Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor

Definitions

• the present invention relates to a method for detecting the presence in a sample, contained in a sterile container, of at least one aerobic or anaerobic microorganism, the sample being in contact with a growth medium.
• the invention also relates to a support which can be used in such a method.
• document US-A-5,672,484 which relates to a container for detecting strict aerobic microorganisms in a sample, which comprises a chamber containing a culture medium therein, and defining a free space above the middle.
• This document also relates to a method of manufacturing such a container and a method of growing strict aerobic microorganisms.
• a non-toxic insert hydrated with said growth medium has a free surface which at least partially extends beyond the free space, in order to allow the supply of oxygen to the growth medium, and therefore to increase the oxygenation of the microorganisms introduced into the sample, and improve their metabolic process.
• This insert is chosen from the following group of materials: sponge, cotton, glass fiber balls and resin.
• this apparatus and method is limited to the detection of strict aerobic microorganisms. Moreover, and very explicitly, the need for increasing the oxygen exchanges between the support and the free and confined space of the container is mentioned. To do this, the insert is positioned at the interface constituted by the culture medium, on the one hand, and the free space, on the other hand. All these features
• the present invention seeks to protect a process which is much more versatile, since it can be used with all microorganisms, whatever their respiratory metabolism.
• the arrangement of the inserts or supports within the container does not have to follow a framework as restrictive as that which is necessary for the document of the state of the art.
• the present invention relates to a method for detecting the presence in a sample, contained in a sterile container, of at least one microorganism, whatever its respiratory metabolism, the sample being in contact with a medium. growth, characterized in that it consists of:
• the characteristic observed is due to a variation of at least one indicator added to the container before incubation, for example a colored or fluorescent indicator, and / or at least one physico-chemical or electrical parameter, for example the CO 2 production pressure, turbidity, redox potential and / or pH.
• at least one indicator added to the container before incubation for example a colored or fluorescent indicator, and / or at least one physico-chemical or electrical parameter, for example the CO 2 production pressure, turbidity, redox potential and / or pH.
• the sample is either biological, for example blood, cerebrospinal fluid, pleural fluids, urine, or non-biological, for example water, food products, pharmaceutical products.
• biological for example blood, cerebrospinal fluid, pleural fluids, urine
• non-biological for example water, food products, pharmaceutical products.
• the variation of the indicator (s) is read optically through all or part of at least one of the walls of the container, which is transparent, and / or the modification of the parameter (s) is read.
• (s) is carried out by means of physico-chemical (s) or electrical (s) sensor (s).
• the method applies to microorganisms with anaerobic metabolism.
• This process is used in a sterility check.
• the present invention also relates to a solid, inert and sterile support, used in the process, defined above, which, in a second embodiment, is characterized by the fact that it consists of natural materials, for example:
• the solid, inert and sterile support used in the above process consists of artificial materials, for example:
• the solid, inert and sterile support used in the above process consists of an element of any shape made of polyethylene. Whatever the embodiment, this support consists of beads or grains with a diameter between 1 ⁇ m and 10 mm, and in particular between 0.1 mm and 5 mm.
• the attached figures are given by way of explanatory example and have no limiting character. They will allow a better understanding of the invention. In each of these figures, curve 1 corresponds to the insertion of a cork support, curve 2 to the insertion of a polyethylene support, and curve 3 to the absence of support, thus serving as a reference .
• FIG. 1 represents a curve of the growth kinetics in the case of Haemophilus influenzae.
• FIG. 2 represents a curve of the growth kinetics in the case of Kingella denitrificans.
• FIG. 3 represents a curve of the growth kinetics in the case of
• FIG. 4 represents a curve of the growth kinetics in the case of Bactero ⁇ des fragilis.
• FIG. 5 represents a curve of the growth kinetics in the case of Veillonella parvula.
• FIG. 6 represents a curve of the growth kinetics in the case of Clostridium sporogenes.
• the present invention relates to a method for detecting the presence in a sample, contained in a sterile container, of at least one aerobic or anaerobic microorganism, the sample being in contact with a growth medium.
• the supports added in a single container may consist of a mixture of at least two artificial or natural supports, mentioned above, or else of a mixture of at least one artificial support and of at least one natural support, mentioned above.
• bottles are then placed under vacuum and gassed so as to reconstitute an atmosphere adapted to the type of broth concerned (aerobic for NITAL AER or anaerobic for NITAL A ⁇ A). They are then capped and capped to be ready for use.
• NITAL AER and A ⁇ A bottles without support underwent the same manufacturing process, except for the preparation and the addition of the support. .
• the microorganisms studied are previously cultivated on an appropriate growth medium such as an agar, generally a Columbia agar with 5% sheep blood. For each of them, a suspension is made in distilled water by removing one or more colonies. This suspension is calibrated at 10 8 cells / ml and diluted to a millionth. Using a sterile syringe, one milliliter of this dilution is introduced into each NITAL bottle with or without support previously inoculated with 5 ml of horse blood or human blood. Thus, about one hundred microorganism cells are introduced per bottle.
