Patent Grant
10626421
The present invention relates to the culture in an acellular medium of bacteria, the growth of which is sensitive to oxygen tension, notably bacteria which poorly tolerate high oxygen tensions and for which optimum growth of said bacterium requires an incubation atmosphere with a relatively reduced oxygen tension with respect to the oxygen tension of air or even strict anaerobic bacteria for which oxygen is toxic and which should be cultivated in total absence of oxygen or only tolerating low concentrations of oxygen.
Therefore among the bacteria sensitive to oxygen are distinguished:
From among strict anaerobic bacteria, are more particularly mentioned extracellular bacteria, i.e. bacteria which can only thrive outside cells.
More particularly, the present invention relates to the cultivation of anaerobic bacteria and to the cultivation of micro-aerophilic bacteria in an aerobic atmosphere.
In WO 2014/064359, it is proposed to improve and facilitate conditions of growth in an acellular culture of bacteria, the growth of which is sensitive to the oxygen content and notably bacteria which poorly tolerate high oxygen tensions and for which optimum growth of said bacterium requires an incubation atmosphere with a relatively reduced oxygen content with respect to the oxygen content of air, said bacteria being selected from among the following bacteria:
By «micro-aerophilic atmosphere» is meant here air depleted in oxygen with a molar oxygen proportion of less than 10%, preferably 5%, still preferably less than 2.5%. For strict anaerobic bacteria, the oxygen content should be close to 0%, notably 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, the addition of certain antioxidant compounds is proposed, i.e. ascorbic acid, glutathion and sodium hydrosulfide in an acellular culture medium which may give the possibility of:
In WO 2014/064359, a method for cultivating in vitro bacteria in an acellular culture medium of bacteria, the growth of which is sensitive to the oxygen content, is therefore provided, the optimum growth of said bacteria requiring an incubation atmosphere with a relatively reduced oxygen content or even zero content with respect to the oxygen content of air, said bacteria being selected from among anaerobic bacteria and intracellular micro-aerophilic bacteria, characterized in that an antioxidant compound selected from ascorbic acid, glutathion and sodium hydrosulfide is added, and said bacterium is cultivated in said culture medium in the presence of oxygen.
In WO 2014/064359, ascorbic acid and glutathion are preferred since they are capable at specific doses of allowing cultivation at a higher oxygen level.
In WO 2014/064359, still more particularly, said antioxidant compounds, are applied at a concentration from 0.1 g/L to 2 g/L, or at a molar concentration from 10−6 M to 10−2 M.
Other antioxidant compounds such as sodium hydrosulfide (NaHS) or cysteine are less effective and require higher concentrations.
The addition of an antioxidant compound gives the possibility of tolerating growth in the presence of relatively higher oxygen content, or even in an ambient air atmosphere notably for intracellular micro-aerophilic bacteria such as Coxiella burnetii or Helicobacter cinaedi and strict anaerobic bacteria such as Bacteroides. And, this addition also gives the possibility for certain bacteria such as Mycobacterium tuberculosis which may be cultivated at higher oxygen tensions in the absence of an antioxidant compound, of improving their growth at reduced oxygen tensions in the presence of an antioxidant compound.
Under certain conditions, ascorbic acid appears to be too toxic and/or too acid with respect to certain bacteria and/or for certain culture media with strong doses. This was shown notably for Mycobacterium tuberculosis (4). On the other hand, glutathion is very expensive.
According to the present invention, the inventors randomly discovered that an equivalent effect on the growth of said anaerobic bacteria or sensitive to oxygen may also be obtained by adding into said culture medium uric acid (7,9-dihydro-1H-purine-2,6,8(3H)-trione or 2,6,8-trioxypurine) in the place of antioxidant compounds as described in WO 2014/064359, uric acid being applied in similar concentrations or less than those of ascorbic acid and of glutathion 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 since under certain conditions, it exhibits on the contrary oxidant properties (5).
The inventors within the scope of research work on Kwashiorkor disease, a form of children malnutrition, discovered the importance of uric acid for the growth of anaerobic bacteria. They observe in these children a very particular microbial flora of the digestive tract in that it includes very few anaerobic bacteria on the one hand and a deficiency in uric acid on the other hand. This deficiency in uric acid stems from a diet not including any meat and few vegetables while uric acid stems from the degradation of purines which are for example found in a significant amount in meat-containing foods. In order to explain the specificity of the flora of these patients, they wanted to check whether uric acid would be sufficient for allowing cultivation under aerobic conditions of theoretically anaerobic bacteria, which led them to the discovery that small doses of uric acid normally present in the digestive tract may actually reestablish the growth of anaerobic bacteria, absent in patients deficient in uric acid.
