial consortium as described herein.
In some embodiments, the compositions described herein can be administered in a form containing one or more pharmaceutically acceptable carriers. Suitable carriers are well known in the art and vary with the desired form and mode of administration of the composition. For example, pharmaceutically acceptable carriers can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, glidants, lubricants, and the like. The carrier may be a solid (including powder), liquid, or combinations thereof. Each carrier is preferably “acceptable” in the sense of being compatible with the other ingredients in the composition and not injurious to the subject. The carrier may be biologically acceptable and inert (e.g., it permits the composition to maintain viability of the biological material until delivered to the appropriate site).
Oral compositions can include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, pastilles, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared by combining a composition of the present disclosure with a food. In one embodiment a food used for administration is chilled, for instance, iced flavored water. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, orange flavoring, or other suitable flavorings. These are for purposes of example only and are not intended to be limiting.
The compositions comprising a microbial consortium can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. Suppositories can comprise one a number of species in a microbial consortium (e.g., a suppository can comprise a C. scindens and a C. bifermentans, or a suppository can comprise a C. scindens or a C. bifermentans). Suppositories comprising only one species of the microbial consortium can be co-administered with another composition comprising another species of the consortium, such that co-administration will equal the sum of the given consortium. The compositions can be prepared with carriers that will protect the consortium against rapid elimination from the body, such as a controlled release formulation, including implants. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from, for instance, Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
In some embodiments, a composition can be encapsulated, e.g., enteric-coated formulations). For instance, when the composition is to be administered orally, the dosage form is formulated so the composition is not exposed to conditions prevalent in the gastrointestinal tract before the small intestine, e.g., high acidity and digestive enzymes present in the stomach. The encapsulation of compositions for therapeutic use is routine in the art. Encapsulation can include hard-shelled capsules, which can be used for dry, powdered ingredients soft-shelled capsules. Capsules can be made from aqueous solutions of gelling agents such as animal protein (e.g., gelatin), plant polysaccharides or derivatives like carrageenans and modified forms of starch and cellulose. Other ingredients can be added to a gelling agent solution such as plasticizers (e.g., glycerin and or sorbitol), coloring agents, preservatives, disintegrants, lubricants and surface treatment.
In one embodiment, a microbial consortium as described herein is formulated with an enteric coating. An enteric coating can control the location of where a microbial consortium is released in the digestive system. Thus, an enteric coating can be used such that a microbial consortium-containing composition does not dissolve and release the microbes in the stomach, which can be a toxic environment for many microbes, but rather travels to the small intestine, where it dissolves and releases the microbes in an environment where they can survive. An enteric coating can be stable at low pH (such as in the stomach) and can dissolve at higher pH (for example, in the small intestine). Material that can be used in enteric coatings includes, for example, alginic acid, cellulose acetate phthalate, plastics, waxes, shellac, and fatty acids (e.g., stearic acid, palmitic acid). Enteric coatings are described, for example, in U.S. Pat. Nos. 5,225,202, 5,733,575, 6,139,875, 6,420,473, 6,455,052, and 6,569,457, all of which are herein incorporated by reference in their entirety. The enteric coating can be an aqueous enteric coating. Examples of polymers that can be used in enteric coatings include, for example, shellac (trade name EmCoat 120 N, Marcoat 125); cellulose acetate phthalate (trade names AQUACOAT™, AQUACOAT ECD™, SEPIFILM™, KLUCEL™, and METOLOSE™); polyvinylacetate phthalate (trade name SURETERIC™); and methacrylic acid (trade name EUDRAGIT™).
In one embodiment, an enteric coated prebiotic composition that additionally comprises members of a microbial consortium as described herein is administered to a subject. In another embodiment, an enteric coated probiotic and prebiotic composition is administered to a subject.
Formulations suitable for rectal administration include gels, aqueous or oily suspensions, dispersible powders or granules, emulsions, dissolvable solid materials, enemas, and the like. The formulations are preferably provided as unit-dose suppositories comprising the active ingredient in one or more solid carriers forming the suppository base, for example, cocoa butter. Suitable carriers for such formulations include petroleum jelly, lanolin, polyethyleneglycols, alcohols, and combinations thereof, provided they are compatible with the bacterial species preparation being administered.
