Patent Application
20190240269
The contents of the text file named “4294_0000002_st25.txt”, which was created on Oct. 22, 2018 and is 13,837 bytes in size, are hereby incorporated by reference in their entireties.
The invention relates to methods and groups of bacterial isolates relevant to bacteriotherapy. In particular, the invention relates to methods for identifying bacterial isolates suitable for bacteriotherapy, to bacterial isolates identified by such methods and to the use of such bacterial isolates in bacteriotherapy.
Clostridium difficile, an anaerobic, Gram-positive bacterium, is a major cause of antibiotic-associated diarrhea and challenges healthcare infection control measures by producing highly infectious and resistant spores. Antibiotic treatment, advanced age and hospitalization are the major risk factors for C. difficile colonization, leading to a spectrum of outcomes ranging from asymptomatic carriage, severe diarrhea, pseudomembranous colitis or even death. Current first line treatments for C. difficile disease are vancomycin or metronidazole, although in 20-35% of these cases recurrent disease (relapse or re-infection) follows the cessation of antibiotic therapy. Recurrent C. difficile disease is associated with a pathological imbalance within the resident intestinal microbial community, or “dysbiosis”, so therapies that restore a healthy microbiota are viewed as promising alternatives. Recurrent Clostridium difficile disease in humans is associated with a pathological imbalance within the resident intestinal microbiota, referred to as dysbiosis.
Fecal bacteriotherapy, the administration of homogenized feces from a healthy donor, has been investigated as an alternative therapy for recurrent C. difficile disease in humans. However, the mechanism of bacteriotherapy using fecal flora and specific probiotic mix in the feces which are of use in bacteriotherapy has so far been unclear.
In a first aspect, the invention provides a method, for the identification of bacterial isolates suitable for use in bacteriotherapy, the method comprising:
(i) preparing a suspension of material collected from a host harbouring microbiota;
(ii) addition of an activator of bacterial spores sufficient to allow growth of bacteria from spores present in the suspension;
(iii) culturing the suspension; and
(iv) identification of at least one bacterial isolate within the culture
In a second aspect, the invention provides method for preparing fecal material suitable for bacteriotherapy or a method for identification of bacterial isolates suitable for use in bacteriotherapy the method comprising preparing a suspension of fecal material followed by incubation of the suspension in a standing culture under anaerobic or aerobic conditions.
In one aspect, the invention provides a method for preparing material suitable for use in bacteriotherapy, the method comprising:
(i) preparing a suspension of fecal material;
(ii) addition of an activator of bacterial spores sufficient to allow growth of bacteria from spores present in the suspension; and
(iii) culturing the suspension.
In one aspect, the invention provides a group of bacterial isolates suitable for bacteriotherapy obtainable or identifiable according to any of the methods described above and in the rest of this application.
In another aspect, the invention provides a group of bacterial isolates obtainable or identifiable according to the method of any previous aspect of the invention for use in bacteriotherapy.
In another aspect, the invention provides the use of a group of bacterial isolates obtainable or identifiable according to the method of any previous aspect of the invention in the manufacture of a medicament for providing bacteriotherapy.
In another aspect, the invention provides cultured fecal material for use in bacteriotherapy, and/or for identification of bacterial strains suitable for use in bacteriotherapy, wherein the bacteriotherapy is to facilitate repopulation of the gut and/or prevention or treatment of diseases associated with infections or the microbiota, or diseases related thereto, and/or prevention of transmission of infection. In one embodiment the infection is a bacterial infection. In one embodiment the infection is a viral infection. In one embodiment the infection is C. difficile infection.
In another aspect, the invention provides a subset of bacteria obtainable or identifiable from fecal material for use in facilitating repopulation of the gut and/or in prevention or treatment of bacterial or viral infections, dysfunction associated with the microbiota or diseases related thereto and/or in prevention of transmission of bacterial or viral infection, wherein the subset comprises 3 to 9, optionally no more than 6, isolates of bacteria.
In another aspect, the invention provides use of 3 to 9, optionally no more 6, bacterial isolates in the preparation of a medicament to facilitate repopulation of the gut and/or in prevention or treatment of bacterial or viral infections, dysfunction associated with the microbiota or diseases related thereto and/or in prevention of transmission of bacterial or viral infection.
In another aspect, the invention provides a composition comprising or consisting essentially of a group of bacterial isolates according to any of the previous aspects. The composition is suitable for providing bacteriotherapy.
In another aspect, the invention provides a method of providing bacteriotherapy, the method comprising delivering to a human or non-human animal a group of bacterial isolates according to any of the previous aspects.
The preferred features may be combined as appropriate, as would be apparent to a skilled person, and may be combined with any of the aspects of the invention.
