WO2015159004A1 - Method and liquid medium for transporting and preserving bacteria - Google Patents
Method and liquid medium for transporting and preserving bacteria Download PDFInfo
- Publication number
- WO2015159004A1 WO2015159004A1 PCT/FR2015/050980 FR2015050980W WO2015159004A1 WO 2015159004 A1 WO2015159004 A1 WO 2015159004A1 FR 2015050980 W FR2015050980 W FR 2015050980W WO 2015159004 A1 WO2015159004 A1 WO 2015159004A1
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- WIPO (PCT)
- Prior art keywords
- cfu
- transport
- bacteria
- medium
- sample
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- 229940012969 lactobacillus fermentum Drugs 0.000 description 1
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- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/22—Means for packing or storing viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention relates to a method for transporting and preserving a sample of bacteria that may be anaerobic and / or aerobic bacteria, in particular a sample of clinical specimens containing anaerobic bacteria, allowing the preservation of the bacterium with a view to its culture and / or or subsequent isolation in the context of a test for detecting and quantifying said bacteria in a biological analysis laboratory.
- the present invention more particularly relates to a transport and preservation medium composition useful for this process.
- the present invention relates to a medium for transport and / or preservation under aerobic conditions at cold or at ambient temperature which must preserve the strict anaerobic bacteria after at least 48 hours, preferably at least 72 hours, or even more preferably at least 7 hours. days in said transport and storage medium to allow the isolation and detection or even subsequent cultivation of strict anaerobic bacteria for which the oxygen is toxic and which must be transported or kept cold in an oxygen-free medium.
- a bacterial sample can not be preserved in a bacterial culture medium because in such a medium, in the absence of culture conditions, especially at an appropriate temperature generally above room temperature, the bacteria are destroyed by the culture medium. .
- the AMIES or STUART liquid media dedicated to the transport of GRAM- bacteria are known to ensure viability for at least 48 hours at room temperature or preferably at 4-8 ° C. and compatible with a subsequent culture of aerobic bacteria.
- these media are only compatible with detection by molecular biology (DNA) analysis because they do not protect against the effect of oxygen.
- DNA molecular biology
- a solid agar medium is used, in particular based on agar containing a phosphate salt and sodium and calcium salts whose supposed interest is to have a buffer effect that avoids pH variations.
- the present invention relates to the transport and preservation of bacteria whose growth is sensitive to the oxygen tension, in particular bacteria that are poorly tolerant of high oxygen tensions and for which optimal preservation of said bacteria requires an atmosphere of 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%, especially between 1% and 20%, most commonly at about 2-2.5%, and
- the strict anaerobic bacteria that is to say that they are not able to cultivate in the presence of oxygen or in concentrations lower than microaerophilic concentrations, especially strictly 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.
- intracellular bacteria Among bacteria that can be cultivated in a microaerophilic atmosphere, intracellular bacteria are more particularly distinguished, but also extracellular bacteria.
- extracellular bacteria a bacterium that has the ability to multiply within a host cell.
- Intracellular bacteria capable of growing under certain conditions in acellular media, are referred to as “facultative intracellular bacteria”.
- intracellular bacterium is intended herein to mean a bacterium that has the capacity to multiply within a host cell and in a cell-free medium.
- extracellular bacterium is meant here a bacterium that does not have the capacity to multiply within a host cell and is grown exclusively in acellular medium.
- 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%.
- 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.
- the object of the present invention is to propose a novel transport and / or liquid storage medium which makes it possible to preserve bacteria, including strict anaerobic bacteria, in order to allow them to be cultured and subsequently isolate both aerobic and non-aerobic bacteria.
- anaerobic bacteria and this after transport or storage under aerobic conditions at room temperature or cold, or at an oxygen tension which is that of the ambient air for at least 48 hours, preferably at least 72 hours, or even 7 days.
- the inventors have fortuitously discovered that the mixture of several mineral compounds and substances with antioxidant effect, and in particular the addition of antioxidant compound in a liquid medium transporting standard bacteria, allowed to preserve cold or at room temperature strict anaerobic bacteria and cultivate them and isolate after transport under aerobic conditions, ie at an oxygen level of about 21%.
- the present invention therefore provides a method of transporting samples containing strict anaerobic bacteria in the form of a liquid medium preserving the viability and the possibility of culturing these strict anaerobic bacteria after transport under an oxygen atmosphere, even at room temperature. .
- antioxidant compound is meant herein a compound with an antioxidant property that is to say which decreases or prevents the oxidation of other substances involved in the process and the medium of the present invention under the conditions of the process of the invention. present invention.
- the inventors have discovered the mixture of all these compounds imparts a buffering effect with respect to oxygen content for strict anaerobic bacteria.
- the inventors have in fact tested several combinations of different molecules at different concentrations, some of which have antioxidant activity and have discovered that certain compounds mixed together at certain concentrations allowed the transport in a liquid medium in an aerobic atmosphere (ambient air) of said bacteria.
- the present invention provides a method for transporting and / or preserving bacterial samples capable of preserving the viability of anaerobic bacteria, for at least 48 hours, preferably at least 72 hours, or even 7 days, at cold or ambient temperature, in particular from -20 ° C to + 30 ° C, preferably at room temperature from 4 ° C to 20 ° C, characterized in that said sample of bacteria is conditioned in a container containing a transport medium and liquid preservation, under oxygen-containing atmosphere, said transport and storage medium being buffered at pH 7 to 7.5, comprising at least in distilled water:
- said pH buffered transport and storage medium of 7 to 7.5 comprises at least: - a phosphate salt
- Ca and at least one antioxidant compound chosen from ascorbic acid, glutathione, and uric acid, at a concentration of at least 1 g / l, and
- PH 7-7.5 pH regulating buffering substance which is potassium hydroxide (KOH).
- said antioxidant compound is used at a concentration of 1 ⁇ g / ml to 2 mg / ml, or molar concentration of 10 -6 to 10 -2 M, preferably at least 100 ⁇ g / ml or at least 10 ⁇ g / ml. "5 M.
- said antioxidant compound comprises at least ascorbic acid, preferably at a concentration of at least 1 g / l.
- composition of the transport and preservation medium comprises:
- NaCl sodium chloride
- said antioxidant compound consists of uric acid alone preferably at a concentration of at least 0.4 g / L.
- composition of the transport and preservation medium comprises:
- NaCl sodium chloride
- said transport and liquid preservation medium further comprises a color redox indicator capable of visibly changing the state of the medium, in particular its color, in the event that the latter has lost its antioxidant capacity, preferably resazurin at a concentration of at least 0.0015g / L.
- This indicator makes it possible to verify the absence of prior oxidation of the antioxidant compounds in said medium which could affect the viability of anaerobic bacteria and thus render it unfit for use.
- said liquid medium is mixed with said sample containing bacteria in a proportion of at least 2 volumes of said medium for 1 volume of said sample of bacteria containing a bacterial count of 1 than 10 15 cfu / mL, particularly February 10-October 10 cfu / mL.
- said sample of bacteria is a sample of clinical biological sample. More particularly, said sample of bacteria is a clinical biological fluid sample of biological fluid such as urine, blood or secretion such as saliva, stool, and other body secretions of mucous membranes or other body organs, pus, various collections and effusions, and biopsies, said sample being possibly supported by a material to perform the sampling of a sample such as a swab of cotton or synthetic fiber.
- said bacterial sample comprises or may comprise an anaerobic extracellular bacterium, in particular anaerobic digestive tract bacteria, which normally only transport in special transport media for anaerobic bacteria, said strict anaerobic bacteria being selected from bacteria belonging to the genera Clostridium, Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Lactobacillus, Actinomyces, Clostridium, Bacteroides, Fimmietes, Porphyromonas, Prevotella, Fusobacterium, Atopobium, Ruminococcus, Solobacterium, Acidaminococcus, Alistipes, Amazonia, Anaerosalibacter, Anaerococcus, Barnesiella , Bifidobacterium, Blautia, Col I insella, Dielam, Flavonifractot, GemellaGodonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevo
- Acidaminococcus intestine / Alistipes finegoldii, Alistipes indistinctus, Alistipes putredinis, Alistipes shahii, Amazonia massi / iensis, Anaerococcus vaginalis, Anaerosalibacter bizertensis, Anaerosalibacter massi / iensis, Atopobium vaginae, Bacteroides caccae, Bacteroides fragilis, Bacteroides intestalis, Bacteroides massi / iensis, Bacteroides nordii, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides timonensis, Bacteroides uniformis, Bacteroides vulgatus, Barnesiella intestinihominis, Bifidobacterium adolescentis, Bifidobacterium brief,
- said bacterial sample comprises or may comprise a bacterium selected from Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli and Clostridium jeddamassiliensis.
- bacterium selected from Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli and Clostridium jeddamassiliensis.
- aerobic bacteria with oxygen deficiency or naerobia which tolerate the oxygen content of growth known as "aero-naerobia", especially bacteria of the genus.
- aero-naerobia Aero-naerobia, Actinomyces, Actinomyces urogenitalis, Aerococcus viridans, Aneurinibacillus migulanus, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus aquimaris sp.
- the transport and preservation medium is universal in that it can also preserve strict microaerophilic or aerobic bacteria or aerobic anaerobes, said bacterial sample can therefore also include microaerophilic or aerobic bacteria.
- said bacterial sample comprising or may comprise an intracellular microaerophilic bacterium capable of being cultured in a said cell-free culture medium, under a microaerophilic incubation atmosphere with a molar proportion of oxygen of not more than 5%, preferably not more than 2.5% in the incubation atmosphere, in the absence of said antioxidant compound.
- said bacterium is chosen from facultative intracellular microaerophilic bacteria selected from bacteria of the genera Bartonella, Rickettsia, preferably R. conorii and R. africae, Coxiella, preferably Coxiella burnetii, Tropheryma, preferably Tropheryma whipplei, mycobacteria, preferably Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium ulcerans and Orientia spp., Preferably Orientia tsutsugamushi, and Propionibacterium including Propionibacterium acnes and Propionibacterium avidum.
- aerobic bacteria of the genera Acinetobacter, Brevibacterium, Gracilibacillus, Halobacillus, Kocuria, Micrococcus, Neisseria, Oceanobaciilus, Paenibacilus, Pseudomona, Roseomonas, Rothia, Virgibacillus and Weissella are also mentioned.
- Acinetobacter Iwoffii Acinetobacter radioresistens, Brevibacterium casei, Brevibacterium luteolum, Brevibacterium ravenspurgense, Gracilibacillus dipsosauri, Halobacillus trueperi, Kocuria rhizophila, Micrococcus luteus, Neisseria elongata, Neisseria flavescens, Neisseria maccacae, Neisseria mucosa, Oceanobaciilus picturae, Oceanobaciilus sojae, Paenibaciilus barengoltzii, Paenibaciilus lactis , Paenibaciilus thiaminolyticus, Paenibaciilus timonensis, Propionibacterium acnes, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Ps
- the transport of a sample which may contain a bacterial extract in a transport and preservation medium, at room temperature and, after transport, storage in storage of the cold sample of preferably at + 4 ° C to + 8 ° C, or frozen, preferably at -20 ° C to -80 ° C under an ambient air atmosphere.
- the present invention also provides a container for a transport and storage fluid medium capable of preserving the viability of aerobic bacteria and / or anaerobic bacteria useful in a process as defined above under a controlled atmosphere. oxygen, said transport and storage medium being buffered at pH 7 to 7.5, comprising at least in distilled water:
- the container may be for example a tube or a bottle.
- said container further contains in said liquid medium, a clinical specimen which may contain a said bacterial sample and, where appropriate, collection equipment for said clinical specimen such as a swab.
- the present invention also relates to a method of manufacturing a container according to the invention containing a said liquid medium for transporting and preserving bacteria, comprising the following successive steps in which:
- said transport medium and incomplete preservation are cooled to ambient temperature and said anti-oxidant compounds previously filtered are added through a filter which retains microorganisms such as bacteria, in particular with a filter which retains elements of more than 0.2 pm or even more than ⁇ , ⁇ (this is at say a filter with pores less than 0.2 ⁇ m, size of the smallest bacteria).
- step 1) said antioxidant compounds are excluded because they are capable of being degraded under the conditions of sterilization in said autoclave. And, in step 2) the said antioxidant compounds are sterilized in solution by filtering them.
- the clinical specimen is immersed in the transport and preservation medium, and then stored for detection of anaerobic bacteria, in the presence of a molar proportion of oxygen less than or equal to that of air.
