Method for detecting and directly identifying a microorganism in a biological sample by an optical route

Abstract

A method for detecting at least one microorganism present in a sample, that includes: a) in a first container, bringing the sample into contact with at least one culture medium, b) placing the first container in suitable conditions to permit growth of the microorganism or microorganisms, c) bringing some or all of the mixture being made of the sample and the culture medium into contact with a reaction mixture and a substrate for capturing the microorganism(s) in the first container or in a second container, the reaction mixture having a device for detecting the microorganism(s); d) detecting, within the first or second container, the presence of the microorganism or microorganisms detected by the detecting device and fixed on the capture substrate.

Claims

1. A method for detecting a target microorganism present in a sample, the method comprising: incubating, in a homogenizing bag, a sample mixture containing the sample and a culture medium for 6 to 48 hours to permit growth of the target microorganism; transferring at least a fraction of the incubated sample mixture from the homogenizing bag to a secondary container that contains a reaction mixture to detect microorganisms in the combined reaction mixture and incubated sample mixture, the reaction mixture comprising a dye, membrane stain, or a fluorescent compound, that colors or causes to fluoresce the microorganisms present in the combined reaction mixture and incubated sample mixture; immersing, in the combined reaction mixture and incubated sample mixture, a substrate configured to specifically capture the target microorganism; and monitoring for an appearance of coloration or fluorescence of the capture substrate, wherein coloration or fluorescence of the capture substrate indicates that the target microorganism is fixed on the capture substrate.

2. The method according to claim 1, further comprising: incubating the capture substrate in the combined reaction mixture and incubated sample mixture in suitable conditions to permit growth of the target microorganism before monitoring for the appearance of coloration or fluorescence of the capture substrate.

3. The method according to claim 1, further comprising: confirming detection of the target microorganism after the monitoring.

4. The method according to claim 1, wherein at least one specific binding partner of the target microorganism is fixed on the capture substrate.

5. The method of detection according to claim 4, wherein the specific binding partner is selected from the group consisting of antibodies, Fab fragments, Fab fragments, aptamers, recombinant or non-recombinant phage proteins, and phages.

6. The method according to claim 1, wherein the monitoring is carried out in real time.

7. The method of detection according to claim 1, wherein the secondary container is a flask, a bottle, a tablet container, or a tube.

8. The method according to claim 1, wherein the capture substrate is a single-piece or particulate substrate, optionally porous.

9. The method according to claim 8, wherein the capture substrate is protected by a protective film.

10. The method according to claim 8, wherein the capture substrate is a particulate substrate of sensitized particles.

11. The method according to claim 10, wherein the sensitized particles are magnetic.

12. The method according to claim 8, wherein the capture substrate is a compressible single-piece substrate.

13. A method for detecting a target microorganism present in a sample, the method comprising: incubating, in a homogenizing bag, a sample mixture containing the sample and a culture medium for 6 to 48 hours to permit growth of the target microorganism; removing a fraction of the incubated sample mixture from the homogenizing bag; introducing the fraction of the incubated sample mixture to an unconnected secondary container that contains a reaction mixture that colors or causes to fluoresce microorganisms present in the combined reaction mixture and incubated sample mixture, the reaction mixture comprising a dye, membrane stain, or a fluorescent compound; monitoring for a first appearance of coloration or fluorescence from the microorganisms in the combined reaction mixture and incubated sample mixture; and when the first appearance of coloration or fluorescence is detected, immersing, in the combined reaction mixture and incubated sample mixture, a substrate configured to specifically capture the target microorganism, and monitoring for a second appearance of coloration or fluorescence from the microorganisms on the capture substrate, wherein the second appearance of coloration or fluorescence indicates that the target microorganism is fixed on the capture substrate.

14. The method of detection according to claim 13, wherein the secondary container is a flask, a bottle, a tablet container, or a tube.

15. The method according to claim 13, wherein the capture substrate is a single-piece or particulate substrate, optionally porous.

16. The method according to claim 15, wherein the capture substrate is protected by a protective film.

17. The method according to claim 15, wherein the capture substrate is a particulate substrate of sensitized particles.

18. The method according to claim 17, wherein the sensitized particles are magnetic.

19. The method according to claim 15, wherein the capture substrate is a compressible single-piece substrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The aims and advantages of the present invention will be better understood in light of the detailed description given below, in conjunction with the drawings, where:

(2) FIG. 1 is a schematic representation of the different steps of the method according to a first embodiment of the invention.

