EP3717627A1

EP3717627A1 - Isolation and pure culture of an archaebacterium of order methanomassiliicoccales

Info

Publication number: EP3717627A1
Application number: EP18811549.7A
Authority: EP
Inventors: Wajdi Ben Hania; Jean-François BRUGERE; Marie-Laure Fardeau; Bernard Ollivier
Original assignee: Lesaffre et Cie SA; Institut de Recherche pour le Developpement IRD; Universite Clermont Auvergne
Current assignee: Lesaffre et Cie SA; Institut de Recherche pour le Developpement IRD; Universite Clermont Auvergne
Priority date: 2017-12-01
Filing date: 2018-11-30
Publication date: 2020-10-07
Also published as: CN111655840A; FR3074501A1; FR3074501B1; US11560544B2; JP2021503964A; US20210171898A1; WO2019106181A1; JP7424989B2

Abstract

The present invention concerns a method for isolating an archaebacterium of order Methanomassiliicoccales of the commensal clade, the use of a culture and/or isolation medium comprising a sterile extract of a bacterial culture of a bacterium of the Eggerthella genus, for isolating and/or culturing an archaebacterium of order Methanomassiliicoccales of the commensal clade, and a pure isolated Methanomethylophilus alvus Mx-05 archaebacterium. It also concerns a method for culturing an archaebacterium of order Methanomassiliicoccales of the commensal clade.

Description

Isolation and pure culture of an arche of the order of Methanomassiliicoccales

The present invention relates to a method of isolating an archaea of the order Methanomassiliicoccales commensal clade, the use of a culture medium and / or isolation comprising a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella, for the isolation and / or cultivation of an archaea of the order Methanomassiliicoccales of the commensal clade, and an archaea Methanomethylophilus alvus Mx-05 pure isolated. It also relates to a culture method of an arche of the order Methanomassiliicoccales clade commensal.

The taxonomic order of the Methanomassiliicoccales, prokaryotic microorganisms of the Archaea type, consists of 2 large clades (Sollinger et al, 2015, Borrel et al, 2017). One of them is formed almost exclusively of micro-organisms living in the digestive tract of animals (insects to humans), called "host-associated cluster" or clade commensal. This clade includes in particular natural members of the gut microbiota of humans, and among them some capable of transforming trimethylamine (TMA) into methane using hydrogen as electron donor. TMA is a pro-atherogenic compound derived from intestinal microbial metabolism of various nutrients. The possible use of these organisms as probiotics, in particular to prevent cardiovascular diseases ("archaebiotics"), by lowering the level of intestinal TMA has been described (Brugère et al, 2014). This consequently leads to a decrease in its plasma derivative (plasma TMA oxide or pTMAO) resulting from its hepatic passage after intestinal absorption (hepatic oxidation by flavins monoxygenases), a metabolite involved in various mechanisms contributing to atherosclerosis. There is currently no other component of the human gut microbiota known to be able to cure TMA in a metabolite considered to be inert to humans, methane.

However, no representative of this group of microorganisms (commensal clade), possessing this bio-remediation activity, has so far been isolated and obtained in pure culture.

Beyond the technical problems related to the anaerobic microbiology of these microorganisms known to be highly sensitive to oxygen (EOS, Extremely Oxygen Sensitive), the usual practice for the isolation of this type of microorganism requires operating conditions. special. After obtaining an enrichment where methanogenic archaea become dominant, two approaches are then carried out: (i) "cloning" of the culture through solid media using the technique of Roll-tubes (Hungate, 1969) or (ii) dilutions / extinctions of the enrichment in a liquid medium to eliminate in particular the non-methanogenic fermentative microorganisms.

Until now, these techniques have proved ineffective in isolating the Methanomassiliicoccales from the commensal clade in axenic culture, irrespective of the isolation conditions used (reported in various scientific papers, notably Solleringer et al, 2015).

However, having a pure strain and controlling its growth are prerequisites to consider an application for therapeutic purposes.

In the context of the present invention, the species Methanomethylophilus alvus strain Mx-05 pure was isolated by applying a completely innovative approach.

The present invention thus relates to an archaea (Archaea domain) Methanomethylophilus alvus Mx-05 isolated pure, strain deposited according to the Treaty of Budapest, on September 18, 2017, at the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM32684 (applicant : Clermont Auvergne University, France).

It also relates to the use of a culture medium and / or isolation comprising a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella for the isolation and / or the pure culture (axenic) of a archée of the order of the Methanomassiliicoccales of the commensal clade. Advantageously, said culture and / or isolation medium comprises a growth factor (s) obtained from said culture of a bacterium of the genus Eggerthella.