Embodiments of the invention will be described, by way of example, with reference to the following drawings, in which:
The invention provides methods for identification of desirable bacterial isolates suitable for bacteriotherapy. The invention also provides methods for preparing fecal material suitable for bacteriotherapy.
In one aspect the method may comprise the steps of preparing a suspension of material collected from a host harbouring microbiota, adding an activator of bacterial spores sufficient to allow growth of bacteria from spores present in the suspension and culturing the suspension. The suspension may be cultured under aerobic or anaerobic conditions. The cultured suspension may be incubated in a standing culture under aerobic or anaerobic conditions. In one embodiment, the standing culture is under aerobic conditions.
In one aspect, the method may comprise the steps of preparing a suspension of material followed by incubation of the suspension in a standing culture under aerobic or anaerobic conditions. In one embodiment, the standing culture is under aerobic conditions. An activator of bacterial spores sufficient to allow growth of bacteria from spores may be added to the suspension before the suspension is incubated in a standing culture under aerobic conditions.
The invention also provides methods for identification of bacterial strains suitable for use in bacteriotherapy comprising the steps of any of the previous aspects and comprising the additional step of identification of at least one, preferably a group of, bacterial isolates within the culture.
In one aspect, the invention provides a method for preparing material comprising desirable bacterial isolates, the method comprising preparing a suspension of material collected from a host harbouring microbiota, such as fecal material, by diluting the material in sterile PBS; plating on nutrient agar plates; adding anaerobic culture media and an activator of bacterial spores sufficient to allow growth of bacteria from spores present in the suspension and growing either aerobically or anaerobically at a suitable temperature, such as about 37° C., for 24-72 hours. Distinct colony types may be isolated and culture purified.
In one embodiment, the material being cultured is serially passaged and the material prepared after 1st or 2nd passage is selected for use in the methods according to the invention. The material collected from a host harbouring microbiota, such as healthy feces, may be passaged overnight in nutrient broth at a suitable temperature, such as about 37° C., to reduce the bacterial community complexity and to enrich for readily culturable bacteria.
In one embodiment, the methods of preparation of identification according to the invention includes incubation of the suspension in a standing culture under aerobic conditions to provide the microbes with an oxygen gradient.
In one embodiment, the culture media used during culturing of the suspension in the methods of preparation of identification according to the invention may be anaerobic culture media.
In one embodiment bacterial isolates according to any previous aspect can be identified from such prepared material by isolating genomic DNA from the distinct colonies and species-level profiling of the intestinal microbiota. The species level profiling may be sequencing specific genes, such as the 16S rRNA gene, and comparing to the GenBank and RDP databases to identify the bacterial species. Whole genome sequencing and phylogenetic analysis of intestinal bacteria can also be carried out to identify common genes between the isolates of interest. The species diversity in each sample may be measured by calculating the Shannon Diversity Index (such as described in P. D. Schloss et al. referenced in the examples section).
In one aspect the bacterial isolate comprises a DNA sequence that encodes 16S rRNA which is one of the following 6 sequences, or which has homology or identity to one of the following 6 sequences, suitably at a level of greater than 85%, such as greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% or more, across the sequence.
In addition the present invention relates to a bacterial isolate per se comprising a DNA sequence that encodes 16S rRNA having the sequence of sequence SEQ ID Nos. 13, 14 or 18 below.
In one embodiment, the individual isolates identified in the resulting cultured suspension are assessed in combinations to identify subsets of the cultured suspension for use in, or suitable for use in, bacteriotherapy. Suitability of a bacterial isolate for bacteriotherapy may be assessed by administration of the isolates or groups of isolates to the recipient and measuring a shift in the recipients' microbiota to a composition similar to that of a healthy microbiota. The shift in recipients' microbiota is linked to increase in species diversity which can be measured by calculating the Shannon Diversity Index described in the example below.
In one embodiment, bacterial isolates suitable for bacteriotherapy according to any previous aspect can be identified by in vivo assessment, for example in an animal model, such as a mouse model, or in a human challenge model with an intestinal phase. In one embodiment, bacterial isolates suitable for bacteriotherapy according to any previous aspect can be identified by assessment of the UTI tract. Such identified isolates can be sequenced as described herein to determine speciation and phylogenetic position.
In one embodiment, the bacterial isolates are initially identified according to a method described above while the actual isolates used for bacteriotherapy are previously characterized isolates of the identified isolates. The previously characterized isolates may be obtained from a biobank of previously identified bacteria assessed to be suitable for bacteriotherapy using the procedures described herein. Restoration of a healthy microbiota with bacteriotherapy is viewed as a promising alternative treatment for recurrent C. difficile disease and other forms of intestinal dysbiosis but it is not widely used because of the time required to identify a suitable donor, the risk of introducing opportunistic pathogens as well as a general patient aversion. The inventors have demonstrated that it is also possible to eradicate C. difficile disease and contagiousness using a simple mixture of defined, culturable, components of the microbiota.
