FR2977589A1 - New Archaea Methanomassiliicoccus luminyensis isolated and established in culture, comprising 16S ribosomal DNA gene-specific sequence or a complementary sequence, useful for diagnostic purposes - Google Patents

Abstract

Archaea Methanomassiliicoccus luminyensisisolated and established in culture, comprising 16S ribosomal DNA (rDNA) gene-specific sequence of SEQ ID NO. 1 of the Sequence Listing or a complementary sequence, is new, where the sequence not defined here may be found at ftp://ftp.wipo.int/pub/published-pct-sequences/publication. Independent claims are included for: (1) the preparation of Archaea; (2) a method (I) of determining the presence of Archaea in a biological sample, preferably in a sample of human or animal feces, comprising extracting the DNA from the biological sample and detecting, preferably specifically quantifying Archaea DNA by enzymatic amplification of PCR type, preferably of real-time PCR with a pair of primers, preferably probe, sequences from the 16S rDNA sequence of SEQ ID NO: 1; and (3) a kit useful for the above method (I), comprising reagents for amplifying DNA comprising amplification primers and probe for detecting Archaea Methanomassiliicoccus luminyensis, including constituents of the oligonucleotides primers and probes derived from SEQ ID NO: 1, preferably SEQ ID NOs: 2 (5'-gttcgtagccggtctggtaa-3'), 3 (5'-ccaagtctggcagtctcctc-3') or 4 (5'-tcgggaaggttaactttccgacttcc-3'), preferably any specific primers of Archaea of sequence comprising SEQ ID NOs: 5 (5'-agccgccgcggtaayac-3') and 6 (5'-cyggcgttgavtccaatt-3'), and reagents for extracting DNA comprising at least one pulverulent abrasive product, preferably glass powder and a reagent of chemical and/or enzymatic lysis.

Description

The present invention relates to a new Archaea isolated and established in culture, a culture medium of said Archaea, a method of preparation of said culture medium and methods for culturing and detecting said Archaea. The Archaea are prokaryotes constituting one of the four areas of life alongside eukarya, bacteria and giant DNA viruses [16]. Initially isolated from the environment and in particular the extreme environments that earned them their name extremophiles, Archaea were then detected in various invertebrate and vertebrate animals including birds and mammals. In mammals, it is the methanogenic Archaea capable of detoxifying hydrogen with methane synthesis that have been essentially detected and these Archaea are therefore of considerable importance now since it is considered that the methane emission by the cattle report constitutes the essential source of destruction of the ozone layer in the environment. In humans as well, meta genomic analyzes have shown the presence of specific DNA signatures of several species of Archaea that belong essentially to both phyla Euryarcheaota and Archaea halophiles; more particularly, the sequences detected in humans indicate the presence of Methanosarcina, Thermop / asma, Crenarchaeaota, and halophilic Archaea as well as a last order of methanogens 1 to 6). Of all these Archaea, only three species of Archaea have been isolated from humans: Methanobrevibacter smithu [7J, Methanosphaera stadtmaniae [8], and Methanobrevibacter ora / is [9] and only M. smithii and M. stadtmanae have been isolated. from the digestive tract. In humans, these Archaea methanogens participate in the homeostasis of the digestive flora by detoxifying hydrogen with methane production and thus participate in the regulation of pH. Also, some Archaea are involved in oral pathology in polymicrobial infection during periodontal disease. It has been shown that the digestive carriage of Archaea methanogens was constant in all individuals [12], and previous work by metanogenomic studies and molecular analyzes of human digestive flora showed a prevalence of Archaea of about 30%. tested individuals and a variety of DNA sequences explained above.

To isolate M. smithii, the authors (7) used a faecal sample inoculated in Balch's medium 1 (composition shown in Table 3 below) [10] under an atmosphere of H2-CO2 (80: 20) pressurized to 2 atmospheres (202.6 kPa), with stirring. After 2 to 3 hours, 0.5 ml samples of the atmosphere were analyzed for the presence of methane by gas chromatography. Methane-producing and fluorescent isolates for factor-420 (as determined by epifluorescence microscopy) were subcultured on Balch medium 1 supplemented with 2% agar until pure culture was obtained. ; purity criteria were uniform cell morphology, methane production, factor-420 fluorescence of all cells, and absence of growth in a complex medium (SMS) containing substrateless carbohydrates for methanogenesis. To isolate M. stadtmanae, the authors (9) used an E1 culture medium (Table 4) modified by the addition of 30% (v / v) rumen fluid, clindamycin and cephalothin, respectively, of 0.4 % methanol and an atmosphere composed of N2,80% and CO2, 20%. The culture was maintained by monthly transfers (10% vol.) In the same medium. A fluorescent bright spherical morphology of factor 420 was the dominant morphotype. At day 60, the culture was subcultured in Balch's Modified Medium 1 by addition of rumen fluid, NH4Cl, cepthalothin and clindamycin (7) with methanol under an atmosphere of 80% H2: and CO2, 20%. A pure culture of a spherical-methanogenic archaea has been obtained. To isolate M. ora / is, the authors used substantially the same culture medium as that used to isolate M. smithii (9). Archaea are microorganisms of medical and veterinary interest given their role in physiology and possibly in pathology. The purpose of the present invention was therefore to isolate and establish in culture a new Archaea whose existence had been presumed in the digestive flora in humans by previous work (12). To do this, the inventors have determined the specific media and conditions for isolating and culturing said Archaea, which has enabled them to have enough DNA from said Archaea to determine the complete sequence of the 16S rDNA gene and thereby confirm its specificity and novelty of said Archaea. More specifically, the present invention provides a new isolated and cultured Archaea comprising the specific sequence of the 16S rDNA (SEQ ribosomal DNA) gene. ID. No. 1 in the sequence listing which the inventors have named Methanomassi / iicoccus / uminyensis. More particularly the subject of the present invention is an Archaea deposited at the DSMZ microorganism depot collection (Germany), in accordance with the Budapest Treaty, under number DSM 24529 on June 23, 2011, by the University of the Mediterranean, Faculty of Medicine Timone, Marseille, France. This new Archaea could not be isolated and established in culture using the media and culture conditions previously described for Archaea previously isolated and established in culture, including Archie methanogens.