The present invention furthermore relates to a method of isolating an archaea of the order Methanomassiliicoccales from the commensal clade comprising:

a) inoculating a biological sample that may comprise an archaeon of the order Methanomassiliicoccales clade commensal, preferably a sample of animal or human feces under anaerobic conditions under an atmosphere gas containing dihydrogen H ₂ under conditions mesothermic media in a culture medium comprising (i) a base medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, acetate of sodium, Cysteine-HCl, resazurin, Widdel micro-nutrient solution, selenite-tungstate solution, (ii) 2% Na ₂ S and 10% NaHCO ₃ , a mixture of vitamins (by example vitamin solution of Balch), to which is added (iii) one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract, and (iv) compounds methylated such as methylamines or methanol; b) replacing the culture medium of step a) with an identical culture medium depleted in compounds providing amino acids and in which vitamins are added;

c) detecting a positive enrichment in an archaea of the order of Methanomassiliicoccales commensal clade in said inoculated sample, in particular by microscopy, methane production and consumption of H ₂ , PCR, qPCR or sequencing; and

d) performing series of liquid dilutions or by the roll tube technique of said positive enrichment obtained in step c) using a medium as defined in step a) or b) to which a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella according to the invention, until obtaining an archaeal clone of the order Methanomassiliicoccales clade commensal.

Finally, it relates to a culture method of an archaea of the order Methanomassiliicoccales commensal clade, pure or present in a microbial consortium, comprising:

a) the inoculation of a biological sample comprising an archaea of the order Methanomassiliicoccales commensal clade, preferably a sample of animal or human stool in a culture medium, or a pure archaea of the order of Methanomassiliicoccales comprising (i) a basal medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, Cysteine - HCI, resazurin, a solution of Widdel trace elements, a solution of selenite-tungstate, Na ₂ S 2% and / or NaHCO ₃ 10%, to which is added (ii) one or more compounds providing acids amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract, (iii) methylated compounds such as methylamines or methanol and (iv) a sterile extract of a bacterial culture of the bacterium of the genus Eggerthella, according to the invention,

b) placing the inoculum obtained in step a) under anaerobic conditions under a gaseous atmosphere comprising dihydrogen H ₂ , and

c) incubation under mesothermal conditions, to reach an exponential growth phase with production of methane in at least 2 days, preferably not more than 15 days.

Archée Methanomethylophilus alvus Mx-05

As mentioned above, the present invention relates to an archaea Methanomethylophilus alvus Mx-05 pure isolated strain deposited according to the Treaty of Budapest, September 18, 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM32684 (applicant: Clermont Auvergne University, France).

This deposit was made by Clermont Auvergne University, 49 boulevard François Mitterrand - 63000 Clermont-Ferrand - France.

By "pure" is meant the meaning commonly used by those skilled in the field of the invention. In particular, by pure is meant the absence of contaminants and the mere presence of a strain, colony or clone of the Archaea Methanomethylophilus alvus Mx-05. The purity criteria are those usually used by those skilled in the art. In particular, in the context of the present invention, the criteria are the uniform morphology of the cells, the production of methane, and the absence of growth in a complex medium (SMS) containing carbohydrates not consumed by the methane Archaea , but allowing to highlight methanogenic contaminants if necessary.

By "isolated" is meant the meaning commonly used by those skilled in the technical field of the invention. An archaea Methanomethylophilus alvus Mx-05 isolated will be an archaea separated from other archaea and / or bacteria and / or yeasts or other eukaryotic contaminants potentially present in the starting sample.

As mentioned above, Methanomassiliicoccales are Archean-type prokaryotic microorganisms consisting of two large clades, including the so-called "commensal" clade to which the aforementioned strain belongs, formed almost exclusively of microorganisms living in the digestive tract of animals ( insects to humans), said strain belonging to those capable of transforming TMA into methane using hydrogen as electron donor.

The archaea Methanomethylophilus alvus Mx-05 pure isolated strain deposited under the Budapest Treaty, on September 18, 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM32684 (applicant: University Clermont Auvergne, France) such that aforementioned can in particular be obtained by the method of isolating an archaea of the order Methanomassiliicoccales commensal clade according to the invention and / or be the subject of the method of culture of an arche of the order Methanomassiliicoccales commensal clade according to the invention and / or be isolated using a culture medium of a culture medium and / or isolation comprising a sterile extract of a bacterial culture of a bacterium of the Eggerthella kind, as demonstrated in the "examples" part of the present description. Use of a culture medium and / or isolation a sterile extract of a bacterial culture of a bacterium of the genus Eaaerthella,

As mentioned above, the present invention also relates to the use of a culture and / or isolation medium comprising a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella, for isolation and / or pure culture. an archée of the order Methanomassiliicoccales clade commensal.

As mentioned above, advantageously, said culture and / or isolation medium comprises a growth factor (s) obtained from said culture of a bacterium of the genus Eggerthella.