In some embodiments, the microbial consortium can be formulated in a food item. Some non-limiting examples of food items to be used with the methods and compositions described herein include: popsicles, cheeses, creams, chocolates, milk, meat, drinks, yogurt, pickled vegetables, kefir, miso, sauerkraut, etc. In other embodiments, the food items can be juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish, hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauce, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, fermented beverages, and pickles; bean products; various confectionery products including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; and the like. It is preferred that food preparations not require cooking after admixture with the microbial consortium to avoid killing the microbes.
Formulations of a microbial consortium can be prepared by any suitable method, typically by uniformly and intimately admixing the consortium with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting in mixture into the desired shape. In addition, the microbial consortium can be treated to prolong shelf-life, preferably the shelf-life of the pre-determined gut flora will be extended via freeze drying.
In some embodiments, the microbial consortium as described herein is combined with one or more additional probiotic organisms prior to treatment of a subject.
A nutrient supplement comprising the microbial consortium as described herein can include any of a variety of nutritional agents, including vitamins, minerals, essential and nonessential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements, short chain fatty acids and the like. Preferred compositions comprise vitamins and/or minerals in any combination. Vitamins for use in a composition as described herein can include vitamins B, C, D, E, folic acid, K, niacin, and like vitamins. The composition can contain any or a variety of vitamins as may be deemed useful for a particularly application, and therefore, the vitamin content is not to be construed as limiting. Typical vitamins are those, for example, recommended for daily consumption and in the recommended daily amount (RDA), although precise amounts can vary. The composition can preferably include a complex of the RDA vitamins, minerals and trace minerals as well as those nutrients that have no established RDA, but have a beneficial role in healthy human or mammal physiology. The amount of material included in the composition can vary widely depending upon the material and the intended purpose for its absorption, such that the composition is not to be considered as limiting.
Also contemplated herein are kits comprising, at a minimum, a biotherapeutic microbial species or a consortium prep or formulations comprising the members of the consortium, e.g., C. scindens and C. bifermentans species, in an admixture or comprising the members of the consortium in sub-combinations or sub-mixtures. In some embodiments, the kit further comprises empty capsules to be filled by the practitioner and/or one or more reagents for enteric coating such capsules. It is also contemplated herein that the microbe preparation is provided in a dried, lyophilized or powdered form. In one embodiment, the kit comprises a strain of C. bifermentans. In another embodiment, the kit comprises a strain of C. scindens and a strain of C. bifermentans. The C. scindens strain comprised in the kit can be a C. scindens strain comprising a 16S rRNA sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1. The C. bifermentans strain comprised in the kit can be a C. bifermentans strain comprising a 16S rRNA sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 2. In another embodiment, the kit comprises at least one reducing agent such as N-acetylcysteine, cysteine, or methylene blue for growing, maintaining and/or encapsulating the microbes under anaerobic conditions. The kits described herein are also contemplated to include cell growth media and supplements necessary for expanding the microbial preparation. The kits described herein are also contemplated to include one or more prebiotics as described herein.
Prior to administration of the bacterial composition, the patient may optionally have a pretreatment protocol to prepare the gastrointestinal tract to receive the bacterial composition. In these instances, the pretreatment protocol can enhance the ability of the bacterial composition to affect the patient's microbiota balance. In an alternative embodiment, the subject is not pre-treated with an antibiotic.
Generally, the defined therapeutic microbiota described herein can be administered after the completion of a course of antibiotics for the treatment of C. difficile infection. However, use of the defined therapeutic microbiota alone to prevent or, for that matter, directly combat the C. difficile infection is specifically contemplated. If a patient has received antibiotics for treatment of an infection other than C. difficile or for C. difficile, in one embodiment the antibiotic should be stopped in sufficient time to allow the antibiotic to be substantially reduced in concentration in the gut before the bacterial composition is administered. In one embodiment, the antibiotic may be discontinued 1, 2, or 3 days before the administration of the bacterial composition. In one embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the bacterial composition.