- the preservation of these bacteria is done at room temperature or at + 4-8 ° C for a period of 48 hours or up to 72 hours or more than 1 week frozen at -20 ° C before inoculation under culture conditions anaerobic bacteria for the performing a said detection test.
- Example 1 Preparation of a liquid transport medium and preservation of bacteria.
- This suspension is sterilized in an autoclave preferably in an autoclave at a temperature above 121 ° C. for at least 20 minutes.
- this solution is filtered on a filter at 0.2 ⁇ m.
- the autoclaved medium is cooled to room temperature and 1 ml of the filtered suspension is mixed with 99 ml of the autoclaved suspension.
- Disodium phosphate Na 2 HPO 4 : 1.15g / L
- the final pH of these media A and B must be between 7 to 7.5
- the bacteria tested to illustrate the invention are all strict anaerobic bacteria particularly known to be difficult to service during their transport and storage, namely Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli, Clostridium jeddamassiliensis, Bacteroides fragilis, Bacteroides intestinalis, Bacteroides thetaiotaomicron, Ruminococcus gnavus, Fusobacterium necrophorum, Fusobacterium nucleatum and Alistipes finegoldii.
- the AMIES medium includes in distilled water:
- the liquid CARY-BLAIR medium in distilled water comprises:
- Bacteroides intestinalis 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 7 cfu / ml 10 6 cfu / mL
- Fine alyssis egoldii 10 8 cfu / ml 10 8 cfu / ml 10 8 cfu / ml 10 5 cfu / ml 10 5 cfu / ml
- Fine alyssis egoldii 10 8 cfu / ml 10 8 cfu / ml 10 8 cfu / ml 10 5 cfu / ml 10 5 cfu / ml
- Clostridium tertium 9.10 cfu / mL 3.10 cfu / mL 5.10 cfu / mL 3.10 cfu / mL 10 cfu / mL Clostridium butyricum 9.10 cfu / mL 5.10 cfu / mL 7.10 cfu / mL 9.10 cfu / mL 0 cfu / mL
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Abstract
The invention relates to a liquid medium and to a method allowing the transport and the preservation of samples containing anaerobic and/or aerobic bacteria in ambient air, that can preserve the viability of said bacteria with a view to the subsequent culturing and isolation thereof.
Description
Procédé et milieu liquide de transport et conservation de bactéries. Method and liquid medium for transporting and preserving bacteria
La présente invention concerne un procédé de transport et conservation d'échantillon de bactéries pouvant être des bactéries anaérobies et/ou aérobies, notamment d'échantillon de prélèvements cliniques contenant des bactéries anaérobies, permettant la préservation de la bactérie en vue de sa culture et/ou isolement ultérieure dans le cadre d'un test de détection et quantification desdites bactéries dans un laboratoire d'analyse biologique. La présente invention concerne plus particulièrement une composition de milieu de transport et conservation utile pour ce procédé. The present invention relates to a method for transporting and preserving a sample of bacteria that may be anaerobic and / or aerobic bacteria, in particular a sample of clinical specimens containing anaerobic bacteria, allowing the preservation of the bacterium with a view to its culture and / or or subsequent isolation in the context of a test for detecting and quantifying said bacteria in a biological analysis laboratory. The present invention more particularly relates to a transport and preservation medium composition useful for this process.
Plus particulièrement, la présente invention concerne un milieu de transport et/ou de conservation en conditions aérobies au froid ou à température ambiante qui doit préserver les bactéries anaérobies strictes après au moins 48h, de préférence au moins 72h, voire de préférence encore au moins 7 jours dans ledit milieu de transport et conservation pour permettre l'isolement et la détection voire la culture ultérieure de bactéries anaérobies strictes pour lesquelles l'oxygène est toxique et qui doivent être transportées ou conservées au froid dans un milieu dépourvu d'oxygène. More particularly, the present invention relates to a medium for transport and / or preservation under aerobic conditions at cold or at ambient temperature which must preserve the strict anaerobic bacteria after at least 48 hours, preferably at least 72 hours, or even more preferably at least 7 hours. days in said transport and storage medium to allow the isolation and detection or even subsequent cultivation of strict anaerobic bacteria for which the oxygen is toxic and which must be transported or kept cold in an oxygen-free medium.
On ne peut pas conserver un échantillon bactérien dans un milieu de culture de bactérie car dans un tel milieu en l'absence de conditions de culture, notamment à température appropriée en général supérieur à la température ambiante, les bactéries sont détruites par le milieu de culture. A bacterial sample can not be preserved in a bacterial culture medium because in such a medium, in the absence of culture conditions, especially at an appropriate temperature generally above room temperature, the bacteria are destroyed by the culture medium. .
C'est pourquoi, dans le cas où l'on veut transporter les prélèvements bactériens destinés à une inoculation en vue de rechercher les bactéries anaérobies strictes éventuelles qu'il contient, pour une chance d'isolement optimale, on est obligé de faire deux échantillonnages, un pour les bactéries aérobies et un autre pour les bactéries anaérobies strictes. En effet, pour le cas des bactéries
aérobies, on utilise généralement un milieu de transport liquide à base de tampon Phosphate ou glycérophosphate et de sels de sodium et calcium. Therefore, in the case where we want to transport the bacterial samples intended for inoculation to search for any strict anaerobic bacteria it contains, for a chance of optimal isolation, it is necessary to do two sampling one for aerobic bacteria and another for strict anaerobic bacteria. Indeed, for the case of bacteria aerobic, generally uses a liquid transport medium based on phosphate or glycerophosphate buffer and sodium and calcium salts.
On connaît, en particulier, les milieux AMIES ou STUART liquides dédiés au transport de bactéries GRAM- en assurant la viabilité pendant au moins 48h à température ambiante ou de préférence à 4-8°C et compatible avec une culture ultérieure de bactéries aérobies. En revanche pour les bactéries anaérobies, ces milieux ne sont compatibles qu'avec une détection par analyse de biologie moléculaire (ADN) car ne protègent pas de l'effet de l'oxygène. Pour être efficaces pour le transport des bactéries anaérobies, ils doivent contenir de l'agar permettant aux bactéries anaérobies d'être protégées de l'oxygène. Ainsi pour les bactéries anaérobies strictes, on utilise un milieu solide gélosé, notamment à base d'Agar contenant un sel de phosphate et des sels sodium et calcium dont l'intérêt supposé est d'avoir un effet tampon qui évite les variations de pH. In particular, the AMIES or STUART liquid media dedicated to the transport of GRAM- bacteria are known to ensure viability for at least 48 hours at room temperature or preferably at 4-8 ° C. and compatible with a subsequent culture of aerobic bacteria. On the other hand, for anaerobic bacteria, these media are only compatible with detection by molecular biology (DNA) analysis because they do not protect against the effect of oxygen. To be effective for the transport of anaerobic bacteria, they must contain agar allowing anaerobic bacteria to be protected from oxygen. Thus, for strict anaerobic bacteria, a solid agar medium is used, in particular based on agar containing a phosphate salt and sodium and calcium salts whose supposed interest is to have a buffer effect that avoids pH variations.
Ce milieu solide dans lequel on dépose (on « pique ») le prélèvement bactérien permet de soustraire les bactéries anaérobies présentes à l'effet de l'oxygène. On connaît en particulier le milieu CARY BLAIR ou AMIES gélosé avec ou sans charbon. Ces milieux de transport de bactéries anaérobies sont relativement coûteux et donc finalement pas utilisés de façon systématique. Après transport, les échantillons bactériens sont inoculés dans les milieux de cultures en conditions adaptées à la culture des bactéries anaérobies. Pour le transport et la conservation au froid, le problème est similaire, que cette conservation soit faite à température ambiante de 0 à 30°C, plus généralement à 20°C +/- 5°C, ou refroidi et isolé au froid de +4°C à +8°C, voire congelé à -20°C qui sont les températures usuelles de conservation permettant la préservation des bactéries aérobies et anaérobies.
Plus particulièrement, la présente invention concerne le transport et la conservation de bactéries dont la croissance est sensible à la tension en oxygène, notamment des bactéries qui tolèrent mal des tensions élevées d'oxygène et pour lesquelles une conservation optimale de ladite bactérie requière une atmosphère d'incubation à teneur en oxygène relativement réduite par rapport à la teneur en oxygène de l'air voire des bactéries anaérobies strictes pour lesquelles l'oxygène est toxique et qui doivent être cultivées en absence totale d'oxygène ou ne tolérant que de faibles concentrations d'oxygène. Il convient en effet de noter que certaines bactéries considérées comme anaérobie strictes peuvent parfois tolérer de faibles concentrations en oxygène. This solid medium in which the bacterial sample is deposited ("spades") makes it possible to subtract the anaerobic bacteria present to the effect of oxygen. CARY BLAIR or AMIES agar medium with or without charcoal is particularly known. These transport media of anaerobic bacteria are relatively expensive and therefore not used systematically. After transport, the bacterial samples are inoculated into the culture media in conditions suitable for the culture of anaerobic bacteria. For transportation and cold storage, the problem is similar, whether this preservation is done at room temperature from 0 to 30 ° C, more generally at 20 ° C +/- 5 ° C, or cooled and cold-insulated from + 4 ° C to + 8 ° C, or even frozen at -20 ° C which are the usual storage temperatures allowing the preservation of aerobic and anaerobic bacteria. More particularly, the present invention relates to the transport and preservation of bacteria whose growth is sensitive to the oxygen tension, in particular bacteria that are poorly tolerant of high oxygen tensions and for which optimal preservation of said bacteria requires an atmosphere of oxygen. relatively low oxygen content incubation with respect to the oxygen content of the air or even strict anaerobic bacteria for which oxygen is toxic and which must be cultured in the total absence of oxygen or tolerating only low concentrations of oxygen. 'oxygen. It should be noted that some bacteria considered strict anaerobic can sometimes tolerate low oxygen concentrations.
On distingue donc parmi les bactéries sensibles à l'oxygène : One can therefore distinguish among oxygen-sensitive bacteria:
- les bactéries microaérophiles c'est-à-dire qu'elles ne sont pas capables de cultiver sous une atmosphère comprenant la concentration d'oxygène ambiante qui est de environ 21%, notamment entre 1% et 20%, le plus communément à environ 2-2,5%, et 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%, especially between 1% and 20%, most commonly at about 2-2.5%, and
- les bactéries anaérobies strictes c'est-à-dire qu'elles ne sont pas capables de cultiver en présence d'oxygène ou dans des concentrations inférieures aux concentrations de microaérophilie, notamment strictement inférieure à 1%, le plus communément inférieure à 0.1%, idéalement 0%. Pour cultiver les bactéries anaérobies strictes, il faut soit les cultiver dans des étuves ne comportant pas d'oxygène, soit dans des tubes qui ont été désoxygénés et elles ne poussent alors qu'au fond du tube. the strict anaerobic bacteria, that is to say that they are not able to cultivate in the presence of oxygen or in concentrations lower than microaerophilic concentrations, especially strictly 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.
Parmi les bactéries anaérobies strictes, on cite plus particulièrement les bactéries extracellulaires, c'est à dire des bactéries qui qui ne peuvent vivre qu'à l'extérieur de cellules. Among the strict anaerobic bacteria, mention is made more particularly of extracellular bacteria, ie bacteria which can live only outside cells.
Parmi les bactéries cultivables en atmosphère microaérophile, on distingue plus particulièrement, les bactéries intracellulaires, mais aussi des bactéries extracellulaires.
On entend ici par «bactérie intracellulaire», une bactérie qui a la capacité de se multiplier au sein d'une cellule hôte. Les bactéries intracellulaires, ayant la faculté de croître dans certaines conditions dans des milieux acellulaires, sont dénommées «bactéries intracellulaires facultatives». Among bacteria that can be cultivated in a microaerophilic atmosphere, intracellular bacteria are more particularly distinguished, but also extracellular bacteria. Here is meant by "intracellular bacteria", a bacterium that has the ability to multiply within a host cell. Intracellular bacteria, capable of growing under certain conditions in acellular media, are referred to as "facultative intracellular bacteria".
On entend ici par «bactérie intracellulaire facultative», une bactérie qui a la capacité de se multiplier au sein d'une cellule hôte et en milieu acellulaire. The term "optional intracellular bacterium" is intended herein to mean a bacterium that has the capacity to multiply within a host cell and in a cell-free medium.
On entend ici par «bactérie extracellulaire», une bactérie qui n'a pas la capacité de se multiplier au sein d'une cellule hôte et se cultive exclusivement en milieu acellulaire. By "extracellular bacterium" is meant here a bacterium that does not have the capacity to multiply within a host cell and is grown exclusively in acellular medium.