The subject of the present invention is also a sterilized culture medium useful for the cultivation of an Archaea according to the invention comprising a methanogenic Archaea culture base medium, characterized in that it further comprises: a-methanol and b- a tungsten salt and / or a selenium salt.

The inventors emit the hypothesis without being bound by this theory, that methanol acts here as electron acceptor and the tungsten and selenium salts as cofactors of an enzyme involved in electron transfer. Preferably, said culture medium comprises: a- methanol, and b- a tungsten salt and a selenium salt. According to other particular characteristics: the methanol is present at a concentration of 1 to 100 mM, preferably 10 to 50 mM, and the said tungsten salt is a sodium tungstate, and the said selenium salt is a sodium selenite. and said tungsten salt is present at a concentration of 0.3 to 3 M, preferably 0.5 to 1.5 M; and said selenium salt is present at a concentration of 0.2 to 2M, preferably 0.5 to 1M. In the concentration values above: 1M = 1mol / I More particularly, said medium Archaea culture base comprises at least: - several sources of carbon and nitrogen, preferably selected from a yeast extract, an acetate salt and a tryptic peptone, - a source of phosphorus, preferably a salt phosphate, - a source of nitrogen, preferably an ammonium salt, - at least one salt of each of the metals K, Mg, Na and Ca, - a reducing substance, more particularly chosen from cysteine-HCI and Na2S a pH-regulating buffer substance for adjusting the pH of 7 to 8; trace elements, preferably salts selected from B, Fe, Cu, Zn, Mn, Co, Ni and Mo salts, more preferably trace elements of Widdel, - vitamins, preferably vitamins in the middle of Balch, and - preferably, a substance orant control of anaerobic, preferably resazurin.

The Widdel trace elements are listed in Table 6 and include: FeCIZ, CoCIZ, MnCIZ, ZnCIZ, H3BO3, Na2MoO4, NiCIZ and CuCIZ. Vitamins from Balch's medium include biotin, folic acid, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, nicotinic acid, DL-calcium pantothenate, vitamin B12, p-aminobenzoic acid, lipoic acid. More particularly, said culture medium according to the invention contains a base medium comprising the following species: KH 2 PO 4, MgSO 4, NaCl, NH 4 Cl, CaCl 2 .2H 2 O, NaCOOCH 3 (Na acetate), cysteine, yeast extract, trypticase peptone, NaOH , Na2S, NaHCO3 (Na carbonate), NaHCO2 (Na formate), Na2SeO3, Na2WO4.5H2O and preferably resazurin, widdel oligoelements, Balch vitamins, the pH being adjusted to 7.5 with KOH. More particularly, said culture medium comprises, in addition to said base medium: a-methanol at a concentration of 10 to 100 mM, preferably 40 mM, and b- a salt of Nat SeO3 at a concentration of 1 to 10 mg / I, preferably 2 mg / l, and c- a salt of Na2WO4.5H2O at a concentration of 1 to 10 mg / l, preferably 4 mg / l. 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. The subject of the present invention is also a method for preparing a sterilized culture medium according to the invention, characterized in that the following successive steps are carried out in which: one sterilizes in an autoclave a said culture medium of Incomplete archaea not containing degradable components in an autoclave comprising methanol, said vitamins, labile sodium salts comprising NaHCO3, NaHCO2 and Na2S, preferably in an autoclave at a temperature above 120 ° C for at least 15 min, or a temperature of 110 ° C for at least 45 minutes, and 2-the oxygen is removed, preferably by bringing said culture medium to boiling under a nitrogen atmosphere, and 3-place said medium of incomplete sterilized culture in an anaerobic atmosphere, and 4-is cooled to room temperature and added in said incomplete culture medium, said components likely to be degraded by sterilization in an autoclave comprising methanol, said vitamins, labile sodium salts including NaHCO3, NaHCO2 and NaZS. The subject of the present invention is also a process for cultivating an Archaea with a culture medium according to the invention in which the following successive steps are carried out: a biological sample which can contain a said Archaea is inoculated in the said medium; in anaerobic culture, preferably in a nitrogen atmosphere, and 2 said culture medium is placed under an atmosphere of anaerobiosis and containing at least 50% hydrogen at a pressure of less than 2 atm, and 3- incubation at a temperature of 20 to 40 ° C, preferably at 37 ° C to reach an exponential growth phase with evolution of methane in at least 5 days, preferably not more than 15 days, more preferably not more than 10 days days. Preferably, in step 2, said culture medium is placed under a HZ / CO2 compound atmosphere in a volume proportion of 80/20 at a pressure of 1 atm.

The media and method of cultivation above made it possible to establish in culture said archaea, that is to say that it can thus be under cultivated indefinitely.

More particularly, it is possible to obtain an exponential growth phase culture in no more than 15 days, preferably 5 to 10 days, this growth phase being characterized by: (1) a methane production of at least 10 μM, as determined by gas chromatography, and (2) fluorescence microscopic visualization of spherical microorganisms of size 0.5 to 1 μm, and self fluorescent at 420 nm. More particularly, the subcultures will be carried out by transplanting 1/5 to 1/10 of the culture into said growth phase.

