Patent Application
20200353018
The invention relates to preventing and treating antibiotic resistance.
According to the World Health Organization (WHO), bacterial “[a]ntibiotic resistance is one of the biggest threats to global health, food security, and development today” (who.int/mediacentre/factsheets/antibiotic-resistance/en). Antibiotic resistance can occur naturally in bacteria but is greatly exacerbated by increasing use and misuse of antibiotics. Adding to the problem is the diversity of antibiotic resistance mechanisms; a variety of disparate genes can confer resistance, and unrelated bacteria can horizontally transmit antibiotic resistance genes. The WHO has also stated “[w]hile there are some new antibiotics in development, none of them are expected to be effective against the most dangerous forms of antibiotic-resistant bacteria (ibid).”
Accordingly, it is imperative that new methods of reducing antibiotic resistance be developed and made available.
The invention relates to the discovery that bacterial compositions can be used to reduce the abundance of antibiotic resistance genes in the gastrointestinal (GI) microbiome of a subject.
The invention provides compositions including a therapeutically effective amount of a microbiome composition comprising spore-former bacteria for use in reducing the abundance of at least one antibiotic-resistance gene in the microbiome of a subject.
The invention also provides compositions including a therapeutically effective amount of a microbiome composition comprising Firmicutes derived from one or more healthy human subjects for use in treating a subject at risk for or diagnosed with an undesirable level or population of drug-resistant bacteria.
The invention further provides ROAR compositions for use reducing the abundance of antibiotic-resistant bacteria in the microbiome of a subject.
The invention additionally provides ROAR compositions for use in disrupting a transmission cycle of antibiotic resistance genes, by administration to at least two individuals with a high likelihood of direct or indirect contact, including optionally contact with a bodily fluid or waste.
In addition, the invention provides methods of reducing the abundance of at least one antibiotic-resistance gene in the microbiome of a subject, the methods including administering a therapeutically effective amount of a microbiome composition containing spore-former bacteria.
The invention also provides methods of treating a subject at risk for or diagnosed with an undesirable level or population of drug-resistant bacteria, the methods including administering a therapeutically effective amount of a microbiome composition containing Firmicutes derived from one or more healthy human subjects.
The invention further provides methods of reducing the abundance of antibiotic-resistant bacteria in the microbiome of a subject, the methods including administering a therapeutically effective amount of a ROAR composition.
The invention additional provides methods of disrupting a transmission cycle of antibiotic resistance genes, the methods including administering a ROAR composition to at least two individuals with a high likelihood of direct or indirect contact including contact with a bodily fluid or waste.
Various embodiments of any of the methods noted above are as set forth below.
In some embodiments, the antibiotic-resistance gene(s) is selected from those listed in Table 1.
In some embodiments, the abundance of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibiotic resistance genes of Table 1 is reduced.
In some embodiments, the drug or antibiotic is from a drug class listed in Table 2.
In some embodiments, the methods further include, or the compositions for use further result in, reducing the abundance of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more antibiotic resistant genes or drug classes.
In some embodiments, the composition includes one or more species listed in Table 3. In some embodiments, the species listed in Table 3 is not also listed in Table 4. The composition may be administered by a method described above or elsewhere herein.
In some embodiments, the composition includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more species listed in Table 3. In some embodiments, the species listed in Table 3 is not also listed in Table 4. The composition may be administered by a method described above or elsewhere herein.
In some embodiments, the species include a 16S rDNA, V4, and/or V6 sequence listed in Table 5, or a sequence having at least 95% identity thereto. The species may be administered by a method described above or elsewhere herein.
In some embodiments, the composition does not include one or more species of Table 4. In some embodiments, the species listed in Table 4 that is not included is not listed Table 3. The composition may be administered by a method described above or elsewhere herein.
In some embodiments, the species does not include a 16S rDNA, V4, and/or V6 sequence listed in Table 6, or a sequence having at least 95% identity thereto. The species may be administered by a method described above or elsewhere herein.
In some embodiments the bacteria in the composition are in the form of spores.
In some embodiments, the bacteria in the composition are cultured.
In some embodiments, the composition includes Firmicutes and one or more species of Bacteroides.
In some embodiments, the subject has a reduced abundance of one, two, or three bacteria selected from the group consisting of quinolone resistant, beta-lactam-resistant, and carbapenem-resistant bacteria.
In some embodiments, the subject has been unresponsive to antibiotic treatment.
In some embodiments, the subject has or is at risk of developing C. difficile infection or disease, or a colitis wherein, in some embodiments, the colitis is optionally Crohn's disease or ulcerative colitis (e.g., mild to moderate ulcerative colitis). In some embodiments, the subject does not have (or is not risk of developing) C. difficile infection or disease, or a colitis (such as Crohn's disease or ulcerative colitis, e.g., mild to moderate ulcerative colitis).
The invention also provides compositions including bacterial species associated with a decrease in the abundance of at least one antibiotic resistance gene in a subject.
In some embodiments, the antibiotic-resistance gene(s) is selected from those listed in Table 1.
In some embodiments, the abundance of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibiotic resistance genes of Table 1 is decreased.
In some embodiments, the antibiotic is from a class listed in Table 2.
In some embodiments, the composition includes one or more species listed in Table 3. In some embodiments, the species listed in Table 3 is not also listed in Table 4.
In some embodiments, the composition includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more species listed in Table 3. In some embodiments, the species listed in Table 3 is not also listed in Table 4.
In some embodiments, the species include a 16S rDNA, V4, and/or V6 sequence listed in Table 5, or a sequence having at least 95% identity thereto.
In some embodiments, the composition does not include one or more species of Table 4. In some embodiments, the species of Table 4 that is not included is not listed in Table 3.
In some embodiments, the species do not include a 16S rDNA, V4, and/or V6 sequence listed in Table 6, or a sequence having at least 95% identity thereto.
In some embodiments, the composition includes one or more species of Firmicutes.
In some embodiments, the composition includes one or more species of Bacteroides.
In some embodiments, the bacteria in the composition are in the form of spores.
In some embodiments, the bacteria in the composition are obtained from a spore fraction derived from feces of a healthy subject.
In some embodiments, the composition includes one or more species of Firmicutes, and optionally one or more species of Bacteroides, as the sole bacterial species in the composition.
In some embodiments, the bacteria in the composition are cultured.
The invention further provides compositions including bacteria for use in decreasing the abundance of at least one antibiotic resistance gene in a subject to whom the composition is administered.
In some embodiments, the bacteria in the composition are obtained from a spore fraction derived from feces of a healthy subject.
In some embodiments, the bacteria are selected for being associated with a decrease in the abundance of one or more antibiotic resistance genes in a subject.
In some embodiments, the antibiotic resistance gene(s) is selected from Table 1.
In some embodiments, the composition includes at least one species selected from Table 3.
In some embodiments, the composition does not include a species of Table 4.
The invention also provides methods for identifying a bacterial composition useful for decreasing the abundance of at least one antibiotic resistance gene in a subject to whom the composition is administered, the methods including screening the composition for the presence of one or more bacterial species of Table 3, wherein detection of one or more species of Table 3 in the bacterial composition indicates the identification of a composition that can be used to decrease the abundance of at least one antibiotic resistance gene. In some embodiments, the species of Table 3 is not also listed in Table 4.