In another embodiment, the bacterial compositions described herein are administered before or concurrently with an antibiotic. In one embodiment, administration of therapeutic microbiota before or concurrently with antibiotic might be contemplated where the administered species are at least somewhat resistant to the effects of the antibiotic administered. In another embodiment, an antibiotic is administered before the administration to the bacterial composition (e.g., less than 1 day, 1, 2, 3, 4, 5, 6, or 7 days before administration of the bacterial composition). Longer times can help to prevent the antibiotic from killing the administered bacteriotherapeutic organism(s). In one embodiment, the antibiotic administered is an antibiotic used to treat C. difficile infection (e.g., metronidazole (Flagyl), vancomycin (Vancocin), or fidaxomicin (Dificid)). In another embodiment, the antibiotic is not specific to treatment of a C. difficile infection, but is an antibiotic known in the art to have therapeutic effects on the intestinal system (e.g., norfloxacin, cephalexin, trimethoprim-sulfamethoxazole, or levofloxacin). Antibiotics listed herein are for purposes of example only and are not intended to be limiting.
In one embodiment, the bacterial compositions described herein are administered with an antacid or proton pump inhibitor (PPI). An antacid works to neutralize the stomach acid, which can interfere to efficient delivery of the bacterial compositions described herein. An antacid can be administered prior to, in combination with, or after the administration of the bacterial compositions described herein. In one embodiment, the bacterial composition can be formulation in a composition that further comprises an antacid. Antacids are known in the art and can comprise the following active ingredients: calcium carbonate, aluminum, magnesium, sodium bicarbonate, and/or alginic acid. Proton pump inhibitors block activity of the H+/K+ ATPase proton pump in stomach epithelium.
Any of the preparations described herein can be administered once on a single occasion or on multiple occasions, such as once a day for several days or more than once a day on the day of administration (including twice daily, three times daily, or up to five times daily). Or the preparation can be administered intermittently according to a set schedule, e.g., once weekly, once monthly, or when the patient relapses from the primary illness. In another embodiment, the preparation can be administered on a long-term basis to assure the maintenance of a protective or therapeutic effect.
Efficacy
Typically, a C. difficile infection can manifest with one of more of the following clinical symptoms or indicators: (i) mild (at least 3 times a day) to severe (4 or more times a day) watery diarrhea, (ii) abdominal pain, (iii) blood and/or pus in the stool, (iv) fever, and (v) loss of appetite. Quantitatively, a C. difficile infection can be assessed by quantitative factors (i) detectable levels of C. difficile toxin, and (ii) detectable levels of C. difficile bacteria. Thus, efficacious treatment and/or prevention of a C. difficile infection using the methods and compositions described herein can reduce or eliminate at least one of the symptoms or indicators associated with a C. difficile infection, as described above. Methods for the measurement of each of these parameters (e.g., measuring the levels of C. difficile toxins) are known to those of ordinary skill in the art and/or described herein.
Effective treatment can be determined by an overall decrease in the Bristol Score from 7 or 6 to a lower value. Alternatively, or in addition, effective treatment can be determined by a decrease, as the term is used herein, in C. difficile biomass or relative abundance in the stool, or by a decrease in C. difficile toxin in the stool.
Efficacy can also be measured by failure of a subject to worsen as assessed by need for medical interventions (e.g., progression of infection is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Example methods include PCR-based or Enzyme-linked immunosorbent assay (ELISA) to detect C. difficile toxin. Treatment includes: (1) inhibiting the infection, e.g., arresting, or slowing symptoms of the infection, for example watery diarrhea; or (2) relieving the infection, e.g., causing regression of symptoms, reducing the symptoms by at least 10%, and/or reducing C. difficile toxin levels by at least 10% compared to a reference level (e.g., a C. difficile toxin level prior to administration; and (4) restoring healthy intestinal flora, thus preventing future C. difficile infection or production. It is expected that the levels of C. difficile toxin and/or C. difficile bacteria present in a subject's intestine should be reduced to levels seen in healthy individuals or below detectable levels at least 1 week, at least 2 weeks, at least 3, weeks, at least 4 weeks following administration of any of the therapeutic compositions described herein.