This discovery is of particular importance and advantageous since uric acid is less acid and less toxic than ascorbic acid and much less expensive than glutathion i.e. about 1 Euro/g for uric acid, instead of 100 Euros/g for glutathion.
The present invention therefore has the object of an in vitro cultivation method in an acellular culture medium, of a bacterian selected from anaerobic bacteria and intracellular micro-aerophilic bacteria, wherein said bacterium is cultivated in said culture medium in the presence of oxygen, characterized in that uric acid at a concentration of at least 0.1 g/L is added into said culture medium.
Said bacteria are bacteria, the growth of which is sensitive to the oxygen content, the optimum growth of said bacterium requiring an incubation atmosphere with a relatively reduced oxygen content with respect to the oxygen content of air.
More particularly, uric acid is applied at a concentration of at least 0.2 g/L, preferably from 0.4 to 2 g/L, notably from 0.2 to 0.5 g/L, in order to obtain an equivalent or even greater effect on the growth of said bacteria sensitive to oxygen, in an aerobic atmosphere and at concentrations less than or equal to those of ascorbic acid and of glutathion alone or in a mixture allowing a similar effect on the growth of said bacteria under the same aerobic ambient atmospheres.
Uric acid may be applied as a mixture with ascorbic acid and/or glutathion but uric acid may be applied without any additional antioxidant compound in said culture medium.
As illustrated in the exemplary embodiments of the description detailed hereafter:
Preferably, the uric acid is applied in combination with ascorbic acid, still preferably with ascorbic acid and glutathion.
More particularly, said culture medium comprises the components which are again found in culture base media able to cultivate an anaerobic bacterium, comprising at least:
More particularly, said culture medium is an acellular medium and selected from an axenic medium consisting of chemical or biological substances defined qualitatively and quantitatively, and an acellular medium comprising an extract of milled material or lyzed material of pluricellular tissue. Preferably, said medium comprises a pH-regulating buffer substance for adjusting the pH from 7 to 7.5.
More particularly, said culture medium is a conventional acellular culture medium of a micro-aerophilic or anaerobic bacterium, preferably a medium comprising component selected from an extract of milled or lyzed material of a pluricellular tissue, an enzymatic digested material, notably an enzymatic digested material of casein, soya and/or animal tissue, a peptone, a yeast extract, a sugar such as dextrose or glucose, an NaCl salt and/or an Na2PO4 salt.
Still more particularly, said culture medium is a filtrate of said milled or lyzed material, notably of blood tissue or heart and/or lung tissue, when said bacterium is an extracellular bacterium such as so-called broth media of the heart-brain type, Columbia media with 5% of sheep blood or Schaedler medium as described hereafter. Other suitable conventional media are the Brucella or Wilkins-Chagren media. Such acellular culture media are well known to one skilled in the art. These media may be used with agar (solid or semi-solid) or without agar (liquid).
In particular polyvalent culture media may be used for anaerobic microorganisms, notably Schaedler medium.
Such acellular culture media whether they are liquid, solid or biphasic are well known to one skilled in the art. More particularly, said culture medium for anaerobic bacteria may be found as a liquid or solid or semi-solid, notably with gelosed or semi-gelosed medium.
More particularly, said bacterium is cultivated in an said incubation atmosphere comprising a greater oxygen molar proportion than the maximum tolerated tension in the absence of uric acid or of any antioxidant compound for a same growth level in a same culture period.
In practice, still more particularly, said bacterium is cultivated in an said incubation atmosphere comprising a molar oxygen proportion of less than or equal to 20%.
Still more particularly, said bacteria according to the present invention are cultivated in an atmosphere comprising an oxygen content of more than 5%, notably in air containing 5% of CO2 (i.e. an oxygen content of less than 16%), or even in an aerobic atmosphere of ambient air.
Still more particularly, in certain cases, as explained hereafter, said intracellular micro-aerophilic bacteria is cultivated in a said micro-aerophilic incubation atmosphere comprising a molar oxygen proportion of less than 5%, preferably from 2 to 5%, still preferably 2.5%.
