EP4623090A1 - Minimal inhibitory concentration (mic) test strip - Google Patents

Abstract

The subject of the invention is a test strip for detecting the minimum inhibitory concentration (MIC), intended to be placed on a culture medium in a microbial culture vessel, and said test strip (1 ) is provided in the form of a layered composite having at least a support layer (2) and combined with the support layer (2) is the base layer (3), where both layers bonded together have the form of rectangular plates corresponding in shape and size, and a microbial growth inhibitor is applied to the surface of the support layer (2), the concentration gradient of which is planned along the longer side support layer (2), and the base layer (3) is equipped with a printed scale (4) indicating the concentration of the said growth inhibitor placed on the support layer, and according to the invention the support layer is made of a fibrous composite.

Description

MINIMAL INHIBITORY CONCENTRATION (MIC) TEST STRIP

The subject of the invention is a strip for detecting the minimum inhibitory concentration (MIC).

Test strips for detecting the minimum inhibitory concentration (MIC) of the growth of microorganisms are well known in the art. They work by transferring antibiotics or other substances placed on the test strip to the surface of the culture medium inoculated with a specific microorganism and determining as based on the inhibition of the growth of the microorganism, the lowest concentration of the substance causing growth inhibition. For this purpose, an inhibitory substance in various concentrations is placed on the test strip, which allows the determination of the minimum concentration at which the growth of microorganisms in the culture medium ceases.

Solutions based on the above-described concept were presented in the specification of the invention EP0157071 , which disclosed a carrier containing a biologically active substance in various concentrations, which, when transferred to the culture medium, inhibited the growth of bacteria, allowing the determination of the Minimum Inhibitory Concentration (M.I.C.). The way the commercially available OXOID strip, M.LC Evaluator work is based on a similar principle.

As indicated in the patent specification of the invention EP2480682, the problem in the above-described solutions is that in the known strips the carriers are made of plastic, which creates difficulties when removing them from the package containing the same, since they are very thin and sensitive to the electrostatic attraction forces typical of for thin plastic layers. Moreover, it was indicated that to solve this problem it is necessary to use specific and expensive equipment. At the same time, EP2480682 indicates an additional problem of solutions in which the carrier is made of plastic. It was disclosed that the plastic from which the carrier is made is impermeable to air, so incorrect positioning on the culture medium may cause the formation of air bubbles, which negatively affect the correctness of the test. Therefore, according to the analysis of the state of the art, the main problem in the production of properly functioning strips for determining the minimum inhibitory concentration is the selection of the material constituting the support layer of the strip.

The support layer made of plastic in accordance with the teaching of in EP2480682 is not a material ensuring optimal performance, therefore the patent proposes replacing the plastic with a paper strip. Patent EP 3399050 suggests replacing the plastic support layer with a tape made of cotton fabric, linen fabric, silk fabric or fabrics made of a mixture of cotton and polymers. The description of said invention indicates that the material used in the strip is easy to supply, has a low cost, is manufactured from materials resistant to wear and tear, and allows for easier and faster diffusion of the antibiotic material.

Although the state of the art pointed out the disadvantages of plastic strips while indicating the advantage of test strips in which the support layer containing the inhibitory substance is made of paper or natural fabrics such as cotton, linen or silk, possibly with the addition of polymer, such strips do not have the same durability and strength as strips made of plastic. There is therefore a further need for minimum inhibitory concentration detection strips that increase ease of use, have improved durability characteristics, and overcome the prior art drawbacks associated with the use of plastics in the test strip materials.

Surprisingly, the inventors of the present invention have found that the optimal parameters of the support layer in a minimum inhibitory concentration test strip can be obtained by using a fiber composite in the support layer.

The subject of the invention is a test strip for detecting the minimum inhibitory concentration (MIC) for the growth of microorganisms, intended to be placed on a culture medium in a microbial culture vessel, said test strip being provided in the form of a layered composite having at least a support layer and a support layer bonded with said base layer, wherein both layers bonded together are provided in the form of corresponding in shape and size rectangular plates, on the surface of the support layer there is applied a microbial growth inhibitor of which concentration gradient is provided along the longer side of the support layer, the base layer is equipped with a print of a scale indicating the concentration of the antibiotic placed on the support layer, wherein the strip according to the invention is characterized in that the support layer is made of a fiber composite.

