METHOD FOR DETECTING AND/OR IDENTIFYING AT LEAST ONE TARGET MICROORGANISM PRESENT ON A SURFACE

Abstract

A method for detecting and/or identifying at least one target microorganism present on a surface, including the following steps: a) depositing, on the surface, a composition including a synthetic, water-soluble film-forming polymer, the composition being a liquid and/or viscous composition or a foam composition; b) drying the composition in order to allow the formation of a polymer film, c) removing, from the surface, the polymer film including the at least one target microorganism; d) dissolving the polymer film with an aqueous diluent in order to form a solution including the at least one microorganism, e) detecting and/or identifying the at least one target microorganism in all or part of the solution, using at least one detection means.

Claims

1. A process for detecting and/or identifying at least one target microorganism present on a surface, comprising the following steps: a) depositing on the surface, a composition comprising a film-forming water-soluble synthetic polymer, the composition being a liquid and/or viscous composition or a foam composition; b) drying the composition to allow the formation of a polymer film, c) removing from the surface the polymer film comprising the at least one target microorganism, d) dissolving the polymer film with an aqueous diluent to form a solution comprising the at least one microorganism, e) detecting and/or identifying the at least one target microorganism in all or part of the solution using at least one detection means.

2. The process as claimed in claim 1, wherein the aqueous diluent is a semi-solid culture medium, the polymer film being dissolved by contact with the semi-solid culture medium.

3. The process as claimed in claim 1, wherein the detection means is the culture medium in which the polymer film has been dissolved.

4. The process as claimed in claim 1, wherein the surface is an inert surface.

5. The process as claimed in claim 1, wherein the process comprises a step of incubating the culture medium, before or during the detection step, at a temperature and for a period of time sufficient to allow the growth of the at least one microorganism.

6. The process as claimed in claim 1, wherein the polymer is selected from polyethylene oxide, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacrylamide, poly(2-hydroxypropyl methacrylamide), poly(2-ethyl-2-oxazoline).

7. The process as claimed in claim 6, wherein the polymer is polyvinyl alcohol with an average molecular weight comprised between 9 000 and 200 000 g/mol and at a concentration comprised between 2 and 60% (m/v).

8. The process as claimed in claim 1, wherein the composition comprises a surfactant.

9. The process as claimed in claim 8, wherein the surfactant is selected from sodium dodecyl sulfate, cholic acid, dicyclohexyl sulfosuccinate sodium, benzalkonium chloride, diethylene glycol, polysorbate 80, saponin, 3-(N,N-dimethyltetradecylammonio)propanesulfonate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate.

10. The process as claimed in claim 9, wherein the surfactant s polysorbate 80 at a concentration equal to or less than 5%.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0085] FIG. 1 is a graph representing on the ordinate the decrease in thickness of the liquid and/or viscous composition during drying (in μm) as a function of the drying time (in hours).

[0086] FIG. 2 is an ordinate graph representing the loss modulus (G″) and the elastic modulus (G′) of the liquid and/or viscous composition expressed in pascals as a function of the drying time (in hours).

[0087] FIG. 3 is a graph representing on the ordinate the viscosity of the liquid and/or viscous composition expressed in mPa per second as a function of the PVOH polymer concentration expressed in % (m/v).

DETAILED DESCRIPTION OF THE INVENTION

[0088] The following examples will allow the present invention to be better understood. However, these examples are given by way of illustration only and must in no case be regarded as limiting the scope of said invention in any way.

Example 1a: Preparation of Liquid and/or Viscous Polymer Compositions

[0089] Different concentrations of water-soluble polymer were used: from 2 to 50% m/v. Different concentrations of surfactants were used: from 0 to 5% v/v or m/v. [0090] 1) The water-soluble polymer is weighed in glass bottles and diluted with demineralized water. [0091] 2) The whole is shaken and heated in order to completely dissolve the polymer in solution. [0092] 3) If a surfactant is present, it is added to the solution by pipetting (if liquid) or by weighing (if solid). [0093] 4) The composition is autoclaved in a liquid cycle, with a plateau at 120° C. for 16 min. [0094] 5) The composition is stored at room temperature

Example 1b: Preparation of Foaming Polymer Compositions

[0095] The preparation of foaming compositions differs only by an additional step (step 6) in which a gas is added. [0096] 1) The water-soluble polymer is weighed in glass bottles and diluted with demineralized water. [0097] 2) The whole is shaken and heated in order to completely dissolve the polymer in solution. [0098] 3) If a surfactant is present, it is added to the solution by pipetting (if liquid) or by weighing (if solid). [0099] 4) The solution is autoclaved in a liquid cycle, with a plateau at 120° C. for 16 min. [0100] 5) The composition is stored at room temperature [0101] 6) The composition is added in a device containing CO.sub.2 in order to create the foam composition by mixing during deposition.