More particularly, uric acid is applied with an antioxidant compound preferably selected from among ascorbic acid and glutathion (γ-L-Glutamyl-L-cysteinylglycine) or even sodium hydrosulfide. Ascorbic acid and glutathion are preferred since they are capable at specific doses of allowing cultivation at a higher oxygen level. Still more particularly, said antioxidant compound is applied at a concentration of 1 mg/L to 2 g/L, preferably at least 100 mg/L.
Preferably, said antioxidant compound is ascorbic acid and/or glutathion, preferably at a concentration of at least 100 mg/L.
More particularly, said bacterium is a bacterium which may be cultivated in a said culture medium in the absence of uric acid or of said oxidizing compound under an atmosphere comprising an oxygen molar proportion less than the oxygen molar proportion in air, preferably less than 20%, and said bacterium is cultivated in the presence of said uric acid in said culture medium under an incubation atmosphere comprising an oxygen content of less than or equal to the oxygen proportion in air, preferably less than 20%, still preferably greater than 5%.
According to a first embodiment, said bacterium is an extracellular anaerobic bacterium which may be cultivated in an anaerobic atmosphere in the absence of said uric acid or said antioxidant compound, and growth of said bacterium is obtained in the presence of oxygen with a molar proportion less than or equal to the oxygen proportion in air.
Anaerobic bacteria may be strict anaerobic bacteria or optional anaerobic bacteria also-called aero-anaerobic bacteria, i.e. anaerobic bacteria which tolerate oxygen but do not need it for growth or aerobic bacteria which support the absence of oxygen for growth.
Among the strict anaerobic bacteria, more particularly let us mention bacteria belonging to the genera Acidaminococcus, Alistipes, Anaerococcus, Anaerosalibacter, Amazonia, Atopobium, Bifidobacterium, Blautia, Bacteroides, Bamesiella, Clostridium, Collinsella, Dielma, Eggerthella, Finegoldia, Flavonifractor, Fusobacterium, Gordonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevotella, Peptostreptococcus, Peptoniphilus, Porphyromonas, Prevotella, Solobacterium, Tissierella, Tuncibacter, Ruminococcus and Veillonella.
From among the optional anaerobic bacteria, more particularly let us mention the bacteria belonging to the genera Actinomyces, Aerococcus, Aeromonas, Aneurinibacillus, Bacillus, Bartonella, Cedecea, Citrobacter, Corynebacterium, Derambacter, Eikenella, Enterobacter, Enterococcus, Escherichia, Eubacterium, Gardnerella, Gemella, Granulicatella, Hafnia, Haemophilus Kingella, Klebsellia, Lactobacillus, Lactococcus, Lysinibacillus, Morganella, Paenibacillus, Pasteurella, Pediococcus, Propionibacterium, Proteus, Providencia, Serratia, Raoultella, Rothia, Staphylococcus, Streptococcus and Weissella.
According to this first more particular embodiment, an anaerobic extracellular bacterium is cultivated, notably a bacterium of the digestive tract in humans or animals in a so-called acellular medium in the presence of a molar oxygen proportion less than or equal to that of air and in the presence of said uric acid, preferably in an atmosphere of ambient air.
This first embodiment is illustrated in the example hereafter by the aerobic cultivation of Bacteroides thetaiotaomicron in the presence of uric acid.
These are strict anaerobic bacteria of the digestive tract, which normally are only cultivated under strictly anaerobic conditions. This is expressed when said bacteria is inoculated into a deoxygenated tube by the fact that a culture veil only appears in the low portion of the tube, while the upper portion remains untouched by any culture. If uric acid according to present invention at 200 μg/ml is added, the cultivation practically occurs up to the surface, or even completely up to the surface at the concentration of 500 μg/ml and even better at 1 g/l with uric acid like for a mixture of ascorbic acid at 500 μg/ml and of glutathion at 500 μg/ml. The bacterium produces in 24 h colonies after having been sown on gelose.
In another embodiment, said bacterium is an intracellular micro-aerophilic bacterium capable of being cultivated in a said acellular culture medium, under a micro-aerophilic atmosphere with a molar oxygen proportion of not more than 5%, preferably not more than 2.5% in the incubation atmosphere, in the absence of uric acid or of an antioxidant compound, and said bacterium is cultivated in the presence of uric acid in a said culture medium under a micro-aerophilic incubation atmosphere comprising a molar oxygen proportion between 2.5% and 20%, preferably between 5% and 16%, notably air optionally enriched with 5% of CO2.
