DEVICES FOR DETECTING AND/OR IDENTIFYING MICROBIOLOGICAL INFECTIONS, FOR NON-IMPLANTABLE MEDICAL DEVICES

Abstract

A biosensor intended to be integrated in the actual structure of a dressing, a compress or any other similar absorbent article. This biosensor makes it possible to monitor the onset of a microbiological infection of a wound and/or of a skin lesion. The biosensor includes a piece of absorbent hydrophilic material on which, on the surface and/or within the thickness thereof, a composition of agglomerated powders is immobilized, the composition including ethylene-vinyl acetate (EVA) particles having a surface partially coated at least with a salt of orthophosphoric acid and a visual indicator of microbiological growth.

Claims

1. A biosensor comprising a piece of absorbent hydrophilic material on which, on the surface and/or within the thickness thereof, a composition of agglomerated powders is immobilized, said composition comprising ethylene-vinyl acetate (EVA) particles having a surface partially coated at least with a salt of orthophosphoric acid and a visual indicator of microbiological growth.

2. The biosensor as claimed in claim 1, in which the salt of orthophosphoric acid is selected from potassium dihydrogen phosphate (KH.sub.2PO.sub.4) and sodium dihydrogen phosphate (NaH.sub.2PO.sub.4).

3. The biosensor as claimed in claim 1, in which EVA and KH.sub.2PO.sub.4 are present in a KH.sub.2PO.sub.4/EVA weight ratio between 1:25 and 1:8.

4. The biosensor as claimed in claim 1, in which EVA and NaH.sub.2PO.sub.4 are present in an NaH.sub.2PO.sub.4/EVA weight ratio between 1:15 and 1:3.

5. The biosensor as claimed in claim 1, in which the EVA has a vinyl acetate content by weight from 10 to 40%.

6. The biosensor as claimed in claim 1, in which the surface of the EVA particles is also coated with a gelling agent.

7. The biosensor as claimed in claim 6, in which the gelling agent is selected from agar, agarose, guar gum and xanthan gum.

8. The biosensor as claimed in claim 1, in which the surface of the EVA particles is also coated with MnCl.sub.2.

9. The biosensor as claimed in claim 1, in which the surface of the EVA particles is also coated with a selective agent for antibiotic and/or antifungal effects.

10. The biosensor as claimed in claim 1, in which the piece of absorbent hydrophilic material is a piece of nonwoven.

11. A composition of agglomerated powders comprising EVA particles having a surface partially coated at least with a salt of orthophosphoric acid and a visual indicator of microbiological growth.

12. The composition as claimed in claim 11, in which the salt of orthophosphoric acid is selected from potassium dihydrogen phosphate (KH.sub.2PO.sub.4) and sodium dihydrogen phosphate (NaH.sub.2PO.sub.4).

13. The composition as claimed in claim 11, in which EVA and KH.sub.2PO.sub.4 are present in a KH.sub.2PO.sub.4/EVA weight ratio between 1:25 and 1:8.

14. The composition as claimed in claim 11, in which EVA and NaH.sub.2PO.sub.4 are present in an NaH.sub.2PO.sub.4/EVA weight ratio between 1:15 and 1:3.

15. The composition as claimed in claim 11, in which the EVA has a vinyl acetate content by weight from 10 to 40%.

Description

[0058] Other aims, features and advantages of the invention will become clear from the description given hereunder and the examples presented below, which relate to the appended figures, where:

[0059] FIG. 1 is a graphical representation of the bacteriostatic effect of EVA on the development of bacteria, and inhibition of this bacteriostatic effect by the salts of orthophosphoric acid;

[0060] FIG. 2 corresponds to two photographs, taken by scanning electron microscopy, showing a pulverulent composition of dissociated particles;

[0061] FIG. 3 corresponds to two photographs, taken by scanning electron microscopy, showing a composition of agglomerated powders according to the invention.

EXAMPLES

Example 1: Demonstration of the Bacteriostatic Effect of EVA and Inhibition of this Effect by Adding Salts of Orthophosphoric Acid (KH.SUB.2.PO.SUB.4., NaH.SUB.2.PO.SUB.4.)

