FR3020378A1 - USE OF URIC ACID FOR THE CULTURE OF OXYGEN TENSION-SENSITIVE BACTERIA - Google Patents

Abstract

The present invention relates to a method of cultivation in vitro in acellular culture medium, of bacteria whose growth is sensitive to the oxygen content, the optimal growth of said bacteria requiring an incubation atmosphere with oxygen content relatively reduced compared to the oxygen content of the air, said bacterium being chosen from anaerobic bacteria and intracellular microaerophilic bacteria, characterized in that uric acid is added to said cell culture medium, and cultivation is carried out said bacterium in said culture medium in the presence of oxygen.

Description

The present invention relates to the cultivation in acellular medium of bacteria whose growth is sensitive to the oxygen tension, in particular bacteria that do not tolerate high voltages. oxygen and for which optimal growth of said bacterium requires an incubation atmosphere with a relatively low oxygen content relative to the oxygen content of the air or even strict anaerobic bacteria for which the oxygen is toxic and which must grown in the absence of oxygen or tolerating only low concentrations of oxygen. Thus, the following are distinguished from bacteria that are sensitive to oxygen: microaerophilic bacteria, that is to say that they are not capable of cultivating under an atmosphere comprising the ambient oxygen concentration which is about 21%, in particular between 1% and 20%, most commonly about 2-2.5%, and strict anaerobic bacteria that is to say that they are not able to grow in the presence of oxygen or in concentrations lower than microaerophilic concentrations, especially less than 1%, most commonly less than 0.1%, ideally 0%. To cultivate strict anaerobic bacteria, it is necessary either to cultivate them in incubators not containing oxygen, or in tubes which have been deoxygenated and they only grow at the bottom of the tube. Among the strict anaerobic bacteria, mention is made more particularly of extracellular bacteria, ie bacteria which can live only outside cells.

More particularly, the present invention relates to the cultivation of anaerobic bacteria and the cultivation of microaerophilic bacteria in an aerobic atmosphere. In WO 2014/064359, it is proposed to improve and facilitate the growth conditions in acellular culture of bacteria whose growth is sensitive to the oxygen tension and in particular bacteria that are poorly tolerant of high oxygen tensions and for which growth optimal of said bacterium requires a relatively low oxygen content incubation atmosphere with respect to the oxygen content of the air, said bacteria being selected from the following bacteria: - anaerobic bacteria, including strict anaerobic bacteria, and microaerophilic bacteria of the type mentioned above.

The term "microaerophilic atmosphere" is used herein to mean oxygen-depleted air with a molar proportion of oxygen of less than 10%, preferably 5%, more preferably less than 2.5%. For strict anaerobic bacteria, the oxygen content must be close to 0%, especially less than 0.1%, as mentioned above, the tolerance to very small amounts of oxygen being variable according to the species of anaerobic bacteria. In WO 2014/064359, it is proposed to add certain antioxidant compounds namely ascorbic acid, glutathione and hydrogen sulfide in an acellular culture medium could: - improve the growth of said bacteria by obtaining faster bacteria in sufficient concentration to be detectable after multiplication and / or by increasing the concentration of bacteria after a given crop time, ie per unit of time, and - cultivate with at least the same level of growth or even at a level higher, strict anaerobic bacteria in the presence of higher amounts of oxygen than in the absence of antioxidant compound, that is to say in a microaerophilic atmosphere, especially with oxygen contents of 2 to 5%, but also oxygen contents greater than 5%, or even aerobic, that is to say in the presence of an oxygen level equivalent to the oxygen tension close to the ambient air is about 2 1%, and cultivating with at least the same level of growth, in an atmosphere containing higher oxygen tensions, said microaerophilic intracellular bacteria, cultivable in the absence of antioxidant compound, in a microaerophilic atmosphere, or even with a level of higher growth as is the case for the bacterium Coxiella burnetii; and cultivating, with a higher level of growth, in a microaerophilic atmosphere, cultivable bacteria, in the absence of antioxidant compound, in an atmosphere containing a higher oxygen tension, but lower than the oxygen content of the air , as is the case for the facultative intracellular bacterium Mycobacterium tuberculosis. In WO 2014/064359, a method is thus provided for in vitro culture of bacteria in acellular culture medium, of bacteria whose growth is sensitive to the oxygen content, the optimal growth of said bacteria requiring an incubation atmosphere with a content of relatively reduced oxygen, or even zero, with respect to the oxygen content of the air, said bacteria being chosen from anaerobic bacteria and intracellular microaerophilic bacteria, characterized in that a compound is added to said cell-free culture medium; antioxidant selected from ascorbic acid, glutathione and sodium hydrosulfide, and said bacterium is cultured in said culture medium in the presence of oxygen.