Plus particulièrement, la présente invention concerne la conservation de bactéries anaérobies strictes et des bactéries microaérophiles. On entend ici par «atmosphère microaérophile», de l'air appauvri en oxygène avec une proportion molaire en oxygène inférieure à 10%, de préférence 5%, de préférence encore inférieure à 2,5%. Pour les bactéries anaérobies strictes, la teneur en oxygène doit être proche de 0%, notamment inférieure à 0.1%, comme mentionné ci-dessus, la tolérance à de très faibles quantités d'oxygène étant variable selon les espèces de bactéries anaérobies. More particularly, the present invention relates to the conservation of strict anaerobic bacteria and microaerophilic bacteria. 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.
Le but de la présente invention est de proposer un nouveau milieu de transport et/ou de conservation liquide qui permette de préserver les bactéries, y compris les bactéries anaérobies strictes en vue de permettre de les cultiver et isoler ultérieurement aussi bien les bactéries aérobies que les bactéries anaérobies, et ce après un transport ou conservation en conditions aérobie à température ambiante ou au froid, soit à une tension en oxygène qui est celle de l'air ambiant pendant au moins 48h, de préférence au moins 72h, voire 7 jours.
Les inventeurs ont découvert de façon fortuite que le mélange de plusieurs composés minéraux et de substances à effet antioxydant, et notamment l'ajout de composé anti-oxydant dans un milieu liquide de transport de bactéries standard, permettait de préserver au froid ou à température ambiante des bactéries anaérobies strictes et de les cultiver et isoler après transport en conditions aérobies, soit à un taux d'oxygène d'environ 21%. The object of the present invention is to propose a novel transport and / or liquid storage medium which makes it possible to preserve bacteria, including strict anaerobic bacteria, in order to allow them to be cultured and subsequently isolate both aerobic and non-aerobic bacteria. anaerobic bacteria, and this after transport or storage under aerobic conditions at room temperature or cold, or at an oxygen tension which is that of the ambient air for at least 48 hours, preferably at least 72 hours, or even 7 days. The inventors have fortuitously discovered that the mixture of several mineral compounds and substances with antioxidant effect, and in particular the addition of antioxidant compound in a liquid medium transporting standard bacteria, allowed to preserve cold or at room temperature strict anaerobic bacteria and cultivate them and isolate after transport under aerobic conditions, ie at an oxygen level of about 21%.
La présente invention fournit donc un procédé de transport de prélèvements contenant des bactéries anaérobies strictes sous forme d'un milieu liquide préservant la viabilité et la possibilité de culture de ces bactéries anaérobies strictes après un transport sous atmosphère d'oxygène et ce même à température ambiante. The present invention therefore provides a method of transporting samples containing strict anaerobic bacteria in the form of a liquid medium preserving the viability and the possibility of culturing these strict anaerobic bacteria after transport under an oxygen atmosphere, even at room temperature. .
On entend ici par « composé antioxydant », un composé à propriété antioxydante c'est-à-dire qui diminue ou empêche l'oxydation d'autres substances impliquées dans le procédé et le milieu de la présente invention dans les conditions du procédé de la présente invention. By the term "antioxidant compound" is meant herein a compound with an antioxidant property that is to say which decreases or prevents the oxidation of other substances involved in the process and the medium of the present invention under the conditions of the process of the invention. present invention.
Les inventeurs formulent l'hypothèse non encore totalement élucidée et démontrée que l'effet du mélange de tous ces composés pourrait provenir d'une limitation de l'apparition de radicaux libres oxygénés toxiques et/ou d'une réaction des composés antioxydants avec lesdits radicaux libres oxygénés toxiques, ayant, dans les deux cas, un effet inhibiteur de la toxicité desdits radicaux à rencontre de la croissance de ladite bactérie. Ces radicaux libres oxygénés résultant eux-mêmes de l'action de l'oxygène sur des substances des bactéries et/ou du milieu de transport seraient responsables des difficultés de conservation en présence d'oxygène et de culture ultérieure. The inventors formulate the hypothesis not yet fully elucidated and demonstrated that the effect of the mixture of all these compounds could come from a limitation of the appearance of toxic oxygen free radicals and / or a reaction of the antioxidant compounds with said radicals. toxic oxygenated free, having, in both cases, an inhibitory effect of the toxicity of said radicals against the growth of said bacterium. These oxygenated free radicals themselves resulting from the action of oxygen on bacteria substances and / or transport medium would be responsible for the difficulties of conservation in the presence of oxygen and subsequent culture.
Plus particulièrement, les inventeurs ont découvert le mélange de tous ces composés confère un effet tampon au regard de la teneur en oxygène pour les bactéries anaérobies strictes.
Les inventeurs ont en effet testé plusieurs combinaisons de différentes molécules à différentes concentrations dont certaines présentant une activité antioxydante et ont découvert que certains composés mélangés entre eux à certaines concentrations autorisaient le transport en milieu liquide en atmosphère aérobie (air ambiant) des dites bactéries. More particularly, the inventors have discovered the mixture of all these compounds imparts a buffering effect with respect to oxygen content for strict anaerobic bacteria. The inventors have in fact tested several combinations of different molecules at different concentrations, some of which have antioxidant activity and have discovered that certain compounds mixed together at certain concentrations allowed the transport in a liquid medium in an aerobic atmosphere (ambient air) of said bacteria.
Plus précisément, la présente invention fournit un procédé de transport et/ou conservation d'échantillons de bactéries capable de préserver la viabilité de bactéries anaérobies, pendant au moins 48h, de préférence au moins 72h, voire 7 jours, à température froide ou ambiante notamment de -20°C à +30°C, de préférence à température ambiante de 4°C à 20°C, caractérisé en ce que l'on conditionne ledit échantillon de bactéries dans un récipient contenant un milieu de transport et conservation liquide, sous atmosphère contenant de l'oxygène, ledit milieu de transport et conservation étant tamponné à PH de 7 à 7,5, comprenant au moins dans de l'eau distillée : More specifically, the present invention provides a method for transporting and / or preserving bacterial samples capable of preserving the viability of anaerobic bacteria, for at least 48 hours, preferably at least 72 hours, or even 7 days, at cold or ambient temperature, in particular from -20 ° C to + 30 ° C, preferably at room temperature from 4 ° C to 20 ° C, characterized in that said sample of bacteria is conditioned in a container containing a transport medium and liquid preservation, under oxygen-containing atmosphere, said transport and storage medium being buffered at pH 7 to 7.5, comprising at least in distilled water:
- une source de phosphore, et a source of phosphorus, and
- au moins un sel de métal alcalin ou alcalino-terreux de K, Mg, Na, Ca, et - au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion, et l'acide urique, et at least one alkali metal or alkaline earth metal salt of K, Mg, Na, Ca, and at least one antioxidant compound selected from ascorbic acid, glutathione, and uric acid, and
- une substance tampon régulateur de pH. a buffer substance that regulates pH.
Plus particulièrement, ledit milieu de transport et conservation tamponné à PH de 7 à 7.5, comprend au moins : - un sel de phosphate, et More particularly, said pH buffered transport and storage medium of 7 to 7.5 comprises at least: - a phosphate salt, and
- au moins un sel de chlorure de chacun des métaux K, Mg, Na,at least one chloride salt of each of the metals K, Mg, Na,
Ca, et
- au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion, et l'acide urique, à une concentration d'au moins lg/L, et Ca, and at least one antioxidant compound chosen from ascorbic acid, glutathione, and uric acid, at a concentration of at least 1 g / l, and
- une substance tampon régulateur de pH à PH de 7-7.5 qui est l'hydroxyde de potassium (KOH). a PH 7-7.5 pH regulating buffering substance which is potassium hydroxide (KOH).
Plus particulièrement encore, ledit composé antioxydant est mis en œuvre à une concentration de 1 pg/ml à 2 mg/ml, ou concentration molaire de 10"6 à 10"2 M, de préférence au moins 100 pg/ml ou au moins 10"5 M. More particularly, said antioxidant compound is used at a concentration of 1 μg / ml to 2 mg / ml, or molar concentration of 10 -6 to 10 -2 M, preferably at least 100 μg / ml or at least 10 μg / ml. "5 M.
Dans un mode de réalisation, ledit composé antioxydant comprend au moins l'acide ascorbique, de préférence à une concentration d'au moins 1 g/1. In one embodiment, said antioxidant compound comprises at least ascorbic acid, preferably at a concentration of at least 1 g / l.
Plus particulièrement encore, la composition du milieu de transport et conservation comprend : More particularly, the composition of the transport and preservation medium comprises:
- chlorure de sodium (NaCL) : 3g/L sodium chloride (NaCl): 3g / L
- chlorure de potassium (KCI) : 0.2g/L - potassium chloride (KCl): 0.2g / L
- chlorure de calcium (CaCI2) : O.lg/L calcium chloride (CaCl 2 ): O.lg / L
- chlorure de magnésium (MgCI2) : O.lg/L magnesium chloride (MgCl 2 ): O.lg / L
- phosphate monopotassique (KH2P04) : 0.2g/L - monopotassium phosphate (KH2P04): 0.2g / L
- phosphate de sodium (Na2HP04) : 1.15g/L sodium phosphate (Na 2 HPO 4 ): 1.15 g / L
- acide ascorbique (C6H806) : lg/L ascorbic acid (C 6 H 8 O 6 ): lg / L
- glutathion (C10H17N3O3S) : O.lg/L glutathione (C 10 H 17 N 3 O 3 S): O.lg / L
- acide urique (C5H4N403) : O.lg/L uric acid (C 5 H 4 N 4 O 3 ): O.lg / L
- hydroxyde de potassium (KOH) 0.3 - 0.6 - potassium hydroxide (KOH) 0.3 - 0.6
- eau distillée qsp : 1 litre
Dans un autre mode de réalisation, ledit composé antioxydant consiste dans l'acide urique seul de préférence à une concentration d'au moins 0.4 g/L. - distilled water qsp: 1 liter In another embodiment, said antioxidant compound consists of uric acid alone preferably at a concentration of at least 0.4 g / L.
Plus particulièrement encore, la composition du milieu de transport et conservation comprend : More particularly, the composition of the transport and preservation medium comprises:
- chlorure de sodium (NaCL) : 3g/L sodium chloride (NaCl): 3g / L
- chlorure de potassium (KCI) : 0.2g/L - potassium chloride (KCl): 0.2g / L
- chlorure de calcium (CaCI2) : O.lg/L calcium chloride (CaCl 2 ): O.lg / L
- chlorure de magnésium (MgCI2) : O.lg/L magnesium chloride (MgCl 2 ): O.lg / L
- phosphate monopotassique (KH2P04) : 0.2g/L - monopotassium phosphate (KH2P04): 0.2g / L
- phosphate de sodium (Na2HP04) : 1.15g/L sodium phosphate (Na 2 HPO 4 ): 1.15 g / L
- acide urique (C5H4N403) : 0.4g/L uric acid (C 5 H 4 N 4 O 3 ): 0.4 g / L
- hydroxyde de potassium (KOH) : 0.3 - 0.6 g/L - potassium hydroxide (KOH): 0.3 - 0.6 g / L
- eau distillée qsp : 1 litre De préférence, ledit milieu de transport et conservation liquide comprend en outre un indicateur coloré d'oxydoréduction apte à changer visiblement l'état du milieu, notamment sa couleur, dans le cas où celui-ci aurait perdu sa capacité anti-oxydante, de préférence la résazurine à une concentration d'au moins 0,0015g/L. Cet indicateur permet de vérifier l'absence d'oxydation préalable des composés antioxydants dans ledit milieu pouvant affecter la viabilité de bactéries anaérobies et le rendrait donc impropre à une utilisation. distilled water qs: 1 liter Preferably, said transport and liquid preservation medium further comprises a color redox indicator capable of visibly changing the state of the medium, in particular its color, in the event that the latter has lost its antioxidant capacity, preferably resazurin at a concentration of at least 0.0015g / L. This indicator makes it possible to verify the absence of prior oxidation of the antioxidant compounds in said medium which could affect the viability of anaerobic bacteria and thus render it unfit for use.
De préférence encore, ledit milieu liquide est mélangé au dit échantillon contenant des bactéries à raison d'une proportion d'au moins 2 volumes de dit milieu pour 1 volume de dit échantillon de
bactéries contenant un nombre de bactéries de 1 à 1015 cfu/mL, notamment de 102 à 1010 cfu/mL. More preferably, said liquid medium is mixed with said sample containing bacteria in a proportion of at least 2 volumes of said medium for 1 volume of said sample of bacteria containing a bacterial count of 1 than 10 15 cfu / mL, particularly February 10-October 10 cfu / mL.