In some embodiments, the screening includes detection of one or more 16S rDNA, V4, and/or V6 sequences of Table 5 in the composition, or a sequence having at least 95% identity thereto.
In some embodiments, the methods further include screening for the presence of one or more bacterial species of Table 4. In some embodiments, the species screened for from Table 4 is also not listed in Table 3.
In some embodiments, the screening includes detection of one or more 16S rDNA, V4, and/or V6 sequences of Table 6 in the composition, or a sequence having at least 95% identity thereto.
The invention also includes methods for screening a potential donor of feces for use in therapeutic methods, the methods including testing a feces sample from the potential donor for the presence of a bacterial species from Table 3 or Table 4, wherein detection of one or more species from Table 3 indicates that the potential donor may proceed to become a donor, while detection of one or more species from Table 4 indicates that the potential donor should not become a donor. In some embodiments, the species from Table 3 tested for is not listed in Table 4. In some embodiments, the species from Table 4 tested for is not listed in Table 3.
The invention further includes methods for determining whether a subject could benefit from treatment with a ROAR composition, the methods including determining whether a sample from the subject includes one or more species of bacteria from Table 4, wherein detection of one or more species from Table 4 indicates that the subject may benefit from treatment with a ROAR composition. In some embodiments, the species from Table 3 tested for is not listed in Table 4. In some embodiments, the species from Table 4 tested for is not listed in Table 3.
The invention further provides compositions (e.g., the compositions described above and elsewhere herein), wherein the bacteria in the compositions demonstrate the ability to decrease the abundance of one or more antibiotic resistance gene(s) in an animal model of antibiotic resistance. In some embodiments, the animal model is a mouse treated with one or more antibiotics and then colonized with one or more bacteria harboring one or more antibiotic resistance genes. In some embodiments, the mice are colonized with vancomycin resistant bacteria or vancomycin and carbapenem resistant bacteria.
The invention also includes use of the compositions described herein for the purposes specified in the methods described herein, as well as the use of these compositions for the preparation of medicaments for these uses.
The entire disclosure of each patent document and scientific article referred to herein, and those patent documents and scientific articles cited thereby, is expressly incorporated by reference herein for all purposes.
Additional features and advantages of the invention are more particularly described below.
Antibiotic resistance (referred to herein as “AbxR”) is a growing global public health issue. Resistance to all classes of antibiotics has been reported in all parts of the world (2014, Antimicrobial Resistance, Global Report on Surveillance, Summary, WHO) and development of conventional antibiotics has been relatively slow compared to the demand. For example, no new classes of antibiotics have been commercialized since the discovery of the lipopeptide antibiotic daptomycin in the 1980s. Furthermore, antibiotic stewardship programs that limit antibiotic use do not immediately reverse the trend of increasing resistance.
Applicants have discovered that treating a subject with a composition comprising bacteria, for example, spore-former bacteria derived from a healthy human, can reduce the abundance of AbxR genes (collectively, the “gut resistome”) in a human subject. Such compositions are referred to herein as reduction of antibiotic resistance compositions (ROAR compositions).
Furthermore, such treatment can reduce the abundance of AbxR genes associated with resistance to multiple classes of antibiotics, e.g., β-lactams (including carbapenems), quinolones, glycopeptides (e.g., vancomycin), and tetracyclines. In addition, it was found that non-pathogenic commensals can harbor antibiotic resistance genes, and treatment results in a decrease in the abundance of antibiotic resistance genes in non-pathogenic commensal organisms. Without committing to any particular theory, these data support a method of decreasing the presence of antibiotic resistance genes by, at least in part, a mechanism of decreasing horizontal transfer of antibiotic resistance genes from non-pathogenic commensal organisms to pathogenic bacteria. Such a mechanism could be related at least in part to decreasing the abundance of bacteria harboring antibiotic resistance genes by out-competing these bacteria for nutrients; decreasing the abundance of bacteria harboring antibiotic resistance genes by inducing a response in the host that limits the growth of potential pathogens harboring AbxR genes; decreasing the abundance of bacteria harboring antibiotic resistance genes by introducing bacteria that are directly antagonistic to potential pathogens harboring AbxR genes; reducing opportunistic pathogens, and/or decreasing the potential for the spread of antibiotic resistance genes between antibiotic resistant and susceptible organisms. In some embodiments, the decrease in the presence of antibiotic resistance genes occurs by reducing colonization in the host of microbes that harbor AbxR genes.
In some embodiments, a ROAR composition is used to break or prevent an antibiotic resistance transmission cycle. In such methods, a ROAR composition can be administered to a subject entering an environment in which there is a risk of an antibiotic resistance cycle or an environment in which the existence of an antibiotic resistance cycle has been identified, e.g., an environment in which antibiotic resistance has been identified as spreading or in which there is a risk of such spreading. Examples of such environments include hospitals, intensive care units, nursing homes, rehabilitation centers, group homes, and the like. In some embodiments, the dosing in such an environment is chronic. In some embodiments, a subject is treated to prevent or reduce colonization by one or more AbxR genes. In a subject that harbors one or more AbxR genes, a composition can be administered, thereby reducing the risk of AbxR infection in that subject and reducing the risk that subject will transmit AbxR genes after treatment of C. difficile disease, as such subjects may be susceptible to infection with or harbor antibiotic resistant bacteria. In some embodiments, a composition is administered to workers in environments in which there is a particular risk associated with spreading AbxR genes.
In some environments, “selective decontamination” of patients has been recommended, for example, in hospitals (particularly intensive care units) and nursing homes (for example, see Silvestri and van Saene, “Selective decontamination of the digestive tract: an update of the evidence,” HSR Proc. Intensive Care Cardiovasc. Anesth. 4:21-29, 2012; Oostdijk et al., “Selective decontamination in European intensive care patients,” Intensive Care Med. 38:533-538, 2012). A ROAR composition can be a useful alternative or supplement to such selective decontamination, adding to the armamentarium of methods available to decrease the presence of antibiotic resistance genes in these and other settings.
ROAR Compositions
ROAR compositions generally comprise Firmicutes. In some embodiments, the Firmicutes are capable of forming spores (spore-formers). Bacteria in a composition can be in the form of spores, mixed vegetative and spore forms, or vegetative form. In some cases, a composition consists of Firmicutes. In some embodiments, a ROAR composition comprises one or more species of Bacteroidetes. In some embodiments, a ROAR composition consists of at least one species from each of Firmicutes and Bacteroidetes. Compositions comprising or consisting of Firmicutes (or Firmicutes and Bacteroidetes) as the sole bacterial species of the compositions are not naturally occurring.
As a non-limiting example, a ROAR composition can be prepared from bacteria that are isolated from stool, e.g., human stool. In general, the stool is from a healthy animal (e.g., a human) of the same species as the animal to be treated. Bacteria can be prepared, for example, in a method using an amount of ethanol suitable to kill any non-spore form bacteria present in the stool. An example of such a preparation can be found in, for example, WO 2014/121302.
In some embodiments, a ROAR composition comprises or consists of selected bacterial species associated with a decrease in the abundance of antibiotic resistance genes in a subject (see, e.g., Table 3, below). Such compositions are referred to herein as “designed Abx compositions.” The number of species (e.g., from Table 3) in a designed composition can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments the number of species is at least one and less than or equal to 50, for example, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5. In some embodiments, the species selected from Table 3 do not include species that are also listed in Table 4.
