EP1135520A1

EP1135520A1 - Method for detecting micro-organisms and support used in said method

Info

Publication number: EP1135520A1
Application number: EP99958239A
Authority: EP
Inventors: Daniel Monget; Jean-Marc Roche
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 1998-12-04
Filing date: 1999-12-03
Publication date: 2001-09-26
Also published as: WO2000034508A1; CA2352481A1; US20010051354A1; FR2786782A1; FR2786782B1; AU773782B2; JP2002531140A; AU1565100A

Abstract

The invention concerns a method for detecting the presence in a sample, contained in a sterile receptacle, at least a micro-organism, whatever its breathing metabolism (aerobic or anaerobic), the sample being in contact with a culture medium. The invention also concerns a support used in said method. The method consists in: adding into the receptacle at least a solid inert sterile support; incubating at a suitable temperature; and observing the variation in at least one characteristic related to the presence of the micro-organism(s) to be detected in said receptacle. The invention is particular applicable in the field of diagnosis.

Description

Method for detecting microorganisms and support usable in such a method DESCRIPTION

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.

// it is known that aerobic micro-organisms (bacteria, yeasts) adhere and colonize the majority of the surfaces offered to them. This fixation improves nutritional exchanges and therefore the level of growth of microorganisms. Thus, the presence of a solid phase influences many metabolic processes, such as nitrogen fixation, alcoholic oxidation, nitrification and denitrification, etc. This feature is widely used in industrial microbiology.

Thus, the state of the art can be defined by 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.

However, 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

COPY OF C0NFIR ATI0W could only divert the skilled person from the interest of using, within said container, a solid, inert and sterile support, even under anaerobic conditions.

The results obtained by the plaintiff during this study work were therefore unexpected.

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. In addition, 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.

To this end, 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:

- add at least a solid, inert and sterile support to the container,

- incubate at an appropriate temperature, and

- observe a variation of at least one characteristic linked to the presence of the micro-organism (s) to be detected within said container. 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.

In all cases, the sample is either biological, for example blood, cerebrospinal fluid, pleural fluids, urine, or 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). Preferably, 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:

- silica beads,

- small glass beads (solid, hollow or porous),

- quartz particles, - grains of sand,

- vermiculite, zeolite and / or feldspar grains,

- glass and / or rock wool,

- clay balls, and

- cork particles. According to a second embodiment, the solid, inert and sterile support used in the above process consists of artificial materials, for example:

- polystyrene beads,

- polyethylene beads,

- polypropylene beads, - agglomerated small polyethylene beads, of variable porosity and thickness,

- growth supports in the form of small beads used in cell culture,

- late balls,

- beads coated with gelatin, and

- resin grains. According to a third embodiment, 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 μm 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

Staphylococcus saprophyticus.

FIG. 4 represents a curve of the growth kinetics in the case of Bacteroïdes fragilis.

FIG. 5 represents a curve of the growth kinetics in the case of Veillonella parvula.

Finally, 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.

This detection is in fact accelerated by the fact that a solid, inert and sterile support is introduced into the container.

The list below brings together supports allowing to accelerate the time of detection of micro-organisms present in a sample. This list is of course not exhaustive.

In the case of so-called natural supports, one can use:

- silica beads,

- small glass beads which can be full, hollow or porous, - quartz particles,

- grains of sand, - grains of vermiculite, zeolite and / or feldspar,

- glass wool or rock wool,

- clay balls, and

- cork particles. In the case of so-called artificial supports, one can use:

- polystyrene beads,

- polyethylene beads,

- polypropylene balls,

- agglomerated small polyethylene beads, of variable porosity and thickness,

- growth supports in the form of beads, of small size, used in cell culture of registered trademark CYTODEX of type 1 and 3,

- latex beads,

- beads covered with gelatin, and - grains of di-ethyl-amino-ethyl resin (DEAE) for anion exchange resins or registered trademark SEPHADEX (CM = Carboxy Methyl cellulose) for cation exchange resins.

Of course, 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.

Implementation :

The implementation presented relates to a particular embodiment, there are many other possibilities. This implementation cannot therefore be considered as limiting the scope of the protection sought.