[0062] A. Demonstration Performed on Agar Culture Media

[0063] The effects of EVA and of the salts of orthophosphoric acid (more particularly, KH.sub.2PO.sub.4 and/or NaH.sub.2PO.sub.4) on bacterial growth and development were evaluated, firstly, on agar culture media, namely: [0064] a tryptone soy agar (TSA), a universal medium known to be suitable for culture and enrichment of most bacteria, whether they are aerobic or anaerobic; [0065] SAID agar (bioMrieux, France), a culture medium marketed for the specific detection of S. aureus,

[0066] For this purpose, strains of S. aureus obtained from the applicant's collection were used. Before inoculating them in the two agars mentioned above, the bacteria (100 mL of a suspension of 10.sup.3 CFU.mL.sup.1) were resuspended beforehand in human serum prepared and supplied by Etablissement Francais du Sang (EFS, the French National Blood Service), with or without supplementing with EVA and/or with salt of orthophosphoric acid as follows: [0067] s01: serum (EFS, reference 4566200/4566201) [0068] s02: serum s01 with addition of EVA (to a final concentration of 53 g.Math.L.sup.1) [0069] s03: serum s01 with addition of EVA and NaH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1 and 4.5 g.Math.L.sup.1 respectively) [0070] s04: serum s01 with addition of EVA and NaH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1 et 9.0 g.Math.L.sup.1 respectively) [0071] s05: serum s01 with addition of NaH.sub.2PO.sub.4 (to a final concentration of 4.5 g.Math.L.sup.1) [0072] s06: serum s01 with addition of NaH.sub.2PO.sub.4 (to a final concentration of 9.0 g.Math.L.sup.1) [0073] s07: serum s01 with addition of EVA and KH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1 and 10.6 g.Math.L.sup.1 respectively) [0074] s08: serum s01 with addition of EVA and KH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1 and 1.5 g.Math.L.sup.1 respectively) [0075] s09: serum s01 with addition of EVA and KH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1 et 3.0 g.Math.L.sup.1 respectively) [0076] s10: serum s01 with addition of KH.sub.2PO.sub.4 (to a final concentration of 1.5 g.Math.L.sup.1) [0077] s11: serum s01 with addition of EVA, NaH.sub.2PO.sub.4 and KH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1, 4.5 g.Math.L.sup.1 and 1.5 g.Math.L.sup.1 respectively) [0078] s12: serum s01 with addition of EVA, NaH.sub.2PO.sub.4 and KH.sub.2PO.sub.4 (to final concentrations of 53 g.Math.L.sup.1, 9.0 g.Math.L.sup.1 and 3.0 g.Math.L.sup.1 respectively) [0079] s13: serum s01 with addition of NaH.sub.2PO.sub.4 and KH.sub.2PO.sub.4 (to final concentrations of 4.5 g.Math.L.sup.1 and 1.5 g.Math.L.sup.1 respectively) [0080] s14: serum s01 with addition of NaH.sub.2PO.sub.4 and KH.sub.2PO.sub.4 (to final concentrations of 9.0 g.Math.L.sup.1 and 3.0 g.Math.L.sup.1 respectively).

[0081] The EVA used in these tests is marketed by the company DAKOTA, Belgium, in the form of a powder with the reference UNEX EVA (EVA T1). Its vinyl acetate content by weight is 28%.

[0082] The results obtained are presented in Table 1 below.

TABLE-US-00001 TABLE 1 Sera TSA SAID s01 bacterial lawn bacterial lawn s02 1 colony Nothing s03 >300 colonies >300 colonies s04 300 colonies 300 colonies s05 bacterial lawn bacterial lawn s06 bacterial lawn bacterial lawn s07 nothing Nothing s08 bacterial lawn bacterial lawn s09 bacterial lawn bacterial lawn s10 bacterial lawn bacterial lawn s11 nothing Nothing s12 nothing Nothing s13 bacterial lawn bacterial lawn s14 bacterial lawn bacterial lawn

[0083] B. Demonstration Performed on an Absorbent, Hydrophilic Culture Substrate

[0084] Evaluation of the effects of EVA and of the salts of orthophosphoric acid (more particularly, KH.sub.2PO.sub.4) on bacterial growth and development was carried out on an absorbent, hydrophilic culture substrate incorporating EVA and impregnated with a nutrient solution comprising, among other things, a salt of orthophosphoric acid.