Individual species in a designed ROAR composition may be cultured separately and then combined to create the composition. In some embodiments, a ROAR composition is produced by co-culturing all or subsets of the composition and then combining the components that were not co-cultured to create the composition. In some embodiments, a ROAR composition is produced by culturing a spore fraction derived from feces of a healthy human. The resulting culture can be used as a ROAR composition.
In some embodiments, the species selected for inclusion in a ROAR composition are associated with a decrease in the abundance of a particular class of antibiotic resistance genes, e.g., genes associated with resistance to quinolones, beta-lactams, and/or carbapenems (also see Table 2). In specific examples of, e.g., the compositions described above, bacterial species in a ROAR composition (e.g., designed Abx compositions) are selected from Table 2, and optionally may be identified by analysis of 16S rDNA sequences (or fragments thereof, e.g., a fragment comprising the 16S rDNA V4 and/or V6 regions; also see below and Table 5 for exemplary sequences).
In some embodiments, a species useful in a ROAR composition is a species having a full-length 16S rDNA with at least 95% sequence identity (“identity”) (e.g., at least 96%, 97%, 98%, or 99%, or 100%) to the 16S rDNA of a reference species, e.g., a species identified in Table 3 (see, e.g., Table 5 for sequence information). In some embodiments, a species useful in a ROAR composition is a species having a full-length 16S rDNA with at least 97% sequence identity (“identity”) to the 16S rDNA of a reference species, e.g., a species in Table 3 (see, e.g., Table 5 for sequence information). In some embodiments, a useful species has a V4 and/or V6 region 16S rDNA sequence having at least 95% identity (e.g., at least 96%, 97%, 98%, or 99%, or 100%) to a V4 and/or V6 region of 16S rDNA sequence of a reference species, e.g., a species identified in Table 3 (see, e.g., Table 5 for sequence information). In some embodiments, a useful species has a V4 and/or V6 region 16S rDNA sequence having at least 97% identity to a V4 and/or V6 region of 16S rDNA sequence of a reference species, e.g., a species identified in Table 3 (see, e.g., Table 5 for sequence information). In some embodiments, a useful species has a genomic sequence having at least 95% identity (e.g., at least 96%, 97%, 98%, or 99%, or 100%) to the full-length genomic DNA of a reference species, e.g., a species identified in Table 3. In some embodiments a useful species has a genomic sequence having at least 97% identity to the full-length genomic sequence of a reference species, e.g., a species identified in Table 3. In the event that a sequence is not provided herein, e.g., a 16S rDNA, V4, or V6 sequence, methods are well known in the art for identifying such sequences. Table 5 provides non-limiting examples of full-length 16S rDNA sequences that can be used as reference sequences. In general, identity or percent identity with a reference species means identity or percent identity with at least one 16S rDNA sequence (or fragment; e.g., V4 and/or V6) found in an organism.
In some cases, strains of bacterial species useful in the invention, e.g., species disclosed herein, can be obtained from a public biological resource center such as the ATCC (atcc.org), the DSMZ (dsmz.de), or the Riken BioResource Center (en.brc.riken.jp). 16s rDNA sequences useful for identifying species or other aspects of the invention can be obtained from public databases, e.g., the Human Microbiome Project (HMP) web site or GenBank.
Methods of determining sequence identity are known in the art and examples are provided infra.
In some embodiments, species excluded from ROAR compositions are species that are positively correlated with the presence of a particular class of antibiotic resistance genes (see, e.g., Table 4). These species can be identified using methods such as those described above (also see Table 6 for corresponding sequence information).
It is to be understood that “consisting of” in these examples refers to bacteria types that are present in a composition. A bacterial formulation “consisting of” listed species may thus contain additional non-bacterial materials such as one or more excipients (including, for example, one or more capsules), an aqueous or non-aqueous medium (e.g., glycerol, polyethylene glycol, cocoa butter, water, and/or a buffer), and/or one or more prebiotics or small molecule drugs.
Species/Naming Information
Names and classification of bacteria are subject to changes that may not be reflected in the literature. For convenience, alternate names for some bacterial species may be provided herein but are not intended to be a comprehensive set of alternative names. In some embodiments, species are identified by sequence identity of all or a portion (e.g., V4 or V6 regions) of a 16S rDNA sequence, e.g., at least 90%, 93% 95%, 96%, 97%, 98%, 99%, or 100% identity.
Determination of Identity
Clades, operational taxonomic units (OTUs), species, and strains are, in some embodiments, identified by their 16S rDNA sequence. The relatedness of clades, OTUs, species, and strains can be determined by the percent identity between clades, OTUs, species, or strains. Percent identity between a reference and query sequence can be determined using methods known in the art. Non-limiting examples of methods for such determinations are provided below. As used herein, the relatedness between two nucleotide sequences is described by the parameter “identity.”
In one embodiment, the degree of sequence identity between a query sequence and a reference sequence is determined by (1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty, (2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical nucleotide in the two aligned sequences on a given position in the alignment, and (3) dividing the number of exact matches with the length of the reference sequence.
In another embodiment, the degree of sequence identity between a query sequence and a reference sequence is determined by (1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty, (2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical nucleotide in the two aligned sequences on a given position in the alignment, and (3) dividing the number of exact matches with the length of the longest of the two sequences.
In another embodiment, the degree of sequence identity between the query sequence and the reference sequence is determined by (1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty, (2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid or nucleotide in the two aligned sequences on a given position in the alignment, and (3) dividing the number of exact matches with the “alignment length,” where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences.
Sequence identity comparisons are carried out, generally, with the aid of a sequence comparison program. These commercially or publicly available computer programs use complex comparison algorithms to align two or more sequences that best reflect the evolutionary events that might have led to the difference(s) between the two or more sequences. Therefore, these algorithms operate with a scoring system rewarding alignment of identical or similar amino acids and penalizing the insertion of gaps, gap extensions, and alignment of non-similar amino acids. The scoring system of the comparison algorithms include:
i) assignment of a penalty score each time a gap is inserted (gap penalty score),
ii) assignment of a penalty score each time an existing gap is extended with an extra position (extension penalty score),
iii) assignment of high scores upon alignment of identical amino acids, and
iv) assignment of variable scores upon alignment of non-identical amino acids.
In general, the default values of the alignment program are used for sequence comparisons.
Suitable computer programs useful for determining identity include, for example, BLAST (blast.ncbi.nlm.nih.gov).
In an embodiment of the present invention, the alignment program optimizes the alignment over the full-length of selected sequences, e.g., full-length, V4, or V6 16S rDNA sequence. For example, the global alignment program is based on the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 443-453, 1970). Non-limiting examples of such programs are EMBOSS Needle and EMBOSS Stretcher programs, available at ebi.ac.uk/Tools/psa/.
In one embodiment, the sequences are aligned by a global alignment program and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the “alignment length,” where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences. In a further embodiment, the global alignment program uses the Needleman-Wunsch algorithm and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the “alignment length,” where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences.
In yet a further embodiment, the global alignment program is selected from the group consisting of EMBOSS Needle and EMBOSS stretcher and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the “alignment length,” where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences.