The material collected from a host may be fecal material or material obtained by biopsy or sampling from the gut of the host. The material may be from the intended recipient of bacteriotherapy prior to the need for bacteriotherapy or from a healthy donor. The donor may be a spouse or a member of the bacteriotherapy recipient's immediate family. A healthy donor for the purpose of this invention is an individual not suffering from an infection, such as C. difficile infection, resulting in decreased hetrogenity of intestinal flora.
In one embodiment, the prepared suspension is administered to the recipient or used for identification of bacterial strains within 10 minutes to 2 hours after preparation. In one embodiment the prepared suspension is administered or used within about 30 minutes after preparation. In one embodiment, no more than 6 hours should have elapsed between material collection and administration to the recipient or identification of bacterial strains.
In one embodiment, the cultured suspension or subset thereof comprises a spore forming bacteria.
Addition of an activator of bacterial spores sufficient to allow growth of bacteria from spores is an embodiment of the methods according to the invention. The activator may be a cholate derivative or may comprise one or more cholate derivatives such as taurocholate and/or glycocholate. In one embodiment the activator may comprise a cholate derivative, such as taurocholate, and glycine.
An activator of bacterial spores is expected to stimulate metabolically dormant spores to begin growth. Therefore, addition of an activator of bacterial spores to the medium increases the chances of isolating such fastidious bacteria from the sample.
The invention also relates to a group of bacterial isolates suitable for bacteriotherapy obtainable or identifiable by the method of any of the previously described aspects.
The group may comprise 3, 4, 5, 6, 7, 8 or 9 bacterial isolates. In one embodiment, the group comprises 4, 5 or 6 bacterial isolates. In one embodiment, the group comprises 6 bacterial isolates. In one embodiment, the group comprises no more than 6 bacterial strains. In one embodiment, the group comprises at least 4 bacterial strains.
The group of bacterial isolates may comprise one or more of the following: Barnesiella intestinihominis, Lactobacillus reuteri, Enterococcus hirae/faecium/durans, Anaerostipes caccae/Clostridium indolis, Staphylococcus warneri/pasteuri, Adlercreutzia equolifaciens, Anaerostipes caccae/Clostridium indolis, Staphylococcus warneri/pasteuri and Barnesiella intestinihominis. In one embodiment, the group of bacterial isolates comprises Barnesiella intestinihominis, Lactobacillus reuteri, Enterococcus hirae/faecium/durans, Anaerostipes caccae/Clostridium indolis and Staphylococcus warneri/pasteuri. In one embodiment, the group of bacterial isolates comprises Staphylococcus warneri, Enterococcus hirae, Lactobacillus reuteri, Anaerostipes sp., Bacteroidetes sp. and Enterorhabdus sp. In one embodiment, the group of bacterial isolates comprises or consists 2, 3, 4, 5, 6, 7, 8 or 9, such as 5 or 6, of these isolates. In one embodiment the group of bacterial isolates comprises or consists of 4, 5 or 6 isolates and includes Enterococcus hirae, Lactobacillus reuteri, and Bacteroidetes sp. In one embodiment the group of bacterial isolates comprises or consists of 4, 5 or 6 isolates and includes members of the phyla Firmicutes and Bacteroidetes optionally with members of the phyla Actinobacteria and Proteobacteria.
In another aspect, the invention provides a composition comprising or consisting essentially of a group of bacterial isolates according to any of the previous aspects. The composition is suitable for providing bacteriotherapy.
In one aspect, the group of bacterial isolates or composition according to the invention is for use in bacteriotherapy.
In one aspect, the invention provides a subset of bacteria obtainable or identifiable from fecal material for use in bacteriotherapy wherein the subset comprises 3 to 9, such as 4 to 6, such as no more than 6, isolates of bacteria.
In one aspect, the invention relates to a method of providing bacteriotherapy, the method comprising delivering to a human or non-human animal a group of bacterial isolates or a composition according to any aspect of the invention.
In one aspect, the invention provides use of a group of bacterial isolates according to the invention in the manufacture of a medicament for providing bacteriotherapy. In one embodiment no more 6 bacterial strains are used in the preparation of the medicament.
In one aspect, the invention provides aerobically cultured fecal material for use in bacteriotherapy, and/or for identification of bacterial isolates suitable for use in bacteriotherapy.
