WO2022112526A1 - Procédé - Google Patents

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Publication number
WO2022112526A1
WO2022112526A1 PCT/EP2021/083235 EP2021083235W WO2022112526A1 WO 2022112526 A1 WO2022112526 A1 WO 2022112526A1 EP 2021083235 W EP2021083235 W EP 2021083235W WO 2022112526 A1 WO2022112526 A1 WO 2022112526A1
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Prior art keywords
bacteria
composition
phage
therapeutic
ncimb
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PCT/EP2021/083235
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English (en)
Inventor
Christophe Rene Leonard CARITE
Antonio Fernández MEDARDE
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4D Pharma Leon, S.L.U.
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Application filed by 4D Pharma Leon, S.L.U. filed Critical 4D Pharma Leon, S.L.U.
Priority to AU2021386691A priority Critical patent/AU2021386691A1/en
Priority to KR1020237021517A priority patent/KR20230112700A/ko
Priority to CA3200126A priority patent/CA3200126A1/fr
Publication of WO2022112526A1 publication Critical patent/WO2022112526A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to processes for analysing a composition comprising therapeutic bacteria.
  • therapeutic bacteria are defined as live bacteria which are used for the prevention, treatment or cure of a disease or condition in a mammal, preferably a human.
  • the invention also relates to phages for use in such a process as well as a kit for conducting such a process.
  • the mammalian intestine is thought to be sterile in utero but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, there is a dynamic period of microbial colonization and succession which provides the gut microbiota.
  • the composition of the gut microbiota is influenced by factors such as delivery mode (e.g. via caesarean section or natural birth), environment, diet and host genotype, particularly during early life. Subsequently, the gut microbiota stabilizes and becomes adult-like.
  • the human gut microbiota contains between about 500 and 1,000 different phylotypes belonging essentially to two major bacterial phyla, the Bacteroidetes and the Firmicutes.
  • the successful symbiotic relationships arising from microbial colonization of the gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions.
  • the enhanced metabolic activities of the colonized gut ensure that otherwise- indigestible dietary components are degraded, releasing by-products which provide an important nutrient source for the host.
  • EP-A-1 280 541 discloses the use of hydrogenotrophic organisms in the treatment of a range of conditions, including human irritable bowel syndrome
  • EP-A-1448995 discloses the use of Bacteroides thetaiotamicron in the treatment of inflammatory diseases
  • EP-A-2 763 685 discloses the use of Roseburia hominis as an immunoregulatory agent
  • WO 2017/085520 discloses the use of Enterococcus gallinarum as an anti-cancer therapy
  • EP- A-3206700 discloses the use of Bifidobacterium in the treatment of a range of autoimmune / inflammatory conditions, including severe asthma.
  • LBP Live Biotherapeutic Products
  • LBP While, superficially, keeping the size of the populations of unwanted organisms small or very small may appear to be attractive, in practice this is actually problematic.
  • LBP must pass through the rigorous assessments of healthcare regulators.
  • the European Pharmacopeia Chopent 2.6.38
  • LBP comprise less than 1000 colony forming units (CFU) of bacteria other than the therapeutic bacteria per gram of drug product.
  • CFU colony forming units
  • LBP developers are able to demonstrate conclusively that these requirements have been complied with.
  • the unwanted organisms are present at low levels in a finished product owing to their growth being suppressed by the conditions to which they are exposed during the fermentation / manufacturing of that product, when delivered to the Gl tract (as the majority of LBP are), the conditions in the gut may permit the unwanted organisms to flourish and their population exponentially to increase.
  • the unwanted organisms may produce metabolites or express compounds whose presence in the finished products or Gl tract may be undesirable or unacceptable from a regulatory perspective. Even where the population sizes of the unwanted organisms may be low, the metabolites / products they produce may accumulate to unacceptable levels.
  • WO 2014/153194 concerns, inter alia, enriching for a contaminant in a composition. Multiple alternatives and speculative assays are suggested. However, no worked example of any of these assays is provided, nor does this disclosure enable a skilled practitioner to selectively enrich for contaminants in an LBP.
  • the present invention provides new and improved analytical processes which allow for the fast and accurate detection of unwanted microorganisms even where the contaminants are present at low levels or where the nature of the contaminants is unknown.
  • step (b) analysing the cultured composition of step (a) to determine whether an unwanted organism was present in the composition.
  • the inventors have found that selective inhibition of the growth of the therapeutic bacteria advantageously enables and facilitates analysis of any unwanted organisms in the microbial composition.
  • the growth of the therapeutic bacteria only is selectively inhibited.
  • selectively inhibited preferably refers to embodiments where only the growth of the therapeutic bacteria is inhibited.
  • the growth of one or more contaminants may also be inhibited.
  • the growth of therapeutic bacteria may be inhibited at least 5 times more than the growth of one or more unwanted organisms is inhibited, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 times more than the growth of one or more unwanted organisms.
  • the growth of the unwanted organism is not inhibited.
  • the growth of any unwanted organism is not inhibited.
  • step (a) selective inhibition it is meant that the growth of the therapeutic bacteria is preferentially inhibited in culturing step (a) compared to the growth inhibition of the unwanted organism.
  • the growth inhibition of the therapeutic bacteria in step (a) may be at least two fold, at least three fold, at least four fold, at least five fold, at least ten fold, at least twenty fold, at least fifty fold higher than the growth inhibition of the unwanted organism. As a skilled person will appreciate, this is to be assessed relative to the growth rate before the selective growth conditions.
  • the growth rate for any unwanted organism in step (a) is at least two fold higher at least three fold higher, at least four fold higher, at least five fold higher, at least ten fold higher, at least twenty fold higher, or at least fifty fold higher than the rate at which the therapeutic bacteria grow.
  • the reference to "unwanted organism” in this context can refer to a single unwanted organism (e.g. a single bacterial species) or it can refer to a multitude of unwanted organisms.
  • the growth inhibition of the therapeutic bacteria may be inhibited relative to all unwanted organisms in the composition or relative to only one of the unwanted organisms.
  • the process may include the step of identifying one or more unwanted anaerobic or aerobic bacteria.
  • the bacteria may be pathogenic bacteria, such as bacteria from the Clostridia and Salmonella genera, for example bacteria from the Clostridium difficile, Clostridium tetani, or Salmonella enterica species, or bacteria from the Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli species, or the like, or mixtures of such unwanted organisms.
  • the expression "unwanted organism” refers to any organism in the composition other than the therapeutic bacteria.
  • the unwanted organism is an unwanted bacterium or unwanted bacteria.
  • An unwanted organism may also be any bacteria that is a mammalian pathogen, such as a human pathogen.
  • the expression "unwanted organism” is interchangeable with "contaminant” or "contaminating organism” in the context of the present specification.
  • the at least one unwanted organism is not E. coli or S. aureus. In most preferred embodiments, the unwanted organism has not been deliberately added to the composition.
  • the ratio of the number of cells of the therapeutic bacteria to the number of cells of the one or more unwanted organisms in the microbial composition prior to the culturing step may be 1,000 : 1 or higher, 10,000 : 1 or higher, 100,000 : 1 or higher, 1,000,000 : 1 or higher, 10,000,000 : 1 or higher, 100,000,000 : 1 or higher, 1,000,000,000 : 1 or higher, 10,000,000,000 : 1 or higher.
  • Selective inhibition of the growth of the therapeutic bacteria may be achieved by selecting the pH, temperature and/or ionic strength of the culture medium.
  • the relationship between pH and temperature and bacterial growth is well understood and methods for controlling pH and/or temperature to control bacterial growth are well known (see for example "Modelling the combined effect of temperature and pH on the rate coefficient for bacterial growth", Davey, International Journal of Food Microbiology (1994) 23 (3-4), pp 295-303).
  • controlling the concentration of salts in growth media is known to inhibit the growth of certain bacteria, thus selectively inhibiting growth of sensitive bacteria (see for example "Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology", Bonnet et al., New Microbes New Infect (2020) 34: 100622).