Example 2: Testing of Families of Water-Soluble Polymers

[0102] Different families of water-soluble polymers were tested for the following properties: [0103] Dissolution of the polymer in water and obtaining a homogeneous solution [0104] Ability to form a film by drying [0105] Peeling of the film from a surface (crystal polystyrene plastic, glass, 316L stainless steel) [0106] Water-solubility of the film in contact with an aqueous diluent [0107] Detachment and then detection of at least one microorganism

[0108] The polymers were prepared according to the following protocol: [0109] 1) The polymer is weighed in glass bottles and diluted with demineralized water. [0110] 2) The whole is shaken and heated in order to completely dissolve the polymer in solution. [0111] 3) A surfactant is added to the solution by pipetting at a concentration of 0.5% v/v. [0112] 4) The solution is stored at room temperature

[0113] The water-soluble synthetic polymers having demonstrated all the properties described above are preferentially: [0114] Poly(ethylene oxide) (PEO) with an average molecular weight comprised between 100 000 and 8 000 000 g/mol (Sigma Aldrich; item: 181986, 372781, 372838) at concentrations comprised between 2 and 30% (m/v) [0115] Poly(ethylene glycol) (PEG) with an average molecular weight comprised between 600 and 20 000 g/mol at concentrations comprised between 2 and 30% (m/v) [0116] Poly(vinyl pyrrolidone) (PVP) with an average molecular weight comprised between 10 000 and 1 300 000 g/mol (Sigma Aldrich; item: PVP10, PVP360, 437190), at concentrations comprised between 2 and 30% (m/v) [0117] Poly(vinyl alcohol) (PVA or PVOH) with an average molecular weight comprised between 9 000 and 200 000 g/mol (Sigma Aldrich; item: 360627; Merck Millipore; item: 8.43866.1000, 8.43867.1000), at concentrations comprised between 2 and 50% (m/v) [0118] Poly(acrylic acid) (PAA) with an average molecular weight comprised between 450 000 and 4 000 000 g/mol (Sigma Aldrich; item: 181285, 306231), at concentrations comprised between 1 and 2% (m/v) [0119] Poly(acrylamide) with an average molecular weight comprised between 40 000 and 150 000 g/mol (Sigma Aldrich; item: 738743, 749222) at concentrations comprised between 2 and 20% (m/v) [0120] Poly(2-hydroxypropyl methacrylamide) with an average molecular weight comprised between 30 000 and 50 000 g/mol (Sigma Aldrich; item: 804746) at concentrations comprised between 2 and 30% (m/v) [0121] Poly(2-ethyl-2-oxazoline) with an average molecular weight of 5 000 g/mol (Sigma Aldrich; item: 773379) at concentrations comprised between 2 and 10% (m/v)

Example 3: Test of Families of Surfactants

[0122] Different families of surfactants were tested for the following properties: [0123] Dilution of the surfactant in the polymer solution and obtaining a homogeneous solution [0124] Little or no negative impact on the cohesion of the film, the water solubility of the film, the formation of a film by drying. [0125] Facilitates the peeling of the film from a surface (crystal polystyrene plastic, glass, 316L stainless steel) [0126] Detachment and then detection of at least 1 microorganism

[0127] Polymer solutions containing a surfactant were prepared according to the following protocol: [0128] 1) The polymer is weighed in glass bottles and diluted with demineralized water. [0129] 2) The whole is shaken and heated in order to completely dissolve the polymer in solution. [0130] 3) The surfactant is weighed (if in solid form) or measured (if in liquid form) and then added to the solution at a concentration of 0.5% m/v or 0.5% v/v. [0131] 4) The solution is stored at room temperature

[0132] The families of surfactants having demonstrated all the properties described above are preferentially: [0133] Anionic surfactants, such as sodium dodecyl sulfate (Sigma Aldrich, item L3771), cholic acid (Sigma Aldrich, item C1129) and sodium dicyclohexyl sulfosuccinate (Sigma Aldrich, item 86141) [0134] Cationic surfactants, such as benzalkonium chloride (Sigma Aldrich, item 12060) [0135] Non-ionic surfactants, such as diethylene glycol (Sigma Aldrich, item 93171), polysorbate 80 (Acros Organics; item 278630000) and saponin (Sigma Aldrich, item 84510) [0136] Zwitterionic surfactants, such as 3-(N,N-dimethyltetradecylammonio)propanesulfonate (Sigma Aldrich, item 40772) and CHAPS or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (Sigma Aldrich, item C9426)

Example 4: Polymer Drying Speed (Base 25 cm.SUP.2.)