It should be observed that, in the media and culture conditions according to the present invention, certain Archaea isolated and cultivated to date and in particular M. smithii can not be established in culture. On the other hand, it is possible that other Archaea than M. uminyensis may be isolated and established in culture with the media and culture conditions according to the present invention.

Finally, the present invention also has a method for determining the presence of an Archaea in a biological sample, preferably in a sample of animal or human stools according to the invention or an Archaea grown according to the process of the invention. characterized in that the DNA is extracted from said biological sample and the DNA of said Archaea is detected and preferably specifically quantified by PCR-type PCR type enzyme amplification in real time with a pair of primers and preferably a probe of sequences derived from the 16S gene rDNA sequence SEQ.ID No. 1. More particularly, said pair of primers and preferably said probe have the sequences chosen from the following sequences and complementary sequences derived from the 16S rDNA gene: 5 'direct primer: SEQ.ID. No. 2 = 5'-GTTCGTAGCCGGTCTGGTAA-3 'Reverse 3' primer: SEQ.ID. No. 3 = 5'-CCAAGTCTGGCAGTCTCCTC-3 'Probe: SEQ.ID. No. 4 = 5'-TCGGGAAGCTTAAC 1 I I CCGACTTCC-3.

More particularly, PCR-type enzymatic amplification is carried out beforehand specifically of the DNA of all Archaea contained in said DNA extracted from the sample with a pair of consensus primers of sequences chosen from the following sequences and complementary sequences derived from 16S rDNA gene Direct 5 'primer: SEQ.ID. No. 5 = 5'-AGCCGCCGCGGTAAYAC-3 'Reverse 3-primer: SEQ.ID. No. 6 = 5'-CYGGCGTTGAVTCCAATT-3 '. These universal primers of all Archaea were initially determined by the inventors to enable identification and determination of the 16S gene of the rDNA of said Archaea. The present invention therefore also relates to a kit for detecting an Archaea according to the invention for the implementation of a detection method according to the invention characterized in that it comprises: - a said culture medium of preferably lyophilized or in solid form, and reagents for carrying out an enzymatic amplification of DNA of the PCR type, and oligonucleotides constituting said primers and probes derived from SEQ ID NO: 1, preferably selected from SEQ.ID.No. 2-6. Other features and advantages of the present invention will be apparent from the detailed examples which follow. Example 1: Molecular technique for the detection of the partial ribosomal 16S gene of the new Archaea Methanomassi / iicoccus / uminyensis The inventors first of all developed a molecular technique based on the detection of the 16S ribosomal gene by using a PCR technique followed by cloning of the amplification products and sequencing of the clones. To do this, the prokaryotic DNA was extracted according to a procedure described in WO 2010/130914 as follows: approximately 1 gram of stool was resuspended in 10 ml of buffer and filtered (Diagnostica-Fumouze-Orion Division, Levallois Perret , France).

The filtrate (250 ml) was transferred to a sterile Eppendorf tube containing 0.3 g of acid-treated glass beads (# 106 mm, Sigma, Saint-Quentin Fallavier, France) and shaken to perform mechanical lysis at room temperature. using the FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) used at level 6.5 (at maximum speed) for 90 seconds. The supernatant was incubated overnight at 56 ° C with 180 ml of NucleoSpinH tissue kit T1 buffer (Macherey Nagel, Hoerdt, France) and 25 ml of proteinase K (20 mg / ml). After a second cycle of mechanical lysis as described above, the filtrate was incubated for 15 minutes at 100 ° C. The total DNA was then extracted using the NucleoSpinH kit according to the manufacturer's recommendations. The extracted DNA was eluted in 100 μl of elution buffer (15) [Dridi B et al 2009]. To do this, the inventors have determined a pair of consensus PCR primers (SEQ.ID.No. 5 and 6 and Table 1) for amplifying by PCR a fragment of the 16S ribosomal DNA gene of any Archaea after aligning the sequences of the 16S ribosomal DNA gene from 20 EuryArchaeaota organisms. The PCR amplification program included 95 ° C for 15 min followed by 45 cycles of 95 ° C for 30 seconds and 60 ° C for 1 min. All DNA samples were examined twice. The results were expressed as the number of copies of the 16 S rDNA genes per gram of stool. Plasmid quantification was performed by inserting a chimeric nucleotide fragment into the plasmid of the ACP II (TA cloning kit (pCRII) Invitrogen, Carlsbad, CA, USA). The chimeric fragment contained real-time PCR targets for the rpoB gene of M. smithii and M. stadtmanae for quantification of these two Archaea as well as a fragment of the 16S gene of the Streptomyces sudanensis SD504 r (number GenBank accession number EF515876) as an internal control to ensure the absence of PCR inhibition The PCR amplification products were cloned into competent Escherichia coli / JM109 bacteria using the vector pGEM-T Easy Vector System ( Promega, Charbonnières-les-Bains, France) and 100 recombinant colonies were verified for the presence of an insert using the universal PCR primers M13d / M13r (Table 1) .The PCR products were purified and sequenced using the kit BigDye terminator and sequencer 3130 (Applied BioSystem) Negative controls were used for each analysis.The sequences were analyzed using the program Seqscape (Applied BioSystem), and similarity values were determined using NCBI's BLAST online program.