  • step (a) in the methods of the invention is performed at a pH ⁇ 7, for example ⁇ 6.5, ⁇ 6, ⁇ 5.5, ⁇ 5, ⁇ 4.5, ⁇ 4, ⁇ 3.5, ⁇ 3, or ⁇ 2.5, for example between 2.5-6.5, 2.5-6, 2.5-5.5, 2.5-5, 2.5-4.5, 2.5-4, 2.5-3.5, 2.5-3, 3-6.5, 3-6, 3-5.5, 3-5, 3-4.5, 3-4, 3-3.5, 4-6.5, 4-6, 4-5.5, 4-5, 4-4.5, 5-6.5, 5-6, 5-5.5, or 6-6.5.
  • step (a) may be conducted at a pH of > 8, for example >
  • the pH may be between 8.5-11.5, 9-11.5, 9.5-
  • the temperature in step (a) may be selected to be ⁇ 10°C, ⁇ 9°C, ⁇ 8°C, ⁇ 7°C, ⁇ 6°C, ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C. Where a low growth temperature is chosen it will be evident to a skilled person that the conditions need to be chosen such that the growth medium in which step (a) is conducted does not freeze.
  • the temperature may be chosen to be in a range selected from 0.5°C-10°C, 0.5°C-9°C, 0.5°C-8°C, 0.5°C-7°C, 0.5°C-6°C, 0.5°C- 5°C, 0.5°C-4°C, 0.5°C-3°C, 0.5°C-2°C, 0.5°C-1°C, 1°C-10°C, 1°C-9°C, 1°C-8°C, 1°C-7°C, 1°C-6°C, 1°C-5°C, 1°C-4°C, 1°C-3°C, 1°C-2°C, 2°C-10°C, 2°C-9°C, 2°C-8°C, 2°C-7°C, 2°C-6°C, 2°C-5°C, 2°C-4°C, 2°C-3°C, 3°C- 10°C, 3°C-9°C, 3°C-8°C, 3°C-7°C, 3°C-6°C, 3°C-9°
  • the temperature in step (a) may be selected to be > 50°C, > 55°C, > 60°C, > 65°C > 70°C, > 75°C, > 80°C, > 85°C, or > 90°C. It will be evident to a skilled person that it is preferable to avoid culturing beyond the boiling point.
  • the temperature may be chosen to be in a range selected from 50°C-99°C, 55°C-99°C, 60°C-99°C, 65°C-99°C, 70°C-99°C, 75°C-99°C, 80°C-99°C, 85°C-99°C, 90°C-99°C, 50°C-95°C, 55°C-95°C, 60°C-95°C, 65°C-95°C, 70°C-95°C, 75°C-95°C, 80°C-95°C, 85°C-95°C, 90°C-95°C, 50°C-90°C, 55°C-90°C, 60°C-90°C, 65°C-90°C, 70°C-90°C, 75°C-90°C, 80°C-90°C, 85°C-90°C, 50°C-85°C, 55°C-85°C, 60°C-85°C, 65°C-85°C, 65
  • enterococcal bacteria are known to survive in temperatures between 5 and 65°C, and in environments with a pH between 4.5 and 10.0, and can survive in high salt concentrations (see “The Ecology, Epidemiology, and Virulence of Enterococcus", Fisher and Phillips, Microbiology (2009) 155 (6), pp 1749-1757); when the therapeutic bacteria is enterococcal, the selective inhibition may therefore be achieved by lowering the temperature to below 5°C and/or lowering the pH to below 4.5.
  • lactic acid bacteria e.g.
  • lactobacilli are intolerant to very high pH (see for example "Diversity and Mechanisms of Alkali Tolerance in Lactobacilli", Sawatari and Yokota, Appl Environ Microbiol (2007) 73(12) pp 3909-3915); as such, when the therapeutic bacteria is a lactic acid bacteria, such as a species of lactobacilli, the selective inhibition may be achieved by raising the pH to 8.0 or higher.
  • optimum growth conditions for other therapeutic bacteria are also known in the art.
  • optimum growth conditions for Blautia hydrogenotrophica are at a pH range of 6.0-7.0, and a temperature of 35-37 ° C. (Bernalier et al. Archives of Microbiology, 1996. Volume 166(3). p. 176-83) and so a process of the invention can be practised using a pH of ⁇ 6.0 or >7.0 and/or a temperature of ⁇ 35 ° C or >37 ° C.
  • Inhibitory agents are also known in the art.
  • optimum growth conditions for Blautia hydrogenotrophica are at a pH range of 6.0-7.0, and a temperature of 35-37 ° C.
  • selective inhibition may be achieved by addition to the culture medium of an agent or agents which selectively inhibit(s) the growth of the therapeutic bacteria.
  • the agent may prevent the therapeutic bacteria from multiplying or may reduce multiplication, for example by at least 80%, 85%, 90%, 95% 96%, 97%, 98%, 99%, 99.5% or 99.9%.
  • the agent actively kills at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or (most preferably) 100% of the therapeutic bacteria.
  • agents include bacteriophages (usually known as "phages"), lysozymes and antibiotics.
  • phages are especially preferred because it is possible to select phages which are specific for the therapeutic bacteria and which therefore will not inhibit the growth of any unwanted organisms, even if they are also bacteria. If specific phage(s) are used, the therapeutic bacteria will be killed off and the unwanted organisms will be able to grow.
  • the data presented in the examples demonstrate the ability of phages to rapidly inactivate high concentrations of therapeutic bacteria, thus facilitating the convenient and straightforward identification of any unwanted organisms (in those examples, bacterial organisms).
  • the use of phages, as compared to lysozyme, is also advantageous as this avoids the additional process steps of isolating and purifying the lysozyme.
  • the agent is not LysA2, or is not an isolated phage lytic enzyme, or is not a recombinant phage lytic enzyme.
  • a phage lytic enzyme is any enzyme deriving from a bacteriophage that is capable of inducing lysis in a bacterium.
  • the agent is not an isolated phage enzyme or protein.
  • the agent is not a recombinant phage enzyme or protein.
  • the selective inhibition is not achieved by addition of an isolated and/or recombinant phage lytic enzyme to the culture medium.
  • Suitable phages for use in the process of the present invention include naturally occurring phages, for example any phage for which the genome is publicly available (for example via the European Nucleotide Archive (ENA) database, accessible at: https://www.ebi.ac.uk/ena/browser/home; or via NCBI GenBank accessible at: https://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi).
  • the phage may be a naturally occurring phage or non-naturally occurring phage, for example a genetically engineered or genetically modified phage.
  • a known phage may be identified by using a microbe-phage database (for example "MVP: a microbe-phage interaction database", Gao et al., Nucleic Acids Res (2016) 46 (Database Issue): D700- D707; accessible at: http://mvp.medgenius.info/home; or the Virus-Host Database accessible here: https://www.genome.ip/virushostdb/).
  • MVP microbe-phage interaction database
  • the specificity of a phage may be determined using any known techniques (see for example "More Is Better: Selecting for Broad Host Range Bacteriophages", Ross et al., Front Microbiol (2016) 7: 1352); for example, one may observe whether a phage is able to form plaques on a particular species or strain of host bacteria, or one may use spot testing to more rapidly determine which bacteria are susceptible to the phage. Ross et al. also describe techniques for isolating phages from natural environments.
  • a phage may be genetically engineered to be more specific to a particular strain of bacteria compared to the wildtype strain, or may be engineered to be less selective compared to the wildtype strain such that it may infect any strain of a certain species.
  • Techniques for genetically modifying and engineering phages are known in the art (see for example “Engineering Bacteriophages as Versatile Biologies", Kilcher and Loessner, Trends in Microbiology (2019) 27(4) pp 355-367; “Approaches to optimize therapeutic bacteriophage and bacteriophage-derived products to combat bacterial infections", Reuter and Kruger, Virus Genes (2020) 56(2) pp 136-149; “Creation of synthetic bacterial viruses", Ando, Nihon Saikingaku Zasshi (2016) 73(4) pp 201-210; “Phage Therapy in the Era of Synthetic Biology", Barbu et al., Cold Spring Harb Perspect Biol (2016) 8(10):a023879; "Reprogramm
  • a plurality of phages may be employed to achieve selective inhibition of the therapeutic bacteria, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 phages may be utilised.