(3) FIG. 2 is a schematic representation of a sensitized capture substrate.

(4) FIG. 3 is a schematic representation of the sensitized capture substrate shown in FIG. 2, after analysis with a positive result.

DETAILED DESCRIPTION OF EMBODIMENTS

(5) According to a first embodiment of the present invention, the method for detecting or for identifying microorganism(s) consists of employing a sterile plastic homogenizing bag, conventionally called Stomacher bag. Such a bag is referenced 10 in FIG. 1A. This bag 10 consists of two roughly rectangular sheets of plastic, joined together on three sides, so as to define an internal space intended to receive the culture medium and the sample to be analysed. It additionally comprises a filter 12 of roughly rectangular shape, joined to the sheets on one side, separating the internal space into two.

(6) During step A, the closed homogenizing bag 10 is incubated with a food sample 14, consisting in this case of a sample of unpasteurized milk cheese. This food sample 14 is immersed in an enrichment medium 16, which can be selective or non-selective. Incubation can be carried out at temperatures between 25 and 44 C. for 6 to 48 h.

(7) A fraction of the enrichment medium is then taken in the homogenizing bag 10 and transferred during step B to a secondary container, consisting here of a tube 20. This tube 20 comprises a reaction mixture 22. Said reaction mixture 22 can consist of a diluent (e.g. tryptone salt broth) suitable for maintaining integrity of the target microorganisms, and at least one detecting means. The detecting means can be a dye that is able to stain the microorganisms present in the fraction of enrichment medium transferred, obtained from the food sample 14. The detecting means can also be a fluorescent compound that makes the microorganisms fluorescent. When we wish to perform a subculture in tube 20, the reaction mixture can contain, in addition to the nutrients, a selective system allowing the population of target bacteria to increase.

(8) According to a particular example, the detecting means is based on the reduction of triphenyl 2-3-5-tetrazolium chloride (TTC) by the microorganisms. Simultaneously with growth of the microorganisms, TTC (colourless in its non-reduced form) is internalized by said microorganisms, and then reduced within the cytoplasm by the latter to triphenyl-formazan (red), thus staining said microorganisms red so that they can then be detected on the substrate. Other tetrazolium salts can be used (CTC, MTT, etc.). Moreover, compounds for speeding up the reaction of reduction of the tetrazolium salts can be added to the reaction mixture.

(9) It is also conceivable to use membrane stains, such as gentian violet or fuchsin.

(10) In the case when the detecting means is a fluorescent compound, it can be acridine orange or fluorescein diacetate.

(11) In step C, a sensitized capture substrate 24 is put in tube 20 and is kept immersed in the reaction mixture 22 by any suitable means. The sensitized capture substrate 24 is functionalized with at least one specific binding partner of a target microorganism to be detected. The capture substrate can consist of any substrate suitable for fixation of specific binding partners and well known by a person skilled in the art. As a non-limiting example, a suitable capture substrate can be made of irradiated polystyrene, such as that marketed by the company Nunc/Thermo Scientific (Cat. No. 472230). A capture substrate of this kind is shown schematically in FIG. 2, with the reference 24. According to a preferred embodiment, the lower portion can advantageously be divided into two. The zone referenced 241 can be sensitized with a solution of binding partners (polyclonal antibodies, monoclonal antibodies, Fab or Fab2 fragments, aptamers, phage proteins), whereas the upper portion 242 remains free from any binding partner and thus plays a role of negative control. The techniques for sensitizing substrates with specific binding partners are well known by a person skilled in the art.

(12) According to one alternative of the method according to the invention, it may be advantageous to carry out a step of subculture, once the capture substrate 24 is immersed in the reaction mixture 22. This subculture consists of incubating tube 20 for 1 to 18 h, at temperatures between 25 and 44 C. According to this alternative, the intensity of staining of the microorganisms increases simultaneously with their growth owing to the detecting means contained in the reaction mixture. Analysis can then be carried out in real time.