The present invention more particularly applies to micro-aerophilic bacteria because, inter alia of the addition of said uric acid.
From among the intracellular micro-aerophilic bacteria, mention may more particularly be made of bacteria of the genera Coxiella, Mycobacterium, Helicobacter, Campylobacter and Vagococcus.
This embodiment is illustrated by the aerobic cultivation of the bacterium Mycobacterium tuberculosis and Helicobacter cinaedi cultivated in ambient air.
Other species of micro-aerophilic bacteria, strict anaerobic bacteria and optional anaerobic bacteria mentioned in example 3 i.e. about 250 species, were tested with improved growths obtained under aerobic conditions in polyvalent culture media additived with uric acid.
Other features and advantages of the invention will become apparent in the light of the detailed description of the following exemplary embodiments.
A strain of the anaerobic bacterium Bacteroides thetaiotaomicron was obtained through the “culturomics” study of the inventors (2) also accessible in diverse deposit collections (CSUR P766 also deposited according to the Budapest Treaty on the deposit collection of DSMZ micro-organisms (Germany) on May 19, 2014 under the number DSM 28808, other strains are also accessible in diverse deposit collections such as the strains DSM 2079, ATCC 29148 and NCTC 10582).
For their production in a sufficient amount, B. thetaiotaomicron was cultivated in an anaerobic atmosphere at 37° C. in a polyvalent culture medium. The suitable Schaedler medium (Reference 42098; BioMérieux, La Balmes-les-Grottes, France) was tested also for cultivating anaerobic bacteria.
The Schaedler medium (marketed by BioMérieux, Marcy l'etoile, France) had the following composition for 1 liter:
This Schaedler medium was supplemented by adding hydrocarbon compounds, i.e. 1 g/L of rice starch and 1 g/L of glucose (Sigma-Aldrich, Saint-Quentin Fallavier, France) and by the addition of uric acid and anti-oxidant compounds, i.e. supplemented by adding:
The addition of hydrocarbon starch and glucose compounds aimed here to produce H2 for controlling the growth of the bacterium.
Resazurin is applied as an oxidation-reduction indicator at a concentration of 0.1 mg/ml for controlling the presence of oxygen (oxidized resazurin has a pink color, and becomes transparent in the absence of oxygen).
The aerobic culture in ambient air of B. thetaiotaomicron was carried out by inoculation of 105 organisms/ml in a container incubated at 37° C. containing the culture medium supplemented with the addition of anti-oxidant compounds and carbon source compounds. The pH was adjusted to 7.5 by adding 10M KOH.
The strain was cultivated in parallel in an aerobic condition and by the inoculation of 105 organisms/ml with the culture medium supplemented according to the present invention and with the Schaedler medium supplemented with the hydrocarbon compounds mentioned above but on the other hand without any anti-oxidant compounds.
The culture medium supplemented by adding 1 g/L of rice starch and 1 g/L of glucose inoculated under anaerobic conditions with 108 cells/L of B. thetaiotaomicron was introduced as a positive control and for checking the production of H2 by B. thetaiotaomicron in an anaerobic culture. These controls were carried out in parallel in an ambient atmosphere (aerobic condition). The non-inoculated culture medium was introduced as a negative control.
The growth of B. thetaiotaomicron was daily evaluated by the production of hydrogen. The measurement of hydrogen was carried out by means of a gas chromatograph GC-8A (Shimadzu, Champs-sur-Marne, France) equipped with a heat conductivity detector and a Chromosorb WAW 80/100 meshes column SP100 (Alltech, Carquefou, France). The nitrogen N2 at a pressure of 100 kPa was used as a carrier gas. The detector and the temperatures of the injector were 200° C. and the temperature of the column was 150° C.
The negative controls remained negative without any growth occurring after one week of incubation indicating that the results reported here are not simply the result of a contamination by other microorganisms.
The positive controls were positive, a production of hydrogen was observed in the anaerobic culture of B. thetaiotaomicron. The B. Thetaiotaomicron culture inoculated under aerobic conditions without any antioxidant compounds remained negative and the hydrogen was not produced.
After incubation for 24 hours at 37° C. in ambient air (under aerobic conditions), a culture medium without any anti-oxidant compounds kept its pink color indicating the presence of oxygen and the culture remained negative for the tested strain. The aerobic culture medium with uric acid or said anti-oxidant compounds became transparent indicating the absence of oxygen.