In this work, the inventors identified in four samples out of 20 samples analyzed (ie in 89 clones) a partial sequence of the 16S rDNA gene of 461-bp deposited by the inventors in GenBank under the number GenBank F3823135. This sequence has only 78.8% similarity to the homologous sequence of the M. smithii 16S rDNA gene. This similarity was compared by aligning the F3823135 sequence with the corresponding region of the M. smithii 16S DNA sequence available in GenBank under the number U55233. The alignment was done using the online program MULTALIN of the Lyon bioinformatics cluster (http://npsapbil.ibcp.fr/cgi bin / npsa_automat.pl? Page = / NPSA / npsa_multalinan.html). Using the same comparison technique, the inventors observed that the original GenBank sequence F3823135 determined by the inventors had a similarity percentage of 98.7% with a sequence previously deposited in GenBank under the title "human faeces clone E18_1652" and under number (F3752571); and exhibited 98% sequence similarity with a sequence previously deposited in GenBank under the title "human faeces clone E20 16S1" and accession number F3752572, both of which correspond to a published work (6) by Mihajlovski and a / ., 2010. This sequence has been identified as a partial 16S ribosomal DNA sequence described in the literature as representative of a new order, a new family, a new genus, and a new species. 'Archaea detected independently by two research teams in Ireland (1.14) and France (5.6) without being isolated and cultivated. The inventors have furthermore carried out a phylogenetic analysis of the partial sequence which they have determined using a maximum likelihood analysis and by plotting the phylogenetic tree by the PhyML software. This phylogenetic analysis indicated that this new sequence was included in a group of sequences deposited in GenBank by different authors who had obtained these sequences from stool of different animals and other environmental sources, corresponding to uncultivated and non-cultured Archaea. isolated, but indicating that it was a new order of Archaea (Figure). From the demonstration of this partial indicative sequence of the 16S rDNA gene of a new Archaea species, the inventors have developed a real-time PCR specific for this clone after having determined M.-specific primers and probe. / uminyensis (SEQ.ID.No. 2-4 and Table 1), allowing them to determine the prevalence of this clone in a large number of stool samples (500). The specificity of these primers and probes was experimentally verified by incorporating DNA extracted from a bacterial representative of 43 species of common gut inhabitant microbes and enteric pathogens, including M. stadtmanaeet and M. smithiien both positive controls (Table 2). The real-time PCR protocol was as follows. Taqman primers and probes used for the real-time gPCR of the total bacterial DNA with the consensus primers and probes described in Dridi B. et al., PIoS ONE, 2009; 4; e7063 primers and "all bacteria" probe and listed in Table 1. Real-time PCR assays were performed with an MX3000TM system (Stratagene, Amsterdam, The Netherlands) using the QuantiTect probe PCR kit (Qiagen, Courtaboeuf, France) with 5 pmol of each primer and probe labeled with FAM or VIC, and 5 μl of DNA in a final volume of 25 μl. The copy number of the rDNA 16S gene was measured in duplex analysis, and total bacterial DNA detection was used in the real-time gPCR as a DNA extraction control. Thus, the inventors were able to demonstrate the presence of this 16S ribosomal gene sequence in only 4% of the stool samples they analyzed by PCR according to the following protocol: 15 min at 95 ° C., followed by 50 cycles comprising 95 ° C for 30 seconds, 56 ° C for 45 seconds and 72 ° C for 1 min; followed by a final 5-min elongation step at 72 ° C. The complete sequence of the 16S rDNA gene of the new Archaea SEQ.ID.No.1 could only be determined after the isolation and pure culture of this new Archaea according to Example 2 below and determination. of the sequence of its genome as described in Example 3 below. EXAMPLE 2 Culture of Archaea Methanomassi / iicoccus / uminyensis The inventors have selected three stool samples that do not have Methanosphaera stadtmanae and that present, by a real-time PCR technique, an optimum ratio between Methanobrevibacter smithii (in the lowest possible concentration). ) measured by the largest possible number of real-time PCR cycles and the lowest number of real-time PCR detection cycles for the new non-isolated Archaea. During this work, the primers, the probes (Eurogentec, Seraing, Belgium) of M. smithii and M. stadtmanae used were already described (15) and are summarized in Table 1.

The specificity of said primers and probes was then also experimentally verified by incorporating DNA extracted from a bacterial representative of 43 species of common intestinal inhabitants and enteric pathogenic microbes of Table 2. The same protocol as above was implemented to perform a real-time PCR. The Taqman primers and probes used for the real-time gPCR of the total bacterial DNA were adapted from the method previously described by Palmer et al. Real-time PCR analyzes were performed with an MX3000TM system (Stratagene, Amsterdam, The Netherlands) using the QuantiTect Probe PCR Kit (Qiagen, Courtaboeuf, France) with 5pmol of each primer and probe labeled with FAM or VIC, and 5 μl of DNA in a final volume of 25 μl. The copy number of the 16S rDNA gene was measured in a duplex analysis, and the total bacterial DNA detection was used in the real-time gPCR as a DNA extraction control. The inventors then used these three stool samples to attempt the isolation and cultivation of the new methanogenic Archaea species which they called Methanomassi / iicoccus / uminyensis. Archaea culture media and conditions described in the literature , especially for the 3 human species M. smithii, M. stadtmanae and M. ora / is among others did not allow to cultivate the new species. Some of the many different culture tests of said Archaea are reported hereinafter. Attempts to isolate Methanomassi / iicoccus / uminyensis began with the inoculation of 200 μL of the stool samples in question into a base medium comprising constituents separately described in the literature for previously isolated archaea containing in g / L: KH2PO4 (0.3), K2HPO4 (0.3), MgCl2 30x6H2O (0.5), KCl (0.1), NaCl (1) NH4Cl (1), CaCl2 x2H2O (0.1), cysteine-HCl (0.1), yeast extract (1), trace element solution of Balch (2.5 ml / L), resazurin (0.001 g / L) and distilled water (qsp); the pH was adjusted to 7 with KOH (10 M) before autoclaving. The medium was boiled under N2 gas to remove the maximum oxygen and cooled to room temperature. The medium was dispensed in 5 ml Hungate tubes or in anaerobic vials under N2 / CO2 (80:20, v / v) at atmospheric pressure. The tubes and / or flasks are then autoclaved for 45 minutes at 110 ° C. After autoclaving, the medium is supplemented with NaHCO 3 (4 g / l), Na 2 S x 9 H 2 O (0.5 g / l), Na-formate (2 g / l) and 10 ml / l of a filtered solution. using a 0.22 μm filter to sterilize it, Balch vitamins (Balch et al., 1979) (10).