Said culture and / or isolation medium may furthermore comprise any compound that may be useful for culturing or isolating an Archaea of the order Methanomassiliicoccales of the commensal clade.

In particular, the medium further comprises one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, and yeast extract.

For example, the medium further comprises from 0.5 to 50 g / l of Biotrypcase, peptone, casamino acids, yeast extract (in particular 10 g / l of each of these compounds). In addition, the medium may comprise coenzyme M. This co-factor is indispensable for methanogenesis. In the case of the Archaea Methanomethylophilus alvus Mx-05, coenzyme M can be omitted.

Even more particularly, the medium additionally comprises KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, cysteine- HCI, resazurin, a solution of Widdel trace elements, a selenite-tungstate solution, 2% Na ₂ S and / or 10% NaHCO ₃ .

Always more particularly; the medium comprises methyl compounds such as methylamines or methanol.

By "methylamines" is meant in particular mono-, di- or tri-methylamine.

An example of such a medium called basal medium is given in Table 3 presented in the examples.

All these compounds are compounds available commercially and / or well known to those skilled in the art.

The base medium is for example prepared under a gaseous atmosphere N ₂ / CO ₂ (80: 20% v / v).

A solution of tungstate-selenite for example the following composition per liter: 0.4 g NaOH, 2 mg Na ₂ Se0 ₃ and 4 mg Na ₂ W0 ₄ .5H ₂ 0. A trace element solution of Widdel has for example the following composition and can be prepared as follows:

Dissolve ferrous chloride in hydrochloric acid. Add the bidistilled water and then the salts of the various trace elements. Adjust the pH between 7.1 and 7.3 with HCl or Na ₂ CO ₃ .

This micronutrient solution may for example be used at a rate of 1 ml per liter of culture medium.

Table 1: Composition of a solution of Widdel trace elements

The culture medium according to the invention may be in the liquid state in which case it comprises distilled water or in a freeze-dried state capable of being regenerated by addition of distilled water or in the solid state, in particular under agar form, more particularly by adding agar, more particularly agar at 2% final.

By "Eggerthella" is meant a bacterial genus of Actinobacteria of the family Coriobacteriaceae. Members of this genus are anaerobic, non-sporulating, non-motile, Gram-positive bacilli that grow in isolation, in pairs or in short chains. They are found especially in the human colon. In the context of the present invention, the sterile extract of a bacterial culture of a bacterium of the genus Eggerthella originates in particular from a bacterium chosen from Eggerthella tenta or the strain called Eggerthella sp. Eg01 -Mx05 deposited under the Budapest Treaty on July 13, 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM 32565.

By "sterile extract" is meant the meaning commonly used by those skilled in the art in the technical field of the invention, that is to say devoid of any viable organism.

In particular in the context of the present invention, the sterile extract is obtained by any technique known to those skilled in the art as long as it does not alter the biological activity of the extract. Sterilization by filtration, for example on a 0.22 μm filter, autoclaving, for example direct autoclaving of the bacterial culture of a bacterium of the genus Eggerthella and direct use of the solution thus obtained or direct autoclaving bacterial culture of a bacterium of the genus Eggerthella and the centrifugation of the solution thus obtained or the use of an antibiotic at the end of culture of a bacterium of the genus Eggerthella coupled or not to a centrifugation step and / or filtration, can be cited for example.

In particular, in the context of the present invention, the sterile extract is a bacterial filtrate of a bacterium of the genus Eggerthella.

By "filtrate" is meant the meaning commonly used by those skilled in the art, that is to say here the liquid collected at the end of the filtration step on a filter of a porosity preventing the passage of living cells.

In particular, in the context of the present invention, the filtrate of a culture of the bacterium of the genus Eggerthella is prepared according to the method comprising:

a) culturing a bacterium of the genus Eggerthella in a medium such as for example the conventional ATCC 260 or 1490 and DSMZ 78 or 209, said medium comprising Biotrypcase, yeast extract, peptone, acids amines, meat extract, hemin, and / or vitamin K1 or K3; and simple sugars; b) incubating the culture medium comprising said bacterium at a temperature of 20 to 40 ° C, preferably at 37 ° C, for at least 1 day, preferably not more than 15 days;

c) filtering the culture medium comprising said bacterium, preferably using filters that do not allow the living organisms to pass, in particular on a filter whose pore size is 0.2 μm; and d) recovering the filtrate obtained at the end of step c).

By "simple sugars" is meant monosides or monosaccharides with 5 or 6 carbon atoms, for example glucose.

In particular, the culture medium of step a) of the filtrate preparation process comprises for example from 0.5 to 50 g / L of Biotrypcase, peptone, casamino acids, yeast extract (preferably 10 g / L) and 0 to 100 mM glucose (preferentially 20 mM).

The temperature mentioned in step b) is between 20 and 40 ° C. It is for example between 30 and 40 ° C or between 35 and 40 ° C. It is therefore for example 21, 22, 25, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39 ° C, in particular 37 ° C.