  • These phages may be specific for the same therapeutic bacteria ( e.g . the same bacterial strain or species) or they may be specific for a plurality of therapeutic bacteria (for example a plurality of bacterial species).
  • the latter embodiment is particularly useful where the invention is used for testing compositions comprising bacterial consortia.
  • the invention also provides compositions comprising such phages optionally in combination with a stabiliser, preservative and / or additive.
  • phages which may be used with the invention include: (DCrAssOOl (specific to Bacteroides bacteria), B124-14 (specific to Bacteroides bacteria), B40-8 (specific to Bacteroides bacteria), AUEF3 (specific to Enterococcus bacteria), BC611 (specific to Enterococcus bacteria), Ec-ZZ2 (specific to Enterococcus bacteria), A2 (specific to Lactobacillus bacteria), 521B (specific to Lactobacillus bacteria), c2 (specific to Lactobacillus bacteria), or phiCDHMll (specific to Clostridium bacteria).
  • DCrAssOOl specific to Bacteroides bacteria
  • B124-14 specific to Bacteroides bacteria
  • B40-8 specific to Bacteroides bacteria
  • AUEF3 specific to Enterococcus bacteria
  • BC611 specific to Enterococcus bacteria
  • Ec-ZZ2 specific to Enterococcus bacteria
  • A2 specific to Lactobacillus bacteria
  • 521B specific to Lactobacill
  • the phage may be specific for a Gram positive bacterium, for example bacteria of the genus Enterococcus.
  • the phage may have a DNA genome.
  • the phage may be specific for enterococcal bacteria, and optionally cannot infect non-enterococcal bacteria.
  • the phage may selectively inhibit the growth of only enterococcal bacteria.
  • Enterococcus-specific phages are of significant value to the process of the present invention as, to the inventors' knowledge, no phages of sufficient specificity and potency to be used in the process of the present invention had been known previously, nor had any Enterococcus-specific antibiotics been available.
  • the phage have high specificity and high selectivity for a therapeutic bacterium.
  • the phage(s) inhibits the growth of no more than 10 different species of bacteria, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 species of bacteria, preferably wherein each of the different species is from the same genus.
  • the phage(s) inhibits the growth of only one species of bacterium.
  • the phage(s) inhibits the growth of no more than 10 different strains of bacteria, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 strains of bacteria, preferably wherein each of the different strains is from the same species.
  • the phage(s) inhibits the growth of only one strain of bacterium.
  • the phage(s) has known specificity and/or known selectivity.
  • the phage(s) selectively inhibits the growth of the therapeutic bacteria in the composition. In an embodiment, the phage(s) selectively inhibits the growth of the therapeutic bacteria, and any bacteria of the same species as the therapeutic bacteria. In an embodiment, the phage(s) inhibits the growth of only the therapeutic bacteria in the composition. In an embodiment, the phage(s) does not inhibit the growth of any unwanted organisms in the composition.
  • suitable phages include NCIMB 43666 phage and NCIMB 43667 phage (both deposited at NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland by 4D Pharma Research Ltd. of Life Sciences and Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS), both of which are specific for enterococcal bacteria, particularly Enterococcus gallinarum, such as Enterococcus gallinarum strain NCIMB 42488 (also deposited at NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland by 4D Pharma Research Ltd.
  • phages are highly specific for Enterococcus gallinarum (in particular Enterococcus gallinarum strain NCIMB 42488) and do not affect other, unwanted microorganisms that may be present in the medium. These phages are therefore suitable agents for specifically inhibiting the growth of Enterococcus gallinarum, such as Enterococcus gallinarum strain NCIMB 42488.
  • the phage selectively inhibits the growth of only bacteria of the species Enterococcus gallinarum. In an embodiment, the phage selectively inhibits the growth of Enterococcus gallinarum strain NCIMB 42488 bacteria. In an embodiment, the phage inhibits the growth of only Enterococcus gallinarum bacteria. In an embodiment, the phage inhibits the growth of only Enterococcus gallinarum strain NCIMB 42488 bacteria.
  • the NCIMB 43667 phage is preferred for use in the present invention, preferably when the therapeutic bacteria comprise or consist of bacteria of the genus Enterococcus.
  • this phage is used when the therapeutic bacteria comprise or consist of bacteria of the species Enterococcus gallinarum, most preferably Enterococcus gallinarum strain NCIMB 42488.
  • the nucleic acid sequence for the genome of NCIMB 43667 is set out in SEQ ID NO: 1.
  • Each of SEQ ID NOs: 3 to 68 represent a separate node of the genome of NCIMB 43667, wherein each node is a scaffold or contig assembled during sequencing of the phage genome.
  • the nucleic acid sequence of the genome of NCIMB 43667 comprises at least one, at least two, at least three, at least 4, at least 5 or all of SEQ ID NOs: 3 to 8, in any order.
  • NCIMB 43667 and any variant thereof which has the same or similar activity in the assays described below is also a part of the present invention. Further details of this phage are disclosed in the Examples below.
  • a variant of the NCIMB 43667 phage may have a DNA genome that is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 1.
  • a variant of the NCIMB 43667 phage may have a DNA genome that is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NOs: 3 to 8 or 3 to 68.
  • the process of the invention can also be practised using a phage which is functionally equivalent to the NCIMB 43667 phage (such a functionally equivalent phage may have a DNA genome with sequence identity to SEQ ID NO: 1 as defined in the preceding sentence).
  • “Functionally equivalent” means that the phage can inhibit the growth of Enterococcus gallinarum strain NCIMB 42488 to the same extent, or at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% compared to NCIMB 43667 phage.
  • the growth inhibition can be determined easily in a pilot experiment in which growth inhibition of the NCIMB 43667 and of the phage of interest are measured in parallel and compared.
  • Growth inhibition may be measured by plating a mixture of the phage and Enterococcus gallinarum strain NCIMB 42488 on agar and inspecting for plaque formation in the resulting bacterial lawn (for example, 100 pL of pre-determined phage dilution combined with 500 pL of Enterococcus gallinarum strain NCIMB 42488 culture, added to 3mL top agar at 42°C, then distributed over Casein peptone Soybean peptone (CASO) agar and incubated for 37°C for between 16 and 48 hours, prior to plaque formation inspection). The number of plaques or plaque forming units may then, for example, be used to indicate growth inhibition.
  • a mixture of the phage and Enterococcus gallinarum strain NCIMB 42488 on agar and inspecting for plaque formation in the resulting bacterial lawn (for example, 100 pL of pre-determined phage dilution combined with 500 pL of Enterococcus gallinarum strain NCIMB 4
  • the NCIMB 43666 phage is also preferred for use in the present invention.
  • This phage is particularly useful when the therapeutic bacteria comprise or consist of bacteria of the genus Enterococcus.
  • this phage is used when the therapeutic bacteria comprise or consist of bacteria of the species Enterococcus gallinarum, most preferably Enterococcus gallinarum strain NCIMB 42488.
  • the nucleic acid sequence for the genome of NCIMB 43666 is set out in SEQ ID NO: 2.
  • Each of SEQ ID NOs: 69 to 2626 represent a separate node of the genome, wherein each node is a scaffold or contig assembled during sequencing of the phage genome.
  • the nucleic acid sequence of the genome of NCIMB 43667 comprises at least one, at least two, at least three, at least 4, at least 5 or all of SEQ ID NOs: 69 to 74, in any order.
  • NCIMB 43666 and any variant thereof which has the same or similar activity in the assays described below is also a part of the present invention. Further details of this phage are disclosed in the Examples below.
  • a variant of the NCIMB 43666 phage according to the invention may have a DNA genome that is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 2.
  • a variant of the NCIMB 43666 phage according to the invention may have a DNA genome that is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least one, at least two, at least three, at least 4, or at least 5 or all of SEQ ID NOs: 69 to 74, or one, some or all of 69 to 2626.
  • the process of the invention can also be practised using a phage which is functionally equivalent to the NCIMB 43666 phage (such a functionally equivalent phage may have a DNA genome with sequence
  • “Functionally equivalent” means that the phage can inhibit the growth of Enterococcus gallinarum strain NCIMB 42488 to the same extent, or at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% compared to NCIMB 43666.