This first way of cultivating methanogenic Archaea was unsuccessful, since a small release of methane was only observed after two months of cultivation without reaching an exponential phase of growth. From this basic medium, the inventors have tested a very large number of different culture conditions by trying to add different substrates before or after autoclaving. As an illustration, the conditions approaching the middle of the establishment of a culture were as follows: Condition 1: Acetate added at the concentration of 20 mM. Condition 2: Methanol added at a concentration of 40 mM. Condition 3: Methanol added at the concentration of 40 mM + H2 / CO2 (80/20) at a pressure of 2 bars because it is the optimal pressure of the majority of the methanogens using this substrate: H2 / CO2 (80/20) - Condition 4: H2 / CO2 (80/20) at a pressure of 2 bar without methanol. No methane production was recorded under conditions 1, 2 and while a small amount of methane was detected by gas chromatography one month after inoculation for conditions 3 and 4 without the culture changing. remarkable way. The tubes corresponding to this production of methane were then subcultured in roll tubes containing the same base medium with the same substrates of conditions 2 and 3, ie in the presence of methanol which the inventors then suspected to be a substance necessary for growth, solidified with 0.08g of agar. These tubes were prepared according to the technique of Hungate (13) []. Low methane production was observed 3 months after inoculation in tubes under condition 2 with appearance of some irregularly shaped white colonies.

Several colonies were subcultured in liquid medium, but the production of methane remained low and was only detectable several weeks after transplanting corresponding to a very weak inoculum, too weak to allow for example the deposit of the strain in collections of deposit. It has therefore been necessary to make further modifications in the basal medium, on the one hand, and in supplementation substrates after autoclaving or culture conditions, on the other hand. After a large number of tests with changes in the culture media and changes in culture conditions, the inventors were able to determine a culture medium and culture conditions of Archaea Methanomassi / iicoccus / uminyensis, making it possible to obtain a strain. (clone B10T) filed as DSM 24529, verifying eligibility and viability requirements in a microorganism deposit collection under the Budapest Treaty (DSMZ Germany). In particular this strain allowed a fast and important methane production which starts 48 hours after inoculation and reaches its maximum between the 5th and the 10th day.

The inventors varied the pressure and the gaseous culture medium. But above all it was necessary to supplement the base medium by the addition of Selenite and sodium tungstate before autoclave and addition of methanol (40 mM) after autoclave. The inventors therefore began by using a base medium containing base constituents common to all previously grown Archaea methanogens. The tests led the inventors to test a background medium from Medium 119 (http://www.dsmz.de, Table 4) which is a specific medium to the genus Methanobrewbacter, as a basal medium, methane production. was greater than that obtained with the first basal medium but still insufficient.

In particular, the inventors have tested the addition of a solution of Selenite and sodium tungstate on several previous basal media under different gaseous atmosphere and pressure medium including 1, 2 and 2.5 bars. Only media supplemented with Selenite and sodium tungstate under an atmosphere containing more than 500% hydrogen at a pressure below 2 bar gave a detectable methane production in only 7 days after inoculation. The 119 medium supplemented with methanol and the tungsten and selenium salts and culture conditions above made it possible to establish the new Archaea in culture.

The optimal culture medium derived from medium 119 contained in g / L: KH 2 PO 4 (0.5), MgSO 4 x 7H 2 O (0.4), NaCl (5), NH 4 Cl (1), CaCl 2 x 2H 2 O (0.05), Na-acetate (1.6). ), cysteine-HCl (0.5), yeast extract (1), trypticase peptone (1), Widdel trace element solution (1 ml / L), 2 mL / L of Selenite-Tungstate solution (NaOH 0.4) g / L, Na2SeO3 2 mg / L, Na2WO4.5H2O 4 mg / L), resazurine (0.001 g / L) and distilled water (qsp); the pH is adjusted to 7.5 with KOH (10 M) before autoclaving. The medium is boiled under N2 gas to remove the maximum oxygen and cooled to room temperature. The medium is distributed in 5 ml Hungate tubes or in anaerobiosis flasks obtained with the N2 / CO2 mixture (80:20, v / v) at atmospheric pressure. The addition of an N2 + CO2 mixture in microorganism cultures allows, in a known manner, the establishment of anaerobiosis and supply of CO2 to the anaerobes that metabolize it as a carbon source. The tubes and / or flasks are then autoclaved for 45 minutes at 110 ° C. After autoclaving, the medium is supplemented with NaHCO 3 (4 g / l), Na 2 S x 9 H 2 O (0.5 g / l), Na-formate (2 g / l) and 10 ml / l of a vitamin solution. from Balch (Balch et al., 1979); finally, the methanol is added (40 mM) with a gaseous mixture composed of H2 / CO2 (80/20) at a pressure of only 1 bar constituting the atmosphere on the surface of the culture The gaseous mixture composed of H2 / CO2 (80/20) at 1 bar pressure is introduced into the culture by bubbling through a sterile needle until total disappearance of the bubbles, the content of the tube is then 1 bar pressure of H2 / CO2 (80/20).