[0137] In order to evaluate the rate at which the water-soluble polymer composition is capable of changing from a liquid and/or viscous state to a solid state in the form of a film, tests were carried out under various conditions.

[0138] The polymer composition 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0139] 1 ml of solution is deposited on a 316L stainless steel square surface of 5 cm×5 cm, i.e. 25 cm.sup.2. The deposits were placed in a ventilated oven (30% ventilation) at different temperatures or under a laminar flow hood at room temperature.

[0140] The time required for the entire 25 cm.sup.2 polymer surface to be in film form was timed.

[0141] The results are shown in the following table:

TABLE-US-00001 Laminar flow 22.5° C. 32.5° C. 37° C. Drying condition hood (RT) oven oven oven Average drying speed 1.23 2.50 1.72 2.06 (in hours) Standard deviation 0.06 0.45 0.36 0.14

[0142] Conclusion:

[0143] It has been shown that the drying speed of water-soluble polymer is more dependent on ventilation than on temperature (better ventilation under a flow hood than in an oven).

[0144] The drying speed is comprised between 1.5 h and 3 h, demonstrating the speed with which the polymer goes from a viscous state to a solid state.

Example 5: Rheological Analysis of Compositions According to the Invention During Drying

[0145] Rheological analyses were performed in order to physically characterize the behavior of the polymer solution during drying.

[0146] A 10% m/v PVOH+0.5% v/v polysorbate 80 polymer solution and a 10% m/v PVOH+5% v/v polysorbate 80 polymer solution were manufactured according to Example 1 and then stored at room temperature.

[0147] The rheological study was performed on a DHR-2 rheometer (TA Instruments) with a lower platform (Peltier) for temperature maintenance and an upper platform covered with a plastic “doughnut”. This doughnut-shaped cover allows a slow and uniform drying of the gel along a radial axis, ensuring a homogeneous gel thickness during drying.

[0148] Roughly 1 ml of polymer solution is deposited on the lower platform of the rheometer, then the upper platform is slowly lowered in contact with the solution, to obtain an initial gap of 500 μm. The experiment is performed at a constant temperature of 25° C.

[0149] The height of the upper platform is controlled by the rheometer so as to impose on the sample a constant zero force that does not vary during drying of the gel. The height of the sample (gap) can thus be monitored over time.

[0150] FIG. 1 is a graph representing the decrease in sample thickness (10% m/v PVOH+5% v/v polysorbate 80 polymer solution) during drying.

[0151] The upper platform also applies low amplitude shear oscillations (stress amplitude: y=0.1% and frequency=1 Hz) to measure the viscoelastic properties of the sample during drying.

[0152] The viscoelastic properties of the sample are reflected by the viscous modulus or loss modulus (G″) and the elastic modulus or storage modulus (G′). When the elastic modulus becomes higher than the viscous modulus it is possible to determine the critical PVOH concentration corresponding to the gel point.

[0153] FIG. 2 is a graph showing the change in the viscous (G″) and elastic (G′) moduli during the drying of the 10% m/v PVOH+5% v/v polysorbate 80 polymer solution.

[0154] At the gel point, the critical PVOH concentration obtained is comprised between 10 and 20% m/v.

[0155] The elastic modulus obtained at the end of drying, and which characterizes the solid aspect of the polymer film, is greater than 100 000 Pa.

[0156] Finally, the viscosity of the liquid and/or viscous composition comprising the polymer was calculated as a function of the PVOH polymer concentration, represented in FIG. 3. This viscosity characterizes the polymer solution in its liquid/viscous form.

Example 6: Film Peel Force

[0157] In order to physically characterize the adhesion of the film to the surfaces, film peel force tests were performed on different surfaces.

[0158] The polymer solutions were manufactured according to Example 1 and then stored at room temperature. 1.5 g of solution is deposited on a rectangular surface measuring 6.5 cm by 4 cm (26 cm.sup.2) and then dried for 24 hours at room temperature.

[0159] After drying and formation of the film, an initial section of about 1 cm of film is peeled off the surface over the width of 4 cm. This initial section is inserted into the clamp of an MTS® System traction bench, at an angle of about 90°, at a distance of 6 mm. The peeling speed is 50 mm/min, over a distance of 50 mm. The average force required to peel off the film is calculated by the traction bench and presented in the table below.

TABLE-US-00002 Peel force (in N) SD (standard Composition Surface AVG deviation) 30% PVOH, 0.5% polysorbate 80 Plastic 0.91 0.14 30% PVOH, 0.5% polysorbate 80 Glass 1.99 0.82 30% PVOH, 0.5% polysorbate 80 Stainless steel 2.32 0.52 10% PVOH, 5% polysorbate 80 Plastic 0.39 0.03 50% PVOH Stainless steel 12.72 2.34

Example 7: Film Elongation Force

[0160] In order to characterize the tear resistance of the film, elongation force tests were carried out.