The cultures de B. thetaiotaomicron made under aerobic conditions with uric acid or said anti-oxidant compounds all gave a positive culture for B. thetaiotaomicron with production of hydrogen after 24 hour incubation.
The growth results with uric acid without said antioxidant compound were equivalent to those obtained with a mixture of uric acid with ascorbic acid and of glutathion when the uric acid concentration alone was of at least 0.2 g/L and greater with 0.3 g/L of uric acid alone.
These results indicate that it is possible to cultivate in ambient air (aerobic condition) bacteria notoriously assumed as strictly anaerobic bacteria, in a suitable medium containing a suitable mixture of antioxidants and in particular with uric acid alone without any antioxidant.
The cultivation procedure is carried out on culture medium tubes of the Schaedler type with 0.2% of agar-agar (BioMérieux, Marcy l'etoile, France).
The Schaedler medium had the following composition for 1 liter:
For each bacterium, 2 tubes are inoculated, a regenerated tube without any uric acid and a regenerated tube in which are added 500 μg/ml or 1 mg/ml of uric acid. In order to regenerate the tube, it is placed in a water bath at 100° C. until all the visible gas bubbles in the medium have disappeared. Next, for the tube with uric acid, the cooling of the tube at 50° C. is awaited (schematically until it is possible to hold it in the hand without burning oneself) and the suspension of uric acid is added. Homogenization is then performed by turning it over (3-4 for ensuring a good mixture). For each bacterium, an inoculum of 107 bacteria/ml was inoculated over the whole height of the Schaedler tubes 0.2%, a normal and a supplemented one in 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 the bacteria from the bottom of the tube was observed up to 1.5 cm below the surface of the medium in the absence of uric acid as a witness of the anaerobic nature of this bacterium, and a growth up to the surface in the presence of 500 μg/ml (28×10−4M) of uric acid indicating growth in the presence of a larger oxygen tension than in the absence of uric acid.
Tests, in every point identical were conducted either with glutathion or ascorbic acid at 500 μg/ml. With uric acid alone, the growth is identical to what is observed with ascorbic acid.
Finally, in order to definitively validate the capability of this uric acid compound of allowing growth of strict anaerobic bacteria in the presence of oxygen, solid media were prepared, consisting of Columbia medium with 5% of sheep blood in which was added uric acid at 500 μg/ml or 1 mg/ml, or a mixture of glutathion at 500 μg/ml+ascorbic acid at 500 μg/ml or of ascorbic acid at 1 mg/ml. These geloses inoculated with anaerobic bacteria were incubated either in ambient air or in ambient air enriched with 5% of CO2. Best growth was obtained with uric acid or ascorbic acid at 1 mg/ml equally.
These tests were conducted with a Columbia medium with 5% sheep blood having the following composition for 1 liter:
A strain DSMZ 5359 was used, 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 except that the culture medium was not supplemented by adding hydrocarbon compounds, starch and glucose.
The bacterium was established in a culture within 24 h and confirmed by mass spectrometry of the Maldi tof type.
In this example, the inventors compared the growth of the mycobacterium Mycobacterium tuberculosis, an agent of human and animal tuberculosis, on three solid media, under identical temperature (37° C.) conditions and atmospheric conditions (5% CO2).
The strain of the M. tuberculosis H37Rv type calibrated to 107 colony forming units (CFU) and four clinical strains of M. tuberculosis calibrated to 105 CFU or 106 CFU were inoculated into sterile Petri dishes in a MOD4 medium described hereafter, enriched with ascorbic acid at 100 mg/L (MOD5), and in uric acid at 100 mg/L (MODE) or in uric acid at 200 mg/L (MOD7). Five dishes were sown for each condition.
The results of the detection times with the naked eye of colonies in days are shown in the table hereafter.
These results show that there is an improvement in significant growth by adding uric acid or ascorbic acid and no significant difference in the growth of M. tuberculosis between ascorbic acid and uric acid, showing the possibility of using uric acid as an antioxidant for cultivation in an aerobic atmosphere of mycobacteria, at a concentration at least equal to 100 mg/L.