[0085] 1. Preparation of the Absorbent, Hydrophilic Culture Substrate

[0086] The absorbent, hydrophilic culture substrate used is in this case a piece of Airlaid obtained from the company SCA, France (reference 95NN81) and having a declared weight of 95 g.Math.m.sup.2, for a thickness of 2 mm.

[0087] EVA particles, whose surface had been coated with a pulverulent composition comprising one or more chromogenic substrate(s) (for example, 5-bromo-4-chloro-3-indolyl-alpha-glucopyranoside and/or 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside), xanthan gum and an agent that stimulates the metabolism of bacteria (MnCl.sub.2), were immobilized within the thickness of this cellulose fiber material.

[0088] Xanthan gum is used in order to increase somewhat the absorptivity of Airlaid and to generate a gel that will facilitate implantation and retention of bacteria within the thickness of the fibrous material. MnCl.sub.2 makes it possible to stimulate bacterial metabolism. 5-Bromo-4-chloro-3-indolyl--glucopyranoside and 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside, under the hydrolytic action of an -glucosidase, release chromophores of blue/green color that are visible to the naked eye. These chromogenic substrates thus make it possible to label the bacterial colonies expressing -glucosidase activity which might develop on this culture substrate, for example such as colonies of S. aureus.

[0089] More precisely, the tests were carried out with square pieces of Airlaid, with a side of 2.7 cm. These cellulose fiber pieces were dry-impregnated with a powder composition comprising (for about 100 g of composition): [0090] 80 g of EVA, with an average granulometry of the order of 60-80 m. [0091] 20 g of xanthan gum, with an average granulometry of the order of 20-30 m. [0092] 0.533 g of 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and 0.3 g of 5-bromo-4-chloro-3-indolyl--glucopyranoside, with an average granulometry of the order of 20-30 m, [0093] 0.027 mg of MnCl.sub.2, with an average granulometry of the order of 20-30 m.

[0094] To facilitate handling of this powder composition, in particular to obtain a good, homogeneous distribution of each constituent in the absorbent substrate, the latter was submitted beforehand to a hot mixing process in order to agglomerate the particles together.

[0095] With suitable heating and stirring, the surface of the EVA particles is made sticky and the particles of the other constituents of the powder composition will adhere to it. Heating is carried out at a temperature of the order of 50-60 C., which leads to softening of the EVA particles and makes their surface sticky. Mixing is carried out mechanically.

[0096] This powder composition thus assembled hot contains EVA particles, more or less well separated, whose surface is partially coated with particles of 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and 5-bromo-4-chloro-3-indolyl--glucopyranoside, particles of xanthan gum and particles of MnCl.sub.2. It is then transferred into the thickness of a sheet of Airlaid by a technique of dry impregnation as described for example in WO 2015/044605, WO 2010/001043 and WO 99/22920. This technique consists of dusting the sheet of Airlaid with the powder composition and causing the particles of said composition to vibrate under the action of an alternating electric field; the particles penetrate and then gradually sink into the cavities of the fibrous body.

[0097] Once impregnated, the sheet of Airlaid is calendered between two sheets of parchment paper, the impregnated face being turned away from the heating plate. This calendering is carried out at a pressure of 4 bar and at 80 C., for 1-2 minutes.

[0098] The sheet of Airlaid is cut into pieces with sides of 2.7 cm. Each of these squares of Airlaid contains about 0.06 g of powder, containing about 80% of EVA.

[0099] 2. Bacterial Strains Used, and Preparation of the Nutrient Solution

[0100] Strains of Staphylococcus aureus were used, in this case a methicillin-sensitive strain (MSSA) obtained from the applicant's collection. In the context of this test, the nutrients necessary for the growth and development of these bacteria are supplied by human serum (obtained from EFS) diluted with PBS. This nutrient solution aims to reproduce the nutritional qualities of the wound exudates.

