Methods for assessing biofilm formation

Abstract

The purpose of the prevent invention is to provide: a method for evaluating about the in vivo formation of a biofilm; and an invertebrate animal. Provided is a method for evaluating about the in vivo formation of a biofilm onto a medical tool material (30) using an invertebrate animal, the method comprising the steps of: inserting the medical tool material (30) into a position between a subcutaneous part and the intestine (18) of an invertebrate animal; removing the medical tool material (30) from the invertebrate animal; and evaluating about the formation of a biofilm on the surface of the medical tool material (30). Also provided is an invertebrate animal which can be used for this evaluation about the formation of a biofilm.

Description

[Technical field]

[0001] The present invention relates to a method for evaluating biofilm formation and an invertebrate used for evaluating biofilm formation, and in particular to a method for evaluating biofilm formation in a living body using an invertebrate, and an invertebrate used for evaluating biofilm formation.

[0002] This invention is a patent application that is subject to the application of Article 17 of the Industrial Technology Enhancement Act, and is commissioned research and development by the Japan Agency for Medical Research and Development (AMED) in fiscal year 2020 as part of the "Infectious Disease Practical Application Research Project: Research Project to Promote the Development of Innovative Pharmaceuticals for Emerging and Re-emerging Infectious Diseases" and "Research into the Elucidation of the Pathogenesis of Invasive Yeast Infections and into Epidemiology, Diagnosis, and Control Methods." [Background technology]

[0003] For example, the fungus Candida albicans (hereafter abbreviated as C. albicans) is a pathogenic fungus that causes blood infections and causes severe systemic mycoses in immunocompromised patients. When C. albicans forms a biofilm on the surface of a patient's catheter or other medical device, antibiotics (antifungal drugs) become less effective, making treatment difficult. Therefore, it is believed that the establishment of a method to inhibit fungal biofilm formation will contribute to the prevention and treatment of blood infections and other diseases caused by fungi.

[0004] The host environment has a significant influence on C. albicans biofilm formation, and not only nutrients such as glucose and amino acids, but also host cells and proteins are important components of C. albicans biofilms. Comprehensive gene expression analysis has revealed that gene expression patterns of C. albicans during biofilm formation in vitro and in vivo are different. Therefore, experimental models to evaluate C. albicans biofilm formation in vivo are very important.

[0005] For example, the following Patent Document 1 describes a method for evaluating lysins used in preventing, destroying, and treating biofilm formation on catheters using mice. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2018-87242 A Summary of the Invention [Problem to be solved by the invention]

[0007] Tests using mammals such as mice and rats involve ethical issues, as well as the burden of maintaining the animals and securing the space for maintaining them, and it is difficult to conduct experiments using a large number of individual animals, as it is also difficult to secure the drugs to be administered, etc. Furthermore, tests using mice and rats require surgery accompanied by anesthesia, making the procedures complicated.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a method for evaluating biofilm formation on medical device materials in vivo using invertebrates, which have fewer ethical issues than mice and are advantageous in conducting experiments using a large number of individuals, and an invertebrate animal to be used for evaluating biofilm formation. [Means for solving the problem]

[0009] In order to achieve the object of the present invention, the method for evaluating biofilm formation of the present invention is a method for evaluating biofilm formation on a medical device material using an invertebrate, comprising the steps of inserting the medical device material between the subcutaneous tissue and the intestinal tract of the invertebrate, removing the medical device material from the invertebrate, and evaluating biofilm formation on the surface of the medical device material.

[0010] Preferably, one form of the invention includes the step of infecting the invertebrate into which the medical device material has been inserted with a fungus prior to the step of removing the medical device material.

[0011] Preferably, one form of the invention comprises administering an antimicrobial to the invertebrate and the evaluating step comprises evaluating the antimicrobial.

[0012] In one embodiment of the present invention, after the medical device material is inserted into the invertebrate, it is preferable to retain the medical device material within the invertebrate for a predetermined period of time.

[0013] In one aspect of the present invention, the medical device material is preferably a catheter.

[0014] In one embodiment of the present invention, the invertebrate is preferably a lepidopteran insect larva.

[0015] In one embodiment of the present invention, the invertebrate is preferably a silkworm.

[0016] In one embodiment of the present invention, the silkworms are preferably 5th instar silkworms.

[0017] In one embodiment of the present invention, the thickness of the medical device material is preferably 1 / 5 or less of the thickness of the silkworm body.

[0018] In one embodiment of the present invention, the inserting step preferably includes inserting the medical device material from the abdomen of the silkworm.

[0019] In one embodiment of the present invention, the inserting step preferably inserts the medical device material from the vicinity of the crescent crest of the abdomen of the silkworm.

[0020] In one embodiment of the present invention, the step of inserting preferably includes inserting the medical device material along the intestinal tract of the silkworm.

[0021] In one embodiment of the present invention, the step of evaluating biofilm formation is preferably carried out by a staining method.