  • the growth inhibition can be determined easily in a pilot experiment in which growth inhibition of the NCIMB 43666 and of the phage of interest are measured in parallel and compared.
  • Growth inhibition may be measured by plating a mixture of the phage and Enterococcus gallinarum strain NCIMB 42488 on agar and inspecting for plaque formation in the resulting bacterial lawn (for example, 100 pL of pre-determined phage dilution combined with 500 pL of Enterococcus gallinarum strain NCIMB 4248 culture, added to 3mL top agar at 42°C, then distributed over Casein peptone Soybean peptone (CASO) agar and incubated for 37°C for between 16 and 48 hours, prior to plaque formation inspection). The number of plaques or plaque forming units may then, for example, be used to indicate growth inhibition.
  • a mixture of the phage and Enterococcus gallinarum strain NCIMB 42488 on agar and inspecting for plaque formation in the resulting bacterial lawn (for example, 100 pL of pre-determined phage dilution combined with 500 pL of Enterococcus gallinarum strain NCIMB 4248
  • references to a percentage sequence identity between two nucleotide sequences refers to the percentage of nucleotides that are the same in comparing the two sequences when aligned. This alignment and percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. Current Protocols in Molecular Biology (F.M.
  • the sequence alignment may be local or global, and is preferably global.
  • a preferred local alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM62 matrix.
  • the Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Ad v. Appl. Math. 2: 482-489. Local alignment may also be achieved by the Smith-Waterman homology search algorithm using a BLOSUM50 matrix. A preferred global alignment is determined by the Needleman-Wunsch algorithm, disclosed in Needleman & Wunsch (1970) Journal of Molecular Biology, 48(3):443-453.
  • NCIMB 43667 and NCIMB 43666 phages are particularly preferred for use in the present invention, especially when the therapeutic bacteria comprise or consist of bacteria of the genus Enterococcus, as the inventors have seen particularly good results with the combination of these phages.
  • this combination of phages is used when the therapeutic bacteria comprise or consist of bacteria of the species Enterococcus gallinarum, most preferably Enterococcus gallinarum strain NCIMB 42488.
  • the use of a phage as the agent is especially preferred.
  • the data presented in the examples demonstrate the ability of phage to rapidly inactivate high concentrations of therapeutic bacteria, thus facilitating the convenient and straightforward identification of any unwanted organisms (in those examples, bacterial organisms).
  • the use of phages, as compared to lysozyme, is also advantageous as this avoids the additional process steps of isolating and purifying the lysozyme.
  • Lysozyme also known as a muramidase or an N-acetylmuramideglycanhydrolase, is an antimicrobial enzyme produced by animals that forms part of the innate immune system. Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N- acetyl-D-glucosamine residues in peptidoglycan, which is the major component of Gram-positive bacterial cell walls. This hydrolysis in turn compromises the integrity of bacterial cell walls causing lysis of the bacteria.
  • Specific lysozymes which may be employed in the present invention may be selected from any of the known lysozymes and are of particular use where the therapeutic bacteria are Gram-positive bacteria and the one or more unwanted organisms are not Gram-positive bacteria.
  • the lysozyme may be naturally occurring or non-naturally occurring.
  • the lysozyme may have been engineered, for example to increase specificity for the therapeutic bacteria.
  • the lysozyme may be a c- type lysozyme (chicken-type or conventional-type), a g-type lysozyme (goose-type), or i-type lysozyme (invertebrate type).
  • lysozymes may be used, for example a combination of hen lysozyme and human lysozyme may be used.
  • a non-naturally occurring lysozyme may also be used, for example a lysozyme which has been artificially mutated, a recombinant lysozyme, or a lysozyme fusion protein.
  • the invention can also be practised using antibiotics. These embodiments are particularly suitable where the unwanted organism is not bacterial, but is a virus, for example.
  • Antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes.
  • An antibiotic may be bactericidal or bacteriostatic. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins) or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones and sulfonamides) have bactericidal activities. Protein synthesis inhibitors (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further categorization is based on their target specificity. "Narrow- spectrum" antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive, whereas broad-spectrum antibiotics affect a wide range of bacteria.
  • antibiotics that may be used in the present invention can readily be selected based on the therapeutic bacteria which need(s) to be inhibited and the one or more unwanted organisms which are to be allowed to grow.
  • the antibiotic may be bactericidal or bacteriostatic.
  • the antibiotic may be a naturally occurring antibiotic, a synthetic antibiotic, or a semisynthetic antibiotic.
  • the antibiotic is preferably a narrow spectrum antibiotic to ensure that unwanted organisms are not affected.
  • suitable examples of narrow-spectrum antibiotics include penicillin G, which is mainly effective against Gram-positive bacteria, vancomycin and macrolides, which are also mainly effective against Gram-positive bacteria, particularly staphylococcal bacteria, temocillin, which is mainly effective against Gram-negative bacteria.
  • An antibiotic specific to Gram-negative bacteria is preferably used when the therapeutic bacteria are Gram-negative and the one or more unwanted organisms are not Gram-negative bacteria.
  • An antibiotic specific to Gram-positive bacteria is preferably used when the therapeutic bacteria are Gram-positive and the one or more unwanted organisms are not Gram-positive bacteria.
  • a narrow-spectrum antibiotic specific to Gram-positive bacteria is preferably used.
  • the growth of the therapeutic bacteria is selectively inhibited by addition to the culture medium of an agent which inhibits the growth of the therapeutic bacteria, with the proviso that the agent is not an antibiotic.
  • the agent is not vancomycin.
  • the agent is not sodium phosphate or glycerophosphate.
  • the selective inhibition is not achieved by addition of an antibiotic.
  • a process according to the invention may involve a step of growing the microbial composition at a low pH in the presence of a lysozyme.
  • a process according to the invention may involve a step of growing the microbial composition in the presence of two types of phage.
  • the microbial composition according to the invention is most preferably a LBP. In some embodiments, it may be a vaccine.
  • the microbial composition is pharmaceutically acceptable before step (a) of the process of the invention. Most preferably, no contaminating bacteria and/or viruses are added to the microbial composition.
  • the microbial composition is a finished drug product composition or unit dose (e.g. a single tablet or capsule).
  • the microbial composition may comprise excipients in addition to the therapeutic bacteria, such as lyoprotectants, preservatives, antioxidants, stabilisers, prebiotic compounds, pharmaceutically acceptable carriers or diluents, binders, lubricants, suspending agents, coating agents, solubilising agents, or the like.
  • the microbial composition may comprise 1 x 10 10 CFU or less, 10 11 or less of therapeutic bacteria.
  • the process of the present invention is sufficiently sensitive to enable the presence or absence of unwanted bacteria to be determined in a single dosage form.
  • the single dosage form may be an enteric formulation, i.e. a gastro-resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition to the intestine by oral administration.
  • the single dosage form may comprise as enteric coating.
  • the microbial composition may be a sample taken from a bulk.
  • the sample may then be analysed according to the invention. If the results indicate the absence of unwanted organisms, the bulk may then be processed further, for example to prepare a unit dose of a medicament.
  • the therapeutic bacteria may be pathogenic bacteria, or optionally non-virulent strains of pathogenic bacteria.
  • the therapeutic bacteria in the vaccine microbial composition may therefore also be alive in the finished drug product composition or unit dose.
  • the vaccine microbial composition does not comprise attenuated therapeutic bacteria.
  • the therapeutic bacteria may be genetically engineered non-pathogenic bacteria that comprise one or more antigens from one or more pathogenic bacteria, such that inoculation with the live non-pathogenic bacteria immunises the patient against the one or more pathogenic bacteria.
  • the genetically engineered non-pathogenic bacteria express one or more antigens from one or more pathogenic bacteria.
  • the genetically engineered non-pathogenic bacteria comprise one or more plasmids that comprise nucleic acids encoding one or more antigens from one or more pathogenic bacteria.
  • the process of the invention comprises the step of manufacturing a microbial composition, wherein the microbial composition is a finished drug product composition suitable for administration to a patient, optionally a human patient, prior to the steps of culturing a microbial composition comprising therapeutic bacteria under conditions which selectively inhibit the growth of the therapeutic bacterium or therapeutic bacteria, and analysing the cultured microbial composition to determine whether any unwanted organism was present in the microbial composition.