Once inoculated (100/0 vol), the medium is incubated at 37 ° C. with stirring. The production of methane starts 48 hours after inoculation and the exponential phase of growth is reached between the 7th and 10th day after inoculation. Five strains of this Archaea have been established in culture under conditions allowing their deposit in collection, namely the DSMZ Microorganism Deposit Collection (Germany) under the number DSM 24529 under conditions of viability and accessibility in accordance with the Budapest Treaty. This strain can be cultivated and sub-cultured by transplants indefinitely in time, by subculture of 1 / 5th of a culture, each subculture arriving in the exponential phase with evolution of methane of at least 10 μM and possibility of microscopic visualization of Archaea of spherical shape from 0.5 to 1 micron after at most 15 days, in practice after 5 days. The evolution of methane can be quantified by an appropriate method by gas chromatography, especially as described in BARREDO et al. [18] Said Archaea has a spherical morphology of smaller size than the other Archaea M.smithii and M.stadtmanae. EXAMPLE 3 Sequence of the 16S Gene of the Complete Mr / uminyensis RNA The new Archaea M. / uminyensis strain was inoculated into a volume of 250 ml of the liquid culture medium and cultured until a culture was obtained. Visible to the naked eye in the form of a medium disorder, the DNA was then extracted as previously described [15] from the pellet obtained by centrifugation of the culture at 13,000 rpm for 15 min at room temperature. Genomic DNA sequencing of M. / uminyensis was performed by pyrosequencing using a 454-Roche GSFLX as previously described [17]. The inventors were then able to extract contigs from the complete M. / uminyensis genome sequence, the complete sequence SEQ. ID. No. 1 of the M.sub.1 / uminyensis 16S-DNA gene, as described in the sequence listing appended to the description. Table 1: Organism (a) Target Sequence (5'-3 ') of primers and probes (bp) M. sinithi / 16S rDNA Smit.16S-740F, 5'-CCGGGTATCTAATCCGGTTC-3' 123 M. stadtmanae 16S rDNA Smit. 16S-862R, 5'-CTCCCAGGGTAGAGGTGAAA-3 'FAM (MGB) 97 Smit.16S FAM, 5'-CCGTCAGAATCGTTCCAGTCAG-3' City 16SF, 5'-AGGAGCGACAGCAGAATGAT-3 'M. / uminyensis 16S rDNA Stadt_16SR, 5'-CAGGACGCTTCACAGTACGA 3 'FAM (Tagman) 79 Stadt_16SFAM, 5'-TGAGAGGAGGTGCATGGCCG-3' B10-dir, 5'-GTTCGTAGCCGGTCTGGTAA-3 '16S consensus bacteria rDNA B10-rev, 5'-CCAAGTCTGGCAGTCTCCTC-3' VIC (Tagman) 327 B10- VIC, 5'-TCGGGAAGCTTAACTTTCCGACTTCC-3 'Bact 8FM, 5'-AGAGTTTGATCMTGGCTCAG-3' Bact515R, 5'-TTACCGCGGCKGCTGGCAC-3 'VIC (Tag man) Bact338K, 5'-CCAKACTCCTACGGGAGGCAGCAG-3' Archaea consensus (b) 16S rDNA TArchaea2 ## EQU1 ## Table 2: List of microbes tested Species Bacteroides fragi / is Salmon // a enterica arizonae Streptococcus pyogenes Lactococcus p / antarum Shige // a boyd // Streptococcus ga // o / yticus Enterococcus faeca / is Klebs / e // a oaytoca Pseudomonas aeruginosa Streptococcus entericus Staphy / ococcus aureus Geme // a morbi // orum Corynebacterium jeikeium Enterococcus ga // inarum Bacteroides thetaiotaomicron Streptococcus ora / is Enterococcus faecium Streptococcus aga / actiae Acinetobater haemo / yticus K / uyvera ascorbata Klebs / e // a pneumoniae Mycobacterium avium Enterobacter carcinogenus Yersinia entero / itica Enterobacter cloacae Mycobacterium tubercu / osis Pro videncia stuartii Ha / obacter / um sp Proteus mirabi / is Methanosarc / na acetivorans Propiombacterium acnes Su / fo / obus n / s / fataricus Bifdobacterium Aeropyrum pern / x Escherichia hermanii Methanobrevibacter ora / is Citrobacter freundii Methanobrevibater smithii Citrobacter koseri Methanosphaera stadtmanae Escherichia col / Camp // Salmon // a enterica enterica Table 3,: Composition of Balch medium 1 (a) 50m1 (b) 50m1 (c) ) 10m1 (d) 10m1 (e) 0.002g (0 5g (g) 2.5g (h)) 2.5g (i) 2g a) 2g (k) 0.5g (1) 0.5g The ingredients are added to distilled water to have a final volume of 1 liter. Cysteine and Na2S are added after bubbling the medium with a gas mixture 80% N2-20% CO2, the final gaseous phase of the tube is composed of a mixture 80% H2-20% CO2. (a):) Contains 6g of KH2PO4 per liter of distilled water. (b): Contains in grams per liter of distilled water: KH2PO4 (6), "NH4) 2504 (6), NaCl (12), MgSO4.7H2O (2.6), CaCl2.2H2O (0.16). (c) Contains in grams per liter of distilled water (pH adjusted to 7 with KOH): nitriloacetic acid (1.5), MgSO4.7H2O (3), MnSO4.2H2O (0.5), NaCl (1), FeSO4.7H2O (0.1), CoSO4 or CaCl2 (0.1), CaC12.H2O (0.1), ZnSO4 (0.1), CuSO4.5H2O (0.01), Na2MoO4.2H2O (0.01) ); Dissolve the nitriloacetic acid with KOH (pH 6.5) and add the other minerals. (d): Contains in milligram per liter of distilled water the following vitamins: biotin (2), folic acid (2), pyridoxine hydrochloride (10), thiamine hydrochloride (5), riboflavin (5), nicotinic acid ( 5), DL-calcium pantothenate (5), vitamin B12 (0.1), p-aminobenzoic acid (5), lipoic acid (5). (e) FeSO4 7H2O; (f): NaHCO3; (g): Na acetate; (h): Na formate; (i): DIFCO yeast extract; (j): trypticase BBL; (k): L-cysteine hydrochloride; (1): Na2S, 9H2O Table 4: Medium 119 Amount per liter of H 2 O KH 2 PO 4 0.5 g MgSO 4 x 7H 2 O 0.4 g NaCl 5 g NH 4 Cl 1 g CaCl 2 x 2 H 2 O 0.05 g Na-acetate 1.6 g Cysteine-HCI 0.5 g Yeast extract 1 g Trypticase peptone 1 g Resazurin 0.001 g Widdel trace elements solution 1 mL NaHCO3 4 g Na2S x 9H2O 0.5 g Na-formate 2 g Balch vitamins 10 mL Table 5: Medium E1 Amount per liter of H 2 O KH 2 PO 4 0.3 g K 2 HPO 4 0.3 g NaCl 1 g NH 4 Cl 1 g CaCl 2 x 2 H 2 O 0.1 g KCl 0.1 g MgCl 2 x 6 H 2 O 0.5 g yeast 1 g Cysteine-HCI 0.1 g resazurine 0.001 g Balch trace elements solution 2.5 mL NaHCO3 4 g Na2S x 9H2O 0.5 g Na-formate 2 g Balch vitamins 10 mL 40 mM methanol Table 6 : Widdel Micronutrient Solution Quantity for 1 liter of distilled water HCI 25% 10 mL FeCIZ x 4HZO 1.5 g CoCIZ x 6H2O 190 mg MnCIZ x 4H2O 100 mg ZnCIZ 70 mg H3BO3 62 mg Na2MoO4 x 2H2O 36 mg NiCIZ x 6H2O 24mg CuCIZ x 2H2O 17mg References 1. Scanlan PD, Shanahan F, March esi JR (2008) Human methanogenetic diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis. BMC Microbiol 8: 79; 2. Rieu-Lesme F, Delbes C, Sollelis L (2005) Recovery of partial 16S rDNA sequences suggests the presence of CrenArchaeaota in the human digestive ecosystem. Curr Microbiol 51: 317-321. 3. Oxley AP, Lanfranconi MP, Wurdemann D, Ott S, Schreiber S, McGenity TJ, KN Timmis, Nogales B (2010) Halophilic Archaea in the intestinal mucosa human.