[0101] Various synthetic or reconstituted exudate compositions are described in the scientific literature and may be used for reproducing the test carried out by the inventors. A culture broth with poor nutritional qualities or else peptonized water may also be used for this same purpose.

[0102] For evaluating the effect of the concentration of KH.sub.2PO.sub.4 on the growth and development of bacteria subjected to the presence of EVA (in this case contained within the thickness of the piece of Airlaid), serum diluted with PBS is supplemented with KH.sub.2PO.sub.4: 0 g.Math.L.sup.1, 3 g.Math.L.sup.1 4 g.Math.L.sup.1 5 g.Math.L.sup.1 and 6 g.Math.L.sup.1 (as final concentrations).

[0103] 3. Execution of the Test, and Results Obtained

[0104] 100 L of a cellular preparation concentrated to 10.sup.6 CFU.mL.sup.1 (in tryptone salt) is added to 900 L of serum diluted with PBS, with or without KH.sub.2PO.sub.4. The pieces of Airlaid are soaked in this solution and then incubated in a jar for 17 hours at 32 C., with a little water in the jar to prevent drying-out.

[0105] After incubation, the pieces of culture substrate were examined and the following findings were made: [0106] The pieces of Airlaid seeded with bacteria and a serum diluted with PBS, lacking KH.sub.2PO.sub.4, are colorless. No colony grew there. [0107] A green-colored hue is observed on the pieces seeded with bacteria and a serum diluted with PBS supplemented with KH.sub.2PO.sub.4. A particularly pronounced coloration is found for concentrations of KH.sub.2PO.sub.4 of 3 g.Math.L.sup.1 and 5 g.Math.L.sup.1. This coloration is even more intense at 4 g.Math.L.sup.1 of KH.sub.2PO.sub.4. At 6 g.Math.L.sup.1 of KH.sub.2PO.sub.4, it is barely observable.

[0108] C. Demonstration in Liquid Culture Medium

[0109] Evaluation of the effects of EVA and of the salts of orthophosphoric acid on bacterial growth and development was also carried out in liquid medium. The aim is more particularly to evaluate the effect of EVA and KH.sub.2PO.sub.4 on the kinetics of bacterial growth.

[0110] For this purpose, 1 mL of a suspension of Staphylococcus aureus (300 CFU.mL.sup.1) obtained from the applicant's collection was cultured at 37 C. in 9 mL of different nutrient solutions: [0111] serum diluted with PBS, [0112] serum diluted with PBS, and comprising 18.5 g.Math.L.sup.1 of EVA, [0113] serum diluted with PBS, and comprising 18.5 g.Math.L.sup.1 of EVA and 4 g.Math.L.sup.1 of KH.sub.2PO.sub.4.

[0114] After each hour elapsed, a cell count is performed for a culture tube of each series. For this purpose, the cells of each tube are transferred onto an agar dish chromID S. aureus (bioMrieux, France). The dishes are incubated for 24 hours at 37 C., before counting the colonies formed.

[0115] The three growth kinetics thus measured are presented in the form of curves in FIG. 1.

[0116] These results confirm those obtained previously, on a solid culture medium. EVA has a bacteriostatic effect. This bacteriostatic effect may be inhibited by salts of orthophosphoric acid, such as KH.sub.2PO.sub.4. Using KH.sub.2PO.sub.4 in an amount by weight corresponding to the order of of the amount of EVA can effectively counteract the bacteriostatic effect of EVA.

Example 2: Preparation of Biosensors According to the Invention and Evaluation of their Capacity for Detecting and Identifying Bacteria

[0117] A. Preparation and Assembly

[0118] The following example illustrates the production of a biosensor according to the invention, according to a particular embodiment.

[0119] In this particular embodiment, said biosensor is prepared from a piece made of hydrophilic absorbent fibrous material. Within the thickness of this material and fixed to its fibers, EVA particles retain and expose on their surface a pulverulent mixture combining: [0120] a salt of orthophosphoric acid (KH.sub.2PO.sub.4), [0121] two chromogenic substrates of -glucosidase (5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and 5-bromo-4-chloro-3-indolyl--glucopyranoside) [0122] a gelling agent (xanthan gum), and [0123] an activator of bacterial metabolism (MnCl.sub.2).