The results give the possibility of drawing the conclusion of faster detection of the colonies in decreasing order with a detection at:
1) The following anaerobic and micro-aerophilic bacteria were cultivated under aerobic conditions (under an atmosphere of ambient air) at 37° C. with improved growths in a solid culture medium consisting in a Schaedler medium with 0.2% agar-agar (Sigma-Aldrich, Saint-Quentin Fallavier, France) described in Example 1 supplemented with 1 g/L ascorbic acid (VWR, Louvain, Belgium)+100 mg/L of glutathion (Sigma, Quentin Fallavier, France)+400 mg/L of uric acid (Sigma, Quentin Fallavier, France).
The pH was adjusted to 7.5 with 10M KOH before passing into the autoclave. The antioxidant compounds were dissolved in 10 mL of distilled water, filtered with a microfilter of 0.2 μm and then added into the autoclaved medium at 50° C. so as to be sterilized before being cooled in solid form into gelose.
In the tables hereafter, the growth times correspond to the appearance of the first colonies visible to the naked eye.
Bifidobacterium
Bifidobacterium adolescentis
Bifidobacterium brevis
Bifidobacterium catenulatum
Bifidobacterium longum
Bifidobacterium pseudocatenulatum
Collinsella
Collinsella aerofaciens
Collinsella massilioamazoniensis
Collinsella tanakaei
Eggerthella
Eggerthella lenta
Gordonibacter
Gordonibacter pamelaeae
Alistipes
Alistipes finegoldii
Alistipes indistinctus
Alistipes putredinis
Alistipes shahii
Bacteroides
Bacteroides caccae
Bacteroides fragilis
Bacteroides intestnalis
Bacteroides nordii
Bacteroides ovatus
Bacteroides stercoris
Bacteroides thetaiotaomicron
Bacteroides timonensis
Bacteroides uniformis
Bacteroides vulgatus
Barnesiella
Bamesiella intestinihominis
Odoribacter
Odoribacter splanchnicus
Parabacteroides
Parabacteroides distasonis
Parabacteroides johnsonii
Parabacteroides merdae
Porphyromonas
Porphyromonas asaccharolityca
Prevotella
Prevotella buccalis
Acidaminococcus
Acidaminococcus intestini
Amazonia
Amazonia massiliensis
Anaerococcus
Anaerococcus vaginalis
Anaerosalibacter
Anaerosalibacter bizertensis
Anaerosalibacter massiliensis
Blautia
Blautia coccoides
Clostridium
Clostridium amazonitimonense
Clostridium amylolyticum
Clostridium anorexicamassiliensis
Clostridium anorexicus
Clostridium baratii
Clostridium bartlettii
Clostridium bifermentans
Clostridium bolteae
Clostridium butyricum
Clostridium clostridioforme
Clostridium cochlearium
Clostridium dakarense
Clostridium difficile
Clostridium glycolicum
Clostridium hathewayi
Clostridium jeddahense
Clostridium lituseburense
Clostridium paraputrificum
Clostridium perfringens
Clostridium ramosum
Clostridium rubiinfantis
Clostridium sartagoforme
Clostridium senegalense
Clostridium sordellii
Clostridium sporogenes
Clostridium subterminale
Clostridium symbiosum
Clostridium tertium
Dielma
Dielma fastidiosa
Finegoldia
Finegoldia magna
Flavonifractor
Flavonifractor plautii
Guyana
Guyana massiliensis
Holdemania
Holdemania massiliensis
Parvimonas
Parvimonas micra
Peptoniphilus
Peptoniphilus asaccharolyticus
Peptoniphilus harei
Peptoniphilus senegalensis
Peptostreptococcus
Peptostreptococcus asaccharolyticus
Ruminococcus
Ruminococcus gnavus
Tissierella
Tissierella praeacuta
Turicibacter
Turicibacter sanguinis
Veillonella
Veillonella dispar
Veillonella parvula
Fusobacterium
Fusobacterium necrophorum
Fusobacterium nucleatum
Mycobacterium
Mycobacterium
smegmatis
Campylobacter
Campylobacter
coli
Campylobacter
concisus
Campylobacter
cuniculorum
Campylobacter
fetus
Campylobacter
jejuni
Vagococcus
Vagococcus
fluvialis
Actinomyces
Actinomyces neuii
Actinomyces oris
Actinomyces radingae
Actinomyces urogenitalis
Corynebacterium
Corynebacterium accolens
Corynebacterium afermentans
Corynebacterium amycolatum
Corynebacterium aurimucosum
Corynebacterium efficiens
Corynebacterium jeikeium
Corynebacterium minutissimum
Corynebacterium propinquum
Corynebacterium pseudodiphtheriticum
Corynebacterium simulans
Corynebacterium striatum
Corynebacterium suicordis
Corynebacterium urealyticum
Corynebacterium ihumii
Corynebacterium tuberculostearicum