About Microbiol .12: 2398-2410. 4. Nam YD, Chang HW, Kim KH, SW Roh, Kim MS, Jung MJ, SW Lee, JY Kim, Yoon JH, Bae JW (2008) Bacterial, Archaeal, and eukaryal diversity in the intestines of Korean people. J Microbiol 46: 491-501 5. Mihajlovski A, Alric M, Brugere JF (2008) A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyzes of the mcrA gene. Res Microbiol 159: 516-521 6. Mihajlovski A, Dore J, Levenez F, Alric M, Brugere JF (2010) Molecular Evaluation of the Methanogenic Gut Metabolic Archaeal microbiota reveals an increase in the association of diversity. Envir Microbiol Rep 2: 272-280. 7. Miller TL et al. Appl Environ Microbio% 1982; 43227-232. 8. Miller TL and Wolin MJArch Microbiol. 1985; 141: 116-122. 9. Ferrari A and a /. Curr Microbio / 1994; 29: 7--12. 10. Balch WE et al. Microbiol Rev. 1979; 43: 260-296. 12. Dridi B, Raoult D, Drancourt M .: Archaea as emerging organisms in complex human microbiomes. Anaerobe 2011, 17: 56-63. 23 13. Hungate, R.E. & Macy, J. (1973). The Roll-Tube Method for Cultivation of Strict Anaerobes. Bull Ecol Res Comm 17, 123-125 14. Grabarse W, Mahlert F, Shima S, Thauer RK, Ermler U. Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual 5 amino acid modification, conservation and adaptation. J Mol Biol. 2000; 303: 329-44. 15. Dridi B, Henry M, El Khechin A, Raoult D, Drancourt M. High prevalence of Methanobrevibactermithn and Methanosphaera stadtmanae detected in the human gut using improved DNA detection protocol. PLoS One. 2009; 4 (: e7063). 16. Boyer M, Madoui MA, Gimenez G, La Scola B, Raoult D. Phylogenetic and 10 phyletic studies of informational genes in genomes highlight existence of a 4 domain of life including giant viruses. PLoS One. 2010; 5: e15530. 17. Raoult, D., Audic S., Robert C., Abergel C., Renesto P., Ogata H., Scola B., Suzan M., and Claverie M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306: 1344-1350. 18. Barredo MS, Evison LM. Methanobiribacter smithii, Methanospirillum hungatii at different pH values. Appl Environ Microbiol. 1991 3a; 57 (6): 1764-9.