In a preferred embodiment, the base layer is made of a plastic foil, preferably at least one of the sides of the base layer is corona oriented.

In a preferred embodiment, the plastic foil from which the base layer is made is a polyester foil or a polypropylene foil, preferably a two-sided oriented polyester foil or a two-sided oriented polypropylene foil.

In a preferred embodiment of the invention, the support layer in the strip according to the invention is bonded to the base layer by cold lamination.

Preferably, the fibrous material of the fiber composite consitute polyester, polyamide, glass, carbon, cellulose, nitrocellulose, polyolefin fibers or a mixture thereof. In a preferred embodiment of the invention, the fiber composite matrix is selected from organic or non-organic polyesters, preferably based on polyvinyl alcohol, resins, preferably polyester resin, ethylene-vinyl acetate resin, polyurethane resin, most preferably polyacrylates (acrylic copolymers), adhesive, preferably a polymer adhesive based on polyvinyl acetate, and preferably additionally includes adhesive additives, preferably in the form of acrylonitrile-styrene-acrylate terpolymer (ASA), alkyl ketene dimer (AKD), fixing additives, preferably in the form of the 5-Chloro-2-methyl-2H-isothiazol-3-one and 2-methyl-2H-isothiazol-3-one (CIT/MIT) system, bulking agents, preferably in the form of metal oxides, most preferably titanium oxide, cellulose retention cross-linking agents, preferably polycarboxylic acids, also preferably N-methyl derivatives of ethylene urea, abrasive or non-abrasive additives in the form of organic or non-organic chemical compounds or minerals.

Preferably, the surface of the support layer adjacent to the base layer is corona oriented.

In a preferred embodiment of the invention, the mentioned scale is provided in the form of numbers and lines, and the print is made using UV printing technology.

According to the invention, the test strip is a layered composite having at least two layers placed opposite to each other and bonded together.

The first layer of the above-mentioned layered composite is the support layer, which is spot-soaked with various concentrations of a microbial growth inhibitor, i.e. a substance that inhibits the growth of specific microorganisms. Various types of antibiotics are an example of a microbial growth inhibitor (a substance that inhibits the growth of microorganisms). The support layer is the side of the strip which when using the strip to determine the minimum concentration inhibiting the growth of microorganisms, is applied to the medium containing the culture of microorganisms whose growth is to be inhibited by the said substance.

According to the invention, the support layer is provided in the form of a fiber composite. Within the meaning of the invention, a fiber composite is at least a two-phase system, in which one phase - the dispersed phase, also called reinforcement, is the fibrous material, while the second phase is the matrix, also called the medium, wherein the fibrous material is embedded in the matrix. The fibrous material of the fiber composite may be polyester, polyamide, glass, carbon, cellulose, nitrocellulose, polyolefin fibers or mixtures thereof. The matrix in a fiber composite within the meaning of the invention is a continuous component of the composite which can be provided in the form of an adhesive (such as a polymer adhesive based on polyvinyl acetate) and/or a resin, preferably a polyester resin, preferably an ethylenevinyl acetate resin, also preferably a polyurethane resin, most preferably polyacrylates (acrylic copolymers). The matrix may also be other synthetic materials known in the state of the art, such as polyesters, epoxies, polyamides or polypropylenes (e.g. PO, CoPA, PUR, CR, SC, PU, S, SE PSA, PVA), alone or with natural additives, filling, retention or adhesive agents known in the art. The adhesive additives are preferably selected from acrylonitrile-styrene-acrylate terpolymer (ASA) and alkyl ketene dimer (AKD), and the CIT/MIT system (5-Chloro-2-methyl-2H-isothiazol-3-one and 2-methyl- 2hisothiazol-3-one) is preferably used as the conserving additives, the fillers are preferably provided in the form of metal oxides, most preferably titanium oxide. Polycarboxylic acids and preferably also N-methyl derivatives of ethylene urea are used as retention cross-linking agents for cellulose, and abrasive or non-abrasive additives are organic or non-organic chemical compounds or minerals known in the art.