  • the microbial composition is a finished drug product composition suitable for administration to a patient, optionally a human patient, prior to the steps of culturing a microbial composition comprising therapeutic bacteria under conditions which selectively inhibit the growth of the therapeutic bacterium or therapeutic bacteria, and analysing the cultured microbial composition to determine whether any unwanted organism was present in the microbial composition.
  • the composition may comprise more than one species or strain of therapeutic bacteria. If the bacteria comprise multiple strains, they may be from different phyla, class, order, family, genera, or species, or may be different strains from the same species.
  • the multiple therapeutic bacterial strains may be: of the same phyla but different class; of the same class but different order; of the same order but different family; of the same family but different genera; of the same genera but different species; or of the same species but different strains.
  • Microbial compositionss comprising combinations of these categories are also encompassed by the present invention, for example the therapeutic bacteria may comprise two or more therapeutic bacterial strains of the same genera but different species and two or more therapeutic bacterial strains of the same species but different strains.
  • the microbial composition may comprise multiple different therapeutic bacterial strains of the phylum Bacteroidetes, of the phylum Firmicutes, or both.
  • the microbial composition may comprise multiple species of therapeutic bacteria of the genus Enterococcus.
  • the microbial composition may comprise multiple strains of therapeutic bacteria of the species Enterococcus gallinarum.
  • the microbial composition does not comprise L. jensenii.
  • the therapeutic bacteria does not comprise L. jensenii.
  • the microbial composition may comprise a single therapeutic bacterial strain.
  • the microbial composition may comprises a consortium of therapeutic bacterial strains, for example it may comprise a plurality of therapeutic bacterial strains.
  • the microbial composition may comprise more than one strain from within the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or 45 strains) and, optionally, does not contain therapeutic bacteria from any other species.
  • the therapeutic bacteria may comprise fewer than 50 strains from within the same species (e.g.
  • the microbial composition comprises 1-40, 1-30, 1-20, 1-19, 1-18, 1-171-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1- 2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25 or 31-50 strains from within the same species and, optionally, does not contain therapeutic bacteria from any other species.
  • the microbial composition comprise therapeutic bacterial strains from more than one species (for example more than one species from within the same genus), e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 species and, optionally, does not contain therapeutic bacterial strains from any other genus.
  • the microbial composition comprises therapeutic bacterial strains from fewer than 50 species (for example fewer than 50 species from within the same genus), e.g. fewer than 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species) and, optionally, does not contain therapeutic bacteria from any other genus.
  • the therapeutic bacteria comprises strains from 1-50, 1-40, 1-30, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species from within the same genus and, optionally, does not contain therapeutic bacteria from any other genus.
  • the microbial composition may comprise any combination of the foregoing.
  • the microbial composition comprises a consortium of therapeutic bacterial strains.
  • the consortium of therapeutic bacterial strains comprises two or more therapeutic bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the consortium of therapeutic bacterial strains is not found together in nature.
  • the consortium of therapeutic bacterial strains comprises therapeutic bacterial strains obtained from faeces samples of at least two different organisms. In some embodiments, the two different organisms are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal.
  • the therapeutic bacteria may comprise or consist of anaerobic bacteria, obligate anaerobic bacteria, facultative anaerobic bacteria and / or microaerophilic bacteria.
  • the therapeutic bacteria may comprise or consist of bacteria from the following genera: Enterococcus (e.g Enterococcus gallinarum, Enterococcus caselliflavus, Enterococcus faeca!is or Enterococcus faecium), Blautia (e.g. Blautia hydrogenotrophica, Blautia stercoris, Blautia wexlerae or Blautia producta), Bacteroides (e.g.
  • Eubacterium e.g. Eubacterium contortum, Eubacterium fissicatena, Eubacterium eligens, Eubacterium hadrum, Eubacterium hallii or Eubacterium rectale
  • Ruminococcus e.g. Ruminococcus torques, Ruminococcus gnavus or Ruminococcus bromii
  • Pseudoflavonifractor e.g. Pseudoflavonifractor capillosus
  • Clostridium e.g.
  • Clostridium nexile Clostridium hylemonae, Clostridium butyricum, Clostridium tertium, Clostridium disporicum, Clostridium bifermentans, Clostridium inocuum, Clostridium mayombei, Clostridium bolteae, Clostridium bartletti, Clostridium symbiosum or Clostridium orbiscindens), Coprococcus (e.g. Coprococcus comes or Coprococcus cattus), Acetivibrio (e.g. Acetovibrio ethanolgignens), Dorea (e.g. Dorea longicatena) or any genera of the family Lachnospiraceae.
  • Coprococcus e.g. Coprococcus comes or Coprococcus cattus
  • Acetivibrio e.g. Acetovibrio ethanolgignens
  • Dorea e.g. Dorea longicatena or any genera of the family
  • the microbial composition does not comprise organisms from the Lactobacillus genus and / or the Lactobacillaceae family.
  • the microbial composition may have any therapeutic use, such as use in the treatment or prevention of cancer, gastrointestinal disease (such as inflammatory bowel disease, inflammatory bowel syndrome, Crohn's disease), diabetes, periodontitis, or metabolic diseases (such as metabolic syndrome).
  • the therapeutic bacteria have therapeutic use in the treatment or prevention of cancer.
  • the therapeutic bacteria may be pathogenic or non-pathogenic to mammals, e.g. humans.
  • the therapeutic bacteria may comprise naturally occurring and / or artificial bacteria, such as genetically modified therapeutic bacteria.
  • Any unwanted organisms in the microbial composition are preferably live at the beginning of the culture step.
  • the unwanted organism could be an anaerobic bacterium which may be pathogenic (e.g. a pathogenic bacterium from the Clostridia or Salmonella genera or a Staphylococcus aureus, Pseudomonas aeruginosa or Escherichia coli species), e.g. an anaerobic bacterium which is pathogenic to mammals, such as humans.
  • pathogenic e.g. a pathogenic bacterium from the Clostridia or Salmonella genera or a Staphylococcus aureus, Pseudomonas aeruginosa or Escherichia coli species
  • an anaerobic bacterium which is pathogenic to mammals, such as humans.
  • organisms may be identified using one or more of the following techniques: microorganism phenotype analysis (such as identifying morphology, Gram staining, and / or sensitivity to antimicrobials, see for example Chapter 3: “Classification”, of Medical Microbiology, 4th edition, 1996, edited by Samuel Baron, or "Methods for the detection and identification of pathogenic bacteria: past, present, and future", Varadi et al., Chem Soc Rev (2017) 46(16) pp 4818-4832); mass spectrometry (such as MALDI-TOF-MS; see for example “Rapid and Robust MALDI-TOF MS Techniques for Microbial Identification: A Brief Overview of Their Diverse Applications", Jang and Kim, J Microbiol (2016) 56(4) pp 209-216); nucleic acid testing (such as nucleic acid sequencing, and / or PCR, see for example "
  • the determination of the presence of an unwanted organism in the microbial composition may comprise the identification of cellular products in the cultured microbial composition, preferably cellular products not produced by the therapeutic bacteria and / or which are indicative of the presence of specific pathogenic bacteria such as pathogenic organisms from the Clostridia and Salmonella genera and the Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli species.
  • the analysis may comprise the identification of metabolites such as short chain fatty acids.
  • the analysis of the microbial composition may comprise the identification of polypeptides.
  • the process is sensitive enough to differentiate between an unacceptable level of unwanted organisms and a de minimis level of unwanted organisms, wherein said unacceptable level is predetermined, for example said unacceptable level may be predetermined by the FDA.
  • said unacceptable level is predetermined, for example said unacceptable level may be predetermined by the FDA.
  • unnecessary waste of useful microbial compositions is avoided, for example because the user is able to determine whether the amount of unwanted organism(s) in the microbial composition is unsafe according to predetermined safety regulations.
  • kits comprising an agent which selectively inhibits a first bacterial population and instructions to use the agent in the process as hereinbefore described.
  • the invention also provides a method of preparing a pharmaceutical composition comprising therapeutic bacteria, comprising a step of testing for unwanted organisms by a process according to the invention.