Claims (17)

REVENDICATIONS1. Archaea Methanomassi / iicoccus / uminyensis isolated and established in culture, comprising the specific sequence of the 16S gene of ribosomal DNA SEQ. ID. No. 1 of the sequence listing or a complementary sequence. 2. Archaea according to claim 1, as deposited in the DSMZ microorganism collection collection (Germany) under the number DSM 24529. 3. Sterilized culture medium useful for the cultivation of an Archaea according to claim 1 or 2, comprising a methanogenic Archaea culture base medium characterized in that it further comprises: a- methanol, and b- a tungsten salt and / or a selenium salt. 4. Culture medium according to claim 3, characterized in that it comprises: a- methanol, and b- a tungsten salt and a selenium salt. 5. Culture medium according to claim 3 or 4, characterized in that the methanol is present at a concentration of 1 to 100 mM, preferably 10 to 50 mM. 6. Culture medium according to one of claims 3 to 5, characterized in that said tungsten salt is present at a concentration of 0.3 to 3 M, preferably 0.5 to 1.5 M. 7. Culture medium according to one of claims 3 to 6, characterized in that said selenium salt is present at a concentration of 0.2 to 2 M, preferably 0.5 to 1M. 8. Culture medium according to one of claims 3 to 7 characterized in that said base medium comprises at least: - several sources of carbon and nitrogen, preferably chosen from a yeast extract, an acetate salt and a tryptic peptone, a source of phosphorus, preferably a phosphate salt, a source of nitrogen, preferably an ammonium salt, at least one salt of each of the metals K, Mg, Na and Ca, a reducing substance, more particularly chosen from cysteine-HCl and Na 2 S - a pH-regulating buffer substance for adjusting the pH of 7 to 8; trace elements, preferably salts chosen from B, Fe and Cu salts, Zn, Mn, Co, Ni and Mo, more preferably micronutrients from Widdel's medium, - vitamins, preferably vitamins from Balch's medium, and - preferably, an anaerobic control coloring substance, preferably resazurin. 9. Culture medium according to one of claims 3 to 8, characterized in that it contains said base medium comprises the following species: KH2PO4, MgSO4, NaCl, NH4Cl, CaCl2.2H20, Na COOCH3 cysteine, yeast extract , trypticase peptone, NaOH, Na2S, NaHCO3, NaHCO2, Na2SeO3, Na2WO4.5H2O, widdel oligoelements, Balch vitamins and preferably resazurin; the pH being adjusted to 7.5 with KOH. 10. Culture medium according to one of claims 3 to 9, characterized in that it comprises in addition to said base medium: a- methanol at a concentration of 10 to 100 mM, preferably 40 mM, and b- a Nat SeO3 salt at a concentration of 1 to 10 mg / l, preferably 2 mg / l, and c- a salt of Na2WO4 .5H20 at a concentration of 1 to 10 mg / l, preferably 4 mg / l. 11. A method for preparing a sterilized culture medium according to one of claims 3 to 10, characterized in that the following successive steps are carried out in which: 1- is sterilized in an autoclave undit Archaea culture medium incomplete not containing the components which may be degraded in an autoclave comprising methanol, said vitamins, labile sodium salts comprising Na HCO3, Na HCO2 and Na2S, preferably in an autoclave a temperature above 100 ° C for at least 15 and oxygen is removed, preferably by boiling said culture medium under a nitrogen atmosphere, and placing said incomplete sterilized culture medium under an anaerobic atmosphere, and it cools to room temperature and is added in said incomplete culture medium, said components may be degraded by sterilization in an autoclave comprising methanol, the said vitamins Ines, labile sodium salts including Na HCO3, Na HCO2 and Na2S. 12. A method of culturing an Archaea with a culture medium according to one of claims 3 to 10 wherein the following successive steps are carried out: 1-is inoculated a biological sample capable of containing a said Archaea in said culture medium in anaerobic manner, preferably under a nitrogen atmosphere, and 2-placing said culture medium under an atmosphere of anaerobiosis and containing at least 50% hydrogen at a pressure of less than 2 atm, and 3-incubation is carried out at a temperature of 20 to 40 ° C, preferably at 37 ° C, to reach an exponential growth phase with evolution of methane in at least 5 days, preferably not more than 15 days. 13. The method of claim 12, characterized in that in step 2 said culture medium is placed under a compound H2 / CO2 atmosphere in a volume ratio of 80/20 at a pressure of 1 atm. 14. A method for determining the presence of an Archaea in a biological sample, preferably in a sample of animal or human stool according to claim 1 or 2 or an Archaea grown according to the method of one of claims 11 or Characterized in that the DNA is extracted from said biological sample and the DNA of said Archaea is detected and preferably specifically quantified by enzymatic amplification of PCR type, preferably of the PCR type in real time with a pair of primers. and preferably a probe, sequences derived from the 16S gene rDNA sequence SEQ.ID No. 1. 15. Method according to claim 14, characterized in that a pair of primers and preferably a said probe of sequences selected from the following sequences and complementary sequences derived from the 16S rDNA gene: 5 'direct primer: SEQ. ID. No. 2 = 5'-GTTCGTAGCCGGTCTGGTAA-3 'Reverse 3' primer: SEQ.ID. No. 3 = 5'-CCAAGTCTGGCAGTCTCCTC-3 'Probe: SEQ ID NO: 4 = 5'-TCGGGAAGCTTAAC 1 I 1 CCGACTTCC-3' 16. The method of claim 14 or 15 characterized in that is carried out beforehand a PCR type enzyme amplification preferably of the PCR type in real time, specifically the DNA of all Archaea contained in said DNA extracted from the sample with a pair of consensus primers of sequences selected from the following sequences and complementary sequences derived from the 16S rDNA gene: 5 'direct primer: SEQ.ID. No. 5 = 5'-AGCCGCCGCGGTAAYAC-3 'Reverse 3-primer: SEQ.ID. No. 6 = 5'-CYGGCGTTGAVTCCAATT-3 '. 17. Archaea detection kit according to one of claims 1 or 2 for the implementation of a method according to one of claims 13 to 15 characterized in that it comprises: - a said culture medium preferably lyophilized or solid, and reagents for carrying out an enzymatic amplification of PCR-type DNA, and oligonucleotides constituting said primers and probes derived from SEQ ID NO: 1, preferably chosen among SEQ.ID.No. 2 to 6.