Corynebacterium ureicelerivorans
Dermabacter
Dermabacter hominis
Gardnerella
Gardnerella vaginalis
Propionibacterium
Propionibacterium acnes
Propionibacterium avidum
Rothia
Rothia aeria
Rothia dentocariosa
Aerococcus
Aerococcus urinae
Aerococcus viridans
Aneurinibacillus
Aneurinibacillus migulanus
Bacillus
Bacillus amyloliquefaciens
Bacillus aquimaris
Bacillus arsenicus
Bacillus badius
Bacillus bataviensis
Bacillus cereus
Bacillus circulans
Bacillus clausii
Bacillus coagulans
Bacillus firmus
Bacillus flexus
Bacillus koreensis
Bacillus lentus
Bacillus liqueniformis
Bacillus massilioamazoniensis
Bacillus megaterium
Bacillus oleronius
Bacillus pumilus
Bacillus rubiinfantis
Bacillus siralis
Bacillus subtilis
Bacillus thermoamylovorans
Bacillus vallismortis
Enterococcous
Enterococcus avium
Enterococcus casseliflavus
Enterococcus cecorum
Enterococcus dispar
Enterococcus durans
Enterococcus faecalis
Enterococcus faecium
Enterococcus gallinarum
Enterococcus hirae
Enterococcus malodoratus
Enterococcus phoeniculicola
Enterococcus pseudoavium
Enterococcus raffinosus
Eubacterium
Eubacterium limosum
Eubacterium tenue
Gemella
Gemella morbillorum
Granulicatella
Granulicatella elegans
Lactobacillus
Lactobacillus agilis
Lactobacillus fermentum
Lactobacillus gasseri
Lactobacillus johnsonii
Lactobacillus kalixensis
Lactobacillus mucosae
Lactobacillus paracasei
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillus sakei
Lactococcus
Lactococcus garvieae
Lactococcus lactis
Lysinibacillus
Lysinibacillus boronitolerans
Lysinibacillus fusiformis
Lysinibacillus meyeri
Lysinibacillus sphaericus
Paenibacillus
Paenibacillus lactis
Pediococcus
Pediococcus acidilactici
Pediococcus pentosaceus
Staphylococcus
Staphylococcus aureus
Staphylococcus capitis
Staphylococcus caprae
Staphylococcus cohnii
Staphylococcus epidermidis
Staphylococcus faecalis
Staphylococcus haemolyticus
Staphylococcus hominis
Staphylococcus intermedius
Staphylococcus lugdunensis
Staphylococcus pasteuri
Staphylococcus pettenkoferi
Staphylococcus saprophyticus
Staphylococcus schleiferi
Staphylococcus simulans
Staphylococcus warneri
Streptococcus
Streptococcus agalactiae
Streptococcus anginosus
Streptococcus constellatus
Streptococcus cristatus
Streptococcus dysgalactiae
Streptococcus equinus
Streptococcus gallolyticus
Streptococcus gordonii
Streptococcus intermedius
Streptococcus lutetiensis
Streptococcus mitis
Streptococcus oralis
Streptococcus parasanguinis
Streptococcus pneumonias
Streptococcus pyogenes
Streptococcus salivarius
Streptococcus sanguinis
Weissella
Weissella cibaria
Aeromonas
Aeromonas urinae
Aeromonas hydrophila
Bartonella
Bartonella henselae*
Cedecea
Cedecea lapagei
Cedecea neteri
Citrobacter
Citrobacter braakii
Citrobacter freundii
Citrobacter koseri
Citrobacter sedlakii
Enterobacter
Enterobacter aerogenes
Enterobacter asburiae
Enterobacter cloacae
Enterobacter kobei
Eikenella
Eikenella corrodens
Escherichia
Escherichia coli
Hafnia
Hafnia alvei
Haemophilus
Haemophilus influenzae*
Haemophilus parainfluenzae*
Kingella
Kingella kingae
Klebsiella
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella
Morganella morganii
Pasteurella
Pasteurella multocida
Proteus
Proteus mirabilis
Proteus vulgaris
Providencia
Providencia heimbachae
Providencia rettgeri
Providencia stuartii
Serratia
Serratia liquefaciens
Serratia marcescens
Serratia ureilytica
Raoultella
Raoultella ornithinolytica
2) 13 anaerobic bacteria and 1 micro-aerophilic bacteria (Campylobacter) were tested comparatively cultivated under aerobic conditions (under an atmosphere of ambient air) at 37° C. with improved growths in a sodium culture medium consisting in a Schaedler medium with 0.2% of agar-agar not supplemented or supplemented with:
The tested bacteria were: Bacteroides ovatus, Clostidium massilioamazoniensis; Anaerosalibacter bizertensis, Clostrididum paraperfringens, Clostridium sporogenes, Peptomphllus harei, Anegoldia magna, Tuncibacter sanguinis, Propionibacterium acnes, Bacteroides timonensis, Eikenella corrodens, Clostridium glycolicum, Bifidobacterium brevis, Campylobacter ureolyticus.