In WO 2014/064359, ascorbic acid and glutathione are preferred because they are capable at precise doses to allow culture at a higher oxygen level. In WO 2014/064359, more particularly, said antioxidant compounds are used at a concentration of 0.1 g / L to 2 g / L, or molar concentration of 10-6 M to 10-2 M. D ' other antioxidant compounds such as sodium hydrosulfide (NaHS) or cysteine are less effective and require higher concentrations.

The addition of an antioxidant compound makes it possible to tolerate growth in the presence of a relatively higher oxygen content, or even in an ambient air atmosphere, in particular for intracellular microaerophilic bacteria such as Coxiella burnetii or Helicobacter cinaedi and strict anaerobic bacteria such as Bacteroides. And, this addition also allows for certain bacteria such as Mycobacterium tuberculosis cultivable at higher oxygen tensions in the absence of antioxidant compound, to enhance their growth at decreased oxygen tensions in the presence of antioxidant compound.

Under certain conditions ascorbic acid appears toxic and / or too acid with respect to certain bacteria and / or for certain culture media in high doses. This has been demonstrated in particular for Mycobacterium tuberculosis (4). On the other hand, glutathione is very expensive.

According to the present invention, the inventors have fortuitously discovered that an equivalent effect on the growth of said anaerobic or oxygen-sensitive bacteria can also be obtained by adding in the said culture medium uric acid (7, 9-dihydro-1H-purine-2,6,8 (3H) -trione or 2,6,8-trioxypurine) in place of the antioxidant compounds described in WO 2014/064259, uric acid being used in similar or lower concentrations than those of ascorbic acid and glutathione as described in WO 2014/064359. Uric acid, although having under certain conditions antioxidant properties, is not conventionally used in microbiology as an antioxidant compound because under certain conditions it has on the contrary oxidizing properties (5). The inventors in work on Kwashiorkor disease, a form of infant malnutrition, have discovered the importance of uric acid for the growth of anaerobic bacteria. They observed in these children microbial flora of the digestive tract very particular in that it has very few anaerobic bacteria on the one hand and a lack of uric acid on the other hand. This uric acid deficiency is derived from a diet lacking meat and little vegetable while uric acid is derived from the degradation of purines found in significant amounts in meat foods. To explain the specificity of the flora of these patients, they wanted to check if the uric acid would be sufficient to allow the aerobic culture of theoretically anaerobic bacteria which led them to discover that low doses of uric acid normally present in the digestive tract can effectively restore the growth of anaerobiosis bacteria absent in these patients deficient in uric acid. This discovery is particularly important and advantageous because uric acid is less acidic and less toxic than ascorbic acid and much less expensive than glutathione, ie approximately 1 Euros / g for uric acid, instead of 100 Euros / g for glutathione. More particularly, the uric acid is used at a concentration of at least 0.1 g / l, preferably at least 0.2 g / l, in particular from 0.2 to 0.5 g / l, to obtain an equivalent effect. even greater on the growth of said oxygen-sensitive bacteria in an aerobic atmosphere and at concentrations lower than or equal to those of ascorbic acid and glutathione alone or as a mixture allowing a similar effect on the growth of said bacteria in the same aerobic ambient atmospheres. Uric acid may be used in admixture with ascorbic acid and / or glutathione, but uric acid may be used without additional antioxidant compound in said culture medium.