Bacteriotherapy refers to the use of a mixture of bacteria to resolve a pathological imbalance within the microbiota of an individual. The mixture may be a mixture of live bacteria obtained from an external source (other human, animal, in vitro culture etc.). In the context of this invention bacteriotherapy may also refer to increasing species diversity of the colonic flora by introducing healthy bacterial flora into a recipient. Healthy bacterial flora refers to heterogeneous intestinal flora such as that present in an individual not suffering from a bacterial infection, such as C. difficile infection, resulting in decreased heterogeneity of intestinal flora. The bacteriotherapy may be to facilitate repopulation of the gut with healthy bacterial flora and/or to prevent or treat bacterial or viral infections, or diseases related thereto, and/or to prevent the transmission of bacterial or viral infection.
The bacteriotherapy may be for the prevention or treatment of any disorder influenced by micobiota, such as intestinal disorders. In one embodiment the bacteriotherapy may be for the treatment a C. difficile bacterial infection or prevention of transmission of C. difficile. In one embodiment the bacteriotherapy may be for the treatment C. Difficile syndromes such as recurrent diarrhea, colitis, pseudomembranous colitis. The bacteriotherapy may be for the treatment of intestinal diseases such as inflammatory bowel disease or irritable bowel syndrome. Further, bacteriotherapy can be used to treat obesity. Because the gut micriobiota in obese individuals is different from non-obese individuals, and because gut micriobiota influences energy metabolism, displacing the gut micriobiota of an obese individual with the guy micriobiota of a non-obese individual. In one embodiment the bacteriotherapy may be for restoring intestinal flora disrupted by antibiotic treatment.
The bacterial isolates, medicaments or compositions according to the invention may be delivered by means of a gastro-resistant capsule (e.g., acid-bio resistant to reach the intestinal tract, having a sterile outside) an enteric tube, duodenal tube, nasogastric tube or colonoscope. Capsules may be prepared by techniques such as microencapsulation described in U.S. Pat. No. 5,733,568.
Treatments or specific processes can be applied to improve the stability or viability of the bacterial isolates in the composition. The bacterial isolates can be applied in a dry form or in a wet from. The bacterial isolates may be lyophilized.
The compositions may comprise a dose demonstrated to have a physiological effect, such as between 104 and 1011 colony forming units (CFU) per g of the dry composition. In one embodiment, the composition comprises between 106 and 5×1011 CFU/g.
The bacterial isolates or medicaments according to the invention may be provided at a dose of 1-50 g/day, such as 5, 10, 15, 20 or 25 g/day.
Bacteriothepary according to the invention may be combined with other treatments. The other treatment can include antibiotic treatment, such as with antimicrobials including metronidazole, vancomycin or rifamycin, and treatment with immunoglobulins. In an example, bacteriotherapy to treat C. difficile or one or more other diseases or afflictions of the digestive tract can be provided using a combination of antibiotics and/or antacid and re-population of a healthy or desired bacterial flora.
In one aspect, a kit of parts can be created to aid in the methods of the invention. The donation kit can include equipment for collection of material from the host. Because much of gut micriobiota is anaerobic, many organisms can die with exposure to air. In an example, the kit can include materials to ship the collected material without harming the samples (e.g., quick freeze, dry ice, etc.). The kit may include the processed material or treatment in a sterile container, such as a nasogastric (NG) tube, a vial (e.g., for use with a retention enema), a gastro-resistant capsule (e.g., acid-bio resistant to reach the intestinal tract, having a sterile outside), etc.
Recipients of bacteriotherapy according to the invention may be humans or non-human animals.
It will be understood that particular aspects and embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In one aspect such open ended terms also comprise within their scope a restricted or closed definition, for example such as “consisting essentially of”, or “consisting of”. The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All documents referred to herein are incorporated by reference to the fullest extent permissible. Any element of a disclosure is explicitly contemplated in combination with any other element of a disclosure, unless otherwise apparent from the context of the application.
The present invention is further described by reference to the following examples, not limiting upon the present invention.
Rational Design of a Simple, Defined Bacteriotherapy that Cures Clostridium difficile Disease
Recurrent Clostridium difficile disease in humans is associated with a pathological imbalance within the resident intestinal microbiota, referred to as dysbiosis. We show that infection of mice with epidemic C. difficile (genotype 027/BI) resulted in chronic intestinal disease that was associated with persistent dysbiosis and a highly contagious state. Epidemic C. difficile 027/BI infection was refractory to vancomycin treatment, resulting in recurrent disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly eradicated C. difficile by restoring a diverse, healthy microbiota leading to the resolution of disease and contagiousness. We used this model to design a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and eradicate C. difficile 027/BI from infected mice as effectively as whole fecal transplants. Thus, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities and to refine bacteriotherapy-based treatments for C. difficile disease and potentially other forms of intestinal dysbiosis.