Avantageusement encore, ledit échantillon de bactéries est un échantillon de prélèvement biologique clinique. Plus particulièrement encore, ledit échantillon de bactéries est un échantillon de prélèvement biologique clinique de fluide biologique tel que urine, sang ou sécrétion telle que salives, selles, et autres sécrétions corporelles de muqueuses ou autres organes corporels, pus, collections et épanchements divers, et biopsies, ledit prélèvement étant éventuellement supporté par un matériel pour réaliser le prélèvement d'un échantillon tel qu'un écouvillon en coton ou fibre synthétique. Advantageously, said sample of bacteria is a sample of clinical biological sample. More particularly, said sample of bacteria is a clinical biological fluid sample of biological fluid such as urine, blood or secretion such as saliva, stool, and other body secretions of mucous membranes or other body organs, pus, various collections and effusions, and biopsies, said sample being possibly supported by a material to perform the sampling of a sample such as a swab of cotton or synthetic fiber.
Plus particulièrement encore, ledit échantillon bactérien comprend ou peut comprendre une bactérie extracellulaire anaérobie, notamment les bactéries anaérobies du tube digestif, qui normalement ne se transportent que dans des milieux de transport spéciaux pour bactéries anaérobies, les dites bactéries anaérobies strictes étant choisies parmi les bactéries appartenant aux genres Clostridium, Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Lactobacillus, Actinomyces, Clostridium, Bacteroides, Fi rmi eûtes, Porphyromonas, Prevotella, Fusobacterium, Atopobium, Ruminococcus, Solobacterium, Acidaminococcus, Alistipes, Amazonia, Anaerosalibacter, Anaerococcus, Barnesiella, Bifidobacterium, Blautia, Col I insella, Dielam, Flavonifractot, GemellaGodonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevotella, Senegalmassillia, Tisssierella, Turicibacter, Veillonella. More particularly, said bacterial sample comprises or may comprise an anaerobic extracellular bacterium, in particular anaerobic digestive tract bacteria, which normally only transport in special transport media for anaerobic bacteria, said strict anaerobic bacteria being selected from bacteria belonging to the genera Clostridium, Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Lactobacillus, Actinomyces, Clostridium, Bacteroides, Fimmietes, Porphyromonas, Prevotella, Fusobacterium, Atopobium, Ruminococcus, Solobacterium, Acidaminococcus, Alistipes, Amazonia, Anaerosalibacter, Anaerococcus, Barnesiella , Bifidobacterium, Blautia, Col I insella, Dielam, Flavonifractot, GemellaGodonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevotella, Senegalmassillia, Tisssierella, Turicibacter, Veillonella.
Plus particulièrement, on cite les bactéries anaérobies strictes suivantes : Acidaminococcus intestin/, Alistipes finegoldii, Alistipes indistinctus, Alistipes putredinis, Alistipes shahii, Amazonia massi/iensis, Anaerococcus vaginalis, Anaerosalibacter bizertensis, Anaerosalibacter massi/iensis, Atopobium vaginae, Bacteroides caccae, Bacteroides fragilis, Bacteroides intestnalis, Bacteroides massi/iensis,
Bacteroides nordii, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides timonensis, Bacteroides uniformis, Bacteroides vulgatus, Barnesiella intestinihominis, Bifidobacterium adolescentis, Bifidobacterium brève, Bifidobacterium catenulatum, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Blautia coccoides, Clostidium butyricum, Clostridium amazonitimonense, Clostridium amylolyticum, Clostridium anorexicamassiliensis, Clostridium anorexicus, Clostridium baratii, Clostridium bartlettii, Clostridium bifermentans, Clostridium bolteae, Clostridium butyricum, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium diolis, Clostridium glycolicum, Clostridium hathewayi, Clostridium jeddamassiliensis, Clostridium lituseburense, Clostridium paraputrificum, Clostridium perfringens, Clostridium ramosum, Clostridium rubiinfantis, Clostridium sartagoforme, Clostridium scindens, Clostridium sordellii, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Collinsella aerofaciens, Collinsella massilioamazoniensis, Collinsella tanakaei, Dielma fastidiosa, Finegoldia magna, Flavonifractor plautii, Fusobacterium necrophorum, Finegoldia magna, Gemella morbillorum, Gordonibacter pamelaeae, Guyana massiliensis, Holdemania massiliensis, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides johnsonii, Parabacteroides johnsonii, Parabacteroides merdae, Parvimonas micra, Pre vote/la bucca/is, Pre vote lia nigrescens, Ruminococcus gnavus Senegalemassilia anaerobia, Solobacterium morei, Tissierella praeacuta, Turicibacter sanguin/s, Veillonella dispar, Veillonella parvula. More particularly, the following strict anaerobic bacteria are mentioned: Acidaminococcus intestine /, Alistipes finegoldii, Alistipes indistinctus, Alistipes putredinis, Alistipes shahii, Amazonia massi / iensis, Anaerococcus vaginalis, Anaerosalibacter bizertensis, Anaerosalibacter massi / iensis, Atopobium vaginae, Bacteroides caccae, Bacteroides fragilis, Bacteroides intestalis, Bacteroides massi / iensis, Bacteroides nordii, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides timonensis, Bacteroides uniformis, Bacteroides vulgatus, Barnesiella intestinihominis, Bifidobacterium adolescentis, Bifidobacterium brief, Bifidobacterium catenulatum, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Blautia coccoid, Clostridium butyricum, Clostridium amazonitimonense Clostridium amylolyticum Clostridium anorexicamassiliensis Clostridium anorexicus Clostridium baratii Clostridium bartlettii Clostridium bifermentans Clostridium bolteae, Clostridium butyricum, Clostridium clostridioforme Clostridium cochlearium, Clostridium difficile, Clostridium diolis Clostridium glycolicum Clostridium hathewayi Clostridium jeddamassiliensis Clostridium lituseburense Clostridium paraputrificum, Clostridium Perfringens, Clostridium ramosum, Clostridium rubiinfantis, Clostridium sartagoforme, Clostridium scindens, Clostridium sordellii, Clostridium sporogenes , Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Collinsella aerofaciens, Collinsella massilioamazoniensis, Collinsella tanakaei, Dielma fastidiosa, Finegoldia magna, Flavonifractor plautii, Fusobacterium necrophorum, Finegoldia magna, Gemella morbillorum, Gordonibacter pamelaeae, Guyana massiliensis, Holdemania massiliensis, Odoribacter splanchnicus, Parabacteroides Distasonis, Parabacteroides johnsonii, Parabacteroides johnsonii, Parabacteroides merdae, Parvimonas micra, Pre vote / la bucca / is, Pre vote lia nigrescens, Ruminococcus gnavus Senegalemassilia anaerobia, Solobacterium morei, Tissierella praeacuta, Turicibacter sanguinis, Veillonella dispar, Veillonella parvula.
Plus particu lièrement encore, ledit échantillon bactérien comprend ou peut comprendre une bactérie choisie parmi Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli et Clostridium jeddamassiliensis.
On cite éga lement des bactéries aérobies q u i su pportent l 'a bsence d 'oxygène ou a naerobies q u i tolèrent la p résence d'oxygène pou r leu r croissa nce dénom mées « aéro-a naerobies », nota m ment les bactéries des gen res : Actinomyces, Aerococcus, Aneurinibacillus, Bacillus, Cedecea, Citrobacter, Corynebacterium, Eggerthella, Eikenella, Enterobacter, Escherichia, Eubacterium, Facklamia, Granulicatella, Haemophilus, Ha f nia, Lactobacillus, Leuconostoc, Lysinibacillus, Pediococcus, Providencia, Serra tia, Staphylococcus, Streptococcus. More particularly still, said bacterial sample comprises or may comprise a bacterium selected from Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli and Clostridium jeddamassiliensis. There are also aerobic bacteria with oxygen deficiency or naerobia which tolerate the oxygen content of growth known as "aero-naerobia", especially bacteria of the genus. res: Actinomyces, Aerococcus, Aneurinibacillus, Bacillus, Cedecea, Citrobacter, Corynebacterium, Eggerthella, Eikenella, Enterobacter, Escherichia, Eubacterium, Facklamia, Granulicatella, Haemophilus, Haifia, Lactobacillus, Leuconostoc, Lysinibacillus, Pediococcus, Providencia, Serra tia, Staphylococcus , Streptococcus.
Pl u s pa rticu l ièrement les bactéries aéro-a naérobies, Actinomyces, Actinomyces urogenitalis, Aerococcus viridans, Aneurinibacillus migulanus, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus aquimaris sp. nov, 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 s ira lis, Bacillus subti/is, Bacillus thermoamylovorans, Bacillus vallismortis, Cedecea lapagei, Cedecea neteri, Citrobacter freundii, Citrobacter koseri, Citrobacter sedlakii, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium efficiens, Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium reamassiliensis, Corynebacterium suicordis, Corynebacterium tuberculostearicum, Corynebacterium ureicelerivorans, Eggerthella /enta, Eikenella corrodens, Enterobacter c/oacae, Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcous gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus phoeniculicola, Enterococcus pseudoavium, Enterococcus raffinosus, Escherichia coli, Eubacterium limosum, Eubacterium tenue, Facklamia hominis, Granulicatella elegans, Haemophilus parainfluenzae, Hafnia alvei, Lactobacillus agilis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii,
Lactobacillus kalixensis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactococcus garvieae, Leuconostoc lactis, Lysinibacillus boronitolerans, Lysinibacillus fusiformis, Lysinibacillus meyeri, Lysinibacillus sphaericus, Pediococcus acidilactici, Pediococcus pentosaceus, Peptoniphilus asaccharolyticus, Peptoniphilus harei, Peptoniphilus senegalensis, Providencia heimbachae, Serra tia marcescens, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus faecalis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus simulans, Staphylococcus warneri, Streptococcus cri status, Streptococcus dysgalactiae, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus lutetiensis, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis. More particularly, aero-naerobia, Actinomyces, Actinomyces urogenitalis, Aerococcus viridans, Aneurinibacillus migulanus, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus aquimaris sp. nov., 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 salla, Bacillus subtilis, Bacillus thermoamylovorans, Bacillus vallismortis, Cedecea lapagei, Cedecea neteri, Citrobacter freundii, Citrobacter koseri, Citrobacter sedlakii, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium efficiens, Corynebacterium minutissimum, Corynebacterium Pseudodiphtheriticum, Corynebacterium reamassiliensis, Corynebacterium suicordis, Corynebacterium tuberculostearicum, Corynebacterium ureicelerivorans, Eggerthella / enta, Eikenella corrodens, Enterobacter c / oacae, Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, En Terococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus phoeniculicola, Enterococcus pseudoavium, Enterococcus raffinosus, Escherichia coli, Eubacterium limosum, Eubacterium holding, Facklamia hominis, Granulicatella elegans, Haemophilus parainfluenzae, Hafnia alvei, Lactobacillus agilis , Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactococcus garvieae, Leuconostoc lactis, Lysinibacillus boronitolerans, Lysinibacillus fusiformis, Lysinibacillus meyeri, Lysinibacillus sphaericus, Pediococcus acidilactici, Pediococcus pentosaceus, Peptoniphilus asaccharolyticus, Peptoniphilus Harei, Peptoniphilus senegalensis , Providencia heimbachae, Serra tia marcescens, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus faecalis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus simulans, Staphylococcus warneri, Streptococcus cry status, Streptococcus dysgalactiae , Streptococcus equinus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus lutetiensis, Streptococcus mitis, Streptococcus oralis, Streptococcus salivariu s, Streptococcus sanguinis.
Le milieu de transport et conservation est universel en ce sens qu'il peut aussi préserver les bactéries microaérophiles ou aérobies strictes ou aéro-anaérobies, ledit échantillon bactérien peut donc comprendre aussi des bactéries microaérophiles ou aérobies. The transport and preservation medium is universal in that it can also preserve strict microaerophilic or aerobic bacteria or aerobic anaerobes, said bacterial sample can therefore also include microaerophilic or aerobic bacteria.