1. Preparation of the vials All the supports were sterilized by heating for 15 minutes (min) at 120 degrees Celsius (° C). After drying for 24 hours at 37 ° C, they were distributed sterile in the bottles. The quantity introduced per bottle depends on the support used and is adjusted so as to obtain a layer of material whose surface is substantially equivalent to the section of the bottles. The vials containing the supports are again heated for 15 minutes at 120 ° C. and then sterile receive 40 ml of NITAL AER aerobic nutritive broth (Ref. 52511 from bioMérieux) or anaerobic nutritive broth NITAL AΝA (Ref. 52512 from bioMérieux) containing the tracer microbial growth or indicator, as described and claimed in patent EP-B-0.424.293 of the applicant.

The 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 AΝA). They are then capped and capped to be ready for use.

In order to be able to compare the performances of the addition of the supports according to the present invention on media, NITAL AER and AΝA bottles without support underwent the same manufacturing process, except for the preparation and the addition of the support. .

2. Analysis of samples:

The bottles, as prepared above, then receive a sterile sample (biological or not) in which one wishes to test for the presence of microorganisms.

In the case of a simulation of positive blood cultures as was the case in the laboratory, it was carried out as follows. First, 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.

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.

Examples according to the invention:

EXAMPLE 1 Detection of Microorganisms with Aerobic Metabolism

The test was carried out under the following conditions:

- NITAL AER bottles with and without support, prepared according to the process described in the paragraph "Preparation of the bottles",

- studied supports: polyethylene balls and cork particles,

- strains tested:

• Haemophilus influenzae (ref. BioMérieux: JHI 8006064),

• Kingella denitrificans (ref. BioMérieux: JKD 8311002), and • Staphylococcus saprophyticus (ref. BioMérieux: MSA 54677), - strain isolation medium: Columbia bioMérieux agar supplemented with 5% sheep blood,

- simulation of positive blood cultures as indicated in the paragraph “Analysis of samples”, with sterile addition of approximately one hundred microorganisms and 5 ml of horse blood per vial,

- determination of the positivity times of the bottles by the NITAL system (registered trademark), and

- plot of growth kinetics for each bottle.

The results obtained are well represented in Table 1 which follows.

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.

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:

- NITAL AΝA bottles with and without support, prepared according to the process described in the paragraph "Preparation of the bottles",

- studied supports: polyethylene balls and cork particles,

- strains tested:

• Bacteroïdes fragilis (ref. BioMérieux: HBF 358),

• Veillonella parvula (ref bioMérieux: JKD 8311002), and

• Clostridium sporogenes (ref. BioMérieux: ACO 100651),

- strain isolation medium: Columbia bioMérieux agar supplemented with 5% mutton blood incubated anaerobically,

- simulation of positive blood cultures as indicated in the paragraph "Analysis of samples", with sterile addition of approximately one hundred microorganisms and 5 ml of horse blood per vial,

- determination of the positivity times of the bottles by the NITAL system, and

- plot of growth kinetics for each bottle.

The results obtained are well represented in Table 2 which follows.

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.

The effect of the supports can be visualized on the growth kinetics presented in FIG. 4 in the case of Bacteroïdes fragilis, in FIG. 5 in the case of Veillonella parvula and in FIG. 6 in the case of Clostridium sporogenes.

Whether we refer to Example 1 or to Example 2, 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)

In this example, the AAF strains used are either bacteria or yeasts. In addition, several analyzes were carried out for each species studied. They belong to the following different species or genera:

• Neisseria, * Tweezers,

• Yeasts: Candida albicans, Candida parapsilosis, Candida guillermondii,

• Staphylococcus,

• Pseudomonas, and

• Other strains belonging to: Haemophilus, Kingella, Eikenella and Micrococcus.

The results obtained are well represented in Table 3 which follows. All the values shown correspond to the detection times in the presence of supports expressed as a percentage relative to the detection times obtained with bottles without support and considered to have a value of 100%.

Table 3: Influence of the nature of the supports on the detection of several aerobic microorganisms

This table 3 clearly indicates that the polyethylene support makes it possible to significantly reduce the detection times of all the species or genera tested, these times varying from 60.8% to 83.6% compared to the times obtained with the flasks without support (100%).

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.