Therapeutic microbiota, including defined therapeutic microbiota as described herein, are administered in an amount sufficient, or an amount effective, to provide therapeutic benefit. An effective amount of a composition for the treatment of C. difficile infection means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that infection. Efficacy of the composition can be determined by a physician by assessing physical indicators of C. difficile infection as described above.
The term “effective amount” as used herein refers to the amount of a therapeutic microbiota composition as described herein needed to alleviate at least one or more symptoms or reduces one or more indicator of a C. difficile infection, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of a composition that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the infection, alter the course of a symptom (for example but not limited to, slowing the progression of a symptom of the infection), or reverse a symptom of the infection. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation. The term “effective amount” is used interchangeably with the term “therapeutically effective amount” and refers to the amount of at least one agent, e.g., a bacterial composition that treats a C. difficile infection, at dosages and for periods of time necessary to achieve the desired therapeutic result, for example, to reduce or stop at least one symptom or indicator of such C. difficile infection, in the subject.
Repeated administration of the defined therapeutic microbiota composition may be beneficial to maintain a protective or curative effect.
Effective amounts, toxicity, and therapeutic efficacy of drug agents, e.g., for formulations or treatments using antibiotics in addition to microbes, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vivo assays. It is contemplated that the relevant level for an agent that treat a C. difficile infection may also be the level achieved in the lumen of the gut. The effects of any particular dosage can be monitored by a suitable bioassay or by measurement of administered and stable biomass (engraftment, persistence) and microbial metabolic activities (Stickland metabolite production, free amino acids, protease activities, associate gene content or expression levels)
The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A pharmaceutical composition comprising an oral formulation comprising C. scindens and C. bifermentans bacteria.
2. A composition comprising C. scindens and C. bifermentans bacteria, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form.
3. A pharmaceutical composition comprising a formulation comprising C. scindens and C. bifermentans bacteria, wherein the composition does not comprise Bacteroides species or Escherichia species.
4. The composition of any one of paragraphs 1-3, wherein one or both of the C. scindens and C. bifermentans bacteria are in spore form.
5. The composition of any one of paragraphs 1-3, wherein one or both of the C. scindens and C. bifermentans bacteria are not in spore form.
6. The composition of any one of paragraphs 1-3, wherein the C. scindens and C. bifermentans bacteria are present as a mixture of metabolically active and spore forms.
7. The composition of any one of paragraphs 1-3, wherein the composition comprises a capsule or microcapsule, or a composition formulated for enteric delivery.
8. The composition of paragraph 1 or paragraph 3, wherein one or both of the C. scindens and C. bifermentans bacteria are in dried viable form.
9. The composition of any one of paragraphs 1-3, which does not comprise C. sardiniensis bacteria.
10. The composition of any one of paragraphs 1-3, which does not comprise any other Clostridium species.
11. The composition of either of paragraphs 1 or 2, which does not contain Bacteroides species or Escherichia coli.
12. The composition of any one of paragraphs 1-3, in which the formulation comprises no other bacteria.
13. A pharmaceutical composition comprising an oral formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium.
14. A composition comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium, wherein one or both of the C. scindens and C. bifermentans bacteria is in dried, viable form.
15. A pharmaceutical composition comprising a formulation comprising a first bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. scindens bacterium, and a second bacterial population that has 16S rDNA with a sequence at least 97% identical to a 16S rDNA sequence present in a reference C. bifermentans bacterium wherein the composition does not comprise Bacteroides species or Escherichia species.
16. The composition of any one of paragraphs 13-15, wherein one or both of the C. scindens and C. bifermentans bacteria are in spore form.
17. The composition of any one of paragraphs 13-15, wherein one or both of the C. scindens and C. bifermentans bacteria are not in spore form.
18. The composition of any one of paragraphs 13-15, wherein the C. scindens and C. bifermentans bacteria are present as a mixture of metabolically active and spore forms.
19. The composition of any one of paragraphs 13-15, wherein the composition comprises a capsule or microcapsule, or a composition formulated for enteric delivery.
20. The composition of paragraph 13 or paragraph 15, wherein one or both of the C. scindens and C. bifermentans bacteria are in dried viable form.