On cite notamment, plus particulièrement encore, ledit échantillon bactérien comprend ou peut comprendre une bactérie microaérophile intracellulaire apte à être cultivée dans un dit milieu de culture acellulaire, sous atmosphère d'incubation microaérophile avec une proportion molaire en oxygène de pas plus de 5%, de préférence pas plus de 2.5% dans l'atmosphère d'incubation, en l'absence de dit composé antioxydant. In particular, more particularly, mention is made of said bacterial sample comprising or may comprise an intracellular microaerophilic bacterium capable of being cultured in a said cell-free culture medium, under a microaerophilic incubation atmosphere with a molar proportion of oxygen of not more than 5%, preferably not more than 2.5% in the incubation atmosphere, in the absence of said antioxidant compound.
Plus particulièrement encore, ladite bactérie est choisie parmi les bactéries microaérophiles intracellulaires facultatives choisies parmi les bactéries des genres Bartonella, Rickettsia, de préférence R. conorii et R. africae, Coxiella, de préférence Coxiella burnetii, Tropheryma, de préférence Tropheryma whipplei, mycobactéries, de préférence
Mycobacterium tuberculosis, Mycobacterium leprae et Mycobacterium ulcerans et Orientia spp., de préférence Orientia tsutsugamushi, et Propionibacterium notamment Propionibacterium acnés et Propionibacterium avidum. On cite également, plus pa rticu lièrement, les bactéries aérobies des genres Acinetobacter, Brevibacterium, Gracilibacillus,Halobacillus, Kocuria, Micrococcus, Neisseria, Oceanobaciilus, Paenibaciilus, Pseudomona, Roseomonas, Rothia, Virgibacillus et Weissella. More particularly, said bacterium is chosen from facultative intracellular microaerophilic bacteria selected from bacteria of the genera Bartonella, Rickettsia, preferably R. conorii and R. africae, Coxiella, preferably Coxiella burnetii, Tropheryma, preferably Tropheryma whipplei, mycobacteria, preferably Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium ulcerans and Orientia spp., Preferably Orientia tsutsugamushi, and Propionibacterium including Propionibacterium acnes and Propionibacterium avidum. Also more specifically, aerobic bacteria of the genera Acinetobacter, Brevibacterium, Gracilibacillus, Halobacillus, Kocuria, Micrococcus, Neisseria, Oceanobaciilus, Paenibacilus, Pseudomona, Roseomonas, Rothia, Virgibacillus and Weissella are also mentioned.
Plus particu lièrement encore, on cite les bactéries aérobies su ivantes ; Acinetobacter Iwoffii, Acinetobacter radioresistens, Brevibacterium casei, Brevibacterium luteolum, Brevibacterium ravenspurgense, Gracilibacillus dipsosauri, Halobacillus trueperi, Kocuria rhizophila, Micrococcus luteus, Neisseria elongata, Neisseria flavescens, Neisseria maccacae, Neisseria mucosa, Oceanobaciilus picturae, oceanobaciilus sojae, Paenibaciilus barengoltzii, Paenibaciilus lactis, Paenibaciilus thiaminolyticus, Paenibaciilus timonensis, Propionibacterium acnés, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas saudiphocaensis, Roseomonas mucosa, Rothia aeria, Rothia dentocariosa, Virgibacillus halodenitrificans, Virgibacillus massiliensis, Virgibacillus proomii, Virgibacillus senegalensis, Weissella cibaria, Weissella cibaria, Weissella cibaria. More particularly still, mention is made of the aerobic bacteria which are present; Acinetobacter Iwoffii, Acinetobacter radioresistens, Brevibacterium casei, Brevibacterium luteolum, Brevibacterium ravenspurgense, Gracilibacillus dipsosauri, Halobacillus trueperi, Kocuria rhizophila, Micrococcus luteus, Neisseria elongata, Neisseria flavescens, Neisseria maccacae, Neisseria mucosa, Oceanobaciilus picturae, Oceanobaciilus sojae, Paenibaciilus barengoltzii, Paenibaciilus lactis , Paenibaciilus thiaminolyticus, Paenibaciilus timonensis, Propionibacterium acnes, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas saudiphocaensis, Roseomonas mucosa, Rothia aeria, Rothia dentocariosa, Virgibacillus halodenitrificans, Virgibacillus massiliensis, Virgibacillus proomii, Virgibacillus senegalensis Weissella cibaria, Weissella cibaria, Weissella cibaria.
Plus particu lièrement encore, on effectue le tra nsport d'u n prélèvement pouvant contenir un écha ntil lon bactérien dans u n d it milieu de transport et conservation, à températu re ambia nte et, après transport, on conserve en stockage ledit prélèvement a u froid de préférence à +4°C à +8°C, ou congelé, de préférence à -20°C à -80°C, sous u ne atmosphère d'air ambiant. More particularly still, the transport of a sample which may contain a bacterial extract in a transport and preservation medium, at room temperature and, after transport, storage in storage of the cold sample of preferably at + 4 ° C to + 8 ° C, or frozen, preferably at -20 ° C to -80 ° C under an ambient air atmosphere.
La présente invention fou rnit aussi un récipient contena nt un milieu l iqu ide de tra nsport et conservation apte à préserver la viabil ité de bactéries aérobies et/ou bactéries anaérobies utile da ns u n procédé tel q ue défini ci-dessus, sous atmosphère contenant de l'oxygène, ledit
milieu de transport et conservation étant tamponné à PH de 7 à 7,5, comprenant au moins dans de l'eau distillée : The present invention also provides a container for a transport and storage fluid medium capable of preserving the viability of aerobic bacteria and / or anaerobic bacteria useful in a process as defined above under a controlled atmosphere. oxygen, said transport and storage medium being buffered at pH 7 to 7.5, comprising at least in distilled water:
- une source de phosphore, et a source of phosphorus, and
- au moins un sel de métal choisi parmi K, Mg, Na et Ca, et - au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion, et l'acide urique, et at least one metal salt chosen from K, Mg, Na and Ca, and at least one antioxidant compound chosen from ascorbic acid, glutathione, and uric acid, and
- une substance tampon régulateur de pH. a buffer substance that regulates pH.
Le récipient peut être par exemple un tube ou un flacon. The container may be for example a tube or a bottle.
Plus particulièrement encore, ledit récipient contient en outre dans ledit milieu liquide, un prélèvement clinique pouvant contenir un dit échantillon bactérien et le cas échéant un matériel de collecte du dit prélèvement clinique tel qu'un écouvillon. More particularly, said container further contains in said liquid medium, a clinical specimen which may contain a said bacterial sample and, where appropriate, collection equipment for said clinical specimen such as a swab.
La présente invention a également pour objet un procédé de fabrication d'un récipient selon l'invention contenant un dit milieu liquide de transport et conservation de bactéries comprenant les étapes successives suivantes dans lesquelles : The present invention also relates to a method of manufacturing a container according to the invention containing a said liquid medium for transporting and preserving bacteria, comprising the following successive steps in which:
1) on chauffe dans un autoclave dans des conditions de température pour le stériliser, de préférence à une température supérieure à 121°C pendant au moins 20 mn, ledit récipient contenant un dit milieu de transport et conservation liquide incomplet ne contenant pas lesdits composés antioxydants, sous atmosphère d'air ambiant, et 1) is heated in an autoclave under temperature conditions to sterilize it, preferably at a temperature above 121 ° C for at least 20 minutes, said container containing a said transport medium and incomplete liquid storage not containing said antioxidant compounds , under ambient air, and
2) on refroidit ledit milieu de transport et conservation incomplet, à température ambiante et on y ajoute lesdits composés anti oxydants préalablement filtrés à travers un filtre qui retient les microorganismes tels que les bactéries, notamment avec un filtre qui retient les éléments de plus de 0.2pm voire de plus de Ο,ΐμιτι (c'est à
dire un filtre avec des pores inférieurs à 0,2 μ m, taille des plus petites bactéries). 2) said transport medium and incomplete preservation are cooled to ambient temperature and said anti-oxidant compounds previously filtered are added through a filter which retains microorganisms such as bacteria, in particular with a filter which retains elements of more than 0.2 pm or even more than Ο, ΐμιτι (this is at say a filter with pores less than 0.2 μm, size of the smallest bacteria).
A l'étape 1), on exclut lesdits composés anti-oxydants car ceux-ci sont susceptibles d'être dégradés dans les conditions de stérilisation dans ledit autoclave. Et, à l'étape 2) on stérile les lesdits composés anti-oxydant en solution en les filtrant. In step 1), said antioxidant compounds are excluded because they are capable of being degraded under the conditions of sterilization in said autoclave. And, in step 2) the said antioxidant compounds are sterilized in solution by filtering them.
Selon l'invention, le prélèvement clinique est immergé dans le milieu de transport et conservation, puis conservé en vue d'une détection de bactérie anaérobie, en présence d'une proportion molaire d'oxygène inférieure ou égale à celle de l'air. La préservation de ces bactéries est faite à température ambiante ou à +4-8°C pendant une durée de 48h ou jusqu'à 72h voire plus de 1 semaine congelé à -20°C avant inoculation en conditions de culture des bactéries anaérobies pour la réalisation d'un dit test de détection. D'autres caractéristiques et avantages de l'invention sont présentés dans la description détaillée des exemples qui suivent. According to the invention, the clinical specimen is immersed in the transport and preservation medium, and then stored for detection of anaerobic bacteria, in the presence of a molar proportion of oxygen less than or equal to that of air. The preservation of these bacteria is done at room temperature or at + 4-8 ° C for a period of 48 hours or up to 72 hours or more than 1 week frozen at -20 ° C before inoculation under culture conditions anaerobic bacteria for the performing a said detection test. Other features and advantages of the invention are presented in the detailed description of the examples which follow.
Exemple 1 : Préparation d'un milieu liquide de transport et conservation de bactéries. Example 1 Preparation of a liquid transport medium and preservation of bacteria.
La fabrication du milieu doit être faire selon la séquence suivante On mélange les composés suivants : The production of the medium must be made according to the following sequence. The following compounds are mixed:
Chlorure de Sodium (NaCL) : 3g Sodium Chloride (NaCl): 3g
Chlorure de Potassium (KCI) : 0.2g Potassium Chloride (KCl): 0.2g
Chlorure de Calcium (CaCI2) : 0.1g Calcium Chloride (CaCl 2 ): 0.1g
Chlorure de Magnésium chloride (MgCI2) : 0.1g Phosphate monopotassique (KH2P04) : 0.2g Magnesium Chloride Chloride (MgCl 2 ): 0.1g Monopotassium Phosphate (KH2PO4): 0.2g
Disodium phosphate (Na2HP04) 1.15g
Hydroxyde de potassium (KOH) 0.45 g/L Disodium phosphate (Na 2 HPO 4 ) 1.15g Potassium hydroxide (KOH) 0.45 g / L
Réazurine 0.0015 g/L Rezurine 0.0015 g / L
Eau distillée qsp : 1 L Distilled water qsp: 1 L
On stérilise dans un autoclave cette suspension de préférence dans un autoclave à une température supérieure de 121°C pendant au moins 20 mn. This suspension is sterilized in an autoclave preferably in an autoclave at a temperature above 121 ° C. for at least 20 minutes.
Séparément, on mélange les composés suivants Separately, the following compounds are mixed
Acide ascorbique (C6H806) : 1 g Ascorbic acid (C 6 H 8 0 6 ): 1 g
Glutathion (CioH17N303S) : 0.1g Acide urique (C5H4N4O3) : 0.1g Glutathione (CioH 17 N 3 03S): 0.1g uric acid (C5H4N4O3): 0.1g
Eau distillée qsp : 10 ml_ Distilled water qs: 10 ml_
Après dissolution cette solution est filtrée sur un filtre à 0.2pm. After dissolution, this solution is filtered on a filter at 0.2 μm.
On refroidit ledit milieu autoclavé à température ambiante et on mélange 1 ml de la suspension filtrée avec 99 ml de la suspension autoclavée. The autoclaved medium is cooled to room temperature and 1 ml of the filtered suspension is mixed with 99 ml of the autoclaved suspension.