(13) Once there is effective capture of a certain amount of stained or fluorescent target microorganisms (in the case of a positive sample), there is a change in the optical properties of the substrate through appearance of coloration or fluorescence on the latter (i.e. transduction of the biological signal). This coloration or fluorescence of the capture substrate is then detectable by eye or can be measured using an automatic reader such as a camera. The capture substrate is shown schematically in FIG. 3, after analysis with a positive result. As can be seen, zone 241 appears coloured owing to fixation of the target microorganisms on the specific binding partners. For its part, zone 242, performing the role of negative control, still has the initial colour of the capture substrate.

(14) To facilitate reading, the sensitized capture substrate should preferably no longer be in contact with the reaction mixture. For this purpose, it can be envisaged for example to remove the capture substrate 24 from said reaction mixture by any suitable means, as is clearly shown in step D, in FIG. 1. As explained above, the reading can be taken at the end point, as dotted lines or in real time.

(15) According to another alternative of the method according to the invention, the capture substrate consists of sensitized particles, namely bearing a specific or non-specific binding partner of the microorganism or microorganisms to be detected. Detection is then preferably indicated by appearance of a coloration or fluorescence of the initially colourless sensitized particles, due to binding of the target microorganisms to the latter, during the reaction.

(16) According to a particular embodiment, the sensitized particles can be magnetic particles. Reading can then be done manually and visually, using a magnetizing system that will allow collection of the magnetic particles on which the stained or fluorescent microorganisms are captured, in the form of clusters against the container wall. Movement of the magnetizing system vertically upwards makes it possible to remove the cluster of magnetic particles from the reaction mixture and thus facilitate analysis. It can also be envisaged to immerse the magnetizing system directly in the container, in order to collect the magnetic particles on which the microorganisms are fixed. In this case the magnetic particles will become fixed directly on the magnetizing system, which then becomes coloured or fluorescent.

(17) According to another embodiment of the method according to the invention, the reaction mixture and the capture substrate, as described above, are added directly to the first container at the end of the enrichment step. The detection step is therefore carried out in said first container without transferring some or all of the mixture consisting of the culture medium and the sample to be analysed to a second container. Prior to this detection step, a subculture can optionally be carried out in order to increase the population of target microorganisms.

(18) It should be noted that, advantageously, the capture substrate can be protected using a protective film. The purpose of this film is to prevent fouling of the capture substrate. In fact, such fouling is likely to impair the capture performance of said substrate. Such a film can be placed permanently on the capture substrate. Alternatively, it can be a film that can dissolve after a certain time of contact with the liquid culture medium.

(19) The method according to the invention is particularly advantageous because at the end of the analysis, only the containers identified as positive by the detecting system (described below) are opened in order to carry out additional analyses for confirming the presumptive result obtained. The confirmation step can be carried out by means of technology different from that employed in the method according to the invention.

(20) The aim of the examples presented below is to present different embodiments of the method according to the invention and the results obtained. They do not limit the invention in any way.

EXAMPLES

Example 1: Optical Detection of Salmonella Napoli, by Means of a Sensitized Substrate, in a Food Sample in Subculture in a Reaction Mixture

(21) The aim of this experiment is direct detection of the presence of the target bacterium Salmonella Napoli in a food sample in subculture in a reaction mixture, by means of a sensitized substrate made of irradiated polystyrene, marketed by the company Nunc/Thermo Scientific (Cat. No. 472230) and shown in FIGS. 2 and 3.

(22) As detailed below, detection is performed during the reaction step by immersing the capture substrate sensitized with a recombinant phage protein specific to Salmonella in a tube that contains the enriched sample, diluted to 1/100th in the reaction mixture.

(23) Protocol:

(24) Step 1: Resuspending the Samples in the Primary Enrichment Medium

(25) Two samples are prepared as follows:

(26) Sample A: In a homogenizing bag, 25 g of unpasteurized milk cheese contaminated with 5 colony forming units (CFU) of Salmonella Napoli is resuspended in 225 mL of Buffered Peptone Water (BPW) (bioMrieux, Ref. 42043), supplemented with 1 ml of Supplement SPT (bioMrieux, Ref. 42650);

(27) Sample B: In a homogenizing bag, 25 g of unpasteurized milk cheese not contaminated with Salmonella Napoli is resuspended in 225 mL of BPW (bioMrieux, Ref. 42043) supplemented with 1 mL of Supplement SPT (bioMrieux, Ref. 42650).