The incubation is carried out by any technique known to those skilled in the art under anaerobic conditions, for example those described for Eggerthella species tried by microorganism collection organisms such as ATCC or DSMZ.

The incubation lasts at least 1 day and preferably not more than 15 days. It is therefore for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days, in particular 7 days.

The sterilization by filtration mentioned in step c) of the process for preparing a bacterial filtrate may be replaced by any technique known to those skilled in the art to obtain the extract according to the invention in sterile form, as long as the process used does not alter the biological activity of the extract.

In the context of the bacterial filtrate, it is for example carried out by filtration on a filter, preferably using filters that do not allow the living organisms to pass, in particular on a filter whose pore size is 0.2 μm. , and more particularly on a filter having a pore size of 0.22 μm. These filters are commercially available, eg Fisher Scientific Basix ™ PES (polyethersulfone) filters, 25 mm diameter (ref: 13-100-106, Fisher Scientific) adaptable to syringes (ref: SLGP033RS at Merck).

Stage c) makes it possible in particular to eliminate any living Eggerthella cell or any other viable viable contaminant.

The filtrate is recovered at the end of stage c) under conditions allowing its good conservation, in particular under anaerobic atmosphere at ambient temperature or at 4 ° C. if the storage periods extend to several months.

In particular, in the context of the use according to the present invention, the Archaea of the order Methanomassiliicoccales of the commensal clade is an archaea Methanomethylophilus alvus, in particular an archaea Methanomethylophilus alvus Mx-05 pure isolated, more particularly the strain deposited according to the Budapest Treaty, the 18 September 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM32684 (applicant: University Clermont Auvergne, France).

Method of isolating an archaea from the order of the Methanomassiliicoccales of the commensal clade

As mentioned above, the present invention also relates to a method for isolating an archaea of the order Methanomassiliicoccales commensal clade comprising:

a) inoculating a biological sample that may comprise an archaeon of the order Methanomassiliicoccales clade commensal, preferably a sample of animal or human feces under anaerobic conditions under an atmosphere gas containing dihydrogen H ₂ under conditions mesothermic media in a culture medium comprising (i) a base medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, acetate of sodium, Cysteine-HCl, resazurin, Widdel micro-nutrient solution, selenite-tungstate solution, (ii) 2% Na ₂ S and 10% NaHCO ₃ , a mixture of vitamins (by example, vitamin solution of Balch), to which is added (iii) one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract, and (iv) methylated compounds such as methylamines or methanol;

b) replacing the culture medium of step a) with an identical culture medium depleted in the amino acid-providing compound and in which vitamins are added;

d) carrying out series of liquid dilutions or by the roll tube technique of said positive enrichment obtained in step c) using a medium as defined in step a) or b) to which is added a sterile extract of a bacterial culture of bacteria of the genus Eggerthella according to the invention, until obtaining an archaeal clone of the order Methanomassiliicoccales clade commensal.

In particular, the method according to the invention further comprises a step c ') between step c) and step d) in which the culture medium comprising the biological sample is filtered, preferably on a filter whose pore size is at least 0.45 pm, advantageously 0.45 pm, the filtrate obtained at the end of this step c ') being subsequently used in step d) to carry out the dilution series.

The filtration step, preferably on a filter having a pore size of at least 0.45 μm, is optional: it makes it possible to increase the proportion of Methanomassiliicoccales of the commensal clade compared with the other microbes present, because this clade is composed of small cells (of the order of 0.5 pm +/- 0.2), passing in part through the filter, unlike many unwanted cells. This step promotes / accelerates the enrichment in Methanomassiliicoccales.

By "inoculation" is meant the common sense used by those skilled in the technical field of the invention, in particular the action of seeding the culture medium with the biological sample, here under anaerobic conditions.

By "biological sample" is meant a human or animal sample, in particular human or animal stool.

By "anaerobic conditions" is meant the direction known to those skilled in the technical field according to the invention, in particular, conditions in which there is no oxygen in the form of oxygen.

By "gaseous atmosphere" is meant, for example, an atmosphere formed of dihydrogen (in particular 2 bar).

By "mesothermal conditions" is meant a temperature of between 20 and 40 ° C, for example between 30 and 40 ° C or between 35 and 40 ° C. The temperature is, for example, 21, 22, 25, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39 ° C, in particular 37 ° C.

The term "base medium" is a term commonly used by those skilled in the technical field of the invention and denotes a medium comprising compounds necessary for the culture of the arche under the conditions set forth above. Such a medium is for example described in detail in Table 3 of the examples. A solution of Widdel trace elements and a selenite-tungstate solution are defined in the previous section relating to the use of a culture and / or isolation medium comprising a bacterial filtrate of a bacterium of the genus Eggerthella,

The compounds used in the composition of the base medium are compounds available commercially and well known to those skilled in the art.