EXAMPLE 4 Study of the Influence of the Polyethylene Support on the Detection of Anaerobic Metabolic Microorganisms

In this example, the strains used are anaerobic bacteria belonging to the following genera: * Bacteroids, * Clostridium, 12

* Veptostreptococcus,

* Veillonella, and

* Fusobacterium.

The results obtained are well represented in Table 4 which follows.

All the values shown are percentages which, as for Table 3, correspond to the detection times in the presence of support, expressed as a percentage relative to the detection times without support reported at 100%.

Table 4: Influence of the support on the detection of several anaerobic microorganisms

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.

Thus, as shown in the examples above, the efficiency of the process is proven. This is true for the processes used in blood cultures (detection of microorganisms in the blood), but also for other applications based on the culture of microorganisms.

This process essentially consists of four or five successive stages.

First, add the sample to be analyzed to the container and a growth medium. Secondly, it is also necessary to add within said container at least one solid, inert and sterile support. Third and possibly, the container can be sealed. Fourth, incubate at an appropriate temperature. Finally and fifth, we observe a variation of a characteristic in relation to the presence, growth and / or metabolism of a microorganism to be detected. This characteristic can generally be of two different natures.

On the one hand, it can be a chemical indicator, for example colored, fluorescent, chromogenic, which is added to the container before incubation. On the other hand, it can be a physico-chemical or electrical parameter, which is modified by the simple growth of the microorganisms to be detected. In the latter case, it is not necessary to add any substance to allow the establishment of a variation of this parameter. This observation of a variation of a characteristic can be manual, that is to say visual, or automatic. In the case of automatic observation, 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.

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). The results presented in the examples above make it possible to demonstrate the impact of the growth supports on the detection times of both aerobic and anaerobic microorganisms (the latter requiring for their growth an N ₂ / CO ₂ atmosphere without O ₂ ). The majority of detection times have been significantly reduced.

The examples are limited to the use of certain supports and to the study of their impact on the detection of certain microorganisms. However, similar results have already been obtained with other supports and by extending the study to other microorganisms. The scope of the present invention is therefore not limited to the species and supports studied.

In conclusion, 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.

Claims

1. 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 growth medium, characterized in that 'it consists of :

- add at least a solid, inert and sterile support to the container,

- incubate at an appropriate temperature, and - observe a variation of at least one characteristic linked to the presence of the micro-organism (s) to be detected within said container.

2. Method according to claim 1, characterized in that the characteristic observed is due to a variation of at least one chemical 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 production of CO ₂ , the pressure, the turbidity, the redox potential and / or the pH.

3. Method according to any one of claims 1 or 2, characterized in that the sample is biological, for example blood, cerebrospinal fluid, pleural fluids, urine, or non-biological, for example water, food products, pharmaceutical products.

4. Method according to any one of claims 1 to 3, characterized in that the reading of the variation of the indicator (s) is carried out optically through all or part of at least one of the walls of the container, which is transparent, and / or the reading of the modification of the parameter (s) is carried out by means of physico-chemical (s) or electrical (s) sensor (s).

5. Method according to any one of claims 1 to 4, characterized in that it applies to microorganisms with anaerobic metabolism.

6. Method according to any one of claims 1 to 5, characterized in that it is used in a sterility check.

7. Solid, inert and sterile support, used in the process, defined in any one of claims 1 to 6, characterized in that it consists of natural materials, for example:

- silica beads,

- small glass beads (solid, hollow or porous), - quartz particles,

- grains of sand ,

- vermiculite, zeolite and / or feldspar grains,

- glass and / or rock wool,

- clay balls, and - cork particles.

8. Solid, inert and sterile support, used in the process, defined in any one of claims 1 to 6, characterized in that it consists of artificial materials, for example: - polystyrene beads,

- polyethylene beads,

- polypropylene balls,

- agglomerated small polyethylene beads, of variable porosity and thickness,

- growth supports in the form of small beads used in cell culture, - latex beads,

- beads coated with gelatin, and

- resin grains.

9. solid, inert and sterile support, used in the process, defined in any one of claims 1 to 6, characterized in that it consists of an element of any shape made of polyethylene.

10. Support according to any one of claims 7 or 9, characterized in that it consists of beads or grains with a diameter between 1 μm and 10 mm and in particular between 0, 1 mm and 5 mm.