Particularly advantageous is the variant of the support phase made of a fiber composite, in which the matrix is a synthetic resin (such as ethylene-vinyl acetate resin, polyurethane resin, most preferably polyester resin) or polyacrylates (acrylate copolymers), and the dispersed phase constitute polyester fibers. In the state of the art, this type of composite is called a polyester composite or a polyester membrane. Particularly advantageous in the strip according to the invention is the use of its two- sided version, which is characterized by the fact that one side is corona oriented, while the other side reveals polyester fibers. Corona orientation within the meaning of the invention means a physical or chemical treatment that changes the surface structure making it more suitable for further processing. This allows to obtain a composite that releases the active substances with which it has been previously soaked much better into the solid substrate, and at the same time reduces the risk of interaction of this substance with inks and printing ink solvents contained in the printed base layer. The corona-treated support layer is therefore protected against the adverse effects of the external environment. A test strip having a two-sided fiber composite with one-side corona-oriented allows for the determination of antibiotic sensitivity in a more uniform way and reduces release disturbances related to the structure of the material.

Fiber composites based on the dispersed phase in the form of glass fibers have similar features to the above-mentioned polyester composites, except that the drying time of test strips based on such a composite is significantly reduced, which allows the product to be obtained faster and the drying process to be carried out at a lower temperature and shorter time, thanks to which the active substances are less exposed to thermal degradation, thus extending the durability of the product. Glass fiber-based composites use matrix in the form of synthetic resins (such as those mentioned above). Examples of suitable fiber composites based on glass fibers are commercially available glass fiber membranes (e.g. ReliaFlow™ 8980 - Glass conjugate pad from AHLSTROM-MUNKSJO).

Fiber composites based on polyolefin fibers (preferably with additives such as silicates) are characterized by a highly smooth and durable surface, and thanks to their unique properties, they are ideal for working with more aggressive solvents, such as organic solvents, making it easier to prepare antibiotic solutions for their application, which means that they do not degrade, and thus the life of the test strip can be extended.

Fiber composites based on cellulose fibers, where the fibrous material constitue high-purity cellulose fibers and the matrix constitute acrylic copolymers, are characterized by a high composite capacity, which means that such a composite can absorb a larger amount of solvent than an analogous non-composite material, such as cellulose paper. While the drying time of such a test strip is longer than when using composites with glass or polyester fibers, it is shorter than when using cellulose paper alone. Thanks to the increased capacity, it is possible to prepare solutions with a lower concentration of the active substance, thanks to which the substance does not degrade and the durability of the test strip is extended. High capacity and reduced migration inside the composite allows appropriate concentrations of antibiotic to be retained in designated areas of the strip.

The most advantageous fibrous composites, in which the fibrous material was a mixture of polyamide and cellulose fibers, with a predominance of polyamide fibers, and the matrix was a mixture of polyvinyl alcohol and acrylic copolymer with the addition of the above-mentioned fillers, retention and adhesive agents, allowed to obtain the best results in terms of drying time, capacity, speed and quality of released substances. Thanks to the improved structure of the fibrous material, a smoother composite surface and improved uniformity were obtained, which significantly influenced the repeatability of the results obtained in relation to the fiber composite based on cellulose fibers and in comparison to ordinary cellulose paper. The composite also achieved strength similar to that of plastics, without any problems associated with their use.

The second layer of the layered composite in the test strip according to the invention, opposite the first layer, is a base layer made of a plastic foil impermeable to water and enabling scale printing, preferably a polyester foil, preferably the polyester foil oriented on both sides, which on one hand has a positive effect on the possibility of printing a scale while on the other hand it facilitates bonding with the fiber composite. Two-sided orientation of the foil is ensured by corona orientation, i.e. such a change in the surface structure of non-absorbent, smooth materials, such as plastic foils, which aims at an increase in their suitability for printing and gluing. In the solution according to the invention, one, or preferably both, sides of the foil can be corona treated, which on one hand makes it easier for the foil to stick to the composite while on the other hand makes it easier to print a scale thereon.

The base layer is printed with a scale reflecting a concentration gradient of a microbial growth inhibitor (i.e. a substance that inhibits the growth of microorganisms) which is spot-applied onto the support layer. The printing is performed in a manner known in the art, particularly preferably by printing using UV technology or, less preferably, printing using laser printing technology. The base layer simultaneously reinforces and protects the structure of the test strip. It is impermeable or barely permeable to water.