[0161] The polymer solutions were manufactured according to Example 1 and then stored at room temperature. 1 g or 3 g of solution was deposited on a round template with a surface area of 23 cm.sup.2 and then dried for 24 hours at room temperature. After peeling off the film, a 3 cm×3 cm square was cut in the center of the circle to ensure a homogeneous thickness of the test sample. The cut film is placed between the two clamps of the tensile tester, at a distance of 10 mm. The elongation speed is 10 mm/min until the film breaks. The value selected is the maximum force value required to rupture the film, visible on the elongation curve by a sharp inflection point (sudden drop).

[0162] The results are presented in the following table:

TABLE-US-00003 Cast Thick- Elongation force (in N) Composition weight ness AVG SD 30% PVOH, 0.5% polysorbate 1 g  44 μm 38.76 3.65 30% PVOH, 0.5% polysorbate 3 g 228 μm 83.45 3.17 10% PVOH, 0.5% polysorbate 1 g  56 μm 12.75 3.98 10% PVOH, 5% polysorbate 1 g  88 μm 12.09 1.97 50% PVOH, 0.5% polysorbate 1 g 111 μm 70.11 5.30 50% PVOH, 0.5% polysorbate 3 g 310 μm 69.11 5.71 50% PVOH, 5% polysorbate 1 g 113 μm 53.14 7.40 50% PVOH, 5% polysorbate 3 g 358 μm 59.94 3.79

Example 8: Dissolution Rate of the Polymer Film (Base 25 cm.SUP.2.)

[0163] In order to evaluate the water solubility property of the PVOH polymer film, the dissolution rate of the film was measured for different volumes of demineralized water.

[0164] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0165] 1 ml of solution is deposited on a square polystyrene crystal plastic surface of 5 cm×5 cm, i.e. 25 cm.sup.2. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film.

[0166] After drying, the polymer film is removed from the plastic surface using sterile forceps. The film is then placed in a bottle containing 100, 50, 10 or 5 ml of demineralized water. The bottle is shaken by hand or vortexed to dissolve the film. The time required for complete dissolution of the film is timed.

[0167] The results are shown in the following table:

TABLE-US-00004 Dissolution volume (ml) 100 50 10 5 Average dissolution rate (min) 1.76 1.61 2.04 5.36 Standard deviation 0.36 0.33 0.29 0.70

[0168] Conclusion:

[0169] The dissolution rate of the PVOH film (1 ml) is shown to be identical for a dissolution volume of 100 ml to 10 ml; i.e. 10 to 100 times more volume of demineralized water than volume of deposited film. On the other hand, the dissolution time increases for a lower dissolution volume of 5 ml (i.e. 5 times the volume of deposited film).

[0170] Finally, regardless of the dissolution volume tested, the dissolution of the PVOH film is very fast, less than 10 min even for a very small volume. This demonstrates the excellent water solubility of the PVOH film, which leaves no particles or residues after dissolution.

Example 9: Advantage of Microorganism Sampling Using a Polymer in Liquid and/or Viscous Form According to the Present Invention

[0171] The collection of microorganisms using a polymer in liquid and/or viscous form according to the present invention was compared with collection using a polymer that was first dried before being brought into contact with the microorganism to be collected.

[0172] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0173] Dry Deposit of Microorganisms:

[0174] A known quantity of Staphylococcus aureus is deposited on a glass slide (Rq≈0.5 nm). A BioBall Multishot 550 CFU (bioMérieux; item: 56019) is diluted in 500 μl of rehydration fluid (bioMérieux; item: 56021) and vortexed for 30 seconds. 50 μl of Staphylococcus aureus solution (about 50 CFU [colony forming units]) is deposited on the glass slide. The deposit is dried in a 30% ventilated oven, at 37° C. for 30 minutes.

[0175] In parallel, 50 μl of Staphylococcus aureus solution is deposited on trypticase soy agar (TSA) Petri dishes (bioMérieux; item: 43011 and 43711). These control plates are incubated at 30-35° C. for 48 hours.

[0176] Polymer Deposition: [0177] 1) Liquid form: The PVOH+polysorbate 80 polymer solution is deposited by pipetting onto the dry deposit of Staphylococcus aureus microorganisms, then spread with a sterile loop if necessary to completely cover the dry deposit. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. [0178] 2) Solid form: the PVOH+polysorbate 80 polymer solution is deposited by pipetting onto a glass slide and spread with a sterile loop. The solution is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. The film obtained is then applied for 10 seconds and with a force of 500 g on the dry deposit of Staphylococcus aureus.