The base medium is for example prepared under a gaseous atmosphere N ₂ / CO ₂ (80:20% v / v).

The basic culture medium may be in the liquid state in which case it comprises distilled water or in a freeze-dried state capable of being regenerated in whole or in part by adding distilled water or in the solid state, especially in agar form, more particularly by adding agar, more particularly agar to 2% final.

As mentioned above, the biological sample is inoculated into a base medium to which are added compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract and methylated compounds.

Thus, according to one embodiment of the isolation process according to the invention, the biological sample is inoculated into a base medium to which is added Biotrypcase, peptone, casamino acids, yeast extract, and methylated compounds.

The methylated compounds are, for example, methanol and / or methylamines. By "methylamines" is meant, for example, mono-, di- and / or tri-methylamine.

According to one embodiment of the process according to the invention, 1 g / L of yeast extract and 10 to 80 mM of methylated simple compounds are added to the base medium.

In one embodiment of step a) of the aforementioned isolation method, the biological sample comprises an archaea of the order Methanomassiliicoccales commensal clade.

As indicated in step b) of the isolation process according to the invention, the culture medium of step a) is then replaced by an identical culture medium depleted in compounds providing amino acids such as Biotrypcase , peptone and / or casamino acids, in yeast extract and in which vitamins are added.

By "depleted" is meant that the concentration of compounds providing amino acids such as Biotrypcase, peptone or casamino acids and yeast extract in the medium is lower than the concentration initially present in the step at). This concentration may even be zero, that is to say that the compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, and yeast extract may not be present in this second middle.

The vitamins that can be added are vitamins known to those skilled in the art as being suitable.

For example, a solution of vitamins Balch (Balch et al, 1979).

Such a solution can be prepared as indicated below with the composition mentioned in Table 2 in mg per liter of distilled water:

Table 2

The culture medium according to step a) can be replaced after 3 to 15 days, for example 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 days. or the culture medium of step a) is replaced by that of step b) according to the results of monitoring a growth indicator of Methanomassiliicoccales, for example as soon as the regular methane production record of the strain indicates the appearance of a plateau.

As indicated in the aforementioned isolation procedure, the abundance of the archaea of the order Methanomassiliicoccales of the commensal clade is then verified by one of the techniques mentioned in step c).

These techniques are all techniques well known to those skilled in the art for detecting enrichment.

Microscopy makes it possible to determine the shapes of the cells, the measurement of methane and H ₂ is for example carried out by gas chromatography, the qPCR allows the quantification of the populations, and the sequencing the determination of the dominant species of the enrichment. These techniques can be used individually or together. The sequencing is in particular the sequencing of general or specific molecular markers (for example 16S and mcrA, genes coding respectively for the 16S RNA of the small ribosomal subunit 16S and the alpha subunit of the methyl coenzyme M reductase, which is specific to the methanogenesis), or a metagenomic approach to sequencing without a priori.

The isolation as such is then carried out in step d) of the aforementioned process.

The archival enriched medium of the order Methanomassiliicoccales commensal clade is diluted by liquid or solid dilutions, that is to say using techniques known to those skilled in the art of dilution in a liquid medium, for example in Hungate tubes containing culture medium or dilution in solid medium (roll tube also containing culture medium). Dilutions are carried out until an archaeal clone of the order Methanomassiliicoccales clade commensal. "Clone" is used in the same way as "strain", the isolation process according to the invention for the purpose of isolating in its final step an archaeal strain of the order Methanomassiliicoccales pure clans commensal.

The culture medium used in step d) is a medium as defined in step a) or b) to which is added a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella as defined in the context of of the present invention.

The culture medium may therefore comprise a base medium free of complex organic compounds (compounds providing amino acids) but comprising vitamins or a base medium comprising one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract, for example at a concentration of 2 g / L, and methylated compounds,

In the case of solid dilutions, it is possible to transplant the colonies obtained in a liquid medium and then redilute them in a liquid medium, the last positive liquid dilution representing the isolated strain.

In particular, in the isolation process according to the invention, the determination of obtaining an archaeal clone of the order Methanomassiliicoccales commensal clade is performed by microscopy, methane production and consumption of H ₂ , qPCR or sequencing. The sequencing is in particular the sequencing of general or specific molecular markers (for example 16S and mcrA, genes coding respectively for the 16S RNA of the small ribosomal subunit 16S and the alpha subunit of the methyl coenzyme M reductase, which is specific to the methanogenesis), or a metagenomic approach to sequencing without a priori.

In addition, as mentioned above, in one embodiment of the method according to the invention, the method according to the invention further comprises a step c ') between step c) and step d) in which the medium of culture comprising the biological sample is filtered. The filtration step is carried out by any technique known to those skilled in the technical field of the invention.