Once the software has produced an alignment, it is possible to calculate percent (%) similarity and percent sequence identity.
Methods of Testing Compositions for AbxR Activity or the Presence of AbxR Genes
A ROAR composition can be tested for the ability to reduce the abundance of antibiotic resistance genes in the GI tract of an animal using in vitro and/or in vivo models known in the art (for example, see Schjørring and Krogfelt, Int. J. Microbiol. 2011:312956, 2011; Marra, “Animal models in drug development for MRSA,” Meth. Mol. Biol. 1085:333-345, 2014). Such models can be adapted for use in testing candidate ROAR compositions. Additional examples of suitable models are described in the Examples. In addition, a ROAR composition can be tested for the ability to reduce the abundance of AbxR genes in human subjects enrolled in clinical trials.
Subjects
In some embodiments, a subject suitable for treatment with a composition disclosed herein (a ROAR composition) is an animal, e.g., a mammal such as a human, dog, cat, bovine, ovine, coprine, equine, or other farm, domestic, or zoo animal at risk for, or diagnosed with, having an undesirable level of AbxR microorganisms, e.g., in at least a portion of the gastrointestinal (GI) tract. An undesirable level of AbxR organisms can be identified as a level significantly greater than that in a healthy population, for example, in the context of treating humans, the healthy population documented in the Human Microbiome Project database. Such organisms can be detected by, for example, culturing a stool or other gastrointestinal sample from the subject and using known methods of testing for the presence of antibiotic resistance. The methods include, for example, dilution methods (broth and agar dilution methods), disk-diffusion methods, E-test, automated methods, mechanism-specific tests such as beta-lactamase detection test and chromogenic cephalosporin tests, and genotypic methods such as PCR and DNA hybridization methods. Such methods also include the identification and quantification of antibiotic resistance associated genes by whole metagenomics shotgun (WMS) sequencing. In some embodiments, an undesirable level of AbxR organisms or genes can be identified using molecular methods that identify the presence or absence of genes, gene cassettes, or genetic markers associated with the phenotype of AbxR. Some examples of such molecular methods include but are not limited to DNA sequencing, transcriptional profiling, and methods that utilize a molecular probe.
In some embodiments, a subject suitable for treatment with a ROAR composition is or has been unresponsive to treatment with one or more antibiotics and the presence of an antibiotic resistant microbial population is inferred.
In some embodiments, a subject suitable for treatment with a ROAR composition has or is at risk of developing C. difficile infection or disease. In some embodiments, a subject suitable for treatment with a ROAR composition has or is at risk of developing a colitis (e.g., Crohn's disease or ulcerative colitis (e.g., mild to moderate ulcerative colitis)).
Use of conventional antibiotics, in addition to the potential for the existence or development of antibiotic resistance by a subject's microbiome, can cause serious side effects including, for example, GI effects such as dysbiosis, overgrowth of pathogens, nervous system damage, and allergic reactions. A ROAR composition typically comprises commensal bacteria and has limited side effects, e.g., is well-tolerated by subjects receiving the treatment. A ROAR composition can be administered repeatedly over time, as needed, and may reduce the risk of developing antibiotic resistance to a large group of antibiotics, e.g., by reducing the prevalence of antibiotic resistance genes in a treated individual. In general, a ROAR composition can modulate transmission cycles in which bacteria transmit antibiotic resistance genes. This feature is useful in, for example, health care settings in which the use of the compositions decreases the risk of fragile patients being exposed to or developing antibiotic resistance, both individually and as a patient population.
In some embodiments, an additional advantage of an ROAR composition that administration of the composition may improve the integrity of the epithelial and mucosal barriers, for example, by reducing the amount of local inflammation, and reducing the risk of translocation and infection by GI local microbes.
“Augmentation” of a type of bacterium, e.g., a species, is an effect of treatment with a composition of the invention that is characterized by post-treatment detection of an increased abundance of a species not present in the composition by a nonparametric test of abundance.
“Engraftment” of a type of bacterium, e.g., a species, is an effect of treatment with a composition of the invention that is characterized by post-treatment detection of a species from the administered composition, which is not detected in the treated subject pretreatment. Methods of detection are known in the art. In one example, the method is PCR detection of a 16S rDNA sequence using standard parameters for PCR.
“Operational taxonomic unit,” “OTU” (or plural, “OTUs”) refers to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence is the 16S sequence or a portion of the 16S sequence, such as a variable region, e.g., a V4 region. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. In 16S embodiments, OTUs that share ≥97% average nucleotide identity across an entire 16S or a variable region of a 16S sequence are considered the same OTU (see e.g., Claesson et al., Nucleic Acids Res. 38:e200, 2010; Konstantinidis et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 361:1929-1940, 2006). In embodiments involving the complete genome, MLSTs, specific genes, or sets of genes, OTUs that share ≥95% average nucleotide identity are considered the same OTU (see e.g. Achtman and Wagner, Nat. Rev. Microbiol. 6:431-440, 2008). OTUs can be distinguished, in some embodiments, by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping”genes), or a combination thereof. Such characterization employs, e.g., WGS data or a whole genome sequence. As used herein, the terms “species” and “OTU” are used interchangeably unless otherwise distinguished by context.
A “therapeutically effective amount” of a composition described herein can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter, or amelioration of at least one symptom of the disorder (and optionally, the effect of any additional agents being administered). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A composition as described herein is generally administered in a therapeutically effective amount.
Formulations
ROAR compositions (e.g., designed Abx compositions) described herein can be prepared and administered using methods known in the art. In general, compositions are formulated for oral, colonoscopic, or nasogastric delivery although any appropriate method can be used.
A ROAR formulation can contain one or more pharmaceutical excipients suitable for the preparation or delivery of such formulations. In some embodiments, the formulation is a liquid formulation. In some embodiments, a formulation comprising a ROAR composition can comprise one or more of surfactants, adjuvants, buffers, antioxidants, tonicity adjusters, thickeners, viscosity modifiers, and the like.
In some embodiments, treatment includes administering a ROAR composition in a formulation that includes a pharmaceutically acceptable carrier. In some embodiments, the excipient includes a capsule or other format suitable for providing the ROAR composition as an oral dosage form. When an excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the active ingredient. Thus, the formulations can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft or hard capsules, suppositories, or packaged powders.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, polyethylene glycol, glycerol, and methyl cellulose. The compositions can be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
A ROAR composition can be formulated in a unit dosage form. In general, a dosage comprises about 10e2 to 10e9 viable colony forming units (cfu). In some embodiments, the ROAR compositions comprises or is composed of spore-former bacteria in spore form. In such cases, the dosage may be determined as “spore CFU” or “sCFU”, which quantify the number of viable spores by germination and growth, typically on plates.
The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and/or other mammals, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect, in association with a suitable pharmaceutical excipient. A dosage may be administered in multiple delivery vehicles, e.g., multiple pills or capsules.
The amount and frequency of administering a ROAR composition to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest one or more symptoms of the disease or its complications. Effective doses will depend on the disease or condition being treated, as well as by the judgment of the attending clinician, depending upon factors such as the severity of the disease, the age, weight, and general condition of the patient, and the like.
