Method for detecting, identifying and enumerating micro-organisms in a porous support dry-impregnated with a dehydrated reaction medium

Abstract

A method for the detection, identification and enumeration of microorganisms in a porous support including reaction medium powder throughout its thickness, the support having been dry-impregnated throughout its thickness by a dehydrated reaction medium. Further, a device is used and enables the method to be carried out.

Claims

1. A device comprising a porous support comprising at least one dehydrated reaction medium in powder form distributed throughout the thickness of the porous support, wherein the reaction medium is a visualization medium and/or a culture medium and said porous support has a thickness of between 0.5 and 2 mm and is calendered.

2. The device as claimed in claim 1, wherein the reaction medium in powder form is homogeneously distributed through the thickness of the porous support.

3. The device as claimed in claim 1, wherein the reaction medium in powder form is distributed in a graduated manner through the thickness of the porous support.

4. The device as claimed in claim 1, wherein at least two different reaction media in powder form are distributed in at least two layers, said porous support comprising, at a given point through the thickness, one of the reaction media.

5. The device as claimed in claim 1, wherein the reaction medium is a culture medium.

6. The device as claimed in claim 1, wherein the reaction medium comprises at least one gelling agent, the amount of which is between 1 mg/cm.sup.2 and 2 mg/cm.sup.2.

7. The device as claimed in claim 1, wherein the amount of reaction medium impregnated in the porous support is between 0.10 mg/cm.sup.3 and 0.1 g/cm.sup.3.

8. The device as claimed in claim 1, comprising a plurality of the porous supports.

9. The device as claimed in claim 1, wherein the porous support is integrated into a dressing, a bandage, a sanitary pad or an item of food packaging.

10. The device as claimed in claim 1, further comprising a rod including an end at which said porous support is fixed.

11. The device as claimed in claim 1, wherein the reaction medium is a culture medium for detecting a methicillin-resistant Staphylococcus.

12. The device as claimed in claim 1, wherein the thickness of the porous support is between 0.8 and 2 mm.

13. The device as claimed in claim 1, further comprising: an upper layer that is translucent or transparent and impermeable to bacteria; and a lower layer including channels or pores, wherein the porous support is between the upper and lower layers.

14. The device as claimed in claim 1, wherein the porous support further comprises an encapsulated chromogenic substrate or selective agent.

15. The device as claimed in claim 1, wherein the porous support further comprises a chromogenic substrate.

16. A device comprising: a powder having a particle size from 1 to 200 m that comprises at least one dehydrated reaction medium including elements for survival and/or growth of a microorganism; and a calendered porous support having a thickness between 0.5 and 2 mm that is dry-impregnated with the dehydrated reaction medium throughout the thickness in an amount between 0.10 mg/cm.sup.3 and 10 mg/cm.sup.3 or between 0.01 g/cm.sup.3 and 0.1 g/cm.sup.3, wherein the porous support has a basis weight between 50 g/m.sup.2 and 150 g/m.sup.2 and a surface area between 1 cm.sup.2 and 40 cm.sup.2, and the porous support is able to retain a volume of water greater than 2 ml.

17. A method for the detection and/or identification and/or enumeration of at least one target microorganism in a sample liable to contain it, comprising the following steps: (a) providing the device as claimed in claim 1, (b) placing the sample in contact with the porous support of the device, (c) incubating the device, and (d) detecting and/or identifying and/or enumerating a colony or colonies of at least one target microorganism within the porous support when the target microorganism was present in the sample.

18. The method as claimed in claim 17, further comprising preparation, dilution or concentration of the sample prior to placing the sample in contact with the porous support.

19. The method as claimed in claim 17, wherein step b) is carried out by placing the sample under the porous support.

20. The method as claimed in claim 17, wherein step b) is carried out by taking the sample using the porous support.

21. The method as claimed in claim 17, wherein, when the sample is not aqueous or is insufficiently aqueous, a suitable volume of liquid is added to the sample and/or to the porous support in order to rehydrate the reaction medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an entirely nonlimiting schematic representation of the invention.

(2) FIG. 1 schematically represents a porous support (10) contained in a device according to the invention comprising a plurality of reaction media (11) at different zones, each of these zones then having reaction medium distributed throughout the thickness of the support.

(3) FIG. 1b corresponds to a plan view of the porous support (10) according to the invention.