[0179] Polymer Detachment and Microorganism Detection:

[0180] After drying or direct application, the polymer film is detached from the glass slide using sterile forceps. The film is then placed on a TSA Petri dish (bioMérieux; item: 43011 and 43811). After a few minutes the film is completely solubilized by virtue of the water contained in the Petri dish. The dishes are incubated at 30-35° C. for 48 hours.

[0181] Microorganism Counting and Analysis:

[0182] After 48 hours of incubation, the colonies present on the control dishes and the dishes containing the water-solubilized polymer film are counted. The recovery rate of the polymer is calculated as follows:

Recovery rate: (100×polymer TSA count)/(control TSA count)

[0183] The results are presented in the following table:

TABLE-US-00005 Average recovery Standard rate (%) deviation Liquid form 56.2 0.14 Solid form 18.7 0.08

[0184] Conclusion:

[0185] These results show that applying the water-soluble polymer solution in liquid and/or viscous form and then drying to obtain a film makes it possible to recover three times more microorganisms than the direct application of the film already in solid form.

Example 10: Advantage of Microorganism Sampling Using a Polymer in Foam Form According to the Present Invention

[0186] The collection of microorganisms using a polymer in foam form according to the present invention was compared with a polymer solution deposited in liquid/viscous form, on a stainless-steel surface (Rq≈0.4 μm).

[0187] Dry Deposit of Microorganisms:

[0188] A known quantity of Staphylococcus aureus is deposited on a stainless-steel surface. A BioBall Multishot 550 CFU (bioMérieux; item: 56019) is diluted in 500 μl of rehydration fluid (bioMérieux; item: 56021) and vortexed for 30 seconds. 50 μl of the Staphylococcus aureus solution (about 50 CFU [colony forming units]) is deposited on the stainless-steel surface. The deposit is dried in a 30% ventilated oven, at 37° C. for 30 minutes.

[0189] Polymer Deposition: [0190] 1) Liquid form control: the polymer solution 10% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1a. This solution is deposited by pipetting on the dry deposit of Staphylococcus aureus microorganisms, then spread with a sterile loop if necessary in order to completely cover the dry deposit. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. [0191] 2) Foam form: The polymer foam 10% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1b. This foam is deposited on the dry deposit of Staphylococcus aureus microorganisms. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, the foam solution goes to a liquid state before solidifying as a film.

[0192] Polymer Detachment and Microorganism Detection:

[0193] After drying, the polymer films are detached from the stainless-steel surface using sterile forceps. The film is then placed on a TSA Petri dish (bioMérieux; item: 43011 and 43811). After a few minutes the film is completely solubilized by virtue of the water contained in the Petri dish. The dishes are incubated at 30-35° C. for 48 hours.

[0194] Microorganism Counting and Analysis:

[0195] After 48 hours of incubation, the colonies present on surfaces and dishes containing the water-solubilized polymer film are counted. The recovery rate of the polymer is calculated as follows:

Recovery rate: (100×polymer TSA count)/(control TSA count)

[0196] The results are presented in the following table:

TABLE-US-00006 Average recovery Standard rate (%) deviation Liquid solution deposit 18 0.03 Foam deposit 49 0.12

[0197] Conclusion:

[0198] On a rough surface such as a stainless-steel surface, the application of a foam polymer solution makes it possible to recover about 2.5 times more microorganisms compared with an application of polymer in liquid form.

Example 11: Influence of PVOH Concentration on the Recovery Rate

[0199] In order to study the influence of PVOH polymer concentration on the microorganism recovery rate, a range of solutions was tested: from 10 to 50% m/v PVOH with 0.5% polysorbate 80.

[0200] The solutions were manufactured according to Example 1 and then stored at room temperature.

[0201] Dry Deposit of Microorganisms:

[0202] A known quantity of Staphylococcus aureus is deposited on a glass slide. A BioBall Multishot 550 CFU (bioMérieux; item: 56019) is diluted in 500 μl of rehydration fluid (bioMérieux; item: 56021) and vortexed for 30 seconds. 50 μl of Staphylococcus aureus solution (about 50 CFU) is deposited on the glass slide. The deposit is dried in a 30% ventilated oven, at 37° C. for 30 minutes.

[0203] In parallel, 50 μl of the Staphylococcus aureus solution is deposited on TSA Petri dishes (bioMérieux; item: 43011 and 43711). These control dishes are incubated at 30-35° C. for 48 hours.

[0204] Polymer Deposition:

[0205] The PVOH+polysorbate 80 polymer solution is deposited by pipetting onto the dry deposit of Staphylococcus aureus microorganisms and then spread with a sterile loop if necessary to completely cover the dry deposit. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film.