The dosage can refer, for example, to the total number of cfus of each individual species or strains, or can refer to the total number of microorganisms in the dose. It is understood in the art that determining the number of organisms in a dosage is not exact and can depend on the method used to determine the number of organisms present. For example, the number of spores in a composition may be determined using a dipicolinic acid assay. In some cases, the number of organisms may be determined using a culture assay. When spores are present, the ability of assays relying on culture methods can depend on efficient germination of spores. Quantitative nucleic acid-based methods can depend on whether the nucleic acids from non-viable microorganisms are sufficiently reduced or eliminated. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides methods for identifying bacterial compositions that can be used to decrease the abundance of antibiotic resistance genes in subjects. In these methods, candidate compositions are analyzed for the presence of one or more bacterial species of Table 3. Detection of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of these species indicates that the composition may be useful in decreasing the abundance of antibiotic resistance genes. Additional testing can be done to further screen candidate compositions. For example, candidate compositions can further be analyzed for the presence of one or more species of Table 4. In some embodiments, it may be determined that it is preferable to avoid use of compositions identified as including one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) species of Table 4.
In other screening methods of the invention, potential donors of feces for use in therapeutic methods (e.g., therapeutic methods of the present invention) are screened for their suitability to serve as donors. In these methods, a fecal sample of a potential donor is screened for the presence of one or more bacterial species associated with decreased antibiotic resistance genes (see, e.g., Table 3; see Table 5 for sequences) and/or one or more bacterial species correlated with the presence of antibiotic resistance genes (see, e.g., Table 4; see Table 6 for sequences). Detection of the former may indicate that the potential donor is suitable to proceed to serve as a donor, while detection of the latter may indicate that the potential donor should not proceed, as can be determined by those of skill in the art.
Additional screening methods include those used to determine whether a subject may benefit from treatment with a ROAR composition, as described herein. In these methods, a sample from the subject (e.g., a fecal sample) is tested for the presence of one or more bacterial species correlated with the presence of antibiotic resistance genes (see, e.g., Tables 4 and 6). Detection of one or more of these species can indicate that the subject may benefit from treatment according to the therapeutic methods of the invention, as can be determined by those of skill in the art.
Detection of species in these methods of the invention can be carried out using methods that are known in the art (see, e.g., above and elsewhere herein). For example, methods including, for example, qPCR, whole metagenomics shotgun sequencing (WMS; see, e.g., below), and/or analysis of 16S rDNA sequences or a portion thereof (e.g., the V4 and/or V6 regions; see, e.g., Table 5) can be performed. Sequences from Table 5 can be detected with respect to the identification of negative correlates (i.e., reduction in antibiotic resistance genes). Sequences from Table 6 can be detected with respect to the identification of positive correlates (i.e., increased abundance of antibiotic resistance genes).
All technical features can be individually combined in all possible combinations of such features.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.
The following non-limiting examples further illustrate embodiments of the inventions described herein.
Human subjects at risk for recurrent Clostridium difficile infection (rCDI) were treated with enriched and purified spores prepared from the stool of healthy, screened humans in a randomized, double-blind, placebo-controlled Phase 2 trial (n=89; randomized 2:1; 36 sites). The spores were administered orally in a single dose (of 4 capsules). Dosing was at 10e8 spore equivalents. The GI microbiomes of these subjects, as well as subjects in a Phase 1b open label dose ranging study, were profiled using whole metagenomics shotgun sequencing (WMS). Stool samples were collected before treatment with the spores (after treatment with antibiotics) and after receiving spores or placebo, as applicable.
DNA was extracted from stool samples and used to generate WMS data with the IIlumina sequencing platform. WMS data was screened for the presence and abundance of genetic markers of proteins known to confer antibiotics resistance, as catalogued in the Comprehensive Antibiotic Resistance Database (CARD v.1.1.8) (card.mcmaster.ca). Genetic markers associated with these proteins were constructed using ShortBRED (Kaminski et al., PLoS Comput Biol 11(12):e1004557), an informatics tool designed to identify proteins of interest at high specificity within WMS data (Table 1). For each class of antibiotics, the abundance of genetic markers associated with resistance to that class summed to generate normalized-gene abundance values (referred to herein as drug class marker-abundance or DCMA).
For the Phase 2 study, differential prevalence of spore-former species was observed based on WMS analysis. Gene abundance was assessed using the ShortBRED markers (Kaminski et al., supra) for genes associated with antibiotic resistance identified in ARDB (Liu and Pop, 2009, ARDB—Antibiotic Resistance Genes Database. Nuc Acids Res 37(Database issue):D443-447). Data were analyzed for a significant engraftment signal, defined as subjects in whom a significant number of species were identified as being significantly genetically similar to species identified in the administered ROAR composition. Analysis of the data resulted in the discovery that in subjects with a significant engraftment signal for the composition, the abundance of genes associated with resistance to multiple classes of drugs, i.e., quinolone drugs (
In additional analyses, for each subject and drug class (from both the Phase 1b and Phase 2 studies), we calculated the log 2 fold change (log 2FC) of drug class marker abundance (DCMA) between samples collected at the visit 1 week after receiving placebo or spores, as compared to samples collected one day prior to treatment with placebo or spores. We compared rCDI subjects who received placebo to rCDI subjects who received spores and in whom significant engraftment was detected. We used the non-parametric Mann-Whitney test to determine if the log 2FC in DCMA was significantly greater in subjects with engraftment of spores as compared to subjects who received placebo (Table 2). False discovery rates (FDR), the expected proportion of incorrectly rejected null hypotheses for a given p-value due to running multiple tests, was calculated using the Benjamini-Hochberg procedure (p<0.1)
The data demonstrate that a ROAR composition can be used to reduce the abundance of antibiotic resistance genes in a subject.
Escherichia
coli emrE
Escherichia
coli mdfA
Enterobacter
cloacae acrA
Clostridium
perfringens mprF
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Computational analysis to determine whether the presence of specific bacterial species is predictive of a decrease in the abundance of antibiotic resistance genes was performed. Species identified in these analyses can be used in designed Abx compositions. To identify microbial species that are associated with a reduction of antimicrobial resistance genes, we calculated the Spearman correlation between species abundance and the DCMA of drug classes showing a significant reduction in subjects in whom engraftment of spores was observed relative to placebo. P-values were adjusted to account for multiple hypothesis testing using the Benjamini-Hochberg procedure (p<0.1) as described supra. The set of species associated with significant reduction of at least one drug of antibiotic resistance genes is summarized in Table 3. We have further defined the set of species positively correlated with elevated abundances of at least one class of antibiotic resistance genes, summarized in Table 4. Dose species in all tables are bacterial species identified in dose lots administered to subjects. Full length 16S sequences of species described herein are provided in Tables 5 (negative correlates) and 6 (positive correlates). In some embodiments, the bacterium(a) are from a genus listed below. Those of skill in the art can use standard methods to identify the genera.
Clostridium
Clostridium
Clostridium
Fusicateniba
cter_sacchar
ivorans
Lachnospira
za
Clostridium
Ruminococc
us_sp.
Clostridium
Blautia_faec
is
Clostridium
Clostridium
Clostridium
Flavonifract
or_sp.
Emergencia
Clostridium
Clostridium
Clostridium
Clostridium
Flavonifract
or_sp.
Intestinimon
as_massilien
sis
Clostridium
Blautia_faec
is
Clostridium
Anaerostipe
s_sp.