During the past decade a distinct genetic variant of C. difficile, genotyped as PCR-ribotype 027 or REA group BI, emerged and caused healthcare-associated epidemics within North America, Europe, Australia and beyond (7, 8). Epidemic C. difficile 027/BI is associated with high-level toxin production (9) (
Vancomycin treatment of C. difficile 027/BI persistent supershedders rapidly suppressed C. difficile excretion to below the culture detection limit (
Fecal bacteriotherapy, the administration of homogenized feces from a healthy donor, has been investigated as an alternative therapy for recurrent C. difficile disease in humans (6, 14). Therefore, we tested the ability of fecal bacteriotherapy to suppress the C. difficile 027/BI supershedder state in mice. A single oral treatment of C. difficile 027/BI-7 supershedding mice with homogenized feces from a healthy donor rapidly (4-7 days) and robustly (23 of 25 attempts) suppressed C. difficile shedding levels to below culture detection limits and, in contrast to vancomycin therapy, this lasted for months (
We hypothesized that the persistent supershedder state caused by C. difficile 027/BI-7 is linked to intestinal dysbiosis, which is resolved by health-associated bacteria present within fecal transplants. Therefore, we performed species-level profiling of the intestinal microbiota of mice (based on the 16S rRNA gene) and demonstrated that distinct microbiota profiles are indeed associated with either “healthy/naïve” mice, “persistent supershedders” or mice undergoing “clindamycin treatment” (
Next we monitored changes in the supershedders' microbiota after fecal bacteriotherapy. Suppression of C. difficile shedding levels was associated with a shift in the recipients' supershedder microbiota to a composition similar to that of a healthy microbiota (
Next, we cultured a diverse collection of 18 bacterial species from the Passage 1 fecal derivative, including representatives of the four phyla that constitute the majority of the mammalian intestinal microbiota (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria; Table 2). Then we performed a series of reductive analysis experiments in persistent supershedder mice testing different combinations of bacteria while maximizing the phylogenetic diversity in each mixture (mixtures summarized in table 2). Ultimately we identified a simple, defined mixture of six bacteria that effectively and reproducibly (20/20 mice) suppressed the C. difficile 027/BI supershedder state (“MixB”;
Cholate derivatives (i.e. Taurocholate and glycocholate) stimulate metabolically dormant spores to begin growth. Therefore addition of cholate-derivatives to the medium increases the chances of isolating such fastidious bacteria from the sample. This is how we identified one of the six MixB bacteria (Anaerostipes).
Significantly, and in contrast to the results with MixB, treatment of C. difficile 027/BI supershedder mice with further subdivisions of this bacterial mixture, including the MixB bacteria administered individually, or mixtures containing six or seven other cultured bacterial strains had a negligible impact on the supershedder state (
To gain insight into the genetic composition and fully define the identity of the six bacterial strains present in MixB (Table 2) we sequenced their genomes (and their closest equivalent human-derived species) and performed a phylogenetic comparison to reference intestinal bacterial genomes representative of the mammalian microbiota (
In conclusion, we demonstrate that epidemic C. difficile 027/BI can out compete health-associated bacteria to enhance the contagious period of the host, increasing its likelihood of infecting a susceptible host. Restoration of a healthy microbiota with bacteriotherapy is viewed as a promising alternative treatment for recurrent C. difficile disease and other forms of intestinal dysbiosis (6, 14), but it is not widely used because of the time required to identify a suitable donor, the risk of introducing opportunistic pathogens as well as a general patient aversion (18). For the first time we demonstrate that it is also possible to eradicate C. difficile disease and contagiousness using a simple mixture of defined, culturable, components of the microbiota. Thus, our results open the way to rationally harness the therapeutic potential of health-associated microbial communities to treat recurrent C. difficile disease and transmission in humans, and potentially other forms of disease-associated dysbiosis.
Methods
Bacterial Culturing.
C. difficile strains BI-7 (genotype 027/BI; clindamycinR, thiamphenicolR, erythromycinS), M68 (genotype 017/CF; clindamycinR, thiamphenicolS, erythromycinS) and 630 (genotype 012/R; clindamycinR, thiamphenicolS, erythromycinR) have been described (1, 2). Culturing of C. difficile for infections and from feces was described previously (1). To isolate the intestinal bacteria from mouse feces or passaged fecal derivatives, the samples were serially diluted in sterile PBS, plated on a panel of nutrient agar plates; Luria Bertani, Brain Heart Infusion, Man Rogosa Sharpe, Fastidious anaerobic media, Columbia base media supplemented with 10% defribrinated horse blood, Wilkins-Chalgren anaerobic media (all media from Becton, Dickinson, Oxford, UK) and grown either aerobically or anaerobically at 37 C for 24-72 hours. Distinct colony types were isolated, culture purified and genomic DNA was isolated to sequence the 16S rRNA gene using broad range primers as described in the microbiota section below. 16S rRNA gene sequences were compared to the GenBank and RDP databases to identify the bacterial species.