(4) FIG. 1c corresponds to a plan view according to another embodiment in which the porous support is impregnated by reaction media at zones forming circles on the surface, each of these zones then having reaction medium distributed throughout the thickness of the support.

EXAMPLES

Examples 1

(5) preparation of a porous support according to the invention, comprising powder of culture medium distributed homogeneously within the support

(6) Material:

(7) 95 g/m.sup.2 nonwoven supports (ref: 95NN81, SCA Life) 25 cm.sup.2 in size and 2 mm thick (before calendering): 0.13 g of ChromID CPS 3 dehydrated culture medium (600-04595, bioMrieux)+0.06 g xanthan (ref: 4452073479, Alliance gum)
Protocol:

(8) 0.2 g of powder is sprinkled homogeneously onto the support then the support is placed between two electrodes with application of a voltage of 3200 V/mm for 15 seconds at a relative humidity of between 35 and 45%.

(9) The support is calendered at 60 C. by applying a pressure of 310.sup.5 Pa/cm.sup.2.

(10) The supports are thus passed through by the powder of culture medium.

Example 2

(11) preparation of a porous support according to the invention, comprising reaction medium powder distributed homogeneously within the support and reaction medium powder distributed solely at the surface

(12) Material:

(13) 95 g/m.sup.2 nonwoven supports 25 cm.sup.2 in size and 2 mm thick (before calendering) (ref: 95NN81, SCA Life) Medium 1: ChromID CPS 3 dehydrated culture medium (0.13 g) (600-04595, bioMrieux)+0.06 g xanthan (ref: 4452073479, Alliance gum) Medium 2: ChromID CPS 3 dehydrated culture medium (0.13 g) (600-04595, bioMrieux)+0.06 g xanthan (ref: 4452073479, Alliance gum)+micronized imipenem monohydrate (sp2129, Merck and Co)

(14) A first face is impregnated with reaction medium homogeneously according to the protocol of example 1. Then, the support is turned over and the second face is impregnated with a reaction medium comprising an antibiotic, imipenem, by means of an electric field weaker than 1050 V/mm for 15 seconds, so that the powder penetrates less and remains more on the surface. Calendering is carried out as described in example 1.

Example 3

(15) preparation of a porous support according to the invention, comprising reaction medium powder distributed homogeneously within the support and reaction medium powder distributed in a gradient within the support

(16) Material: identical to example 2.

(17) A first face is impregnated homogeneously with medium 1 according to the protocol of example 1. Then, the support is turned over, and the second face is impregnated with the second medium with the same strong electric field (3169V/mm).

(18) An impregnation gradient of the second reaction medium within a homogeneous distribution of the first reaction medium is therefore obtained.

Example 4

(19) preparation of a porous support according to the invention, comprising different reaction media distributed within the support in two layers

(20) A first face is impregnated homogeneously with the reaction medium with an intermediate electric field (1408 V/mm). Then, the support is turned over and the second face is impregnated with a second reaction medium with the same intermediate electric field (1408 V/mm).

(21) Two separate layers of reaction media are therefore obtained, and the whole thickness of the porous support comprises reaction medium powder.

Example 5

(22) Detection of Escherichia coli and Pseudomonas aeruginosa with a device according to the invention comprising 1 mg/l imipenem as antibiotic

(23) Material:

(24) 1. Reagents Used CPS3 agar (bioMrieux, ref: 43549, 22002) Chrom ID CPS3 dry medium (bioMrieux, ref: 600-04595) Tryptone salt 9 ml vial, (AES, ref: 111499, batch 327601) Xanthan (Alliance Gum FF Pharma, batch FF 2524429), particle size<75 m. Imipenem (bioMrieux INC, ref: 066259-1) 2. Strains Used E. coli ATCC 25922, MIC (minimum inhibitory concentration) of 0.12 mg/ml Pseudomonas aeruginosa ATCC 27853, MIC 2 mg/ml
Method:

(25) To produce the agar media, a liter of water is added to the test specimen of the dry medium, i.e. 38.3 g for ChromID CPS3. The dry medium is then dissolved with stirring, brought to the boil then sterilized by autoclaving. After cooling the agar medium to 55 C., the imipenem sterilized by filtration is added at a concentration of 1 mg/l. To carry out impregnation according to the present invention of the dry medium Chrom ID CPS3, a test specimen of the dry medium corresponding to the manufacture of one liter of medium is taken, i.e. 26 g, to which test specimen xanthan (20 g) and imipenem (1 g) are added. All the constituents are then mixed together in a Turbula. The porous supports are then impregnated by the culture media powder produced as described above and sterilized by gamma radiation between 10 and 17 kGy.