  • a method may comprise the steps of (a) culturing therapeutic bacteria, (b) performing a process according to the invention on at least a fraction of the culture of step (a) and (c) formulating the cultured therapeutic bacteria of step (a) into a pharmaceutical composition.
  • a process according to the invention may be performed following formulation of the pharmaceutical composition.
  • a pharmaceutical composition is prepared, and tested in accordance with the invention, wherein a sample of a bulk therapeutic bacteria culture is tested and, if an acceptable level (preferably, a predefined acceptable level) of unwanted organisms is found, the bulk therapeutic bacteria culture is used in the formulation of a pharmaceutical composition (e.g. by admixing with a pharmaceutically-acceptable excipient). If greater than an acceptable level of unwanted organisms is found (preferably, a predefined acceptable level), the bulk therapeutic bacteria culture is not used in the formulation of a pharmaceutical composition, for example the bulk therapeutic bacteria culture may be destroyed.
  • predefined acceptable level is (i) less than 10 3 CFU/g or CFU/ml total aerobic microbial content (excluding the therapeutic bacteria), or (ii) less than 10 2 CFU/g or CFU/ml total combined yeasts and mould count; preferably both (i) and (ii). More preferably, the predefined acceptable level also requires the absence of any Escherichia coli bacteria (e.g. the culture does not comprise detectable Escherichia coli bacteria).
  • the sample of a bulk therapeutic bacteria culture may comprise less than 1% by volume of the total bulk therapeutic bacteria culture, for example less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% by volume.
  • the sample of a bulk therapeutic bacteria culture may comprise less than 10% by volume of the total bulk therapeutic bacteria culture, for example less than 9%, 8%, 7%, 6%, 5%, 4%,. 3%, 2%, or 1% by volume.
  • the sample of a bulk therapeutic bacteria culture that is tested is not used in the formulation of a pharmaceutical composition.
  • the bulk therapeutic bacteria culture is at least 1 litre, 2 litres, 3 litres, 4 litres, 5 litres, 6 litres, 7 litres, 8 litres, 9 litres, or 10 litres. In an embodiment, the bulk therapeutic bacteria culture is at least 100 litres, 200 litres, 300 litres, 400 litres, 500 litres, 600 litres, 700 litres, 800 litres, or 900 litres. In an embodiment, the bulk therapeutic bacteria culture is at least 1,000 litres, 2,000 litres, 3,000 litres, 4,000 litres, 5,000 litres, 6,000 litres, 7,000 litres, 8,000 litres, 9,000 litres, or 10,000 litres.
  • the invention also provides compositions comprising one or more phages, optionally in combination with a stabiliser, preservative and / or additive.
  • the composition comprises a plurality of phages, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10 phages may be utilised.
  • the one or more phages may be any phage described herein.
  • the one or more phages may be naturally occurring or non-naturally occurring phages, for example the one or more phages may be genetically engineered or genetically modified.
  • the composition may comprise a mixture of naturally occurring phages and non-naturally occurring phages.
  • the composition may comprise one or more phages that have been genetically engineered to be more or less specific for one or more therapeutic bacteria.
  • the composition may comprise at least one phage that is specific for a Gram positive bacterium, and optionally does not comprise phages specific for a Gram negative bacterium.
  • the composition may comprise at least one phage that is specific for a Gram negative bacterium, and optionally does not comprise phages specific for a Gram positive bacterium.
  • the composition may comprise at least one phage having a DNA genome.
  • the composition may comprise one or more phages specific for one therapeutic bacterium, and optionally does not comprise any phages specific for any other therapeutic bacterium.
  • the composition may comprise one or more phages, wherein each phage is specific for one therapeutic bacterium, and optionally each phage is specific for a different therapeutic bacterium.
  • the composition may comprise one or more phages specific for one genus of therapeutic bacteria or one species of therapeutic bacteria.
  • the composition may comprise one or more phages specific for one strain of therapeutic bacteria.
  • the composition does not comprise more than one phage specific for the same genus of therapeutic bacteria. In embodiments, the composition does not comprise more than one phage specific for the same species of therapeutic bacteria. In embodiments, the composition does not comprise more than one phage specific for the same strain of therapeutic bacteria.
  • the composition may comprise phages specific for more than one strain of therapeutic bacteria, and optionally may comprise phages specific for more than one strain of therapeutic bacteria of the same species.
  • the composition may comprise phages specific for more than one species of therapeutic bacteria of the same genus.
  • the composition may comprise one or more phages specific for enterococcal bacteria, and optionally said composition does not comprise any phages that are not specific for enterococcal bacteria.
  • the composition may comprise one or more phages specific for enterococcal bacteria and one or more phages specific for one or more non-enterococcal therapeutic bacteria.
  • suitable phages include NCIMB 43666 phage and NCIMB 43667 phage, both of which are specific for enterococcal bacteria, particularly Enterococcus gallinarum strain NCIMB 42488.
  • the composition comprises NCIMB 43666 phage, NCIMB 43667 phage, or both. It has been found that these phages are highly specific for Enterococcus gallinarum strain NCIMB 42488 and do not affect other, unwanted microorganisms that may be present in the medium. These phages are therefore suitable agents for specifically inhibiting the growth of Enterococcus gallinarum strain NCIMB 42488.
  • the composition comprises a phage that has a DNA genome which is at least 85% identical to SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the composition may comprise a phage which is functionally equivalent to a phage having the genome sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the composition comprises a phage that has a DNA genome which is at least 85% identical to SEQ ID NOs: 3 to 8 or 3 to 68 and/or SEQ ID NOs: 69 to 74 or 69 to 2626.
  • the composition may comprise a phage which is functionally equivalent to a phage having the genome sequence set out in SEQ ID NOs: 3 to 8 or 3 to 68 or SEQ ID NOs: 69 to 74 or 69 to 2626.
  • Figure 1 shows gel electrophoresis results for restriction fragment length polymorphism analysis of phage and bacterial DNA
  • Figure 2 shows the putative gene analysis of NCIMB 43667.
  • one of the phages used is the NCIMB 43667 phage. This phage was analysed in a number of ways.
  • the mixture was further incubated for 30 min at 37°C. The entire volume was transferred into a new 15 mL centrifuge tube. The solution was mixed 1:1 with phenol / chloroform (pH 8). The phases were separated by centrifugation for 5 min at 1500 x g. The aqueous phase was removed into a new 15 mL tube and the step was repeated. The aqueous phase was removed and extracted once with an equal volume of chloroform. The upper phase was removed after centrifugation for 5 min at 6000 x g. 10 pL of 3M sodium acetate and 1 volume of 100 % isopropanol were added. The DNA was left to precipitate at room temperature for 20 min and was then centrifuged at 14000 x g for 20 min. The DNA pellet was washed with 70 % ethanol. The ethanol was removed and the DNA pellet was dried at room temperature for 10 min. The DNA was resuspended in 500 pL 5 mM T ris-HCI, pH 8.5.
  • RFLP Restriction Fragment Length Polymorphism
  • the phage DNA was digested with various restriction enzymes. 500 ng of DNA was used in each reaction. 1 unit of each enzyme was used in the reaction in a volume of 20 pL, as shown in Table 3 below.
  • DNA was then sequenced using conventional protocols for producing DNA libraries. Illumina next-generation sequence data at a minimum coverage of 30x were produced. 224 contigs were obtained of which only three were longer than 1,000 bp. The largest contig representing the DNA sequence of the phage was 57201 bp long, with the mean coverage of 1,756.31. The complete genome sequence is given in SEQ ID NO: 1 in the sequence listing, as described above.
  • the DNA sequence had: an A content of 31.51 %; a C content of 19.56 %; a G content of 21.95%; a T content of 26.98 % and a CG percentage of 41.51%.
  • Putative genes were annotated using the PFIASTER web server for the identification and annotation of prophage sequences within bacterial genomes and plasmids. The termini of the DNA sequence were not analysed. It was therefore not possible to determine whether the phage genome was linear or circular. The putative gene analysis is shown in Figure 2.
  • the 57,202 bp sequence of NCIMB 43667 phage was searched for sequences similar to those in the NCBI databases using the BlastN search tool with a highly similar sequences search algorithm (megablast).