In particular, the filtration takes place on a filter whose pore size is at least 0.45 μm, advantageously 0.45 μm. These filters are available commercially, for example from Fisher Scientific, in the form of sterile 0.45 μm Basix ™ PES (polyethersulfone) filters (reference 13-100-107).

The filtrate obtained at the end of this step c ') is subsequently used in step d) to carry out the dilution series. In particular, in the context of the process according to the present invention, the sterile bacterial culture extract of a bacterium of the genus Eggerthella is a bacterial filtrate of a bacterium of the genus Eggerthella.

More particularly, said filtrate is prepared according to the process comprising:

a) culturing a bacterium of the genus Eggerthella in a medium such as for example the conventional ATCC 260 or 1490 and DSMZ 78 or 209, said medium comprising Biotrypcase, yeast extract, peptone, acids amines, meat extract, hemin, and / or vitamin K1 or K3, and simple sugars. b) incubating the culture medium comprising said bacterium at a temperature of 20 to 40 ° C, preferably at 37 ° C, for at least 1 day, preferably not more than 15 days;

c) filtering the culture medium comprising said bacterium, preferably using filters that do not allow the living organisms to pass, in particular on a filter whose pore size is 0.2 μm; and

d) recovering the filtrate obtained at the end of step c).

This method is defined in the preceding section relating to the use of a culture and / or isolation medium comprising a sterile bacterial culture extract of a bacterium of the genus Eggerthella,

In particular, in the context of the process according to the present invention, the Archaea of the order Methanomassiliicoccales of the commensal clade is an archaea Archaea Methanomethylophilus alvus, in particular an Archaea Methanomethylophilus Alvus Mx-05 pure isolated, strain deposited according to the Treaty of Budapest, 18 September 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM32684 (applicant: Clermont Auvergne University, France).

Method of cultivation of an arche of the order of the Methanomassiliicoccales of the commensal clade

As mentioned above, the present invention also relates to a method for culturing an archaea of the order Methanomassiliicoccales commensal clade, pure or present in a microbial consortium, comprising:

a) the inoculation of a biological sample comprising an archaea of the order Methanomassiliicoccales commensal clade, preferably a sample of animal or human stool, or a pure archaea of the order of Methanomassiliicoccales, in a medium culture medium comprising a basal medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, Cysteine-HCl , resazurin, a solution of trace elements of Widdel, a solution of selenite- tungstate, Na ₂ S 2% and / or NaHCO ₃ 10%, to which is added (ii) one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids and the yeast extract, (iii) methylated compounds such as methylamines or methanol and (iv) a sterile bacterial culture extract of a bacterium of the genus Eggerthella, according to the invention,

b) placing the inoculum obtained in step a) under anaerobic conditions under a gaseous atmosphere containing H ₂ dihydrogen, and

c) incubation under mesothermal conditions, to reach an exponential growth phase with evolution of methane in at least 2 days, preferably not more than 15 days.

The terms "inoculation", "biological sample", "sterile extract", "pure", "anaerobic conditions", "gaseous atmosphere" and "mesothermal conditions" have the same definitions as those mentioned in the previous sections.

By "microbial consortium" is meant a set of microorganisms, consisting of different species (ie not occurring in pure culture) and living in community.

The incubation lasts at least 2 days and preferably not more than 15 days. It is therefore for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.

The incubation is carried out by any technique known to those skilled in the art such as for example in Hungate tube.

As mentioned above, the base medium is composed of KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, Cysteine-HCl , resazurin, a solution of Widdel trace elements, a selenite-tungstate solution, 2% Na ₂ S and / or 10% NaHCO ₃ , to which is added (ii) one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, yeast extract, (iii) methylated compounds such as methylamines or methanol and (iv) a sterile extract from a culture. bacterium of the bacterium of the genus Eggerthella, according to the invention.

In addition, the medium may comprise coenzyme M. This co-factor is indispensable for methanogenesis. In the case of the Archaea Methanomethylophilus alvus Mx-05, coenzyme M can be omitted.

In particular, in the context of the method according to the present invention, the sterile extract of a bacterial culture of a bacterium of the genus Eggerthella is a bacterial filtrate of a bacterium of the genus Eggerthella.

More particularly, said filtrate is prepared according to the process comprising: a) culturing a bacterium of the genus Eggerthella in a medium such as for example the conventional ATCC 260 or 1490 and DSMZ 78 or 209, said medium comprising Biotrypcase, yeast extract, peptone, acids amines, meat extract, hemin, and / or vitamin K1 or K3, and simple sugars; b) incubating the culture medium comprising said bacterium at a temperature of 20 to 40 ° C, preferably at 37 ° C, for at least 1 day, preferably not more than 15 days;

d) recovering the filtrate obtained at the end of step c).