On obtient ainsi la formulation A suivante : This gives the following formulation A:
Chlorure de Sodium (NaCL) : 3g/L Sodium Chloride (NaCl): 3g / L
Chlorure de Potassium (KCI) : 0.2g/L Potassium Chloride (KCl): 0.2g / L
Chlorure de Calcium (CaCI2) : O.lg/L Chlorure de Magnésium chloride (MgCI2) : O.lg/L Calcium Chloride (CaCl 2 ): O.lg / L Magnesium Chloride Chloride (MgCl 2 ): O.lg / L
Phosphate monopotassique (KH2P04) : 0.2g/L Monopotassium phosphate (KH2P04): 0.2g / L
Disodium phosphate (Na2HP04) : 1.15g/L Disodium phosphate (Na 2 HPO 4 ): 1.15g / L
Acide ascorbique (C6H806) : lg/L
Glutathion (CioH17N303S) : 0.1g/L Ascorbic acid (C 6 H 8 0 6 ): lg / L Glutathione (CioH 17 N 3 03S): 0.1g / L
Acide urique (C5H4N4O3) : 0.1g/L Uric acid (C5H4N4O3): 0.1g / L
Hydroxyde de potassium (KOH) : 0.45 g/L Potassium hydroxide (KOH): 0.45 g / L
Réazurine 0.0015 g/L On a également testé une formulation B suivante obtenue en remplaçant dans le procédé de préparation les 3 composés oxydants par le seul acide urique à une concentration supérieure de 0.4 g/L : Reaazurin 0.0015 g / L A subsequent formulation B was also tested by replacing in the preparation process the 3 oxidizing compounds with uric acid only at a concentration of 0.4 g / L higher:
Chlorure de Sodium (NaCL) : 3 g/L Sodium Chloride (NaCl): 3 g / L
Chlorure de Potassium (KCI) : 0.2 g/L Chlorure de Calcium (CaCI2) : 0.1 g/L Potassium Chloride (KCl): 0.2 g / L Calcium Chloride (CaCl 2 ): 0.1 g / L
Chlorure de Magnésium chloride (MgCI2) : 0.1 g/L Phosphate monopotassique (KH2P04) : 0.2 g/L Disodium phosphate (Na2HP04) : 1.15 g/L Magnesium Chloride chloride (MgCl 2 ): 0.1 g / L Monopotassium phosphate (KH 2 PO 4): 0.2 g / L Disodium phosphate (Na 2 HPO 4 ): 1.15 g / L
Acide urique (C5H4N403) : 0.4 g/L Hydroxyde de potassium (KOH) : 0.45 g/L Uric acid (C 5 H 4 N 4 O 3 ): 0.4 g / L Potassium hydroxide (KOH): 0.45 g / L
Réazurine 0.0015 g/L Rezurine 0.0015 g / L
Le pH final de ces milieux A et B doit être entre 7 à 7.5 The final pH of these media A and B must be between 7 to 7.5
Dans les valeurs de concentration ci-dessus : 1 M = 1 mol/L In the concentration values above: 1 M = 1 mol / L
Exemple 2: Effet du milieu de transport et de conservation pour la culture de bactéries anaérobies. Example 2 Effect of the Transport and Preservation Medium for the Culture of Anaerobic Bacteria
Les bactéries testées pour illustrer l'invention sont toutes des bactéries anaérobies strictes particulièrement connues pour être difficiles à viabilisé pendant leur transport et stockage, à savoir
Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli, Clostridium jeddamassiliensis, Bacteroides fragilis, Bacteroides intestinalis, Bacteroides thetaiotaomicron, Ruminococcus gnavus, Fusobacterium necrophorum, Fusobacterium nucleatum et Alistipes finegoldii. The bacteria tested to illustrate the invention are all strict anaerobic bacteria particularly known to be difficult to service during their transport and storage, namely Clostridium tertium, Clostridium butyricum, Clostridium ramosum, Clostridium perfringens, Clostridium sordelli, Clostridium jeddamassiliensis, Bacteroides fragilis, Bacteroides intestinalis, Bacteroides thetaiotaomicron, Ruminococcus gnavus, Fusobacterium necrophorum, Fusobacterium nucleatum and Alistipes finegoldii.
Dans des essais comparatifs, ces bactéries ont été mises d'une part dans des milieux standards de conservation à savoir un tampon PBS, et des milieux CARY BLAIR et AMIES (milieu ESWAB de la société COPAN, Italie) d'autre part et parallèlement dans les milieux de transport A et B de l'exemple 1 conservées à température ambiante comme décrit ci-après. In comparative tests, these bacteria were firstly placed in standard preservation media, namely a PBS buffer, and CARY BLAIR and AMIES media (ESWAB medium from COPAN, Italy), on the other hand and in parallel with the transport media A and B of Example 1 stored at room temperature as described below.
Le milieu AMIES comprend dans de l'eau distillée: The AMIES medium includes in distilled water:
- Chlorure de sodium : 3.0 g/L - Sodium chloride: 3.0 g / L
- Disodium de phosphate : 1.15 g/L - Thioglycolate de sodium : 1.0 g/L - Phosphate disodium: 1.15 g / L - Sodium thioglycolate: 1.0 g / L
- Monopotassium de phosphate : 0.2 g/L - Monopotassium phosphate: 0.2 g / L
- Chlorure de potassium : 0.2 g/L - Potassium Chloride: 0.2 g / L
- Chlorure de calcium : 0.1 g/L - Calcium chloride: 0.1 g / L
- Chlorure de magnésium : 0.1 g/L - Eau distillée qsp 1 litre - Magnesium chloride: 0.1 g / L - Distilled water qs 1 liter
Le milieu CARY-BLAIR liquide dans de l'eau distillé comprend : The liquid CARY-BLAIR medium in distilled water comprises:
- Thioglycolate de sodium : 1.5 g/L - Sodium thioglycolate: 1.5 g / L
- Disodium d'hydrogénophosphate : 1.1 g/L - Hydrogen phosphate disodium: 1.1 g / L
- Chlorure de sodium : 5.0 g/L
- Chlorure de calcium : 0.09 g/L - Sodium Chloride: 5.0 g / L - Calcium chloride: 0.09 g / L
- L-Cystéine : 0.5 g/L - L-Cysteine: 0.5 g / L
- Eau distillée qsp 1 litre PH : 8.2 à 8.6. a) Préparation d'un récipient contenant un prélèvement contenant un échantillon de dites bactéries anaérobies. Le milieu préparé dans l'exemple 1 est aliquoté en tubes à raison de 1ml par tube. Une gélose de chaque bactérie à tester est récoltée sur une boite de gélose préalablement incubée à 37°C en anaérobiose puis diluées dans 1ml de milieu de transport préparé comme dans l'exemple 1. On procède de façon similaire dans des tubes contenant 1ml de PBS et des milieux CARY BLAIR et AMIES. b) Après différentes durées de conservation, à savoir : après 24h, 48h et 72h et 7jours, 10 μΙ de chaque suspension est dilué en jusqu'à 10"12 puis ΙΟΟμΙ de chaque dilutions sont inoculées sur gélose columbia au sang et incubées en anaérobiose. Après 48h d'incubation à 37°C, la dernière dilution permettant de compter moins de 10 colonies par gélose permet d'évaluer la concentration initiale de bactéries vivantes. Distilled water qsp 1 liter PH: 8.2 to 8.6. a) Preparation of a container containing a sample containing a sample of said anaerobic bacteria. The medium prepared in Example 1 is aliquoted into tubes at a rate of 1 ml per tube. An agar of each bacterium to be tested is collected on a plate of agar previously incubated at 37 ° C. under anaerobic conditions and then diluted in 1 ml of transport medium prepared as in Example 1. The procedure is similarly carried out in tubes containing 1 ml of PBS. and CARY BLAIR and AMIES circles. b) After different storage times, namely: after 24h, 48h and 72h and 7days, 10 μl of each suspension is diluted to 10 "12 and then ΙΟΟμΙ of each dilutions are inoculated on columbia agar with blood and incubated anaerobically After 48 hours of incubation at 37 ° C., the last dilution making it possible to count fewer than 10 colonies per agar makes it possible to evaluate the initial concentration of living bacteria.
Les résultats des tests sont indiqués dans les tableaux ci-après différents durés de conservation à différentes températures de conservation. The results of the tests are indicated in the tables below different storage times at different storage temperatures.
On observe que la viabilité de bactéries cultivables dans les milieux de transport selon l'invention est globalement nettement supérieure à celle observée dans le tampon PBS et des milieux CARY BLAIR et AMIES, cette survie est préservée de façon améliorée dans tous les cas et en particulier après conservation à +4°C pendant 7 jours.
1.1) Test avec conservation dans le milieu PBS à température ambiante de 20°C. It is observed that the viability of bacteria which can be cultivated in the transport media according to the invention is generally much greater than that observed in the PBS buffer and with CARY BLAIR and AMIES media, this survival is preserved in an improved manner in all cases and in particular after storage at + 4 ° C for 7 days. 1.1) Test with preservation in PBS medium at room temperature of 20 ° C.
Tableau 1 Table 1
Tableau 2 Table 2
1.3) Test dans le milieu AMIES ESWAB à 20°C. 1.3) Test in the AMIES ESWAB medium at 20 ° C.
Tableau 3
Souches 10 24h 48h 72h 7jTable 3 Strains 10 24h 48h 72h 7d
C. perfringens 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 103 cfu/mLC. perfringens September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / mL March 10 cfu / mL
C. sordellii 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 105 cfu/mLC. sordellii September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / ml May 10 cfu / mL
C. jeddamassiliensis 109 cfu/mL 109 cfu/mL 109 cfu/mL 105 cfu/mL C. jeddamassiliensis 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 5 cfu / ml
Bacteroides fragilis 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 105 cfu/mLBacteroides fragilis September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / ml May 10 cfu / mL
Bacteroides intestinalis 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 106 cfu/mLBacteroides intestinalis 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 7 cfu / ml 10 6 cfu / mL
Bacteroides thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 107 cfu/mL 107 cfu/mLBacteroides thetaiotaomicron 10 cfu / ml 10 cfu / ml 10 cfu / ml 10 7 cfu / ml 10 7 cfu / mL
Ruminococcus gnavus 106 cfu/mL 106 cfu/mL 106 cfu/mL 105 cfu/mL Ruminococcus gnavus 10 6 cfu / mL 10 6 cfu / mL 10 6 cfu / mL 10 5 cfu / mL
Fusobacterium necrophorum 108 cfu/mL 108 cfu/mL 108 cfu/mL 105 cfu/mL 105 cfu/mLFusobacterium necrophorum 10 8 CFU / ml 10 8 CFU / ml 10 8 CFU / mL May 10 cfu / ml May 10 cfu / mL
Fusobacterium nucleatum 109 cfu/mL 109 cfu/mL 109 cfu/mL 106 cfu/mL 105 cfu/mLFusobacterium nucleatum September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL June 10 cfu / ml May 10 cfu / mL
Alistipes fin egoldii 108 cfu/mL 108 cfu/mL 108 cfu/mL 105 cfu/mL 105 cfu/mLFine alyssis egoldii 10 8 cfu / ml 10 8 cfu / ml 10 8 cfu / ml 10 5 cfu / ml 10 5 cfu / ml
Clostridium tertium 109 cfu/mL 109 cfu/mL 109 cfu/mL 103 cfu/mL 102 cfu/mLClostridium tertium September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL March 10 cfu / mL February 10 cfu / mL
Clostridium butyricum 106 cfu/mL 106 cfu/mL 106 cfu/mL 105 cfu/mL 105 cfu/mLClostridium butyricum June 10 cfu / mL June 10 cfu / mL June 10 cfu / ml May 10 cfu / ml May 10 cfu / mL
Clostridium ramosum 109 cfu/mL 109 cfu/mL 109 cfu/mL 105 cfu/mL 105 cfu/mL Clostridium ramosum September 10 cfu / mL September 10 cfu / mL September 10 cfu / ml May 10 cfu / ml May 10 cfu / mL
1 .4) Test dans le m i l ieu de transport A selon l 'i nvention à 20 ° C. 1 .4) Test in the transport medium A according to the invention at 20 ° C.
Tableau 4 Table 4
.5) Test dans le m i l ieu de transport B selon l 'i nvention à 20 ° .5) Test in the transport medium B according to the invention at 20 °
Tableau 5 Table 5
Bacteroides Bacteroides
thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL thetaiotaomicron 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Ruminococcus gnavus 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLRuminococcus gnavus 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium Fusobacterium
necrophorum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL necrophorum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium Fusobacterium
nucleatum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL nucleatum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Alistipes fin egoldii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFinely annealed alistipes 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium tertium 10 cfu/mL 10 cfu/mL 9.10 cfu/mL 9.10 cfu/mL 10 cfu/mLClostridium tertium 10 cfu / mL 10 cfu / mL 9.10 cfu / mL 9.10 cfu / mL 10 cfu / mL
Clostridium butyricum 10 cfu/mL 10 cfu/mL 10 cfu/mL 1.10 cfu/mL 10 cfu/mLClostridium butyricum 10 cfu / mL 10 cfu / mL 10 cfu / mL 1.10 cfu / mL 10 cfu / mL
Clostridium ramosum 10 cfu/mL 10 cfu/mL 10 cfu/mL 1.10 cfu/mL 0 cfu/mL Clostridium ramosum 10 cfu / mL 10 cfu / mL 10 cfu / mL 1.10 cfu / mL 0 cfu / mL
2. 1 ) Test avec conservation dans le m i l ieu P BS à +4 ° C. 2. 1) Test with preservation in the medium PBS at +4 ° C.