The support and base layers are bonded in a manner known in the art, e.g. by a cold lamination process known in the art. It is also possible to glue the layers using a binder such as an acrylic copolymer. The layered composite strip of the invention then includes an additional adhesive layer.

Combining the support layer of a fiber composite and the base layer of a water- impermeable plastic foil, preferably a two-sided oriented polyester foil, ensures the creation of a layered composite that is, on one hand, durable, ensuring the appropriate stiffness of the strip and its appropriate strength parameters (base layer), and on the other hand it is soft, water-permeable and with the desired absorbent properties (support layer). This combination ensures optimal physical parameters of the strip, which becomes more resistant to breaking, and at the same time provides the unexpected benefit of reducing the drying time of the strip in the production process, which will be further explained below.

Test strips in which the support side of the layered composite is a fiber composite based on fibers selected from the group of polyester, polyamide, glass, carbon, cellulose, nitrocellulose or polyolefin fibers or their mixtures in the abovedescribed continuous phase, while the stiffening side is a two-sided oriented polyester foil, provide a one-sided barrier composite, limiting the penetration of moisture into the support layer and protecting antibiotics against non-uniform diffusion into the substrate and undesirable migration inside the support layer.

A preferred embodiment of the strip according to the invention is the variant in which the support layer is the fiber composite described above, in which the dispersed phase is made of a mixture of polyamide and cellulose fibers, and acrylic copolymers, polyvinyl alcohol, filler, retention and adhesive agents are used as the matrix. The base layer in this particularly advantageous variant is a transparent polyester foil oriented on both sides. A particularly advantageous embodiment of the strip according to the invention is the variant in which the support layer is a fiber composite based on high- purity cellulose fibers (e.g. in the form of cotton linters), and an acrylic copolymer is used as the matrix. In another preferred variant of the strip according to the invention, the support layer is a fiber composite based on polyester fibers, and polyester resin is used as the matrix. Both layers, the base layer made of transparent, two-sided oriented polyester foil and the support layer made of the above-mentioned preferred composites, are connected with an acrylic copolymer binder using lamination techniques, and the scale is applied to the base layer using UV printing. The strip constructed in this way provides particularly desirable properties, in particular higher physical strength and moisture resistance thanks to the barrier water-impermeable foil, very good adhesion to the surface thanks to the air-permeable support layer, shortened drying time compared to paper or plastic, higher absorbency of antibiotic solutions, very good and precise release of absorbed antibiotics into the substrate thanks to the fibers used.

Further features and advantages of the invention will become more apparent from the following description of the preferred embodiments, which are presented for illustrative purposes only and do not in any way limit the scope of protection as defined by the claims.

The invention is illustrated in embodiments based on the drawing in which:

Fig. 1 shows schematically an embodiment of the invention with separated layers of a layered composite forming a test strip.

Fig. 2 is a schematic perspective view of the integrated layers of a layered composite forming a test strip according to an embodiment of the invention.

Fig. 3 schematically shows a test strip in an embodiment with separated layers of a layered composite with symbolically marked fields of saturation of the support layer with a substance that inhibits the growth of microorganisms.

Fig. 4 and Fig. 5 schematically show a method for determining the minimum inhibitory concentration using a test strip according to the invention.

Fig. 1 schematically shows a test strip 1 in an embodiment according to the invention, having a support layer 2 and a base layer 3. In Fig. 1 , the support 2 and base 3 layers are shown as rectangular plates separated from each other, corresponding in shape and size. The support 2 and the base 3 layers are bonded together in any manner known in the art, e.g. by cold lamination as indicated above. The support 2 and the base 3 layers bonded together form the layered composite of the test strip according to the invention (as schematically shown in Fig. 2).

As shown in the drawing, the base layer 3 is equipped with a scale 4 indicating the concentration gradient of the substance that inhibits the growth of microorganisms (microbial growth inhibitor) applied to the support layer 2. Fig. 3 shows schematically in the form of circles the points where the appropriately changing concentration of a substance inhibiting the growth of microorganisms is applied, so that this concentration corresponds to the scale 4 provided for on the base layer, which scale corresponds to the arrangement of points on the support layer 2 (one of such points are marked in the figure with reference number 5).