More particularly, said culture medium comprises the components that are found in culture base media capable of cultivating an anaerobic bacterium, comprising at least: several sources of carbon, a source of phosphorus, preferably a salt of phosphate, - a nitrogen source, preferably an ammonium salt, - at least one metal salt selected from K, Mg, Na, Ca, preferably NaCl. More particularly, said culture medium is an acellular medium is selected from an axenic medium consisting of chemical or biological substances defined qualitatively and quantitatively, and an acellular medium comprising a mulate-cell extract or lysate of multicellular tissue. Preferably, said medium comprises a pH regulating buffer substance to adjust the pH from 7 to 7.5. More particularly, said culture medium is a conventional acellular medium of microaerophilic or anaerobic bacterium, preferably a medium comprising components chosen from a mulate-tissue extract or lysate, an enzymatic digest, in particular an enzymatic digest of casein, soybean and or animal tissue, a peptone, a yeast extract, a sugar such as dextrose or glucose, an NaCl salt and / or Na 2 PO 4. More particularly, said culture medium is a filtrate of said ground or lysate, in particular blood tissue or heart tissue and / or lung, when said bacterium is an extracellular bacteria such as the so-called broth-type broth medium, media Columbia 5% sheep blood or Schaedler medium as described below. Other suitable conventional media are Brucella or Wilkins-Chagren media. Such cell-free culture media are well known to those skilled in the art. These media can be used with agar (solid or semi-solid) or without agar (liquid). In particular, polyvalent culture media for anaerobic microorganisms, especially Schaedler's medium, may be used. Such acellular culture media whether liquid, solid or biphasic are well known to those skilled in the art. More particularly, said culture medium of anaerobic bacteria may be in liquid or solid or semi-solid form, especially agar or semi-agarose, agar or semi-agarose. More particularly, the said bacterium is cultured in a so-called incubation atmosphere comprising a molar proportion of oxygen greater than the maximum tolerated tension in the absence of uric acid or of an antioxidant compound for the same level of growth in the same period of time. culture. In practice, more particularly, the said bacterium is cultured in a so-called incubation atmosphere comprising a molar proportion of oxygen of less than or equal to 20%. More particularly, said bacteria according to the present invention are cultivated in an atmosphere comprising a in oxygen greater than 5%, especially in air containing 5% CO2 (ie an oxygen content of less than 16%), or even in an aerobic atmosphere of ambient air. More particularly, in some cases, as explained below, the said intracellular microaerophilic bacterium is cultured in a said microaerophilic incubation atmosphere comprising a molar proportion of oxygen of less than 5%, preferably of 2 to 5%, preferably another 2.5%. More particularly, the uric acid is used with an antioxidant compound is selected from ascorbic acid, glutathione (y-L-Glutamyl-L-cysteinylglycine) and sodium hydrosulfide. Ascorbic acid and glutathione are preferred because they are capable at precise doses of allowing culture to a higher oxygen level. More particularly, said antioxidant compound is used at a concentration of 1 μg / ml to 2 mg / ml, preferably at least 100 μg / ml. Preferably, said antioxidant compound is ascorbic acid and / or glutathione, preferably at a concentration of at least 100 μg / ml. More particularly, the said bacterium is a bacterium cultivable in a said culture medium in the absence of uric acid or said oxidizing compound in an incubation atmosphere comprising a molar proportion of oxygen less than the molar proportion of oxygen in the air, preferably less than 20%, and the said bacterium is cultured in the presence of said uric acid in the said culture medium under an incubation atmosphere comprising an oxygen content less than or equal to the proportion of oxygen in the air, preferably less than 20%, more preferably greater than 5%. According to a first embodiment, said bacterium is an extracellular anaerobic bacterium cultivable in an anaerobic atmosphere in the absence of said uric acid or said antioxidant compound, and one obtains a growth of said bacterium in the presence of oxygen with a molar proportion less than or equal to the proportion of oxygen in the air. Among the anaerobic extracellular bacteria, mention is made more particularly of the bacteria belonging to the genera Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Propionibacterium, Lactobacillus, Actinornyces, Clostridiurn, Bacteroides, Porphyrornonas, Prevotella, Firincinnutes, Solobacterium, Atopobiurn, Rurninococcus and Fusobacterium, plus especially the species selected from Finegoldia magna, Bacteroides rnassiliensis, Fusobacterium necrophorum, Finegoldia magna, Prevotella nigrescens, Solobacterium brnorei, Atopobiurn vaginae and Rurninococcus gna vus. According to this first particular embodiment, an anaerobic extracellular bacterium, in particular a bacterium of the digestive tract, is cultured in humans or animals in an acellular medium in the presence of a molar proportion of oxygen less than or equal to that of air and in the presence of said uric acid, preferably in ambient air atmosphere. This first embodiment is illustrated in the following example by the aerobic culture of Bacteroides thetaiotaornicron in the presence of uric acid. These are strict anaerobic bacteria in the digestive tract, which are normally cultured only under strictly anaerobic conditions. This results when one inoculates a said bacterium in a deoxygenated tube in that a culture veil appears only in the lower part of the tube, while the upper part remains virgin culture. If uric acid according to the present invention is added at 200 μg / ml, the culture is practically to the surface, or even completely to the surface at a concentration of 500 μg / ml and even more preferably at 1 mg / ml. I with uric acid as with a mixture of ascorbic acid at 500 μg / ml and glutathione at 500 μg / ml. The bacterium produces colonies in 24 hours after agar seeding.