(26) Results:

(27) ChromID CPS3 agar

(28) TABLE-US-00001 Starting concen- ChromID CPS3 ChromID CPS3 with trations without antibiotic 1 mg/l imipenem CFU/ml CFU CFU/ml CFU CFU/ml E. coli 10.sup.3 67/56/88 703 0/0/0 0 ATCC 25922 10.sup.5 300 0/0/0 0 colonies Pseudomonas 10.sup.3 122/105/105 1107 69/61/73 677 aeruginosa 10.sup.5 300 300 ATCC 27853 colonies colonies CFU: Colony Forming Unit
Porous Support According to the Invention

(29) TABLE-US-00002 Starting ChromID CPS3 concen- without ChromID CPS3 with trations antibiotic 1 mg/l imipenem CFU/ml CFU CFU/ml CFU CFU/ml E. coli 10.sup.3 28/32/32 307 0/0/0/0/0/0 0 ATCC 25922 10.sup.5 300 0/0/0/0/0/0 0 colonies Pseudomonas 10.sup.3 49/57/61 556 40/53/38/39/46/56 453 aeruginosa 10.sup.5 300 300 ATCC 27853 colonies colonies
Conclusion

(30) Two strains were tested: E. coli ATCC 25922 (MIC 0.12) and Pseudomonas aeruginosa ATCC 27853 (MIC 2 mg/1).

(31) The results obtained with the porous supports according to the invention and with the ChromID CPS3 agar media are consistent with the growth of the Pseudomonas aeruginosa ATCC 27853 strain having an MIC of 2 mg/l in the presence of 1 mg/l imipenem and with the absence of growth of the E. coli ATCC 25922 strain having an MIC of 0.12 mg/l in the presence of 1 mg/l imipenem.

Example 6

(32) Detection of Proteus and of Pseudomonas with a device according to the invention comprising 1.5 mg/l ciprofloxacin as antibiotic

(33) Material:

(34) Mueller Hinton 2 powder base (bioMrieux, ref: 8301143, batch 1002661760) American Agar (ROKO SA, ref: 11000301) European Agar (SETEXAM, ref: TMN2) Ciprofloxacin (HCL TOKU-E COMPANY, ref: C032) ChromID CPS3 agar (bioMrieux, ref: 43549, 22002) Chrom ID CPS3 dry medium (bioMrieux, ref: 600-04595) Tryptone salt 9 ml vial (AES, ref: 111499, batch 327601) Pre-cast MH2 (Muller Hinton 2) agar (bioMrieux, ref: 43301) TSA agar (bioMrieux, ref: 43011) Xanthan (Alliance Gum FF Pharma, batch FF 2524429), particle size<75 m Ciprofloxacin (HCL, ref: 00743041)

(35) The porous supports were impregnated by the following media: MH2 (Muller Hinton 2)+xanthan alliance gum pharma MH2+xanthan alliance gum pharma+1.5 mg/l ciprofloxacin MH2+xanthan alliance gum pharma CPS3+xanthan alliance gum pharma CPS3+xanthan alliance gum pharma+1.5 mg/l ciprofloxacin
Strains Tested: Proteus mirabilis API 8803099, ADM AP3, MIC: 0.125 Proteus mirabilis API 8803080, ADM JS10, MIC: 0.25 Proteus mirabilis API 9406037, bioMrieux collection, MIC: 4 Proteus vulgaris API 8803017, ADM CQ11, MIC: 0.125 Pseudomonas aeruginosa API 9405061, bioMrieux collection, MIC: 0.125 Pseudomonas aeruginosa API 7509005, ATCC 25853, MIC: 0.5 Pseudomonas aeruginosa API 9410075, MIC: 16 Pseudomonas aeruginosa API 9405063, MIC: 4
Method:

(36) To produce the agar media, a liter of water is added to the test specimen of the dry medium, i.e. 38.3 g for ChromID CPS3 and 41.57 g for the Muller Hinton 2 medium. The dry medium is then dissolved with magnetic stirring, brought to the boil then sterilized by autoclaving. After cooling the agar medium to 55 C., the ciprofloxacin sterilized by filtration is added to the agar medium at a concentration of 1.5 mg/l. To carry out impregnation according to the present invention of the dry media MH2 and Chrom ID CPS3, a test specimen of the dry medium corresponding to the manufacture of one liter of medium is taken, i.e. 26 g for Chrom ID CPS3 and 26.07 g for the Muller Hinton 2 medium, to which test specimen xanthan (20 g) and, if appropriate, ciprofloxacin (1.5 g) are added. The whole thing is then mixed together in a Turbula. The porous supports are then impregnated with the culture media powders as described above and sterilized by gamma radiation between 10 and 17 kGy.

(37) Results:

(38) TABLE-US-00003 Impregnation MH2 Impregnation of porous MH2 Agar of porous support with Agar medium + support with MH2 + STRAINS MIC medium 1.5 mg/l Ci MH2 1.5 mg/l Ci Proteus mirabilis 0.032 ++ ++ API 8803099 Proteus mirabilis 0.38-0.5 ++ ++ API 8803080 Proteus mirabilis 8-12 ++ ++ ++ ++ API 9406037 Proteus vulgaris 0.125 ++ ++ API 8803017 P. aeruginosa 0.25 ++ ++ API 9405061 P. aeruginosa 0.25 ++ ++ API 7509005 P. aeruginosa >32 ++ ++ ++ ++ API 9410075 P. aeruginosa 6-8 ++ ++ ++ ++ API 9405063

(39) TABLE-US-00004 Impregnation ChromID Impregnation of porous ChromID CPS3 of porous support with CPS3 Agar support with ChromID Agar medium + ChromID CPS3 + STRAINS MIC medium 1.5 mg/l Ci CPS3 1.5 mg/l Ci Proteus mirabilis 0.032 ++ ++ API 8803099 Proteus mirabilis 0.38-0.5 ++ ++ API 8803080 Proteus mirabilis 8-12 ++ ++ ++ ++ API 9406037 Proteus vulgaris 0.125 ++ ++ API 8803017 P. aeruginosa 0.25 ++ ++ API 9405061 P. aeruginosa 0.25 ++ ++ API 7509005 P. aeruginosa >32 ++ ++ ++ ++ API 9410075 P. aeruginosa 6-8 ++ ++ ++ ++ API 9405063

CONCLUSION

(40) In this example, the Muller Hinton and ChromID CPS3 agar media with or without 1.5 g/l ciprofloxacin were compared to the porous supports according to the present invention impregnated with the same media with or without 1.5 g/l ciprofloxacin for three strains of Proteus mirabilis, one strain of Proteus vulgaris and four strains of Pseudomonas aeruginosa. All these strains had an MIC around the value of 1.5 mg/l of ciprofloxacin. The strains with an MIC of less than 1.5 mg/l (Proteus mirabilis: API 8803099, Proteus mirabilis: API 8803080, Proteus vulgaris: API 8803017, Pseudomonas aeruginosa: API 9405061, Pseudomonas aeruginosa: API 7509005) do not grow on MH2 agar media+substrates+ciprofloxacin (1.5 mg/1) and ChromID CPS3 with ciprofloxacin added (1.5 mg/1). Similarly, these ciprofloxacin-sensitive strains, for an MIC of 1.5 mg/1, do not grow either on the porous supports impregnated with ChromID CPS3 media and MH2+substrate in the presence of 1.5 mg/l ciprofloxacin. The results obtained on agar media and on porous supports impregnated according to the present invention are therefore consistent with one another. All these strains grow on MH2+substrate or ChromID CPS3 agar media and on porous supports impregnated by MH2+substrate and ChromID CPS3 media.

(41) The strains with an MIC of greater than 1.5 mg/l (Proteus mirabilis: API 9406037, Pseudomonas aeruginosa: API 9410075, Pseudomonas aeruginosa: API 940506) grow on all the agar or impregnated media, with or without ciprofloxacin (1.5 g/1). These results confirm that it is possible to produce culture media according to the present invention containing small amounts of active agents such as chromogenic substrates or antibiotics.