Fig. 4 shows a test strip 1 placed on a symbolically presented Petri dish 6. The Petri dish 6 is filled with a culture medium on which microorganisms are cultured, the growth of which is to be inhibited by an inhibitory substance placed in the support layer 2 (not shown in the drawing). Fig. 5 symbolically shows the inhibitory effect of the inhibitory substance placed in the support layer 2. Zone 6 of the Petri dish is marked in gray, wherein after an appropriate incubation time, the growth of microorganisms is not limited by the effect of the substance inhibiting the growth. The area on the Petri dish where the inhibitory substance caused growth inhibition is marked in white. The scale 4 of the concentration gradient of the inhibitory substance provided in the support layer 2 placed on the base layer 3 of the test strip 1 allows the determination of the minimum inhibitory concentration (MIC) for the growth of microorganisms cultured on the Petri dish 6.

In the examplary embodiment, the test strip 1 was made as a layered composite with a fiber composite as the support layer 2, in which the dispersed phase consisted of polyester fibers bonded with a synthetic resin matrix, and the synthetic resins used in the stripes were those mentioned earlier in the description, i.e. polyester resin, ethylene-vinyl acetate, polyurethane resin. The base layer was a two-sided oriented polyester foil. The test strip produced in this way was characterized by high strength and favorable physical properties during processing, especially when cutting the material, allowing for obtaining even edges without frayed fibers. A strip made of such a layered composite dries quickly after being saturated with an inhibitory substance, is characterized by low migration of substances inside the composite and quick and precise release of active substances into the substrate. In another embodiment, the test strip 1 was made as a layered composite with a fiber composite as the support layer 2, in which the dispersed phase material consisted of glass fibers which were bonded with a matrix in the form of a polyacrylate - acrylic copolymer. The base layer was a two-sided oriented polyester foil.

In yet another embodiment, the test strip 1 was made as a layered composite with a fiber composite as the support layer 2, in which the dispersed phase consisted of polyolefin fibers which were bonded with a matrix in the form of a polyacrylate - acrylic copolymer. The base layer was a two-sided oriented polyester foil.

In yet another embodiment, the test strip 1 was made as a layered composite with a fiber composite as the support layer 2, in which the dispersed phase consisted of cellulose fibers which were bonded with an acrylic copolymer matrix, and the base layer was a two-sided oriented polyethylene film.

In yet another embodiment, the test strip was made as a layered composite having a fiber composite as the support layer 2, in which the dispersed phase was a mixture of polyamide and cellulose fibers, with a predominance of polyamide fibers, and the matrix used was a mixture of polyvinyl alcohol and acrylic copolymer with the addition of the described above fillers, retention and adhesive agents. In particular, acrylonitrile-styrene-acrylate terpolymer (ASA) and alkyl ketene dimer (AKD) were used as adhesive additives. CIT/MIT (5-Chloro-2-methyl-2H-isothiazol-3-one and 2- methyl-2H-isothiazol-3-one) were preferably used as conserving additives, and titanium oxide was provided as a filler. Polycarboxylic acids were used as a retention cellulose cross-linking agent.

The strips were tested using a variety of different microbial growth inhibitors. For example, cefuroxime in a solvent in the form of a phosphate buffer with pH 6 at at concentration of 0.1 mol/L was applied to the tested strips in the concentration range from 0.015 to 256 pg/mL, and ampicillin in a solvent in the form of a phosphate buffer with pH 8 at a concentration of 0.1 mol/L was applied in the concentration range from 0.015 to 256pg/mL. Strips with chloramphenicol applied in a solvent in the form of 95% ethyl alcohol were also tested in the concentration range from 0.015 to 256pg/mL.

The best results in terms of drying time, capacity, rate and quality of the substances released were obtained in the disclosed embodiments. Thanks to the improved structure of the cross-linking material, a smoother composite surface and improved uniformity were obtained, which significantly influenced the repeatability of the results obtained in relation to the fiber composite based on cellulose fibers and in comparison to ordinary cellulose paper. The composite also achieved strength similar to that of plastics, without any problems associated with their use.

As a result of the tests carried out, it was found that the test strip according to the invention in the form of the presented layered composite has a larger capacity, which means that it can absorb a larger amount of solvent, and its evaporation time (drying time) is shorter than when using ordinary cellulose paper.

The tests also showed that the solvent was removed much faster from the composite with a support layer of cellulose fibers and a base layer of two-oriented foil than from cellulose paper, but slower than from a layered composite with a support layer based on glass or polyester fiber.