21. The composition of any one of paragraphs 13-15, which does not comprise C. sardiniense bacteria
22. The composition of either of paragraphs 13 or 14, which does not comprise Bacteroides species or Escherichia coli.
23. The composition of any one of paragraphs 13-15, which does not comprise any other Clostridium species.
24. The composition of any one of paragraphs 13-15, in which the formulation comprises no other bacteria.
25. The composition of any one of paragraphs 1-24, further comprising a prebiotic.
26. The composition of any one of paragraphs 1-11, 23, or 25, further comprising a microbe that supports C. scindens and/or C. bifermentans.
27. The composition of paragraph 26, wherein the microbe that supports C. scindens is Ruminococcus obeum.
28. The composition of any one of paragraphs 1-27, for use in the treatment of C. difficile infection.
29. The composition for use of paragraph 28, wherein the use comprises suppressing the expression of C. difficile toxin.
30. The composition for use of paragraph 28, wherein the use comprises promoting a shift towards use of the proline reductase pathway of Stickland fermentation in C. difficile.
31. The composition for use of paragraph 28, wherein the use comprises inducing CodY activity or expression in C. difficile.
32. The composition for use of paragraph 28, wherein the use comprises promoting ethanolamine utilization by C. difficile.
33. A method comprising administering a composition of any one of paragraphs 1-27 to a subject in need thereof.
34. The method of paragraph 33, wherein the subject has or has been diagnosed with C. difficile infection.
35. The method of paragraph 34, wherein the C. difficile infection is recurrent.
36. The method of paragraph 33, wherein the subject is at risk of C. difficile infection or recurrent C. difficile infection.
37. The method of paragraph 33, wherein the administration is oral.
38. A method comprising administering a composition of any one of paragraphs 2, 3, 14 or 15 directly to the colon of a subject in need thereof.
39. The method of paragraph 38, wherein administration is via colonoscope or enema.
40. The method of any of paragraphs 33-39, wherein the subject is receiving or has recently received antibiotic treatment.
41. A method of treating an infection, the method comprising administering an antibiotic and a composition of any one of paragraphs 1-27.
42. The method of paragraph 41, wherein the composition is administered before or concurrently with the antibiotic.
43. The method of paragraph 41, wherein the composition is administered after a course of an antibiotic.
44. A method of predicting recurrence of C. difficile infection in a subject, the method comprising:
The technology described herein is related to the surprising discovery that two human commensal species, Clostridium scindens and Clostridium bifermentans, offer protection from an otherwise lethal infection with the Gram-positive, toxigenic bacterium, C. difficile.
The effect of commensal species on the survival of mice infected with C. difficile spores was assessed using a gnotobiotic colonization model.
As expected, control mice that were challenged with C. difficile alone developed a severe toxin-mediated pathology and died within 5 days of the infection with the Gram-positive toxigenic bacterium C. difficile (
Consistent with the survival data, the body condition scores (BCS) and histopathological assessment of the mice showed that C. bifermentans provided protection against an acute primary C. difficile infection, as shown by a milder acute tissue pathology and lower lymphocytic infiltration as compared to the control group (
The C. difficile biomass and levels of C. difficile toxin were examined in the gnotobiotic mouse model. C. difficile toxin B levels were assessed in cecal samples collected from germfree Swiss-Webster mice infected with 1000 spores of the C. difficile ATCC-43255 strain (
The effects of C. bifermentans and C. sardiniense were examined in conventional mice infected with C. difficile.
An examination of species that provided protective effects against C. difficile was undertaken in order to identify what characteristics of the protective species mediate the protection. Species including C. bifermentans, C. hiranonis, C. sardinense, C. scindens, C. ramosum and C. difficile clinical isolates were assessed for their proteolytic activity using both microbiological and biochemical assays.