TcdA ELISA.
C. difficile cultures were grown in Wilson's broth (1) with shaking for 30 h, pelleted by centrifugation and supernatant was removed for TcdA quantification. Microtitre plates (96 well) were coated with capture antibody by adding 50 μl/well of a 2 μg/ml solution of anti-TcdA (TGCBiomics GmbH, Mainz, Germany) in PBS, and incubating overnight at 4° C. Plates were then washed three times in 0.05% Tween20 in PBS (PBS-T) and blocked with 200 μl 1% BSA (bovine serum albumin) in PBS for 2 h at room temperature. Purified TcdA from C. difficile strain VPI10463 (TGCBiomics GmbH, Mainz, Germany) was diluted in 1% BSA-PBS (50 μl/well) and used to construct a standard curve. Culture filtrates were diluted as above in order to generate readings within the linear range of the standard curve. Plates were then incubated at room temperature for 2 h, followed by washing in PBS-T as above. The detection antibody (rabbit anti-Clostridium difficile toxin A; antibodies-online GmbH, Aachen, Germany) was diluted 1:5000 in 1% BSA-PBS, added to wells (50 μl/well) and incubated for 2 h at room temperature. After washing, polyclonal swine anti-rabbit IgG conjugated to horseradish peroxidase (Dako, Cambridgeshire, UK) was diluted 1:1000 in 1% BSA-PBS, added to the wells (50 μl/well) and incubated for 2 h at room temperature. Finally, plates were washed and 100 μl 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma Aldrich, Dorset, UK) substrate was added for 30 min at room temperature in the dark. 50 μl 0.5 M H2504 was added to stop the reaction. Absorbance was then measured at 450 nm on a FLUOStar Omega (BMG Labtech, Bucks, UK).
Mouse Infections.
Female mice between 5-9 weeks of age and from the genetic backgrounds C57BL/6, C57BL/6 p40−/−, C3H/HeN and C3H/HeJ were routinely used. Mice to be used as C. difficile spore donors were infected with 105 C. difficile cells via oral gavage and immediately clindamycin (250 mg/L; Apollo Scientific Ltd, Chesire, UK) was added to the drinking water for 1 week to induce high-level spore excretion. To infect experimental mice, one petri dish of contaminated bedding was removed from spore donor cages, placed into recipient mice cages and clindamycin (250 mg/L) was added to the drinking water for 1 week to induce the supershedder phenotype. To infect germ-free C3H/HeN mice, the feces of supershedder mice was collected, diluted in serial PBS and inoculated into mice via oral gavage. To suppress infection, vancomycin (300 mg/L; Sigma Aldrich, York, UK) was added to the drinking water for 10 days. To assess impact of infection, mice were sacrificed at indicated times and cecal tissue was aseptically collected and fixed for pathology as described (1), or fixed for RNA extractions by immersing samples in RNA-later (Applied Biosystems, Warrington, UK).
Bacteriotherapy Treatment.
To prepare input for bacteriotherapy, 1 gram of fresh feces was collected from 5 naïve mice, homogenized in 5 ml of sterile PBS and centrifuged for 30 seconds at 14,000 RPM to pellet the particulate matter. The supernatant slurry was collected and 200 μl was gavaged into each mouse within 30 minutes of excretion. To create the defined bacterial mixtures, individual bacteria were grown in Wilkins-Chalgren broth (Lactobacillus in Man Rogosa Sharpe broth) for 48-72 hours under anaerobic conditions at 37 C. Bacteria were harvested by centrifugation and re-suspending the pellet in 2 mls of sterile, pre-reduced PBS. Approximately 1010 of each bacterium was gavaged into each mouse in a 200 μl volume. To passage healthy feces, two fecal pellets (˜50 mg) were collected aseptically and immediately placed into 20 ml of Wilkins-Chalgren Anaerobic broth or Luria broth that was pre-warmed to 37 C under aerobic or anaerobic conditions. Fecal pellets were physically disrupted within the broth using a sterile pipette tip and subsequently incubated standing for 16 hours. For serial passage, 200 μl of the fecal derivative was inoculated into fresh broth and grown as described. For inoculations, the 20 ml cultures were pelleted and then resuspended into 2 ml of sterile PBS pre-warmed to 37 C under aerobic or anaerobic conditions. Based on visual counts, approximately 4×108 (anaerobic passage) and 8×108 (aerobic passage) bacteria were gavaged into each mouse in a 200 μl volume.
Microarrays.