[0206] Polymer Detachment and Microorganism Detection:

[0207] After drying, the polymer film is detached from the glass slide with sterile forceps. The film is then placed on a TSA Petri dish (bioMérieux; item: 43011 and 43811). After a few minutes the film is completely solubilized by virtue of the water contained in the Petri dish. The dishes are incubated at 30-35° C. for 48 hours.

[0208] Microorganism Counting and Analysis:

[0209] After 48 hours of incubation, the colonies present on the control dishes and the dishes containing the water-solubilized polymer film are counted. The recovery rate of the polymer is calculated as follows:

Recovery rate: (100×polymer TSA count)/(control TSA count)

[0210] The results are shown in the following table:

TABLE-US-00007 PVOH concentration 10 15 20 25 30 35 40 45 50 (with 0.5% polysorbate 80) in % m/v Average recovery rate (%) 64% 80% 62% 67% 68% 72% 52% 51% 47% Standard deviation 0.06 0.17 0.14 0.05 0.06 0.07 0.12 0.12 0.08

[0211] Conclusion:

[0212] It has been shown that the PVOH concentration in the polymer solution does not significantly influence the recovery rate of Staphylococcus aureus, except above 35% m/v where the recovery rate tends to decrease. Below 40% m/v PVOH in solution, the Staphylococcus aureus recovery rate is comprised between 60 and 80%.

[0213] Finally, these recovery rates show a very good envelopment of the microorganisms in the film, then a very good peeling by the film and no toxicity of the PVOH+polysorbate solution.

Example 12: Influence of Polysorbate 80 Concentration on the Recovery Rate

[0214] In order to study the influence of the polysorbate 80 concentration on the microorganism recovery rate, a range of solutions were manufactured according to Example 1: from 0 to 5% v/v polysorbate 80 with 30% m/v PVOH.

[0215] The solutions were tested using the same protocol as in Example 11.

[0216] The results are shown in the following table:

TABLE-US-00008 Polysorbate 80 0 0.5 1 2 5 concentration (with 30% m/v PVOH) in % v/v Average recovery rate (%) 12% 73% 73% 64% 45% Standard deviation 0.05 0.12 0.23 0.19 0.19

[0217] Conclusion:

[0218] It has been shown that the polysorbate 80 concentration influences the microorganism recovery rate. In the absence of polysorbate 80, the Staphylococcus aureus recovery rate is strongly decreased. The addition of 0.5 or 1% polysorbate 80 makes it possible to obtain a recovery rate of about 70% which decreases beyond this concentration.

Example 13: Detection on Petri Dishes

[0219] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0220] The polymer solution is deposited on the surface to be sampled and then, after drying, the film is removed from the surface using sterile forceps (see Example 11 protocol).

[0221] In order to detect and count the number of microorganisms present in the film, thus initially on the surface, the film is deposited on a solid culture medium. Very quickly, the water contained in the agar solubilizes the film and it disappears completely from the surface of the culture medium. Different solid culture media can be used according to the requirements of the microorganisms to be detected.

[0222] The culture media are then incubated under the usual culture conditions. After incubation, the isolated colonies can be easily counted in order to give a quantitative result of the surface monitoring.

[0223] Tests on TSA Petri dishes (bioMérieux, item: 43811) showed that the size of colonies at 24/48/72 hours is equivalent between a direct inoculum deposit and a deposit after removal of the water-soluble polymer.

[0224] In addition, tests were carried out with a medium containing a colored indicator (Chapman medium; bioMérieux, item: 46671) and a medium containing a chromogen (SAID medium; bioMérieux, item: 419042). The results show that the staining of colonies, of the medium, and the appearance of the colonies are equivalent between a direct inoculum deposit and a deposit after removal of the water-soluble polymer.

[0225] These results demonstrate the compatibility of the water-soluble polymer with growth methods on culture medium.

Example 14: Detection by Rapid Methods (BacT/ALERT®, Tempo®, Gene-Up®)

[0226] BacT/ALERT®:

[0227] The BacT/ALERT® technology is a kinetic microorganism detection method by colorimetry. Microorganisms present in the medium produce CO.sub.2 during their growth phase. The CO.sub.2 produced induces a decrease in the pH of the culture medium. This change in pH induces the colorimetric shift of a sensor composed of silicone impregnated with liquid emulsion detectors (LES), placed at the bottom of each bottle. An LED sends a light beam on the sensor.

[0228] A photodiode collects the intensity of light reflected by the sensor in the form of a reflectance unit. The reflectance units are analyzed over time.