In another embodiment, said bacterium is an intracellular microaerophilic bacterium capable of being cultured in said cell-free culture medium, in a microaerophilic incubation atmosphere with a molar proportion of oxygen of not more than 5%, preferably not more 2.5% in the incubation atmosphere, in the absence of uric acid or antioxidant compound, and the said bacterium is cultured in the presence of uric acid in a said culture medium under an incubation atmosphere microaerophilic comprising a molar proportion of oxygen between 2.5% and 20%, preferably between 5% and 16%, including air optionally enriched with 5% CO2. The present invention applies more particularly to microaerophilic bacteria due, among other things, to the addition of said uric acid. This embodiment is illustrated by the aerobic culture of the bacterium Helicobacter cinaedi (DSMZ 5359) grown in ambient air. Other characteristics and advantages of the invention will become apparent in the light of the detailed description of the following exemplary embodiments. Example 1: Cultivation of Bacteroides thetaiotaornicron with a Schaedler medium zo. A strain of anaerobic bacterium Bacteroides thetaiotaornicron was obtained through the "culturomics" study of the inventors (2) also available in various depot collections (CSUR P766 also deposited according to the Budapest Treaty at the micro-depot collection). DSMZ organisms (Germany) on May 19, 2014 under the number DSM 28808, other strains are also available in various deposit collections such as strains DSM 2079, ATCC 29148 and NCTC 10582). For their production in sufficient quantity, B. thetaiotaornicron was cultured in an anaerobic atmosphere at 37 ° C. in a polyvalent culture medium. Schaedler's medium (Reference 42098, BioMerieux, La Balmes-les-Grottes, France) was also tested for the culture of anaerobic bacteria. The Schaedler medium (marketed by Biomerieux, Marcy l'étoile, France) had the following composition for 1 liter: Enzymatic digest of casein 5.6 g Enzymatic digest of soybean meal 1 g Enzymatic digest of animal tissues 5 g yeast 5 g - NaCl 1.7 g - Potassium phosphate 0.82 g - Dextrose 5.82 g - Tris (hydroxymethyl) Aminomethane 3 g - Hemine 0.01 g - L-cysteine 0.4g This Schaedler medium was supplemented by the addition of hydrocarbon compounds, namely of 1 g / L of rice starch and 1 g / L of glucose (Sigma-Aldrich, Saint-Quentin Fallavier, France) and the addition of uric acid and anti-oxidant compounds namely complemented by the addition of - either 0.1 g / L of ascorbic acid (VWR International, Leuven, Belgium), 0.1 g / L of uric acid and 0.1 g / L of glutathione (Sigma-Aldrich, Saint-Quentin Fallavier, France ), -soit.0.1; 0.2 or 0.3 g / L of uric acid alone as an antioxidant compound. The addition of starch and glucose hydrocarbon compounds aimed here to produce H2 to control the growth of the bacteria. Resazurin is used as a redox indicator at a concentration of 0.1 mg / ml to control the presence of oxygen (the oxidized resazurin has a pink color, and becomes transparent in the absence of oxygen).

The B. thetaiotaornicron aerobic culture in ambient air was performed by inoculating 105 organisms / mL in a vessel incubated at 37 ° C containing the supplemented culture medium by the addition of antioxidant compounds and carbon source compounds. The pH was adjusted to 7.5 by addition of 10M KOH. The strain was grown in parallel under aerobic conditions and by the inoculation of 105 organisms / ml with the culture medium supplemented according to the present invention and with Schaedler medium supplemented by the hydrocarbon compounds mentioned above but on the other hand without any anti-aging compounds. oxidants. The culture medium supplemented by adding 1 g / L of rice starch and 1 g / L of glucose inoculated anaerobically with 108 cells / L of B. thetaiotaornicron was introduced as a positive control and to verify the production of H2 by B. thetaiotaornicron in anaerobic culture. These checks were carried out in parallel in an ambient atmosphere (aerobic). The non-inoculated culture medium was introduced as a negative control. The growth of B. thetaiotaornicron has been evaluated daily by the production of hydrogen. The measurement of hydrogen was carried out using a GC-8A gas chromatograph (Shimadzu, Champs-sur-Marne, France) equipped with a thermal conductivity detector and a Chromosorb WAW 80/100 column SP100 mesh (Alltech, Carquefou, France). N2 nitrogen at a pressure of 100 kPa was used as the carrier gas. The detector and the injector temperatures were 200 ° C and the column temperature was 150 ° C. Negative controls remained negative with no growth occurring after one week of incubation indicating that the results reported here are not simply the result of contamination by other microorganisms.