RNA purification from cecal mucosal tissue was performed using a Qiagen RNeasy mini kit (Qiagen, Austin, Tex., USA) according to the manufacturer's protocol. Quality control and quantification were performed using Bioanalyzer 2100 (Agilent Technologies, Palo Alto, Calif., USA) and Nanodrop ND100 (Nanodrop Technologies, Wilminton, Del.). RNA samples were then amplified and labelled using the Illumina TotalPrep 96 kit (Ambion, Austin, Tex., USA) and hybridized onto Illumina™ Mouse WG-6-V2 Beadchips (Illumina, San Diego, Calif., USA). The chips were scanned on an Illumina BeadArray Reader and raw intensities were extracted using Illumina BeadStudio Gene Expression Module.
Normalization and analysis of the microarrays were performed using GeneSpring X software (Agilent Technologies, Berkshire, UK). Normalization procedures utilized were quantile normalization and median of all samples baseline correction. For each comparison, differentially expressed genes were defined as having a fold change 2 and a FDR (false discovery rate) corrected p-value 0.05. Adjusted p-values were calculated using the Benjamini and Hochberg method (3).
RT-PCR.
Quantitative expression analysis was performed by real-time TaqMan RT-PCR on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Warrington, UK) as described previously (4). Expression of IL-6, iNOS and Ly6G was normalized to Gapdh mRNA. TaqMan primers and probes were designed to span exon junctions or to lie in different exons to prevent amplification of genomic DNA, as described (4). Primer and probe sequences are shown in Table 3. Probes were labelled with the reporter dye FAM at the 5′- and the quencher dye TAMRA at the 3′-end.
Transmission Experiments.
Protocols to test the contagiousness of infected donors (supershedders or carriers) have been described (1). To compare the contagiousness of different C. difficile strains mice infected with either C. difficile 012 (strain 630), 017 (strain M68) and 027 (strain BI-7) (immediately after cessation of 7 days of clindamycin treatment) were co-housed with 7 naïve recipient mice for 30 days. Experiments were repeated for a total of 14 naïve mice. To determine if recipient mice were infected with C. difficile they were individually placed (aseptically) in sterile cages for 3 days and given clindamycin in their drinking water for 4 days (1). Afterwards, feces was collected from individual mice and C. difficile enumerated by standard methods (1). Antibiotic resistance profiles were used to determine which C. difficile strain had infected mice.
Analysis of Microbiota.
Fecal DNA extraction, clone library construction and sequencing were carried out as described previously (1). Sequences were aligned using the RDP aligner (5) and these alignments were manually curated in the ARB package (6) before further analysis. Otherwise, sequences were checked and classified as described previously (7). In total 19,991 sequences were generated and these were deposited in GenBank (accession numbers JF241944-JF260864 and HE605382-HE608150).
The species diversity in each sample was measured by calculating the Shannon Diversity Index, which takes into account both species richness and relative proportional abundance (evenness), using the mothur software package (8). Rarefaction curves and Chao1 estimates of total bacterial diversity were also calculated in mothur (8).
Cluster dendrograms and PCA plots were based on a master alignment, which was built using the RDP aligner and subjected to manual curation. Using this alignment a distance matrix, with Felsenstein correction, was created using ARB. The distance matrix was then used as an input for DOTUR (9) using a 98% identity cut-off under the default furthest-neighbor setting. Sequences with >98% phylogenetic similarity were regarded as belonging to the same OTU. These OTUs were then used to calculate cluster dendrograms, using the Bray Curtis calculator, in the mothur package (8). 336 OTUs (12,308 clones) contributed to this analysis. Cluster dendrograms, with added bar charts showing the microbial composition of each sample and Shannon Diversity Indices, were visualized using the iTOL web package (10). For the PCA plot OTUs were generated as above but with a 97% identity cut-off. PCA decomposition was performed on the (symmetric) matrix of pairwise sample similarity, where the similarity metric was based on the sum of absolute differences in OTU frequency. 344 OTUs (16,154 clones) contributed to the analysis, which was insensitive to the removal of low frequency OTUs.
To determine the SCFA profile, the cecal contents from 5 mice per group were pooled and then resuspended in sterile PBS at a concentration of 500 mg/ml, homogenized and centrifuged at 14,000 rpm for 10 minutes. Supernatant was collected, acidified and following conversion to t-butyldimethylsilyl derivatives were analyzed by gas chromatography (11).
Whole Genome Sequencing and Phylogenetic Analysis of Intestinal Bacteria.