(28) Step 2: After 16 h of Incubation, Transfer of a 0.1-mL Aliquot from the Homogenizing Bag to the Reaction Tube

(29) 0.1 mL from the Sample A homogenizing bag is transferred to the reaction tube containing 10 mL of SX2 (bioMrieux, Ref. 42121) and 1.6 g/L of TTC (bioMrieux, Cat. No. 04568088). This gives Sample A.

(30) A similar operation is carried out for Sample B.

(31) Step 3: Immersion of the Sensitized Substrates in the Reaction Tubes Before Subculture and Reaction

(32) The sensitized capture substrate is placed in each tube (Samples N and B). The tubes are then closed again and incubated in a stove at 37 C. for 6 h.

(33) Step 4: Reading the Capture Substrates at the End of the Incubation Period

(34) At the end of incubation (6 h at 37 C.) and following non-specific reduction of TTC by all of the bacteria present in the sample (i.e. belonging to the additional flora and the target flora), the reaction mixture has turned red. Thus, in order to observe the capture substrate, revealing whether the sample analysed is positive or negative, the tubes are slanted so as to isolate said capture substrate from the reaction mixture.

(35) In accordance with the experimental design, the capture substrate placed in sample A appears coloured red, confirming that sample A is positive, whereas the capture substrate placed in sample B remains colourless, confirming that sample B is negative. Analysis of these same samples by the VIDAS SPT method, marketed by the applicant (ref. 30707), led to similar results, thus confirming the results obtained by optical reading of the sensitized capture substrate.

Example 2: Optical Detection of Salmonella Napoli in an Enriched Biological Sample, by Means of a Sensitized Substrate Immersed in a Reaction Mixture

(36) The aim of this experiment is direct detection of the presence of the target bacterium Salmonella Napoli in an enriched food sample, by means of a sensitized substrate made of irradiated polystyrene, marketed by the company Nunc/Thermo Scientific (Cat. No. 472230) and shown in FIGS. 2 and 3.

(37) As detailed below, detection is performed during the reaction step by immersing the sensitized capture substrate with an anti-Salmonella recombinant phage protein in a tube containing the enriched sample, diluted to 1/2 in the reaction mixture.

(38) Protocol:

(39) Step 1: Resuspending the Samples in the Primary Enrichment Medium

(40) Two samples are prepared as follows:

(41) Sample A: In a homogenizing bag, 25 g of minced steak contaminated with Salmonella Napoli is resuspended in 225 mL of BPW (bioMrieux, Ref. 42043) supplemented with 1 mL of Supplement SPT (bioMrieux, Ref. 42650);

(42) Sample B: In a homogenizing bag, 25 g of minced steak not contaminated with Salmonella Napoli is resuspended in 225 mL of BPW (bioMrieux, Ref. 42043) supplemented with 1 mL of Supplement SPT (bioMrieux, Ref. 42650);

(43) Step 2: After 16 h of Incubation, Transfer of a 1-mL Aliquot from the Homogenizing Bag to the Reaction Tube

(44) 1 mL from the Sample A homogenizing bag is transferred to the reaction tube containing 1 mL of tryptone salt (bioMrieux, Ref. 42076) supplemented with 10 of gentian violet (bioMrieux, Ref 55545). This gives Sample A.

(45) A similar operation is carried out for Sample B.

(46) Step 3: Immersion of the Sensitized Substrates in the Reaction Tubes Before Reaction

(47) The sensitized capture substrate is placed in each tube (Samples A and B), as described below. The tubes are then closed again during the reaction period.

(48) Step 4: Reading the Capture Substrates at the End of the Reaction Period

(49) At the end of the reaction (40 min at room temperature), all of the bacteria present in the sample (i.e. belonging to the additional flora and the target flora) are stained violet. Thus, in order to be able to observe the capture substrate, revealing whether the sample analysed is positive or negative, the tubes are slanted so as to isolate said capture substrate from the reaction mixture.

(50) In accordance with the experimental design, the capture substrate placed in sample A appears coloured violet, confirming that sample A is positive, whereas the capture substrate placed in sample B remains colourless, confirming that sample B is negative. Analysis of these same samples by the VIDAS SPT method, marketed by the applicant (ref. 30707), led to similar results, thus confirming the results obtained by reading the sensitized capture substrate by eye.