The cecal contents of germfree Swiss-Webster mice were collected 20 hours after infection with C. difficile and untargeted metabolomic analysis was performed. The metabolic profiles of Stickland donor and acceptor amino acids, branched-chain amino acids (
Analyses of Stickland donor amino acids including branch chain amino acids (alanine, leucine, valine, and isoleucine) and the aromatic amino acids (phenylalanine, tryptophan and tyrosine) are consistent with these amino acids participating in Stickland fermentation in C. difficile and in C. bifermentans (
C. difficile Short Chain Fatty Acids (SCFA) produced in in vitro culture was measured (
Gene expression was measured by bacterial RNAseq analysis of cecal contents from C. difficile infected gnotobiotic Swiss-Webster mice at 20 hours post-inoculation, compared with mice colonized with C. bifermentans for 7 days prior to challenge with C. difficile for 20 hours. C. bifermentans colonized mice showed a >48× decrease in C. difficile tcdR sigma factor gene expression with concomitant >10× decreases in toxinA (tcdA) and toxinB (tcdB) gene expression (
Gene expression was measured by bacterial RNAseq analysis of cecal contents from C. difficile-infected gnotobiotic Swiss-Webster mice at 20 hours post-inoculation, compared with mice colonized with C. bifermentans for 7 days prior to challenge with C. difficile for 20 hours. It was found that C. difficile structural proteins for the ethanolamine carboxysome (eutH, eutK, eutL, eutN) were up-regulated >10× when C. difficile is inoculated into a C. bifermentans-colonized mouse (
The results of the experiments described herein demonstrate that the commensal bacterium C. bifermentans not only suppresses toxin production by a mechanism involving inhibition of the C. difficile TcdR sigma factor required for C. difficile toxin expression, but also promotes Stickland fermentation by Gram-positive toxigenic bacteria such as C. difficile in vivo and thereby can treat and/or prevent the development of a toxin-mediated pathology. These in vivo activities infer specific action of C. bifermentans on the C. difficile metabolic regulators codY (primary repressor of tcdR), ccpA (repressor of the tcdAEB opeon, also of tcdR), prdR (activator of proline reductase and repressor of glycine reductase operons), and rex (sensor of NADH/NAD+ energy state). Suppression of toxin production provides an alternative route of treatment for C. difficile-mediated pathology, in that it can be sufficient for treatment to just suppress production of the pathology-generating toxin without necessarily killing the pathogenic microbe. It is contemplated that at least part of this effect is through the strong extracellular proteolytic activity expressed by C. bifermentans, in that this activity can feed amino acids necessary for the generation of energy by C. difficile through Stickland fermentation into the gut environment, thereby keeping C. difficile energetically satisfied and suppressing its expression of toxin. Another part of the effect is likely through promotion of ethanolamine metabolism for energy by C. difficile; ethanolamine derived from diet, host tissues and other commensals is fairly abundant in the gut, and a shift in C. difficile gene expression induced by C. bifermentans that permits C. difficile to gain energy from ethanolamine is contemplated to further contribute to maintaining C. difficile in an energetically satisfied state that suppresses toxin expression.
This application is a 35 U.S.C. § 371 National Phase Entry of International Application No. PCT/US2018/065023 filed Dec. 11, 2018, which designates the U.S., and which claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application Nos 62/597,116 filed Dec. 11, 2017 and 62/665,754 filed May 2, 2018, the contents of each of which are incorporated herein by reference in their entirety.
This invention was made with Government support under Grant Nos. P30DK034854 and T32HL007627 awarded by the National Institutes of Health and under Grant No. HR0011-15-C-0094 awarded by the U.S. Department of Defense. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/065023 | 12/11/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/118510 | 6/20/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 10265349 | Chatila | Apr 2019 | B2 |
| 10391131 | Chatila | Aug 2019 | B2 |
| 10898527 | Chatila | Jan 2021 | B2 |
| 20170087196 | Pamer et al. | May 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2014110493 | Jul 2014 | WO |
| 2016059350 | Apr 2016 | WO |
| 2017079450 | May 2017 | WO |
| WO-2017079450 | May 2017 | WO |
| Entry |
|---|
| Collins et al., (Microbiol Spectr. Sep. 2017; 5(5): pp. 1-25). (Year: 2017). |
| Number | Date | Country | |
|---|---|---|---|
| 20210069264 A1 | Mar 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62665754 | May 2018 | US | |
| 62597116 | Dec 2017 | US |