We sequenced the genomes (and their closest equivalent human-derived species) using the MiSeq platform, and performed de novo assembly using Velvet {(12) and gene prediction using GLIMMER3 (13). We then identified the genes that were in common between the 6 MixB species, and reference intestinal bacterial genomes sourced from the MetaHIT project, the HGMI project, and the Human Microbiome Project (Tables 4 and 5). 44 Common genes were identified using TBLASTN (14) searches against the complete dataset of the reference and assembled genomes for 80 bacteria (Table 5). Although the “true” core genome amongst these samples may be higher—we were limited by the fact that in several cases only draft assemblies were available, and so some genes which may have been expected to be present in the “core” group, were in fact not present, due to their absence in one or more of the draft genome sequences used. A gene was classified as being ‘present’ if it had a minimum percent amino acid identity across the entire gene of 30% compared to the reference. The reference genes used for querying were taken from the strain of Staphylococcus warneri taken from MixB. The common genes so identified were manually checked, translated, extracted, and concatenated together. We then used FastTree 2.1 (15), with its default settings (BLOSUM45 and the Jones-Taylor-Thorton CAT model, with 20 rate categories), to generate a maximum likelihood phylogeny from the concatenated protein sequence, in order to place the bacteria into their correct context and to distinguish species.
C. difficile
C. diff
C. diff
Bacteroides acidifaciens
intestinihominis [87%]
Lactobacillus
taiwanensis/gasseri/johnsonii
Flavonifractor plautii
Bifidobacterium pseudolongum subsp.
globosum/pseudolongum
Escherichia coli
intestinihominis [87%]
Lactobacillus reuteri
Lactobacillus reuteri
Enterococcus hirae/faecium/durans
Enterococcus hirae
Anaerostipes caccae/Clostridium
Anaerostipes novel species
indolis
Staphylococcus warneri/pasteuri
Staphylococcus warneri
Enterorhabdus novel species
equolifaciens (97%)
Parabacteroides distasonis
intestinihominis [87%]
Lactobacillus murinus/animalis
Enterococcus faecalis
Blautia producta
Propionibacterium acnes
Acinetobacter lwoffii/baumannii
Enterorhabdus novel species
equolifaciens (97%)
Anaerostipes caccae/Clostridium
Anaerostipes novel species
indolis
Staphylococcus warneri/pasteuri
Staphylococcus warneri
intestinihominis [87%]
Lactobacillus reuteri
Lactobacillus reuteri
Enterococcus hirae/faecium/durans
Enterococcus hirae
Proteus mirabilis
Escherichia coli
Citrobacter rodentium
Enterobacter cloacae
Klebsiella pneumoniae
Alistipes shahii
Parabacteroides distasonis
Bacteroides fragilis
Bacteroides thetaiotaomicron
Bacteroides xylanisolvens
Bacteroides vulgatus
Bacteroides dorei
Bifidobacterium
pseudocatenulatum
Bifidobacterium bifidum
Bifidobacterium breve
Bifidobacterium longum
Atopobium parvulum
Enterorhabdus sp. nov.
Enterorhabdus mucosicola
Eggerthella lenta
Gordonibacter pamelaeae
Bacillus subtilis
Staphylococcus aureus
Staphylococcus haemolyticus
Staphylococcus epidermidis
Staphylococcus pasteuri
Staphylococcus warneri
Staphylococcus warneri
Listeria monocytogenes
Lactobacillus casei
Lactobacillus rhamnosus
Lactobacillus fermentum
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus brevis
Lactobacillus plantarum
Streptococcus thermophilus
Streptococcus gordonii
Enterococcus faecalis
Enterococcus durans
Enterococcus faecium
Enterococcus hirae
Enterococcus hirae
Enterococcus hirae
Enterococcus casseliflavus
Enterococcus gallinarum
Clostridium difficile
Clostridium bartletti
Clostridium botulinum
Clostridium cellulovorans
Clostridium acetobutylicum
Flavonifractor plautii
Clostridium leptum
Ruminococcus bromii
Eubacterium siraeum
Subdoligranulum variabile
Faecalibacterium prausnitzii
Eubacterium hallii
Coprococcus catus
Anerostipes sp. nov.
Anaerostipes caccae
Clostridium hathewayi
Clostridium clostridioforme
Clostridium bolteae
Clostridium aldenense
Clostridium citroniae
Clostridium indolis
Clostridium saccharolyticum
Clostridium symbiosum
Ruminococcus obeum
Blautia producta
Blautia producta
Blautia coccoides
Ruminococcus torques
Butyrivibrio fibrisolvens
Eubacterium rectale
Roseburia intestinalis
| Number | Date | Country | Kind |
|---|---|---|---|
| 1208845.6 | May 2012 | GB | national |
| 1211961.6 | Jul 2012 | GB | national |
This application is a divisional of U.S. application Ser. No. 14/402,033, which was filed on Nov. 18, 2014, which is U.S. national stage application of International Application No. PCT/GB2013/05198, which was filed on May 20, 2013 with the title “METHODS AND GROUPS,” which claims priority to GB 1211961.6 filed Jul. 5, 2012 and GB 1208845.6 filed May 18, 2012, each of which is incorporated by reference in their entireties for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14402033 | Nov 2014 | US |
| Child | 16167009 | US |