The positive controls were positive hydrogen production observed in the anaerobic B. thetaiotaornicron culture. The culture of B. thetaiotaornicron inoculated aerobically without antioxidant compounds remained negative and hydrogen was not produced.

After incubation for 24 hours at 37 ° C in ambient air (under aerobic conditions), a culture medium without antioxidant compounds retained its pink color indicating the presence of oxygen and the culture remained negative for the strain tested. The aerobic culture medium with uric acid or said antioxidant compounds has become transparent indicating the absence of oxygen. B. thetaiotaornicron cultures grown under aerobic conditions with uric acid or the said antioxidant compounds all gave a positive culture for B. thetaiotaornicron with production of hydrogen after a 24 hour incubation.

Growth results with uric acid without said antioxidant compound were equivalent to those obtained with a mixture of uric acid with ascorbic acid and glutathione when the concentration of uric acid alone was at least 0.2 g / L and higher with 0.3g / L of uric acid alone.

These results indicate that it is possible to cultivate in the ambient air (aerobic condition) bacteria deemed strictly anaerobic, in a suitable medium containing a suitable mixture of antioxidants and in particular with uric acid alone without antioxidant.

The culture procedure is carried out on tubes of culture medium of the Schaedler type with 0.2% agar (Biomerieux, Marcy l'étoile, France). The Schaedler medium had the following composition for 1 liter: Enzymatic casein digest 5.6 g Enzymatic soybean meal digest 1 g Enzymatic enzymatic tissue digest 5 g-Yeast extract 5 g-NaCl 1.7 g-Potassium phosphate 0.82 g -Dextrose 5.82 g -Tris (hydroxymethyl) Aminomethane 3 g -Hemine 0.01 g -L-cysteine 0.4g -Agar (semi-solid medium) 0.2% For each bacterium two tubes are inoculated, a regenerated tube without uric acid and a regenerated tube in which 500 μg / ml or 1 mg / ml of uric acid is added. To regenerate the tube, it is placed in a water bath at 100 ° C. until all the gas bubbles visible in the medium have disappeared. Then, for the tube with uric acid, it is expected to cool the tube at 50 ° C (schematically until it can be held in the hand without burning) and the uric acid suspension is added. Then homogenized by inversion (3-4 to ensure a good mixture). For each bacterium an inoculum of 107 bacteria / ml was inoculated over the entire height of the Schaedler 0.2% tubes, one normal and one supplemented with uric acid. The tubes were incubated at 37 ° C in a strict anaerobic oven for 24-48 hours. Under these conditions, a usual growth of bacteria was observed from the bottom of the tube up to 1.5 cm below the surface of the medium in the absence of uric acid testifying to the anaerobic nature of this bacteria, and a growth up to at the surface in the presence of 500 μg / ml (28 X 10 -4 M) of uric acid indicating growth in the presence of a higher oxygen tension than in the absence of uric acid.

Identical tests were performed with either glutathione or ascorbic acid at 500 μg / I. With uric acid alone, the growth is identical to that observed with ascorbic acid. Finally, in order to definitively validate the ability of this uric acid compound to allow the growth of strict anaerobic bacteria in the presence of oxygen, solid media consisting of Columbia medium with 5% sheep blood blood were prepared. added uric acid at 500 μg / l or 1 mg / l, or a glutathione mixture at 500 μg / l + ascorbic acid at 500 μg / l or ascorbic acid at 1 mg / l. These inoculated agar plates of anaerobic bacteria were incubated either in ambient air or in ambient air enriched with 5% CO2. The best shoot was obtained with uric acid or ascorbic acid at 1 mg / I indifferently. These tests were carried out with a Columbia medium containing 5% sheep blood having the following composition for 1 liter: enzymatic digest of casein 5g Enigmatic enzyme of animal tissues 8g -Peptone enriched with yeast 10g Corn starch 1g-NaCl 5g -Agar (if agar medium) 14 g -Mon sheep 5% Example 2: Effect of uric acid on the culture Helicobacter cinaedi (DSMZ 5359). A strain DSMZ 5359 cultivated in the same Schaedler culture medium as described in Example 1 and under the same aerobic operating conditions at the same concentrations of uric acid was used except that the medium was not supplemented. of culture by the addition of hydrocarbon compounds starch and glucose. The bacterium was established in culture in 24h and confirmed by mass spectrometry type Maldi tof.10 Bibliographical References [1] The Scola B, Fournier PE, Raoult D. Burden of emerging anaerobes in the MALDI-TOF and 165 rRNA gene sequencing era. Anaerobe 2011; 17: 106-112 [2] Lagier JC et al., Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012; 18: 1185-93) [11] D. A. Podkopaeva et al. Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii. Microbiology, vol. 72, No. 5, Io 2003, pp. 534-541. [3] Omsland A, Cockrell DC, Howe D, et al. Host cell-free growth of 3he Q fever bacterium Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4430-4434. (4) Catherine Vilcheze, Travis Hartman, Brian Weinrick, and William R.