• an agar generally a Columbia agar with 5% sheep blood.
• the bottles, containing the sample to be analyzed, are placed in the NITAL brand system (ref .: 99105, 99106 or 99122 from bioMérieux) which plays the role of both an incubator and a reader.
• This system incubates the vials at an appropriate temperature.
• the kinetics of the indicator are followed by an optical measurement through the transparent glass wall of the bottles. If the sample initially contains microorganisms, these will multiply during incubation and the modification of the indicator, present in the reaction medium, will reflect the presence of said microorganisms in the sample.
• the effectiveness of a support will be assessed by comparing the detection times obtained using bottles with and without support.
• Table 1 Detection time in hours and time saving in% of the use of flasks with supports compared to flasks without support in the case of aerobic, optional anaerobic microorganisms
• This table 1 above indicates a gain in detection time compared to the flasks without support of between 22.1 to 45.9% depending on the strains in the presence of polyethylene beads. This range ranges from 20.1 to 66.3% depending on the strains in the presence of cork particles.
• FIG. 1 The effect of the supports can be visualized on the growth kinetics presented in FIG. 1 in the case of Haemophilus influenzae, in FIG. 2 in the case of Kingella denitrificans and in FIG. 3 in the case of Staphylococcus saprophyticus.
• Example 2 Detection of Microorganisms with a Strict Anaerobic Metabolism The test was carried out under the following conditions:
• Table 2 Detection time in hours and time saving in% of the use of flasks with supports compared to flasks without support in the case of anaerobic microorganisms Table 2 above indicates a gain in detection time compared to flasks without support from 39 to 69.6% depending on the strains, in the presence of polyethylene beads, and from 16.3 to 63.4% depending on the strains , in the presence of cork particles.
• Example 1 there is therefore a marked reduction in detection times which reflects the positivity of the bottles containing a support, which is the result of a faster turn of the indicator, and this for both microorganisms with aerobic metabolism and anaerobic metabolism.
• Example 3 Complementary study on the detection of different microorganisms with aerobic metabolism, facultative anaerobic (AAF)
• the AAF strains used are either bacteria or yeasts.
• several analyzes were carried out for each species studied. They belong to the following different species or genera:
• Yeasts Candida albicans, Candida parapsilosis, Candida guillermondii,
• the polypropylene support is not of interest because it slows the detection times in a good number of cases, that is to say that the value is greater than the value of 100% obtained for the reference as regards bottles without support. This value can even reach 174.3% for Pseudomonas.
• strains used are anaerobic bacteria belonging to the following genera: * Bacteroids, * Clostridium, 12
• This table 4 above indicates a gain in detection time compared to the flasks without support of between 2.7 and 72.4% depending on the anaerobic species tested.
• This process essentially consists of four or five successive stages.
• the parameter (s) can be observed by sensors which, for example measure the pressure, the production of CO 2 , the turbidity, the redox potential or the pH. This list is not exhaustive, and it may contain any physico-chemical or electrical characteristic which may vary with the metabolic activity of a microorganism.
• incubation temperature As regards the appropriate incubation temperature, mentioned in the above process, this varies depending on the family, species or genus of the microorganism tested. Thus, this variation is very wide and is between substantially 0 and 100 ° C depending on the microorganism. These temperatures are however well known to those skilled in the art and / or present no difficulty in being determined.
• This method can also be used, in addition to the analysis of samples in which we want to highlight the presence of microorganisms, in the search for sterility (biological fluids: blood, cerebrospinal fluid, pleural fluids, urine , etc., non-biological samples such as water, food or pharmaceutical products).
• biological fluids blood, cerebrospinal fluid, pleural fluids, urine , etc., non-biological samples such as water, food or pharmaceutical products.
• the presence of a support according to the invention significantly improves the speed of detection of the presence of microorganisms in a sample, whatever its metabolism, aerobic or anaerobic. Such a result was obtained without having to seek the effect of better oxygenation or better exposure of microorganisms to the oxygen that may be present in the medium.

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• Chemical & Material Sciences (AREA)
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• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
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• General Health & Medical Sciences (AREA)
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• 1998
  • 1998-12-04 FR FR9815501A patent/FR2786782B1/fr not_active Expired - Fee Related
• 1999
  • 1999-12-03 AU AU15651/00A patent/AU773782B2/en not_active Ceased
  • 1999-12-03 EP EP99958239A patent/EP1135520A1/de not_active Withdrawn
  • 1999-12-03 CA CA002352481A patent/CA2352481A1/fr not_active Abandoned
  • 1999-12-03 JP JP2000586941A patent/JP2002531140A/ja active Pending
  • 1999-12-03 WO PCT/FR1999/003003 patent/WO2000034508A1/fr active IP Right Grant
• 2001
  • 2001-06-01 US US09/870,510 patent/US20010051354A1/en not_active Abandoned

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