  • the sequences which showed the highest similarity to the NCIMB 43667 sequence were the sequences of Enterococcus phage vB_EfaS_Ef7.1 and Enterococcus phage VD13.
  • the megablast hit table for highly similar sequences is shown in Table 4 below.
  • NCIMB 43667 Phage Sequence [0098] The characteristics of NCIMB 43667 phage shows that it is likely to be useful in the process of the present invention. This is shown by the following Example 2.
  • strains of the species Enterococcus gallinarum are effective for treating and preventing cancer.
  • these species are therapeutic bacteria as defined above.
  • a particular strain of Enterococcus gallinarum has been deposited under the accession number NCIMB 42488 and is described in more detail in WO 2017/085520.
  • a microbial composition containing NCIMB 42488 was supplied in capsules. Each capsule contained about 5 x 10 10 CFU of E. gallinarum. The capsules were stored at 5 ⁇ 3°C before use.
  • the phage detection process is based on the ability of phages to create clear zones (plaques) on a lawn of host cells.
  • a double agar overlay assay was applied in this study.
  • phage plating 100 pL of phage dilution in SM buffer is mixed with 500 pL of an overnight culture of NCIMB 42488 and incubated for 15 min at 37°C. The phage-host mixture is added to 3 mL of top agar which had been warmed to 42°C and the evenly distributed over CASO agar. The plates are incubated at 37°C for 16-48 h and inspected for plaque formation. The phage titre was determined from plates with plaque numbers between 10 and 300.
  • NCIMB 43667 phage lysate was produced to be further used for production of liquid and solid media containing phage cocktail against NCIMB 42488.
  • An exponentially growing culture of NCIMB 42488 was prepared by inoculation of 95 mL CASO bouillon with 5 mL overnight culture of the strain. The diluted culture was further incubated for 1 h at 37°C and infected with 10 pL NCIMB 43667 phage.
  • the NCIMB 43667 phage inoculum used for infection was prepared from a filter-sterilized sample. After infection, the culture was further incubated without agitation for 16 h until lysed.
  • the lysate was pooled, 1 mL chloroform was added, mixed using vortex and centrifuged at 3000 x g for 5 min. The supernatant was filter-sterilized. The titre of the phage was determined as described above. The results are given in Table 5.
  • the sterility of the phage was tested using the spread plate method. 100 pL of the NCIMB 43667 solution was spread-plated on CASO agar in duplicate and incubated for 48 h at 32.5 ⁇ 2.5°C. The results are given in Table 6. The NCIMB 43667 solution was stored in two 40 mL aliquots at 5 ⁇ 3°C until use. Table 6 - NCIMB 43667 Sterility Testing
  • the criterion of the lowest acceptable concentration of the phage preparation was set as not less than 50% of the titre determined previously in the method development study.
  • NCIMB 43666 phage lysate was produced to be further used for production of liquid and solid media containing phage cocktail against NCIMB 42488.
  • the decimal dilution series of NCIMB 43666 phage was prepared in SM buffer.
  • the dilutions 10 3 , 10 4 and 10 5 were plated in a volume of 100 pL together with 0.5 mL overnight culture of NCIMB 42488.
  • LB agar plates supplemented with 5mM MgS04 + 5mM CaCh were used. Each dilution was plated in quadruplicate using the method described above. The plates were incubated for 16-48 h at 37°C.
  • the criterion of the lowest acceptable concentration of the phage preparation was set as not less than 50% of the titre determined as set forth above.
  • the two phage stock solutions were mixed in equal volumes directly before use.
  • the final concentration of the phages in the cocktail was 2.85 x 10 ⁇ and 1.2 x 10® PFU/ml of NCIMB 43666 and NCIMB 43667 respectively.
  • the phage cocktail was used to cover the agar plates used for the detection of contaminant microorganisms.
  • Test strains were prepared as cryo stocks with 43% glycerol and are stored at ⁇ -64 °C. The cell counts were determined on an approved batch of media. Dilutions in peptone water were prepared to obtain ⁇ 100 CFUs in 100 pL as indicated in Table 9 below.
  • control contaminant microorganisms were diluted to less than 10 ⁇ CFU /mL in buffered peptone water and 100 pL of the dilutions were spread-plated on CASO or Columbia blood agar. The plates were covered with 100 pL phage cocktail and allowed to dry before plating the control microorganisms. Each strain preparation was plated in duplicate.
  • the Columbia agar plates with C. sporogenes ATCC 19404 were incubated at 32.5 ⁇ 2.5°C for ⁇ 48 h in anaerobic atmosphere created with the Anaerobic Jar GasPack system.
  • the CASO agar plates with S. aureus ATCC 6538 and B. cereus ATCC 11778 were incubated for ⁇ 18 h at 32.5 ⁇ 2.5°C aerobically. The acceptance criteria and results are presented in Table 10.
  • Each tested capsule of NCIMB 42488 drug product was removed from the blister aseptically using flamed tweezers in laminar air flow cabinet and suspended in 10 mL buffered peptone water. The capsule shell was dissolved after about 30 min incubation and the content was homogenized by vortexing. The suspension was stored not longer than 8 h at 2-8°C before use. For testing, the entire volume of the suspension (10 mL) was withdrawn. The residual volume was next collected by single wash step with 10 mL of .appropriate cultivation media.
  • the test for the detection of contaminant bacteria was performed in two steps.
  • the first step was the enrichment of the potential contaminant strain in liquid culture together with the sample and bacteriophage cocktail active against E. gallinarum NCIMB 42488.
  • the second step was the subculture of the enrichments on agar plates previously covered with phage cocktail.
  • the enrichments were made in CASO broth (S. aureus ATCC 6538 and B. cereus ATCC 11778) or in RCM broth (C. sporogenes ATCC 19404). Both media were supplemented with 5 mM MgS04.
  • the CASO cultures were incubated for 24 h at 32.5 ⁇ 2.5°C without shaking and the RCM cultures were incubated for 48 h under the same conditions.
  • Each enrichment consisted of:
  • test strains were prepared as described above. The recovery of a low number of the test strains in the presence of the sample was tested to prove the method suitable for detection. The test was performed as described above. For each tested strain, the enrichment step was repeated three times. The subculture step after each enrichment was performed in triplicate also. The results are presented in Table 13.
  • the described method is suitable for the detection of Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 11778 and Clostridium sporogenes ATCC 19404 in 1 capsule of E. gallinarum NCIMB 42488 drug product (CFU/capsule) according to the demands of Ph. Eur. 2.6.38.
  • the use of the phage cocktail allows for sufficient reduction of f. gallinarum NCIMB 42488 cells to allow for detection of contaminant strains.
  • the phage detection process is based on the ability of phages to create clear zones (plaques) on a lawn of host cells.
  • a double agar overlay assay was applied in this study.
  • phage plating 100 pL of phage dilution in SM buffer is mixed with 500 pL of an overnight culture of NCIMB 42488 and incubated for 15 min at 37°C. The phage-host mixture is added to 3 mL of top agar which had been warmed to 42°C and the evenly distributed over CASO agar. The plates are incubated at 37°C for 16-48 h and inspected for plaque formation. The phage titre was determined from plates with plaque numbers between 10 and 300.
  • the number of plaque forming units in 1 mL was calculated using the equation: * 0,1 where: c weighted average of the plaques number ⁇ c sum of the plaques from all plates used for calculations n 1 number of plates with the lowest dilution n 2 number of plates with the next highest dilution.
  • the 10 4 , 10 5 , 10 s , 10 7 , and 10 8 dilutions of NCIMB 43667 phage in SM buffer were prepared. 100 pi of each dilution was plated in 3ml molten top agar in triplicates with 500 mI overnight culture of NCIMB 42488 (in CASO broth, 37 °C no agitation). Supplemented LB agar was used. The plates were incubated 18h at 37 °C. The plates showing confluent lysis were washed with 5 ml SM buffer/ plate for 4 h on 3D rotator. The phage lysate was pooled, and 0.5 ml chloroform was added.
  • the mix was centrifuged at 3000 x g for 5 min.
  • the supernatant was filter sterilized and sterile DMSO was added to a final concentration of 7 %.
  • Aliquots of 0.5 mL were prepared and were stored at ⁇ -64 °C until use.