Tableau 6 Table 6
Tableau 7 Table 7
Souches 10 24h 48h 72h 7j Strains 10 24h 48h 72h 7d
C. perfringens 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 103 cfu/mLC. perfringens September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / mL March 10 cfu / mL
C. sordellii 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 105 cfu/mLC. sordellii September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / ml May 10 cfu / mL
C. jeddamassiliensis 109 cfu/mL 109 cfu/mL 109 cfu/mL 105 cfu/mL C. jeddamassiliensis 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 5 cfu / ml
Bacteroides fragilis 109 cfu/mL 109 cfu/mL 109 cfu/mL 107 cfu/mL 105 cfu/mL
Bacteroides intestinalis 109cfu/mL 109cfu/mL 109cfu/mL 107cfu/mL 106cfu/mLBacteroides fragilis September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL July 10 cfu / ml May 10 cfu / mL Bacteroides intestinalis 10 9 cfu / ml 10 9 cfu / ml 10 9 cfu / ml 10 7 cfu / ml 10 6 cfu / mL
Bacteroides thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 107cfu/mL 107cfu/mLBacteroides thetaiotaomicron 10 cfu / ml 10 cfu / ml 10 cfu / ml 10 7 cfu / ml 10 7 cfu / mL
Ruminococcus gnavus 106cfu/mL 106cfu/mL 106cfu/mL 105cfu/mL Ruminococcus gnavus 10 6 cfu / mL 10 6 cfu / mL 10 6 cfu / mL 10 5 cfu / mL
Fusobacterium necrophorum 108cfu/mL 108cfu/mL 108cfu/mL 105cfu/mL 105cfu/mLFusobacterium necrophorum 10 8 CFU / ml 10 8 CFU / ml 10 8 CFU / mL May 10 cfu / ml May 10 cfu / mL
Fusobacterium nucleatum 109cfu/mL 109cfu/mL 109cfu/mL 106cfu/mL 105cfu/mLFusobacterium nucleatum September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL June 10 cfu / ml May 10 cfu / mL
Alistipes fin egoldii 108cfu/mL 108cfu/mL 108cfu/mL 105cfu/mL 105cfu/mLFine alyssis egoldii 10 8 cfu / ml 10 8 cfu / ml 10 8 cfu / ml 10 5 cfu / ml 10 5 cfu / ml
Clostridium tertium 109cfu/mL 109cfu/mL 109cfu/mL 103cfu/mL 102cfu/mLClostridium tertium September 10 cfu / mL September 10 cfu / mL September 10 cfu / mL March 10 cfu / mL February 10 cfu / mL
Clostridium butyricum 106cfu/mL 106cfu/mL 106cfu/mL 105cfu/mL 105cfu/mLClostridium butyricum June 10 cfu / mL June 10 cfu / mL June 10 cfu / ml May 10 cfu / ml May 10 cfu / mL
Clostridium ramosum 109cfu/mL 109cfu/mL 109cfu/mL 105cfu/mL 105cfu/mL Clostridium ramosum September 10 cfu / mL September 10 cfu / mL September 10 cfu / ml May 10 cfu / ml May 10 cfu / mL
2.3) Test dans le milieu AMIES ESWAB +4°C. 2.3) Test in the environment AMIES ESWAB + 4 ° C.
Tableau 8 Table 8
Tableau 9 Table 9
Souches 10 24h 48h 72h 7j Strains 10 24h 48h 72h 7d
C. perfringens 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. perfringens 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
C. sordellii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. sordellii 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
C. jeddamassiliensis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. jeddamassiliensis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides fragilis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides fragilis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides intestinalis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides intestinalis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides Bacteroides
thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL thetaiotaomicron 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Ruminococcus gnavus 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLRuminococcus gnavus 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium Fusobacterium
necrophorum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL necrophorum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium nucleatum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFusobacterium nucleatum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Alistipes fin egoldii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL
Clostridium tertium 9.10 cfu/mL 9.10 cfu/mL 9.10 cfu/mL 9.10 cfu/mL 10 cfu/mLFinely annealed alistipes 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL Clostridium tertium 9.10 cfu / mL 9.10 cfu / mL 9.10 cfu / mL 9.10 cfu / mL 10 cfu / mL
Clostridium butyricum 9.10 cfu/mL 9.10 cfu/mL 2.10 cfu/mL 1.10 cfu/mL 10 cfu/mLClostridium butyricum 9.10 cfu / mL 9.10 cfu / mL 2.10 cfu / mL 1.10 cfu / mL 10 cfu / mL
Clostridium ramosum 4.10 cfu/mL 4.10 cfu/mL 10 cfu/mL 1.10 cfu/mL 0 cfu/mL Clostridium ramosum 4.10 cfu / mL 4.10 cfu / mL 10 cfu / mL 1.10 cfu / mL 0 cfu / mL
2.5) Test dans le m i l ieu de tran sport B selon l ' i nvention à +4 ° C. 2.5) Test in the sports equipment B according to the invention at +4 ° C.
Tableau 1 0 Table 1 0
Tableau 1 1 Table 1 1
Souches 10 24h 48h 72h 7j Strains 10 24h 48h 72h 7d
C. perfringens 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mLC. perfringens 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
C. sordellii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mLC. sordellii 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
C. jeddamassiliensis 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mL 0 cfu/mLC. jeddamassiliensis 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL 0 cfu / mL
Bacteroides fragilis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mLBacteroides fragilis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
Bacteroides intestinalis 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mL 0 cfu/mLBacteroides intestinalis 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL 0 cfu / mL
Bacteroides thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mLBacteroides thetaiotaomicron 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
Ruminococcus gnavus 10 cfu/mL 10 cfu/mL O cfu/mL 0 cfu/mL 0 cfu/mLRuminococcus gnavus 10 cfu / mL 10 cfu / mL O cfu / mL 0 cfu / mL 0 cfu / mL
Fusobacterium necrophorum 10 cfu/mL 10 cfu/mL O cfu/mL 0 cfu/mL 0 cfu/mLFusobacterium necrophorum 10 cfu / mL 10 cfu / mL O cfu / mL 0 cfu / mL 0 cfu / mL
Fusobacterium nucleatum 10 cfu/mL 10 cfu/mL 0 cfu/mL 0 cfu/mL 0 cfu/mLFusobacterium nucleatum 10 cfu / mL 10 cfu / mL 0 cfu / mL 0 cfu / mL 0 cfu / mL
Alistipes fin egoldii 10 cfu/mL 10 cfu/mL 0 cfu/mL 0 cfu/mL 0 cfu/mLFinely annealed alistipes 10 cfu / mL 10 cfu / mL 0 cfu / mL 0 cfu / mL 0 cfu / mL
Clostridium tertium 9.10 cfu/mL 3.10 cfu/mL 5.10 cfu/mL 3.10 cfu/mL 10 cfu/mL
Clostridium butyricum 9.10 cfu/mL 5.10 cfu/mL 7.10 cfu/mL 9.10 cfu/mL 0 cfu/mLClostridium tertium 9.10 cfu / mL 3.10 cfu / mL 5.10 cfu / mL 3.10 cfu / mL 10 cfu / mL Clostridium butyricum 9.10 cfu / mL 5.10 cfu / mL 7.10 cfu / mL 9.10 cfu / mL 0 cfu / mL
Clostridium ramosum 4.10 cfu/mL 0 cfu/mL Ocfu/mL 0 cfu/mL 0 cfu/mL Clostridium ramosum 4.10 cfu / mL 0 cfu / mL Ocfu / mL 0 cfu / mL 0 cfu / mL
3.2) Test dans le milieu CARY BLAIR à -20°C 3.2) CARY BLAIR test at -20 ° C
Tableau 12 Table 12
Tableau 13 Table 13
3.4) Test dans le milieu de transport A selon l'invention à -20°C. 3.4) Test in the transport medium A according to the invention at -20 ° C.
Tableau 14 Table 14
Souches 10 24h 48h 72h 7j Strains 10 24h 48h 72h 7d
C. perfringens 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL
C. sordellii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. perfringens 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL C. sordellii 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
C. jeddamassiliensis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. jeddamassiliensis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides fragilis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides fragilis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides intestinalis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides intestinalis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides Bacteroides
thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLthetaiotaomicron 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Ruminococcus gnavus 5.10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLRuminococcus gnavus 5.10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium necrophorum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFusobacterium necrophorum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium nucleatum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mLFusobacterium nucleatum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
Alistipes fin egoldii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFinely annealed alistipes 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium tertium 9.10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLClostridium tertium 9.10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium butyricum 9.10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLClostridium butyricum 9.10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium ramosum 4.10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL O cfu/mL Clostridium ramosum 4.10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL O cfu / mL
3.5) Test dans le m i l ieu de transport B selon l 'i nvention à -20 ° C. 3.5) Test in the transport medium B according to the invention at -20 ° C.
Tableau 1 5 Table 1 5
Souches 10 24h 48h 72h 7j Strains 10 24h 48h 72h 7d
C. perfringens 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. perfringens 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
C. sordellii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. sordellii 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
C. jeddamassiliensis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLC. jeddamassiliensis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides fragilis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides fragilis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides intestinalis 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides intestinalis 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Bacteroides thetaiotaomicron 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLBacteroides thetaiotaomicron 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Ruminococcus gnavus 5.10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLRuminococcus gnavus 5.10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium necrophorum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFusobacterium necrophorum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Fusobacterium nucleatum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL O cfu/mLFusobacterium nucleatum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL O cfu / mL
Alistipes fin egoldii 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLFinely annealed alistipes 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium tertium 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLClostridium tertium 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium butyricum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mLClostridium butyricum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL
Clostridium ramosum 10 cfu/mL 10 cfu/mL 10 cfu/mL 10 cfu/mL 0 cfu/mL
Clostridium ramosum 10 cfu / mL 10 cfu / mL 10 cfu / mL 10 cfu / mL 0 cfu / mL
Claims
1. Procédé de transport et/ou conservation d'échantillons de bactéries capable de préserver la viabilité de bactéries anaérobies pendant au moins 48h, de préférence au moins 72h, à température froide ou ambiante notamment de -20°C à +30°C, de préférence à température ambiante de 4°C à 20°C, caractérisé en ce que l'on conditionne ledit échantillon de bactéries dans un récipient contenant un milieu de transport et conservation liquide, sous atmosphère contenant de l'oxygène, ledit milieu de transport et conservation étant tamponné à PH de 7 à 7,5, comprenant au moins dans de l'eau distillée: 1. Process for transporting and/or preserving bacterial samples capable of preserving the viability of anaerobic bacteria for at least 48 hours, preferably at least 72 hours, at cold or ambient temperature, in particular from -20°C to +30°C, preferably at ambient temperature of 4°C to 20°C, characterized in that said sample of bacteria is packaged in a container containing a liquid transport and preservation medium, under an atmosphere containing oxygen, said transport medium and conservation being buffered at PH from 7 to 7.5, comprising at least in distilled water:
- une source de phosphore, et - a source of phosphorus, and
- au moins un sel de métal choisi parmi K, Mg, Na, Ca, et - at least one metal salt chosen from K, Mg, Na, Ca, and
- au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion et l'acide urique, et - at least one antioxidant compound chosen from ascorbic acid, glutathione and uric acid, and
- une substance tampon régulateur de pH. - a pH regulating buffer substance.
2. Procédé selon la revendication 1, caractérisé en ce que ledit milieu de transport et conservation est tamponné à PH de 7 à 7.5, comprenant au moins : - un sel de phosphate, et 2. Method according to claim 1, characterized in that said transport and storage medium is buffered at PH from 7 to 7.5, comprising at least: - a phosphate salt, and
- au moins un sel de chlorure de chacun des métaux K, Mg, Na,- at least one chloride salt of each of the metals K, Mg, Na,
Ca, et That, and
- au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion et l'acide urique, à une concentration d'au moins lg/L, et
- une substance tampon régulateur de pH à PH de 7-7.5 qui est l'hydroxyde de potassium (KOH). - at least one antioxidant compound chosen from ascorbic acid, glutathione and uric acid, at a concentration of at least lg/L, and - a pH regulating buffer substance at PH of 7-7.5 which is potassium hydroxide (KOH).