Ruminococc
us_bicircula
ns
Ruminococc
us_bicircula
ns
Streptococc
us_gordonii
Streptococc
us_gordonii
Streptococc
us_gordonii
Streptococc
us_gordonii
Akkermansi
a_muciniphi
la
Akkermansi
a_muciniphi
la
Akkermansi
a muciniphi
la
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Eggerthella_
lenta
Eggerthella_
lenta
Eggerthella_
lenta
Burkholderi
ales_bacteri
um_1_1_47
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Ruminococc
us_obeum
Ruminococc
us_obeum
Ruminococc
us_obeum
Ruminococc
us_obeum
Ruminococc
us_obeum
Ruminococc
us_torques
Ruminococc
us_torques
Ruminococc
us_torques
Ruminococc
us_torques
Ruminococc
us_torques
Ruminococc
us_torques
Dorea_longi
catena
Dorea_longi
catena
Anaerostipe
s_caccae
Anaerostipe
s_caccae
Anaerostipe
s_caccae
Anaerostipe
s_caccae
Anaerostipe
s_caccae
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Clostridium
Clostridium
Clostridium
Faecalibacte
rium_prausn
itzii
Faecalibacte
rium_prausn
itzii
Faecalibacte
rium_prausn
itzii
Faecalibacte
rium_prausn
itzii
Faecalibacte
rium_prausn
itzii
Faecalibacte
rium_prausn
itzii
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Anaerotrunc
us_colihomi
nis
Anaerotrunc
us_colihomi
nis
Anaerotrunc
us_colihomi
nis
Anaerotrunc
us_colihomi
nis
Clostridium
Clostridium
Clostridium
Anaerofustis
minis
Anaerofustis
minis
Coprococcu
s_comes
Roseburia_i
ntestinalis
Blautia_hyd
rogenotrophi
ca
Holdemania
Clostridium
me
Blautia_wex
lerae
Blautia_wex
lerae
Blautia_wex
lerae
Blautia_wex
lerae
Blautia_wex
lerae.
Blautia_wex
lerae
Blautia_wex
lerae
Dialister_in
visus
Dialister_in
Dialister_in
visus
Dorea_formi
cigenerans
Dorea_formi
cigenerans
Dorea_formi
cigenerans
Dorea_formi
cigenerans
Dorea_formi
cigenerans
Dorea_formi
cigenerans
Eubacterium
Ruminococc
aceae_bacter
ium_D16
Streptococc
us_vestibula
ris
Streptococc
us_vestibula
ris
Streptococc
us_vestibula
ris
Streptococc
us_vestibula
ris
Streptococc
us_vestibula
ris
Streptococc
us_vestibula
ris
Streptococc
us_thermop
hilus
Streptococc
us_thermop
hilus
Streptococc
us_thermop
Streptococc
us_thermop
Streptococc
us_thermop
hilus
Prevotella_b
uccae
Lachnospira
ceae_bacteri
um_5_1_63
Lachnospira
ceae_bacteri
um_8_1_57
Streptococc
us_australis
Lachnospira
ceae_bacteri
um_4_1_37
Streptococc
us_sanguinis
Lachnospira
ceae_bacteri
um_3_1_46
Lachnospira
ceae_bacteri
um_6_1_63
Lachnospira
ccae bacteri
um_9_1_43
Ruminococc
us_bromii
Coprococcu
s_catus
Dysgonomo
nas_mossii
Lachnospira
ceae_bacteri
um_1_4_56
Lachnospira
ceae_bacteri
um_3_1_57
Lachnospira
ceae_bacteri
um_3_1_57
Lachnospira
ceae_bacteri
um_3_1_57
Lachnospira
ceae_bacteri
um_5_1_57
Lachnospira
ceae_bacteri
um_1_1_57
Lachnospira
ceae_bacteri
um_2_1_58
Roseburia_h
ominis
Roseburia_h
ominis
Roseburia_h
ominis
Roseburia_h
ominis
Erysipelotric
haceae_bact
erium_2_2_
Clostridium
Flavonifract
or_plautii
Lachnospira
ceae_bacteri
um_7_1_58
Erysipelotric
haceae_bact
erium_6_1_
Erysipelotric
haceae_bact
erium_6_1_
Erysipelotric
haceae_bact
erium_6_1_
Erysipelotric
haceae_bact
erium_6_1_
Erysipelotric
haceae_bact
erium_21_3
Erysipelotric
haceae_bact
erium_21_3
Erysipelotric
haceae_bact
erium_21_3
Erysipelotric
haceae_bact
erium_21_3
Erysipelotric
haceae_bact
erium_21_3
Haemophilu
s_parainflue
nzae
Streptococc
us_parasang
uinis
Streptococc
us_parasang
uinis
Streptococc
us_parasang
uinis
Streptococc
us_parasang
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Eubacterium
Streptococc
us_salivariu
S
Streptococc
us_salivariu
s
Streptococc
us_salivariu
s
Streptococc
us_salivariu
S
Streptococc
us_salivariu
s
Streptococc
us_salivariu
s
Streptococc
us_salivariu
S
Parabacteroi
des_distason
is
Parabacteroi
des_distason
is
Parabacteroi
des_distason
is
Parabacteroi
des_distason
is
Parabacteroi
des_distason
is
Clostridiace
ae_bacteriu
m_JC118
Anaerostipe
s_hadrus
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
Clostridium
rme
Clostridium
rme
Clostridium
rme
Lactobacillu
s_fermentu
m
Lactobacillu
s_fermentu
m
Lactobacillu
s_fermentu
m
Lactobacillu
s_fermentu
m
Lactobacillu
s_fermentu
m
Bacteroides
micron
Bacteroides
micron
Bacteroides
micron
Bacteroides
micron
Sutterella_w
adsworthens
is
Sutterella_w
adsworthens
is
Sutterella_w
adsworthens
is
Sutterella_w
adsworthens
is
Clostridium
Clostridium
Eubacterium
Ruminococc
us_gnavus
Ruminococc
us_gnavus
Ruminococc
us_gnavus
Ruminococc
us_gnavus
Ruminococc
us_gnavus
Blautia_wex
lerae
Gemmiger_f
ormicilis
Eisenbergiel
la_massilien
sis
Ruminococc
us_sp.
Roseburia_f
aecis
Butyrivibrio
Blautia_sp_
Eubacterium
Gemmiger_f
ormicilis
Intestinimon
as_butyricip
roducens
Oscillospira
ceae_bacteri
um_VE202_
Clostridium
Ruminococc
us_obeum
Blautia sp.
Blautia_sp.