[0229] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0230] The polymer solution is deposited on a surface previously loaded with a known amount of the microorganism Staphylococcus aureus (roughly 50 CFU), as described in Example 9. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. After drying, the polymer film is detached from the surface using sterile forceps. The film is then diluted in 2 ml of saline solution (bioMérieux, item: 33892). After complete solubilization, the solution is withdrawn by syringe and then injected into a

[0231] BacT/ALERT® SA bottle (bioMérieux, item: 259789). In parallel, control bottles are inoculated directly with the same known quantity of the Staphylococcus aureus strain (roughly 50 CFU). The bottles are loaded onto the BacT/ALERT® VIRTUO® automated system (bioMérieux; item: 411660) for automatic kinetic reading.

[0232] The results show similar detection times and growth curves between the control bottles inoculated directly with the strain and the bottles inoculated with the solubilized polymer film. These results demonstrate the compatibility of the water-soluble polymer with a colorimetric microorganism detection method.

[0233] TEMPO®:

[0234] The Tempo technology is a method for counting microorganisms based on the most probable number (MPN) technique by analyzing series of dilutions of the initial sample. The Tempo technology miniaturizes this test on a specifically designed board. The system consists of: [0235] A preparation station (TEMPO® FILLER) to inoculate the TEMPO® cards with the samples and the specific culture media. TEMPO® culture media allow the rapid growth of bacteria or yeast/molds and contain a fluorescent indicator. The innovative TEMPO® card integrates a 16×3 miniaturized tube MPN method. [0236] An automated reading station (TEMPO® READER) to determine the bacterial concentration in each card and thus determine the number of microorganisms present in the initial sample.

[0237] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0238] The polymer solution is deposited on a surface previously loaded with a known amount of the microorganism Staphylococcus aureus (roughly 50 CFU), as described in Example 9. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. After drying, the polymer film is detached from the surface using sterile forceps. The film is then placed in a TEMPO CTB bottle (bioMérieux, item: 416683). In parallel, control bottles are inoculated directly with the same known quantity of the Staphylococcus aureus strain (roughly 50 CFU). The medium contained in the bottle is rehydrated by adding 4 ml of distilled water per bottle, also allowing the polymer film to solubilize.

[0239] After rehydration and complete solubilization, the TEMPO® CTB bottles and the corresponding TEMPO® CTB cards (bioMérieux, item: 416683) are scanned on the TEMPO® FILLER station. The bottles and cards are placed on the filling rack, placed in the preparation station which will completely fill the card and then cut the straws and thus seal the card.

[0240] The cards are incubated at 30° C.±1° C. for 24 h-28 h (bacterial detection).

[0241] After incubation, the cards are placed in the TEMPO® READER reading station and read automatically. The result is displayed directly in CFU/ml, taking into account the dilution factor.

[0242] Results show equivalent bacterial counts between control bottles inoculated directly with the strain and bottles inoculated with the solubilized polymer film. These results demonstrate the compatibility of the water-soluble polymer with a fluorescence-based microorganism detection method using the MPN method.

TABLE-US-00009 Polymer Polymer Control bottles on glass on plastic Average TEMPO ® count (in 56 65 60 CFU/ml) Standard deviation 0 23.09 10.58

[0243] Gene UP®:

[0244] The Gene-Up® technology is a method for detecting microorganisms by real-time PCR. Following enrichment in culture medium, the DNA of the microorganisms is released by mechanical lysis and then detected by real-time PCR using FRET probes and probe fusion peaks to guarantee the specificity of the test.

[0245] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0246] Different surfaces are loaded with the microorganism Listeria monocytogenes and then dried in a 30% ventilated oven at 37° C., for 30 minutes to obtain dry deposits. The polymer solution is then deposited on the microorganisms and dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. After drying, the polymer film is removed from the surface with sterile forceps and deposited in a tube of LPT enrichment medium (bioMérieux, item: 410845). In parallel, dry deposits of microorganisms are sampled with a swab (bioMérieux, item: 70.6016) and then deposited in a tube of LPT enrichment medium. The tubes are incubated at 37° C. for 18-24 hours.

[0247] After enrichment, 20 μl of sample is deposited in a lysis tube (bioMérieux, Gene-Up® lysis kit, item: 414057). The lysis tubes are placed on a rack and shaken with the Troemner vortex at 2200 rpm for 5 minutes.

[0248] A bottle of lyophilized reagent (bioMérieux, Listeria spp. Kit: item: 414059, Listeria monocytogenes Kit: item: 414058) is reconstituted with 45 μl of reconstitution buffer. Empty PCR tubes are placed on a rack and 5 μl of PCR reagent is placed in each tube. Then 5 μl of lysed sample is added to each tube. A negative control consisting of 5 μl of control buffer is performed for each kit. The tubes are closed with stoppers, sealed and the rack is then centrifuged. The plate is placed in the Gene UP instrument.