3. Procédé selon l'une des revendications 1 ou 2, caractérisé en ce que ledit composé antioxydant est mis en œuvre à une concentration de 1 pg/ml à 2 mg/ml, ou concentration molaire de 10"6 à 10"2 M, de préférence au moins 100 pg/ml ou au moins 10"5M. 3. Method according to one of claims 1 or 2, characterized in that said antioxidant compound is used at a concentration of 1 pg/ml to 2 mg/ml, or molar concentration of 10 "6 to 10 "2 M , preferably at least 100 pg/ml or at least 10 "5 M.
4. Procédé selon la revendication 3, caractérisé en ce que ledit composé antioxydant comprend au moins l'acide ascorbique, de préférence à une concentration d'au moins 1 g/1. 4. Method according to claim 3, characterized in that said antioxidant compound comprises at least ascorbic acid, preferably at a concentration of at least 1 g/l.
5. Procédé selon l'une des revendications 1 à 4, caractérisé en ce que la composition du milieu de transport et conservation comprend : 5. Method according to one of claims 1 to 4, characterized in that the composition of the transport and conservation medium comprises:
- chlorure de sodium (NaCL) : 3g/L - sodium chloride (NaCL): 3g/L
- chlorure de potassium (KCI) : 0.2g/L - potassium chloride (KCI): 0.2g/L
- chlorure de calcium (CaCI2) : O.lg/L - calcium chloride (CaCI 2 ): O.lg/L
- chlorure de magnésium (MgCI2) : O.lg/L - magnesium chloride (MgCI 2 ): O.lg/L
- phosphate monopotassique (KH2P04) : 0.2g/L - monopotassium phosphate (KH2P04): 0.2g/L
- phosphate de sodium (Na2HP04) : 1.15g/L - sodium phosphate (Na 2 HP0 4 ): 1.15g/L
- acide ascorbique (C6H806) : lg/L - ascorbic acid (C 6 H 8 0 6 ): lg/L
- glutathion (C10H17N3O3S) : O.lg/L - glutathione (C 10 H 17 N 3 O 3 S): O.lg/L
- acide urique (C5H4N403) : O.lg/L - uric acid (C 5 H 4 N 4 0 3 ): O.lg/L
- hydroxyde de potassium (KOH) 0.3 - 0.6 - potassium hydroxide (KOH) 0.3 - 0.6
- eau distillée qsp : 1 litre
- distilled water qsp: 1 liter
6. Procédé selon la revendication 3 caractérisé en ce que ledit composé antioxydant consiste dans l'acide urique seul de préférence à une concentration d'au moins 0.4 g/L. 6. Method according to claim 3 characterized in that said antioxidant compound consists of uric acid alone preferably at a concentration of at least 0.4 g/L.
7. Procédé selon la revendication 6, caractérisé en ce que la composition du milieu de transport et conservation comprend : chlorure de sodium (NaCL) : 3g/L chlorure de potassium (KCI) : 0.2g/L chlorure de calcium (CaCI2) : O.lg/L chlorure de magnésium (MgCI2) : O.lg/L phosphate monopotassique (KH2P04) : 0.2g/L phosphate de sodium (Na2HP04) : 1.15g/L acide urique (C5H4N403) : 0.4g/L hydroxyde de potassium (KOH) 0.3 - 0.6 eau distillée qsp : 1 litre7. Method according to claim 6, characterized in that the composition of the transport and storage medium comprises: sodium chloride (NaCL): 3g/L potassium chloride (KCI): 0.2g/L calcium chloride (CaCI 2 ) : O.lg/L magnesium chloride (MgCI 2 ): O.lg/L monopotassium phosphate (KH2P04): 0.2g/L sodium phosphate (Na 2 HP0 4 ): 1.15g/L uric acid (C 5 H 4 N 4 0 3 ): 0.4g/L potassium hydroxide (KOH) 0.3 - 0.6 distilled water qs: 1 liter
8. Procédé selon l'une des revendications 1 à 7, caractérisé en ce que ledit milieu de transport et conservation comprend en outre un indicateur coloré d'oxydoréduction apte à changer visiblement l'état du milieu, notamment sa couleur, dans le cas où celui-ci contiendrait de l'oxygène, de préférence la résazurine à une concentration d'au moins 0.0015g/L. 8. Method according to one of claims 1 to 7, characterized in that said transport and conservation medium further comprises a colored redox indicator capable of visibly changing the state of the medium, in particular its color, in the case where this would contain oxygen, preferably resazurin at a concentration of at least 0.0015g/L.
9. Procédé selon l'une des revendications 1 à 8, caractérisé en ce que ledit milieu liquide est mélangé au dit échantillon de bactéries à raison d'une proportion d'au moins 5 volume de dit milieu de transport et conservation pour 1 volume de dit échantillon de bactéries contenant un nombre de bactéries de 1 à 1015 cfu/mL.
9. Method according to one of claims 1 to 8, characterized in that said liquid medium is mixed with said sample of bacteria in a proportion of at least 5 volume of said transport and conservation medium for 1 volume of said bacterial sample containing a bacterial count of 1 to 10 15 cfu/mL.
10. Procédé selon l'une des revendications 1 à 9, caractérisé en ce que ledit échantillon de bactéries est un échantillon de prélèvement biologique clinique de fluide biologique tel que urine, sang ou sécrétion telle que salives, selles, et autres sécrétions corporelles de muqueuses ou autres organes corporels, pus, collections et épanchements divers, et biopsies, ledit prélèvement étant éventuellement supporté par un matériel pour réaliser le prélèvement d'un échantillon tel qu'un écouvillon en coton ou fibre synthétique. 10. Method according to one of claims 1 to 9, characterized in that said sample of bacteria is a clinical biological sample of biological fluid such as urine, blood or secretion such as saliva, stool, and other body secretions of mucous membranes or other bodily organs, pus, various collections and effusions, and biopsies, said sample possibly being supported by equipment for taking a sample such as a cotton or synthetic fiber swab.
11. Procédé selon l'une des revendications 1 à 10, caractérisé en ce que ledit échantillon bactérien comprend ou peut comprendre : 11. Method according to one of claims 1 to 10, characterized in that said bacterial sample comprises or may comprise:
- des bactéries anaérobies strictes des genres Clostridium, Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Lactobacillus, Actinomyces, Clostridium, Bacteroides, Fi r mi eûtes, Porphyromonas, Prevotella, Fusobacterium, Atopobium, Ruminococcus, Solobacterium, Acidaminococcus, Alistipes, Amazonia, Anaerosalibacter, Anaerococcus, Barnesiella, Bifidobacterium, Blautia, Col I insella, Dielam, Flavonifractot, GemellaGodonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevotella, Senegalmassillia, Tisssierella, Turicibacter, Veillonella., et - des bactéries aéro-anaéorobies des genres Actinomyces,- strict anaerobic bacteria of the genera Clostridium, Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, Lactobacillus, Actinomyces, Clostridium, Bacteroides, Fi r mi eûtes, Porphyromonas, Prevotella, Fusobacterium, Atopobium, Ruminococcus, Solobacterium, Acidaminococcus, Alistipes, Amazonia, Anaerosalibacter, Anaerococcus, Barnesiella, Bifidobacterium, Blautia, Col I insella, Dielam, Flavonifractot, GemellaGodonibacter, Guyana, Holdemania, Odoribacter, Parabacteroides, Parvimonas, Prevotella, Senegalmassillia, Tisssierella, Turicibacter, Veillonella., and - aero-anaeorobic bacteria of the genera Actinomyces,
Aerococcus, Aneurinibacillus, Ba ci II us, Cedecea, Citrobacter, Corynebacterium, Eggerthella, Eikenella, Enterobacter, Escherichia, Eubacterium, Facklamia, Granulicatella, Haemophilus, Hafnia, Lactobacillus, Leuconostoc, Lysinibacillus, Pediococcus, Providencia, Serra tia, Staphylococcus, Streptococcus. Aerococcus, Aneurinibacillus, Ba ci II us, Cedecea, Citrobacter, Corynebacterium, Eggerthella, Eikenella, Enterobacter, Escherichia, Eubacterium, Facklamia, Granulicatella, Haemophilus, Hafnia, Lactobacillus, Leuconostoc, Lysinibacillus, Pediococcus, Providencia, Serra tia, Staphylococcus, Streptococcus.
12. Procédé selon l'une des revendications 1 à 11, caractérisé en ce que l'on effectue le transport d'un prélèvement pouvant contenir un échantillon bactérien dans un dit milieu de transport et conservation, à température ambiante et, après transport, on conserve en stockage ledit prélèvement au froid, de préférence à +4 à- + 8°C ou congelé, de préférence à -20°C, sous une atmosphère d'air ambiant.
12. Method according to one of claims 1 to 11, characterized in that a sample which may contain a bacterial sample is transported in a said transport and storage medium, at room temperature and, after transport, we stores said sample cold, preferably at +4 to - + 8°C or frozen, preferably at -20°C, under an atmosphere of ambient air.
13. Récipient contenant un milieu liquide de transport et conservation apte à préserver la viabilité de bactéries aérobies et/ou bactéries anaérobies utile dans un procédé tel que défini dans l'une des revendications 1 à 12, sous atmosphère contenant de l'oxygène, ledit milieu de transport et conservation étant tamponné à PH de 7 à 7,5, comprenant au moins dans de l'eau distillée : 13. Container containing a liquid transport and conservation medium capable of preserving the viability of aerobic bacteria and/or anaerobic bacteria useful in a process as defined in one of claims 1 to 12, under an atmosphere containing oxygen, said transport and storage medium being buffered at PH from 7 to 7.5, comprising at least in distilled water:
- une source de phosphore, et - a source of phosphorus, and
- au moins un sel de métal choisi parmi K, Na, Ca et Mg, et - at least one metal salt chosen from K, Na, Ca and Mg, and
- au moins un composé anti-oxydant choisi parmi l'acide ascorbique, le glutathion et l'acide urique, et - at least one antioxidant compound chosen from ascorbic acid, glutathione and uric acid, and
- une substance tampon régulateur de pH. - a pH regulating buffer substance.
14. Récipient selon la revendication 13, caractérisé en ce qu'il contient en outre dans ledit milieu liquide, un prélèvement clinique pouvant contenir un dit échantillon bactérien. 14. Container according to claim 13, characterized in that it further contains in said liquid medium, a clinical sample which may contain a said bacterial sample.
15. Procédé de fabrication d'un récipient selon l'une des revendications 12 à 14, comprenant les étapes successives suivantes dans lesquelles : 15. Method of manufacturing a container according to one of claims 12 to 14, comprising the following successive steps in which:
1) on chauffe dans un autoclave dans des conditions de température pour le stériliser, de préférence à une température supérieure à 121°C pendant au moins 20 mn, un dit récipient contenant ledit milieu de transport et conservation liquide incomplet ne contenant pas lesdits composés antioxydants, sous atmosphère d'air ambiant, et 1) a said container containing said incomplete liquid transport and preservation medium not containing said antioxidant compounds is heated in an autoclave under temperature conditions to sterilize it, preferably at a temperature above 121°C for at least 20 minutes. , under an ambient air atmosphere, and
2) on refroidit ledit milieu de transport et conservation incomplet, à température ambiante et on y ajoute lesdits composés anti oxydants en suspension préalablement filtrés à travers un filtre qui retient les microorganismes tels que les bactéries, de préférence avec un filtre qui retient les éléments de plus de 0.2pm.
2) said incomplete transport and conservation medium is cooled to room temperature and said antioxidant compounds in suspension are added thereto, previously filtered through a filter which retains microorganisms such as bacteria, preferably with a filter which retains the elements of more than 0.2pm.
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| FR1453427 | 2014-04-16 | ||
| FR1453427A FR3020069B1 (en) | 2014-04-16 | 2014-04-16 | METHOD AND LIQUID MEDIUM FOR TRANSPORTING AND PRESERVING BACTERIA |
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| CN109456920A (en) * | 2018-11-30 | 2019-03-12 | 江苏大学 | The Halophilic Bacterium bacterial strain seawater bacillus of one plant of raising alec fermentation quality |
| WO2020247846A1 (en) * | 2019-06-07 | 2020-12-10 | Anaerobe Systems | Microbial collection formulations, devices and systems |
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| WO2020247846A1 (en) * | 2019-06-07 | 2020-12-10 | Anaerobe Systems | Microbial collection formulations, devices and systems |
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| FR3020069A1 (en) | 2015-10-23 |
| FR3020069B1 (en) | 2018-01-12 |
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