Lachnospira
za
Lactonifacto
r_longovifor
mis
Eubacterium
contortum
Eubacterium
Eubacterium
Eubacterium
Eisenbergiel
la_tayi
Ruminococc
us_gnavus_s
Clostridium
Clostridium
Clostridium
Blautia_pro
ducta
Clostridium
Roseburia_i
nulinivorans
Streptococc
us_infantis
Streptococc
us_infantis
Streptococc
us_infantis
Clostridium
Streptococcus_mitis_
oralis_pneumoniae
Streptococcus_mitis_
oralis_pneumoniae
Streptococcus_mitis_
oralis_pneumoniae
Streptococcus_mitis_
oralis_pneumoniae
Proteus_mirabilis
Proteus_mirabilis
Proteus_mirabilis
Proteus_mirabilis
Proteus_mirabilis
Proteus_mirabilis
Proteus_mirabilis
Pediococcus_
acidilactici
Bifidobacterium_
dendum
Proteus_penneri
Proteus_penneri
Lactobacillus_
salivarius
Veillonella_dispar
Veillonella_dispar
Veillonella_dispar
Rothia_dentocariosa
Rothia_dentocariosa
Rothia_dentocariosa
Megasphaera_
micronuciformis
Veillonella_atypica
Streptococcus_
thermophilus
Streptococcus_
thermophilus
Streptococcus_
thermophilus
Streptococcus_
thermophilus
Streptococcus_
thermophilus
Lactobacillus_
animalis
Bilophila_
wadsworthia
Bilophila_
wadsworthia
Bilophila_
wadsworthia
Bilophila_
wadsworthia
Veillonella_parvula
Lachnospiraceae_
bacterium_3_1_57FAA_
Lachnospiraceae_
bacterium_3_1_57FAA_
Erysipelotrichaceae_
bacterium_2_2_44A
Rothia_mucilaginosa
Lactobacillus_
rhamnosus
Lactobacillus_
rhamnosus
Klebsiella_oxytoca
Clostridium_
perfringens
Streptococcus_mitis_
oralis_pneumoniae
Bacteroides_ovatus
Bacteroides_ovatus
Bacteroides_ovatus
Bacteroides_ovatus
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_uniformis
Bacteroides_vulgatus
Bacteroides_vulgatus
Bacteroides_vulgatus
Bacteroides_vulgatus
Bacteroides_vulgatus
Bacteroides_vulgatus
Eubacterium_sp_3_1_
Eubacterium_sp_3_1_
Eubacterium_sp_3_1_
Eubacterium_sp_3_1_
Eubacterium_sp_3_1_
Morganella_morganii
Parabacteroides_
distasanis
Parabacteroides_
distasanis
Parabacteroides_
distasanis
Parabacteroides_
distasanis
Parabacteroides_
distasanis
Citrobacter_freundii
Citrobacter_freundii
Lactococcus_lactis
Lactococcus_lactis
Alistipes_onderdonkii
Alistipes_onderdonkii
Campylobacter_
curvus
Lactobacillus_
fermentum
Lactobacillus_
fermentum
Lactobacillus_
fermentum
Lactobacillus_
fermentum
Lactobacillus_
fermentum
Bacteroides_
thetaiotaomicron
Bacteroides_
thetaiotaomicron
Bacteroides_
thetaiotaomicron
Bacteroides_
thetaiotaomicron
Lactobacillus_gasseri
Lactobacillus_casei_
paracasei
Lactobacillus_casei_
paracasei
Enterococcus_faecium
Lactobacillus_casei_
paracasei
Lactobacillus_casei_
paracasei
Lactobacillus_casei_
paracasei
Lactobacillus_casei_
paracasei
Lactobacillus_casei_
paracasei
Lactobacillus_
plantarum
Enterobacter_cloacae
Escherichia_coli
Escherichia_coli
Escherichia_coli
Escherichia_coli
Escherichia_coli
Escherichia_coli
Escherichia_coli
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Klebsiella_pneumoniae
Fusobacterium_
nucleatum
Butyrivibrio_sp.
Ruminococcus_obeum
Human subjects having mild-moderate ulcerative colitis received placebo only (Arm A), weekly doses of enriched and purified spores prepared from the stool of healthy, screened humans (Arm B), a short course of vancomycin followed by weekly administration of the spores (Arm D), or a short course of vancomycin followed by daily administration of the spores (Arm C).
As antibiotics are not the standard of care for ulcerative colitis, this population of subjects is less likely to have been frequently treated with antibiotics as compared to the multiply recurrent C. difficile population (Example 1, above). Therefore, the microbiomes of ulcerative colitis subjects have experienced fewer periods of strong selection for antibiotic resistance. At the pre-antibiotic baseline sample (Visit 1,
The results show that AbxR gene abundances are lower in post-treated time points as compared to post antibiotics baseline samples (Visit 4, day 8). In Arms C and D, treatment with vancomycin results in a spike in antibiotic resistance genes post-treatment (Visit 4, day 8). Treatment with spores daily or weekly resulted in a significant reduction in antibiotic resistance genes at Visit 5 (day 15, 7 days after spore dosing commenced) relative to post-antibiotic baseline (Visit 4, Table 7).
AbxR gene abundances are lower in post-treated time points as compared to pre-antibiotic baseline samples (Visit 1). In Arms C and D, treatment with spores resulted in reduction in antibiotic resistance genes relative to pre-antibiotic baseline (Visit 1, Table 8,
The results show that engraftment of spores drives reduced antibiotic resistance. In Arm D (weekly dosing), spores engrafted rapidly in a subset of subjects (Rapid Early Engrafters=True,
One method of testing a ROAR composition for the ability to reduce antibiotic resistance uses conventional mice (laboratory strains that have a microbiome). The mice are treated with selected antibiotics, then colonized with bacteria harboring one or more antibiotic resistance genes, for example, vancomycin resistant bacteria (vancomycin resistant Enterococcus; VRE) or vancomycin and carbapenem resistant bacteria (carbapenem resistant Enterobacteriaceae; CRE). Following colonization, the mice are treated with a ROAR composition, fecal transplant, or control. Titers of antibiotic resistant bacteria (for example, vancomycin resistant and carbapenem resistant bacteria) are monitored via plating of stool collected from treated animals.
Applicants have established a reproducible and robust vancomycin-resistant enterococci (VRE) colonization model in mice based on a published model (Caballero and Pamer, 2015, Ann Rev Immunol 33:227-256). Prior to VRE inoculation, specific pathogen free (SPF) mice are pre-conditioned with ampicillin to disrupt colonization resistance and allow VRE colonization. Following ampicillin treatment, mice are challenged with VRE and achieve high titer (107 to 1010 CFU/g feces) carriage in the GI tract, a condition that is stable for multiple weeks. Once colonized with VRE, mice are treated with a human FMT or a ROAR resistance composition via daily oral gavage for 3 days. Fresh fecal samples are collected throughout the study to evaluate the ability of compositions to clear VRE from the mice and restore a healthy gut microbiome. VRE viable titers in feces pre- and post-treatment are determined by plating on selective media that contains bile esculin azide agar with vancomycin and streptomycin. The VRE colonization model is able to detect a >4 log difference in colonization over 3 weeks in mice treated with human FMT compared to mice treated only with PBS. The ability to detect a >4 log difference in VRE titers during the study window has also distinguished designed microbial compositions with differential potency. In addition, the abundance of antibiotic resistance genes can be assayed. Carbapenem-resistant Enterobacteriaceae (CRE) resistance can be tested in a similar way, substituting selective media appropriate for CRE.
Using such models, a ROAR composition can be tested for its effects on the abundance of antibiotic resistance genes. A ROAR composition that can reduce the abundance of one or more antibiotic resistance genes in this model is useful for the methods described herein.
Various embodiments of the invention are within the following numbered paragraphs.
1. A method of reducing the abundance of at least one antibiotic-resistance gene in the microbiome of a subject, the method comprising administering a therapeutically effective amount of a microbiome composition comprising spore-former bacteria.