Jacobs Jr * in Nat Commun. 2013, 4: 1881. doi: 10.1038 / ncomms2898 (5) Yuri Y. Sautin and Richard J. Johnson inNucleosides Nucleotides Nucleic Acids. Jun 2008; 27 (6): 608-619.uric acid: oxidant-antioxidant pardox. 20

Claims (10)

REVENDICATIONS1. Process for growing in vitro in a cell-free culture medium, of a bacterium whose growth is sensitive to the oxygen content, the optimal growth of said bacterium requiring an incubation atmosphere with a relatively low oxygen content relative to the oxygen content of the air, said bacterium being chosen from anaerobic bacteria and intracellular microaerophilic bacteria, in which said bacterium is cultured in said culture medium in the presence of oxygen, characterized in that the said medium is added to uric acid culture. 2. Method according to claim 1, characterized in that the uric acid is used at a concentration of at least 0.1 g / l, preferably at least 0.2 g / l, more preferably 0, 2 to 2 g / L. 3. Method according to claim 1 or 2, characterized in that the uric acid is used without additional antioxidant compound in said culture medium. 4. Method according to one of claims 1 to 3, characterized in that said acellular medium is selected from: - an axenic medium consisting of chemical or biological substances defined qualitatively and quantitatively, and - a medium comprising a groundnut extract or lysate of multicellular tissue. 5. Method according to one of claims 1 to 4, characterized in that the said bacterium is cultured in a said incubation atmosphere comprising a molar proportion of oxygen greater than the maximum voltage tolerated in the absence of acid uric or antioxidant compound for the same level of growth in the same culture period. 6. Method according to one of claims 1 to 5, characterized in that said bacterium is a bacterium cultivable in a said culture medium in the absence of uric acid or antioxidant compound in an incubation atmosphere comprising a molar proportion of oxygen less than the molar proportion of oxygen in the air, preferably less than 20%, and said bacterium is cultured in the presence of said uric acid oxidizing in said culture medium under an incubation atmosphere comprising an oxygen content less than or equal to the proportion of oxygen in the air, preferably less than 20%, more preferably greater than 5%. 7. Method according to one of claims 1 to 6, characterized in that said bacterium is an extracellular anaerobic bacterium culturable in anaerobic atmosphere in the absence of uric acid or antioxidant compound, and one obtains a growth of said bacterium with a culture medium comprising uric acid in the presence of oxygen acid with a molar proportion less than or equal to the proportion of oxygen in the air, preferably in the presence of an atmosphere of ambiant air. 8. Method according to one of claims 6 or 7, characterized in that an anaerobic extracellular bacterium is cultivated in an acellular medium, said culture medium is a conventional acellular medium of anaerobic bacterium, preferably a medium comprising component selected from an extract of crushed bromine or lysate of multicellular tissue, an enzymatic digestate, in particular an enzymatic digest of casein, soya and / or animal tissue, a peptone, a yeast extract, a sugar such as dextrose or glucose, a salt NaCl and / or Na2PO4. 9. The culture method according to one of claims 1 to 6, characterized in that said bacterium is a said intracellular microaerophilic bacteria capable of being cultured in said cell-free culture medium, in a microaerophilic incubation atmosphere with a molar proportion in oxygen of not more than 5%, preferably not more than 2.5% in the incubation atmosphere, in the absence of said antioxidant compound, and the said bacterium is cultured in the presence of said oxidizing compound in a said culture medium under an incubation atmosphere comprising a molar proportion of oxygen between 2.5% and 20%, preferably between 5% and 16%. 10. Method according to one of claims 1 or 9, characterized in that said bacterium is selected from Bacteroides thetaiotaornicron and Helicobacter cinaedi.