The results reported in table 2 show that 5 bacteria i.e. about ⅓ of the bacteria only have improved growth with uric acid (AU) alone or in combination with ascorbic acid (AA) and not with ascorbic acid alone.
In Table 2:—o
means: no colony was detected with the naked eye at t=96 h, and—“positive” means: colonies detected with the naked eye at t=96 h.
Peptoniphilus
harei
Finegoldia
magna
Clostridium
glycolicum
Bifidobacterium
brevis
Campylobacter
ureolyticus
Hemocultures of strict anaerobic bacteria were made, inoculated to 105 cfu/mL on a culture medium: BD BACTEC™ Plus Aerobic/F. comprising 25 ml of enriched Trypticase soya broth, with resines (Reference: 442192) at 37° C. in an aerobic atmosphere with uric acid a 400 mg/L and without uric acid and in an anaerobic atmosphere without uric acid. The detection of the growth was ensured by detection of the production of CO2 with the BD BACTEC™ 9000 Series Instrumented Blood Culture Systems device.
The comparative growth results reported in Table B hereafter establish that the growth detection was slightly faster in an aerobic atmosphere with uric acid (UA
in Table B) at 400 mg/L than in an anaerobic atmosphere and considerably faster than under aerobic conditions without uric acid.
Clostridium
tertium
Clostridium
tertium
Clostridium
tertium
Clostridium
perfringens
Clostridium
perfringens
Clostridium
perfringens
Clostridium
butirycum
Clostridium
butirycum
Clostridium
butirycum
| Number | Date | Country | Kind |
|---|---|---|---|
| 14 53652 | Apr 2014 | FR | national |
| 14 55745 | Jun 2014 | FR | national |
| 14 58622 | Sep 2014 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2015/051082 | 4/21/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/162377 | 10/29/2015 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2013110891 | Aug 2013 | WO |
| 2014064359 | May 2014 | WO |
| Entry |
|---|
| Rouf et al. “Degradation of Uric Acid by Certain Aerobic Bacteria” Journal of Bacteriology, Sep. 1968, p. 617-622. |
| Kole et al. “Protease production by Bacillus subtilis in oxygen-controlled, glucose fed-batch fermentations” Appl Microbiol Biotechnol (1988) 28:404-408. |
| Chen et al. “Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions” Appl Microbiol Biotechnol (2003) 63:143-146. |
| International Search Report dated Jul. 21, 2015 for Application No. PCT/FR2015/051082. |
| La Scola, B., et al., “Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report”, Eur. J. Clin. Microbiol. Infect. Dis., vol. 33, No. 10, May 13, 2014, pp. 1781-1783. |
| Krieg, N. R., et al., “Microaerophily and Oxygen Toxicity”, Ann. Rev. Microbiol., vol. 4, No. 1, Oct. 1, 1986, pp. 107-130. |
| Anonymous: “Uric Acid in Cell Culture”, Jun. 4, 2013 (Jun. 4, 2013). XP055177496. |
| Wilkinson, S. P., et al., HucR, a Novel Uric Acid-responsive Member of the MarR Family of Transcriptional Regulators from Deinococcus radiodurans, The Journal of Biological Chemistry, vol. 279, No. 49, Dec. 3, 2004, pp. 51442-51450. |
| Number | Date | Country | |
|---|---|---|---|
| 20170183620 A1 | Jun 2017 | US |