In particular, in the context of the method according to the present invention, the Archaea of the order Methanomassiliicoccales of the commensal clade is an archaea Archaea Methanomethylophilus alvus, in particular Methanomethylophilus Alvus Mx-05 pure isolated strain deposited under the Treaty of Budapest , on September 18, 2017, from the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM DSM32684 (applicant: University Clermont Auvergne, France).

The present invention also relates to an Archaea archaea detection kit according to the invention for the implementation of an isolation method according to the invention or of a culture method according to the invention characterized in that it includes:

one or more culture media such as those mentioned above in the context of the invention, preferably lyophilized or in solid form, and / or

reagents for carrying out an enzymatic amplification of DNA of the PCR type, and / or

a sterile extract of a bacterial culture as mentioned above in the context of the invention.

The present invention will be further illustrated by the following examples.

Examples

Composition of a basic medium (MB) for 1 liter:

Table 3

After autoclaving, add:

Composition of the Balch vitamin solution (Balch et al, 1979)

Table 4 (in mg per liter of distilled water)

Composition and preparation of a Widdel trace elements solution

Ferrous chloride was dissolved in hydrochloric acid. Bidistilled water was added and the salts of the various trace elements. The pH was adjusted between 7.1 and 7.3 with HCl or Na ₂ CO ₃ .

This micronutrient solution was used at a rate of 1 ml per liter of culture medium. Table 5

Enrichment in Archaea Methanomethylophilus alvus Mx-05

The basal medium was prepared anaerobically under a N ₂ / CO ₂ gas atmosphere (80: 20% v / v). The enrichments were carried out in this MB base medium in which 1 g / L of yeast extract and 10 to 80 mM of methyl compounds (methanol, methylamines) were added as final electron acceptors. They were conducted in the presence of an anaerobic gaseous atmosphere, formed of H ₂ dihydrogen as electron donor (2 bar), under mesothermal conditions (incubation of 20 to 40 ° C).

The culture medium subsequently used (containing Balch's vitamin solution) was identical except that it was depleted of yeast extract (until it was totally absent (0 g / l of yeast extract). ) depending on the case) in order to obtain a dominant population of the Archaea Methanomethylophilus alvus Mx-05.

Verification of the abundance of the Archaea Methanomethylophilus alvus Mx-05 was performed by: - Microscopy to determine cell shapes and the degree of purity of our enrichment;

- Measurement of methane production and H ₂ consumption by gas phase chromatography;

qPCR for quantifying populations of bacteria and archaea, using primers and protocols described and known to those skilled in the art (Borrel et al, 2017);

- Sequencing to determine the dominant species of enrichment.

The dilution experiments in a liquid medium (Hungate tubes containing 5 ml of culture medium) or in solid medium (roll tubes also containing 5 ml of culture medium) in the presence of H ₂ and methyl compounds were then carried out. They were carried out by prior filtration (filter 0.45pm) to eliminate a majority of bacilli, the archaea Methanomethylophilus alvus Mx-05 pure isolated in the form of small size shells, including cells smaller than 500pm of diameter. The growth of Archie Methanomethylophilus alvus Mx-05 was only achieved in the presence of a sterile extract from a culture of Eggerthella sp. prepared by filtration (0.22 μm filter) (see preparation of the filtrate below).

Preparation of the filtrate of Eggerthella sp.

Eggerthella sp. strain Eg01 -Mx-05 (strain deposited under the Budapest Treaty on July 13, 2017, at the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM 32565 was grown in the base medium containing 10 g / L of Biotrypcase, 10 g / L of yeast extract, 10 g / L of peptone, 10 g / L of casamino acids and 20 mM of glucose The incubation was carried out at 37 ° C. for one week After filtration of the culture medium (filter of 0.22 μm) of Eggerthella, 0.5 ml of filtrate was added per 5 ml of culture medium contained in the tubes for diluting the archaeal enrichment Methanomethylophilus alvus Mx-05.

Isolation of the Archaea Methanomethylophilus alvus Mx-05 in pure form

In order to obtain the archaea Methanomethylophilus alvus Mx-05 pure isolated in axenic culture, dilution series in liquid or solid media were carried out using MB base medium free of complex organic compounds (eg yeast extract, biotrypcase, etc.) but in the presence of the solution of vitamins Balch and Widdel trace elements solution or on the contrary by adding these compounds (yeast extract, biotrypcase, peptone, casaminoacids at a rate of 2 g / L). This last operation made it easier to identify the possible presence of heterotrophic contaminants in the last positive dilutions. After obtaining colonies in solid medium, they were subcultured in liquid medium and then rediluted in liquid medium, the last positive liquid dilution representing the collection strain to be studied. The archaeal strain Methanomethylophilus alvus Mx-05 pure isolated was deposited with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM32684 (applicant: Université Clermont Auvergne, France) on September 18, 2017.