The active substances applied to the strip are protected by a base layer that provides stiffening and protection against moisture, which limits the migration of substances that inhibit the growth of microorganisms inside the composite and ensures that such active substances are released in a precise manner. Thanks to the printing technique and foil layer used, it is impossible for inks to penetrate the support layer, which results in more repeatable results. The tests also showed that the possibility of air bubbles appearing during the application of the strip was minimized.

The tests carried out allow us to conclude that the test strip according to the invention, made of the disclosed layered composites, has increased physical resistance to tearing and mechanical processing, including cutting, allowing to obtain a smooth, frayed cutting edge, unlike ]paper or fabrics. This feature affects the uniformity of the release of the active substance from the strip, and thus affects the quality of the results obtained and the quality of the product. The appropriate selection of the type of fiber composite fibers allows for optimal results and affects the ease of preparation, durability of the product and the material itself, as well as the repeatability of the results obtained.

Claims

Claims

1. A test strip (1 ) for detecting the minimum inhibitory concentration (MIC), intended to be placed on a culture medium in a microbial culture vessel, said test strip (1 ) being made in the form of a layered composite having at least a support layer (2) and a base layer (3) bonded with the support layer (2), wherein both layers bonded together have the form of corresponding in shape and size rectangular plates, and wherein a microbial growth inhibitor is applied to the surface of the support layer (2) the concentration gradient of which is provided along the longer side of support layer (2), the base layer (3) is provided with a printed scale (4) indicating the concentration of the said growth inhibitor placed on the support layer, characterized in that the support layer is made of a fiber composite.

2. The test strip according to claim 1 , characterized in that the base layer (3) is made of plastic foil.

3. The test strip according to claim 2, characterized in that the base layer (3) is a polyester foil or a polypropylene foil, preferably a polyester foil.

4. The test strip according to any of the preceding claims, characterized in that at least one of the surfaces of the foil constituting the base layer is corona-oriented.

5. The test strip according to one of the preceding claims, characterized in that the base layer is provided in the form of a two-sided oriented foil.

6. The test strip according to any of the preceding claims, characterized in that the fibrous material of the fiber composite constitute fibers selected from the group of polyester, polyamide, glass, carbon, cellulose, nitrocellulose, polyolefin fibers or a mixture thereof, and preferably the fiber material of the fiber composite are cellulose or polyamide fibers, most preferably are cellulose fibers.

7. The test strip according to any of the preceding claims, characterized in that the fiber composite matrix is selected from organic or non-organic polyesters, preferably based on polyvinyl alcohol, resins, preferably polyester resin, ethylene-vinyl acetate resin, polyurethane resin, most preferably polyacrylates (acrylic copolymers), adhesive, preferably polymer adhesive based on polyvinyl acetate, and preferably additionally includes adhesive additives, preferably in the form of acrylonitrile-styrene-acrylate terpolymer, alkyl ketene dimer, fixing additives, preferably the 5-Chloro-2-methyl-2H-isothiazol-3-one and 2-methyl- 2H-isothiazol-3-one system, bulking agents, preferably in the form of metal oxides, most preferably titanium oxide, retention cross-linking agents, preferably cellulose and/or polycarboxylic acids, also preferably N-methyl derivatives of ethylene urea, abrasive or non-abrasive additives in the form of organic or inorganic chemical compounds or minerals. The test strip according to any of the preceding claims, characterized in that the dispersed phase of the fibrous composite constitutes polyester fibers and the continuous phase is ethylene-vinyl acetate resin. The test strip according to any of the preceding claims, characterized in that the dispersed phase of the fibrous composite consists of cellulose fibers and the continuous phase is an acrylic copolymer resin. The test strip according to any of the preceding claims, characterized in that the dispersed phase of the fibrous composite is a mixture of polyamide and cellulose fibers, and the continuous phase is a resin based on an acrylic copolymer and polyvinyl alcohol. The test strip according to any of the preceding claims, characterized in that the surface of the support layer (2) adjacent to the base layer (3) is corona oriented.The test strip according to any of the preceding claims, characterized in that the support layer (2) is bonded to the base layer (3) in a cold lamination process.The test strip according to any of the preceding claims, characterized in that said scale (4) is provided in the form of numbers and lines and the print is made using UV printing technology.