[0124] The EVA particles coated with said pulverulent mixture are prepared beforehand by hot mixing of a pulverulent composition of dissociated particles, comprising (for about 100 g of composition): [0125] 80 g of EVA, [0126] 20 g of xanthan gum, [0127] 6.67 g of KH.sub.2PO.sub.4, [0128] 0.533 g of 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and 0.3 g of 5-bromo-4-chloro-3-indolyl--D-glucopyranoside, and [0129] 0.027 g of MnCl.sub.2.

[0130] This pulverulent mixture is submitted to a hot mixing process in order to obtain a composition of agglomerated powders, used in the same conditions as for the hot mixing described above.

[0131] FIG. 2 shows two photographs taken by scanning electron microscopy of the pulverulent composition of dissociated particles, before hot mixing.

[0132] FIG. 3 shows two photographs taken by scanning electron microscopy of a composition of agglomerated powders according to the invention, clearly showing the EVA particles, on the surface of which finer particles are distributed, in this case particles of KH.sub.2PO.sub.4, 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside, xanthan gum and MnCl.sub.2.

[0133] The composition of agglomerated powders thus prepared is then transferred into the thickness of a sheet of Airlaid by a technique of dry impregnation, as detailed above.

[0134] Once impregnated, the sheet of Airlaid is calendered between two sheets of parchment paper, the impregnated face being turned away from the heating plate. This calendering is carried out at a pressure of 4 bar and at 80 C., for about 1 minute and 30 seconds.

[0135] Biosensors according to the invention are cut from this sheet of Airlaid dry-impregnated with the composition of agglomerated powders, and then calendered. They allow detection of bacteria expressing -glucosidase activity, as is the case notably for the Staphylococcus aureus bacteria.

[0136] Other biosensors very similar in design to the first example were also assembled. In these biosensors according to the invention, the EVA particles also expose cefoxitin on their surface, which is an antibiotic to which the methicillin-sensitive strains of Staphylococcus aureus (MSSA) are particularly sensitive, in contrast to the methicillin-resistant strains (MRSA). This second example of biosensors according to the invention allows detection and identification of strains of MRSA.

[0137] The cefoxitin is fixed on the surface of the EVA particles concomitantly with KH.sub.2PO.sub.4, 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside and/or 5-bromo-4-chloro-3-indolyl--glucopyranoside, xanthan gum and MnCl.sub.2, by a hot mixing process. For this purpose, the aforementioned pulverulent composition of dissociated particles also comprises 6.6 mg of cefoxitin.

[0138] B. Evaluation of the Above Biosensors for Detection and Identification of Staphylococcus aureus

[0139] Biosensors as described above and incorporating a composition of agglomerated powders according to the invention, comprising EVA particles on the surface of which the following are distributed: [0140] KH.sub.2PO.sub.4, [0141] 5-bromo-4-chloro-3-indolyl-N-methyl--glucopyranoside, [0142] 5-bromo-4-chloro-3-indolyl--glucopyranoside, [0143] xanthan gum and [0144] MnCl.sub.2,
were tested for their ability to detect strains of Staphylococcus aureus, in this case methicillin-sensitive strains (MSSA) obtained from the applicant's collection.

[0145] For this purpose, 1 mL of serum diluted with PBS, comprising 10.sup.5 CFU of a strain of MSSA (or 1 mL of serum diluted with PBS free from bacteria, for the control biosensors) was deposited on squares of biosensors, with side of 2.7 cm.

[0146] The pieces of Airlaid are then incubated in a jar for 17 hours at 32 C., with a little water in the jar to prevent drying-out.

[0147] After incubation, the biosensors were examined and the following findings were made: [0148] The control biosensors seeded only with serum diluted with PBS remain colorless. [0149] A green-colored hue is observed on the biosensors without cefoxitin, seeded with MSSA bacteria and the serum diluted with PBS. [0150] The biosensors containing cefoxitin and seeded with MSSA bacteria and the serum diluted with PBS remain colorless.