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Claims

claims

1 Archée Methanomethylophilus alvus Isolated Mx-05 pure, strain deposited under the Budapest Treaty, on September 18, 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM32684.

2.- Use of a culture medium and / or isolation comprising a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella, for the isolation and / or the pure culture of an archaea of the order of the Methanomassiliicoccales of the commensal clade.

3. Use according to claim 2, wherein the medium further comprises one or more compounds providing amino acids such as Biotrypcase, peptone and / or casamino acids, and yeast extract.

4. Use according to any one of claims 2 or 3, wherein the medium further comprises coenzyme M.

5. Use according to any one of claims 2 to 4, wherein the medium further comprises KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ CI, CaCl ₂ .2H ₂ 0, sodium acetate, Cysteine-HCI, resazurin, Widdel trace element solution, selenite-tungstate solution, 2% Na ₂ S and / or 10% NaHCO 3.

6. Use according to any one of claims 2 to 5, wherein the bacterium of the genus Eggerthella is selected from Eggerthella tenta or the strain Eggerthella sp. Eg01 -Mx05 deposited under the Budapest Treaty on July 13, 2017, with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under the number DSM 32565.

7.- A method of isolating an archaea of the order Methanomassiliicoccales commensal clade comprising:

a) inoculating a biological sample that may comprise an archaeon of the order Methanomassiliicoccales clade commensal, preferably a sample of animal or human feces under anaerobic conditions under an atmosphere gas containing dihydrogen H ₂ under conditions mesothermic agents in a culture medium comprising (i) a base medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, Cysteine-HCl, resazurin, a solution of Widdel trace elements, a solution of selenite-tungstate, (ii) 2% Na ₂ S and 10% NaHCO ₃ , a mixture of vitamins, to which is added (iii) one or more compounds providing amino acids such as Biotrypcase, peptone and or casamino acids, and yeast extract, and (iv) methyl compounds such as methylamines or methanol;

b) replacing the culture medium of step a) with an identical culture medium depleted in compounds providing amino acids and in which vitamins are added;

d) carrying out series of liquid dilutions or by the roll tube technique of said positive enrichment obtained in step c) using a medium as defined in step a) or b) to which is added a sterile extract of a bacterial culture of a bacterium of the genus Eggerthella as defined in any one of claims 2 to 6, until obtaining an archaeal clone of the order Methanomassiliicoccales clade commensal.

8. The isolation method according to claim 7, further comprising a step c ') between step c) and step d) in which the culture medium comprising the biological sample is filtered, preferably on a filter whose pore size is at least 0.45 pm, the filtrate obtained at the end of this step c ') being subsequently used in step d) to carry out the dilution series.

9. The isolation method according to any one of claims 7 or 8 wherein the determination of obtaining an archaeal clone of the order of Methanomassiliicoccales clade commensal is carried out by microscopy, production of methane and H ₂ consumption, PCR, qPCR or sequencing.

10.- Method of cultivating an archaea of the order Methanomassiliicoccales commensal clade, pure or present in a microbial consortium, comprising:

a) the inoculation of a biological sample comprising an archaea of the order Methanomassiliicoccales commensal clade, preferably a sample of animal or human stool in a culture medium, or a pure archaea of the order of Methanomassiliicoccales comprising (i) a basal medium comprising KH ₂ PO ₄ , NaCl, MgSO ₄ .7H ₂ O, NH ₄ Cl, CaCl ₂ .2H ₂ O, sodium acetate, Cysteine - HCI, resazurin, a solution of Widdel trace elements, a solution of selenite-tungstate, Na ₂ S 2% and / or NaHCO ₃ 10%, to which is added (ii) one or more compounds providing acids amino acids such as Biotrypcase, peptone and / or casamino acids, and yeast extract, (iii) methylated compounds such as methylamines or methanol and (iv) a sterile extract of a bacterial culture of the bacterium of the genus Eggerthella, as defined in any one of claims 2 to 6,

1 1. The method of claim 10, wherein the medium further comprises coenzyme M.

Use according to any one of claims 2 to 6, process according to any one of claims 7 to 9, or method according to claim 10 or 11, wherein or wherein the sterile extract of a culture bacterium of a bacterium of the genus Eggerthella is a bacterial filtrate of a bacterium of the genus Eggerthella.

The use according to claim 12, the method according to claim 12, or the method of claim 12, wherein or wherein the bacterial filtrate of a bacterium of the genus Eggerthella is prepared according to the process comprising:

Use according to any one of claims 2 to 6 and 12 to 13, method according to any one of claims 7 to 9 and 12 to 13, or method according to any one of claims 10 to 13, wherein in which the archaea of the order Methanomassiliicoccales commensal clade is an archaea Methanomethylophilus alvus, in particular an arche as defined in the subject of claim 1.