  • Phage titre was determined on CASO agar by plating 100 pi dilutions of the phage in SM buffer with 500 mI overnight culture of the NCIMB 42488. Results are in Table 15 below.
  • NCIMB 43666 phage stock solution was prepared using the procedure described above. Aliquots of 0.5 mL were prepared and were stored at ⁇ -64 °C. Phage titre was determined on CASO agar by plating 100 mI dilutions of the phage in SM buffer with 500 mI overnight culture of the NCIMB 42488. Results are shown below in Table 16.
  • Both phage stock solutions were mixed 1:1 directly before use.
  • the final concentration of the phages was 2.6 xlO 6 PFU of NCIMB 43666 and 1.1x10 s PFU of NCIMB 43667 in 10 pi cocktail.
  • NCIMB 42488 An overnight culture of NCIMB 42488 was prepared in CASO broth. The culture was diluted 1:100 and 100 mI aliquots in sterile 96-well titre plate were prepared. Each aliquot was spiked with 10 mI containing 1, 10, 100, and 1000 NCIMB 43667 and NCIMB 43666 each and in combination (as 1:1 cocktail). Additionally undiluted phage stock solutions were tested: 2.2 x 10 s PFU of NCIMB 43667 phage and 5.2 x 10 s PFU NCIMB 43666. The phages were diluted to the desired concentration in SM buffer. The kinetics of the bacterial culture turbidity changes (OD620nm) were measured during 6 h incubation in 30 min intervals.
  • Phage NCIMB 43667 showed significantly better performance against NCIMB 42488 than phage NCIMB 43666. Phage NCIMB 43666 was not able to lyse the liquid cultures irrespective of the phage amount spiked. Slightly slower growth of NCIMB 42488 was observed when the culture was spiked with 5.2 x 10 s PFUs of NCIMB 43666. Cultures spiked with NCIMB 43667 phage were completely lysed after about 4h even when very low numbers of phage were initially in the culture. Cultures spiked with high number of NCIMB 43667 phage (2.2x10 s PFU) did not show growth at all.
  • NCIMB 43667 phage replicates via lytic cycle on NCIMB 42488 and is a very good candidate to be used as an agent for the neutralization of E. gallinarum in microbial examination of compositions containing NCIMB 42488. No evidence of improvement in NCIMB 42488 killing activity was observed when phage cocktail containing both phages was used. Complete lysis and no further growth of bacteria in the presence of NCIMB 42488 specific phages indicate that the sample tested was microbiologically pure.
  • test strains were prepared as cryo stocks with 43% glycerol and are stored at ⁇ -64 °C. The cell counts were determined before. Dilutions in peptone water prepared to obtain ⁇ 100 CFUs in 100 pi as indicated in the table below.
  • test strains [0134] To verify testing conditions, a negative control was performed using the buffered peptone water as diluent in place of the test preparation. There must be no growth of micro-organisms.
  • P. aeruginosa and B. subtilis were tested in the same way and plates A, B, and C were incubated for 3 days at 32.5 °C. Acceptance criteria were as follows: the counts of S. aureus, P. aeruginosa and B. subtilis on plates A and C should not differ more than 50 % the growth obtained on plate A must not differ by a factor greater than 2 from the calculated value for the stable S. aureus, P. aeruginosa and B. subtilis inocula
  • Phage cocktail can be used as an agent for neutralization of NCIMB 42488 in the test for enumeration of microbial contaminants microbial compositions containing NCIMB 42488.
  • the phage cocktail is selectively active against E. gallinarum and none from the test strains was affected.
  • a homogeneous suspension of the LBP was prepared. Two grams of the NCIMB 42488 sample (DS) were suspended in 18 mL CASO broth (10 1 dilution). A control with no test material included was prepared in the same way. The LBP suspension was further diluted 1:10 and 1:100 so dilutions 10 2 , and 10 3 of the LBP were created.
  • test strains were prepared as cryo stocks with 43% glycerol and are stored at ⁇ -64 °C. The cell counts were determined before. Dilutions in peptone water prepared to obtain ⁇ 100 CFUs in 10 pi as indicated in the table below.
  • Phage cocktail was prepared as described before. Tests were conducted as follows:
  • the surface-spread method was chosen for the enumeration of contaminating micro organisms. Plate-counting was performed in duplicate for each test strain/ DS dilution combination. The arithmetic mean was taken of the counts per medium and the number of CFU in the original inoculum was calculated. When verifying the suitability of the plate-count method, a mean count of any of the test organisms not differing by a factor greater than 2 from the value of the control in the absence of the LBP must be obtained.
  • a homogeneous suspension of the LBP was prepared. Seven grams of the NCIMB 42488 sample (DS) were suspended in 63 mL buffered peptone water (10 1 dilution). A control with no test material included was prepared in the same way. The LBP suspension was further diluted 1:10 and 1:100 so dilutions 10 2 , and 10 3 of the LBP were created. [0146] The test strains were prepared as cryo stocks with 43% glycerol and are stored at ⁇ -64 °C. The counts were determined before. Dilutions in peptone water were prepared to obtain ⁇ 100 CFUs in 100 pi (low number of specified micro-organisms) as indicated in the table below. Additionally, undiluted suspensions of the test strains were used to evaluate the overall feasibility of the detection procedure.
  • the phage cocktail was prepared as described above.
  • Test strains suspensions with the counts of ⁇ 100 CFU in the volume of 100 mI each were spread-plated on CASO agar plates covered or not with phage cocktail. The plates were incubated for ⁇ 18 h at 32.5 °C and the counts of micro-organisms were determined. To prepare the plates covered with phages, 100 mI of the phage cocktail was spread-plated on the agar surface and dried shortly before use.
  • Negative controls were prepared by inoculation of 90 mL CASO broth with 10 mL of the 10 _1 , 10 2 , 10 3 dilutions of the test sample and 100 mI phage cocktail. No S. aureus or B. cereus were added.
  • a reference sample with the microbial composition only was produced by inoculation of 90 mL CASO broth with 10 mL of the 10 _1 , 10 2 , 10 3 dilutions of the test sample.
  • Buffered peptone water, CASO broth and phage cocktail were spread-plated each in the volume of 100 mI on CASO agar.
  • one CASO agar plate was placed under the laminar flow bench and was kept open while processing the samples. The plates were incubated for 3 days at 32.5 °C.
  • the described method is suitable for the enumeration of B. subtilis provided that DS is diluted at least 1:100, i.e. the B. subtilis can be reliably enumerated in 0.01 g of DS.
  • DS is diluted at least 1:100, i.e. the B. subtilis can be reliably enumerated in 0.01 g of DS.
  • phage cocktail as an agent neutralising the inhibitory activity of the LBP.
  • the method is not suitable for the enumeration of S. aureus and P. aeruginosa, however the use of phage cocktail and dilution of the DS significantly improved the recovery of these test micro organisms in the presence of the LBP.
  • the method is suitable for the detection of S. aureus and B. cereus in the presence of the LBP provided that the DS is diluted at least 1:1000, i.e. low numbers of deliberately added test micro organisms can be reliably detected in 0.01 g or lower amount of the DS.

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Abstract

La présente invention concerne un procédé d'analyse d'une composition comprenant des bactéries thérapeutiques, le procédé comprenant les étapes consistant à (a) cultiver la composition dans des conditions qui inhibent sélectivement la croissance des bactéries thérapeutiques et (b) analyser la composition cultivée à l'étape (a) pour déterminer si un organisme indésirable a été présent dans la composition. L'invention concerne également un bactériophage approprié pour être utilisé dans l'inhibition de la croissance de bactéries thérapeutiques.
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EP1448995A1 (fr) 2001-11-21 2004-08-25 The Rowett Research Institute Technique de criblage de medicaments candidats pour le traitement de maladies inflammatoires
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EP1448995A1 (fr) 2001-11-21 2004-08-25 The Rowett Research Institute Technique de criblage de medicaments candidats pour le traitement de maladies inflammatoires
EP2763685A1 (fr) 2011-10-07 2014-08-13 Gt Biologics Ltd Bactérie pouvant être utilisée en tant que probiotique dans des applications nutritionnelles et médicales
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