[0249] The samples and associated detection kits are entered into the Gene UP software before starting. The results are automatically interpreted when the PCR cycle is completed. The software interprets the amplification and fusion curve data for each sample and gives a positive, negative or inhibited result.

[0250] The results show equivalent detections between the control samples and the samples collected with the polymer for all surfaces tested. In addition, all internal PCR controls are detected.

[0251] These results demonstrate the compatibility of the polymer with a detection method via the PCR technique. In particular, the polymer does not inhibit the PCR reaction.

TABLE-US-00010 Repli- Plastic Plastic Glass Glass Negative Detection kit cate control polymer control polymer control Listeria spp. 1 Positive Positive Positive Positive Negative 2 Positive Positive Positive Positive Listeria 1 Positive Positive Positive Positive Negative monocytogenes 2 Positive Positive Positive Positive

Example 15: Detection by Biochemical Method (VIDAS®)

[0252] The VIDAS® technology is a method for detecting elements (hormones, viruses, microorganisms, etc.) by immunoassay. The VIDAS® principle consists of the interaction of two elements: the cone (solid phase), whose inner surface is coated with antigens or antibodies, and the strips, composed of several wells and containing the exact quantity of reagents required for the test.

[0253] Reactions occur in the cone in two key steps: [0254] immunological reaction, capturing the desired element; [0255] enzymatic reaction, revealing the presence of the element sought by the ELFA (enzyme-linked fluorescent assay) technique.

[0256] The entire operation is automated: from incubation, to the washes and to the final reading. The incubation time and the number of wash cycles are optimized for each parameter to ensure optimal performance.

[0257] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0258] Different surfaces are loaded with the microorganism Listeria monocytogenes and then dried in a 30% ventilated oven, at 37° C. for 30 minutes to obtain dry deposits. The polymer solution is then deposited on the microorganisms and dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. After drying, the polymer film is removed from the surface with sterile forceps and deposited in a tube of LPT enrichment medium (bioMérieux, item: 410845). In parallel, dry deposits of microorganisms are sampled with a swab (bioMérieux, item: 70.6016) and then deposited in a tube of LPT enrichment medium. The tubes are incubated at 30° C. for 22-30 hours.

[0259] After enrichment, 500 μl of sample is deposited in the sample well of a VIDAS® UP Listeria strip (bioMérieux, item: 30126). The strip is heated for 5 minutes at 95-100° C. on the VIDAS® Heat and Go instrument and left to cool for 10 minutes.

[0260] As positive control, two standards Si are prepared by depositing 500 μl of standard Si in the sample well of a VIDAS® UP Listeria strip (bioMérieux, item: 30126). These strips are not heated.

[0261] The VIDAS® cones and strips are placed in the instrument, the samples and the associated detection parameter are entered into the software before the test is started. Results are obtained in about 62 minutes.

[0262] The results show equivalent positivity between the control samples and the samples collected with the polymer for all surfaces tested. These results demonstrate the compatibility of the water-soluble polymer with an immunoassay detection method.

Example 16: Identification by Biochemical Method (Vitek 2)

[0263] The VITEK® 2 Compact is a microbial identification system based on miniaturized colorimetric biochemical reactions on a specifically designed card. All identification phases, from reading to recording of results, are automated. The VITEK® 2 Compact includes an extensive identification database that allows the identification of numerous microorganisms.

[0264] The polymer solution 30% m/v PVOH+0.5% v/v polysorbate 80 was manufactured according to Example 1 and then stored at room temperature.

[0265] The polymer solution is deposited on a surface previously loaded with a colony of the microorganism Kocuria kristinae. The whole is dried in a 30% ventilated oven, at 37° C. for 1 hour, allowing the polymer solution to solidify as a film. After drying, the polymer film is detached from the surface using sterile forceps. The film is placed in a hemolysis tube and then solubilized by adding 3 ml of saline solution (bioMérieux; item: 21218) for VITEK®. In parallel, a control tube containing 3 ml of saline solution is prepared to obtain 0.5 McFarland of the Kocuria kristinae strain. The tubes are loaded with a VITEK® GP card (bioMérieux; item: 21342) in the Smart Carrier Station™. Once the cassettes are loaded, the system manages the incubation and reading of each card without any further intervention. Results are obtained within 2 to 18 hours after loading.

[0266] The results show equivalent identifications (“Excellent identification”) between the control tube inoculated directly with the strain and the tube inoculated with the solubilized polymer film. These results demonstrate the compatibility of the water-soluble polymer with a colorimetric biochemical identification method.