2. A method of treating a subject at risk for or diagnosed with an undesirable level or population of drug-resistant bacteria, the method comprising administering a therapeutically effective amount of a microbiome composition comprising Firmicutes derived from one or more healthy human subjects.
3. A method of reducing the abundance of antibiotic-resistant bacteria in the microbiome of a subject, the method comprising administering a therapeutically effective amount of a ROAR composition.
4. A method of disrupting a transmission cycle of antibiotic resistance genes, the method comprising administering a ROAR composition to at least two individuals with a high likelihood of direct or indirect contact including contact with a bodily fluid or waste.
5. The method of paragraph 1, wherein the antibiotic-resistance gene(s) is selected from those listed in Table 1.
6. The method of paragraph 5, wherein the abundance of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibiotic resistance genes of Table 1 is reduced.
7. The method of any one of paragraphs 1 to 6, wherein the drug or antibiotic is from a drug class listed in Table 2.
8. The method of any one of paragraphs 1 to 7, further comprising reducing the abundance of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more antibiotic resistant genes or drug classes.
9. The method of any one of paragraphs 1 to 8, wherein the administered composition comprises one or more species listed in Table 3.
10. The method of paragraph 9, wherein the administered composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more species listed in Table 3.
11. The method of paragraph 9 or 10, wherein the administered species comprises a 16S rDNA, V4, and/or V6 sequence listed in Table 5, or a sequence having at least 95% identity thereto.
12. The method of any one of paragraphs 1 to 11, wherein the administered composition does not comprise one or more species of Table 4.
13. The method of any one of paragraphs 1 to 12, wherein the bacteria in the composition are in the form of spores.
14. The method of any one of paragraphs 1 to 13, wherein the bacteria in the composition are cultured.
15. The method of any one of paragraphs 1 to 14, wherein the composition comprises Firmicutes and one or more species of Bacteroides.
16. The method of any one of paragraphs 1 to 15, wherein the subject has a reduced abundance of one, two, or three bacteria selected from the group consisting of quinolone resistant, beta-lactam-resistant, and carbapenem-resistant bacteria.
17. The method of any one of paragraphs 1 to 16, wherein the subject has been unresponsive to antibiotic treatment.
18. The method of any one of paragraphs 1 to 17, wherein the subject has or is at risk of developing C. difficile infection.
19. The method of any one of paragraphs 1 to 18, wherein the subject has or is at risk of developing a colitis.
20. The method of paragraph 19, wherein the colitis is Crohn's disease or ulcerative colitis.
21. The method of paragraph 20, wherein the colitis is mild to moderate ulcerative colitis.
22. A composition comprising bacterial species associated with a decrease in the abundance of at least one antibiotic resistance gene in a subject.
23. The composition of paragraph 22, wherein the antibiotic-resistance gene(s) is selected from those listed in Table 1.
24. The composition of paragraph 23, wherein the abundance of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibiotic resistance genes of Table 1 is decreased.
25. The composition of any one of paragraphs 22 to 24, wherein the antibiotic is from a class listed in Table 2.
26. The composition of any one of paragraphs 22 to 25, wherein the composition comprises one or more species listed in Table 3.
27. The composition of paragraph 26, wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more species listed in Table 3.
28. The composition of paragraph 20 or 27, wherein the species comprises a 16S rDNA, V4, and/or V6 sequence listed in Table 5, or a sequence having at least 95% identity thereto.
29. The composition of any one of paragraphs 22 to 28, wherein the composition does not comprise one or more species of Table 4, and optionally the one or more species of Table 4 comprises a 16S rDNA, V4, and/or V6 sequence listed in Table 6, or a sequence having at least 95% identity thereto.
30. The composition of any one of paragraphs 22 to 29, wherein the composition comprises one or more species of Firmicutes.
31. The composition of any one of paragraphs 22 to 30, wherein the composition comprises one or more species of Bacteroides.
32. The composition of any one of paragraphs 22 to 31, wherein the bacteria in the composition are in the form of spores.
33. The composition of any one of paragraphs 22 to 32, wherein the bacteria in the composition are obtained from a spore fraction derived from feces of a healthy subject.
34. The composition of any one of paragraphs 22 to 33, wherein the composition comprises one or more species of Firmicutes, and optionally one or more species of Bacteroides, as the sole bacterial species in the composition.
35. The composition of any one of paragraphs 22 to 34, wherein the bacteria in the composition are cultured.
36. A composition comprising bacteria for use in decreasing the abundance of at least one antibiotic resistance gene in a subject to whom the composition is administered.
37. The composition of paragraph 36, wherein the bacteria in the composition are obtained from a spore fraction derived from feces of a healthy subject.
38. The composition of paragraph 36, wherein the bacteria are selected for being associated with a decrease in the abundance of one or more antibiotic resistance genes in a subject.
39. The composition of any one of paragraphs 36 to 38, wherein the antibiotic resistance gene(s) is selected from Table 1.
40. The composition of any one of paragraphs 36 to 39, wherein the composition comprises at least one species selected from Table 3.
41. The composition of any one of paragraphs 36 to 40, wherein the composition does not comprise a species of Table 4.
42. A method for identifying a bacterial composition useful for decreasing the abundance of at least one antibiotic resistance gene in a subject to whom the composition is administered, the method comprising screening the composition for the presence of one or more bacterial species of Table 3, wherein detection of one or more species of Table 3 in the bacterial composition indicates the identification of a composition that can be used to decrease the abundance of at least one antibiotic resistance gene.
43. The method of paragraph 42, wherein the screening comprises detection of one or more 16S rDNA, V4, and/or V6 sequences of Table 5 in the composition, or a sequence having at least 95% identity thereto.
44. The method of paragraph 42 or 43, further comprising screening for the presence of one or more bacterial species of Table 4, and optionally the one or more species of Table 4 comprises a 16S rDNA, V4, and/or V6 sequence listed in Table 6, or a sequence having at least 95% identity thereto.
45. A method for screening a potential donor of feces for use in therapeutic methods, the method comprising testing a feces sample from the potential donor for the presence of a bacterial species from Table 3 or Table 4, wherein detection of one or more species from Table 3 indicates that the potential donor may proceed to become a donor, while detection of one or more species from Table 4 indicates that the potential donor should not become a donor.
46. A method for determining whether a subject could benefit from treatment with a ROAR composition, the method comprising determining whether a sample from the subject comprises one or more species of bacteria from Table 4, wherein detection of one or more species from Table 4 indicates that the subject may benefit from treatment with a ROAR composition.
47. A composition of any one of paragraphs 22 to 41, wherein the bacteria in the composition demonstrate the ability to decrease the abundance of one or more antibiotic resistance gene(s) in an animal model of antibiotic resistance.
48. The composition of paragraph 47, wherein the animal model comprises a mouse treated with one or more antibiotics and then colonized with one or more bacteria harboring one or more antibiotic resistance genes.
49. The composition of paragraph 48, wherein the mice are colonized with vancomycin resistant bacteria or vancomycin and carbapenem resistant bacteria.
Other embodiments are within the scope of the following claims
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/058279 | 10/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62578698 | Oct 2017 | US | |
| 62741346 | Oct 2018 | US |