[0022] In order to achieve the objectives of the present invention, the invertebrate of the present invention is an invertebrate used for evaluating biofilm formation, in which a medical device material is inserted between the subcutaneous tissue and the intestinal tract, and used for evaluating biofilm formation on the surface of the medical device material in vivo. Effect of the Invention

[0023] According to the present invention, it is possible to evaluate biofilm formation in vivo using invertebrates, which has the advantage of having few ethical issues and being able to conduct experiments using a large number of individuals. [Brief description of the drawings]

[0024] [Figure 1] 1 is a flow chart illustrating a method for assessing biofilm formation. [Diagram 2] This is a diagram of a silkworm. [Diagram 3] FIG. 13 is a diagram illustrating the step of inserting a medical device material. [Figure 4] 1 is a flow chart illustrating a method for assessing biofilm formation according to another embodiment. [Diagram 5] 1 is a flow chart illustrating a method for assessing biofilm formation according to yet another embodiment. [Figure 6] FIG. 1 is a diagram showing the results of Test Example 1. [Figure 7] FIG. 1 is a diagram showing the results of Test Example 1. [Figure 8] FIG. 1 shows the results of Test Example 2. [Figure 9] FIG. 13 is a graph showing the results of Test Example 3. [Figure 10] FIG. 13 is a graph showing the results of Test Example 3. [Figure 11] FIG. 13 is a diagram showing an experimental flow of Test Example 4. [Figure 12] FIG. 13 is a diagram showing the results of Test Example 4. [Figure 13] FIG. 13 is a diagram showing an experimental flow of Test Example 5. [Figure 14] FIG. 13 is a diagram showing the results of Test Example 5. [Figure 15] FIG. 13 is a diagram showing an experimental flow of Test Example 6. [Figure 16] FIG. 1 shows the results of Test Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Hereinafter, preferred embodiments of the method for evaluating biofilm formation according to the present invention and an invertebrate will be described with reference to the accompanying drawings.

[0026] <Method for evaluating biofilm formation> 1 is a flowchart showing a method for evaluating biofilm formation. The method for evaluating biofilm formation of the present embodiment is a method for evaluating biofilm formation on a medical device material using an invertebrate, and includes a step of inserting the medical device material between the subcutaneous tissue and the intestinal tract of the invertebrate (step S10), a step of removing the medical device material from the invertebrate (step S20), and a step of evaluating biofilm formation on the surface of the medical device material (step S30).

[0027] [Step of inserting medical equipment material] (Step S10) The step of inserting the medical device material is a process of inserting the medical device material between the subcutaneous layer and the intestinal tract of the invertebrate. In the following, a silkworm will be used as an example of the invertebrate.

[0028] Figure 2 is a diagram of a silkworm. Figure 3(a) is a diagram of inserting a medical device material into a silkworm, and Figure 3(b) is a diagram of the inside of the silkworm body with the medical device material inserted.

[0029] As shown in Figure 2, a silkworm 10 is composed of a head 12, a thorax 14, and an abdomen 16. The body is composed of 13 segments, with the next three segments following the head 12 at the front end being called the thorax 14, and the following 10 segments being called the abdomen 16. The bulk of the inside of the body is occupied by the intestine 18, which is the digestive tract, and is broadly divided into the foregut, midgut, and hindgut (not shown).

[0030] When inserting the medical device material 30 into the body of the silkworm 10, it is preferable to insert the medical device material 30 from the abdomen 16 of the silkworm, and more preferably, from the vicinity of the crescent crest 20 located at the second somite from the thorax 14 side of the abdomen 16. When inserting the medical device material 30 into the silkworm 10, it is preferable to make a hole with an injection needle or the like at the place where the medical device material 30 is to be inserted. By inserting the medical device material 30 from the vicinity of the crescent crest 20 along the subcutaneous tissue, the medical device material 30 can be inserted along the intestinal tract 18 between the subcutaneous tissue and the intestinal tract 18. By preventing the medical device material 30 from being inserted into the intestinal tract 18, biofilm formation on the medical device material 30 in vivo can be appropriately evaluated.

[0031] After inserting the medical device material 30 into the silkworm, the medical device material 30 is left inside the silkworm 10 for a predetermined time. This allows evaluation of biofilm formation in vivo. The time for which the medical device material 30 is left inside the silkworm 10 can be changed as appropriate depending on the experimental method and evaluation method, but it is preferable that the time is within a time period in which the silkworm does not die due to experimental treatments such as inoculation of fungi and administration of drugs. Specifically, it is preferable that the time is within 24 hours.

[0032] <Invertebrates> The invertebrate animal used in this embodiment is not limited to silkworms and can be appropriately selected depending on the purpose, but is preferably an insect larva for ease of handling. The larvae of lepidopteran insects such as butterflies and moths are more preferable because they have a caterpillar form and can be accurately administered with a drug by injection or the like.

[0033] Furthermore, as such larvae, silkworms are preferred for the following reasons. (1) It is easy to obtain. (2) The methods for raising them have already been established, and they are convenient to raise. (3) Previous research has revealed to some extent that the internal organs and tissues of the rat are similar to those of mammals such as humans. (4) The genetic lineage has been established, and genetic uniformity has been maintained. (5) They are relatively large, slow-moving, and virtually hairless, facilitating the administration of drugs, e.g., by quantitative injection. (6) Compared to mice and rats, they are less expensive, large numbers of them can be raised in a small space, and there are fewer ethical issues. (7) Evaluation can be performed even when only small amounts of test substance are available. (8) It is easy to collect individuals of the same age and in the same condition. (9) Since they do not escape and cannot survive in nature, there is no risk of biological disasters (biohazards).

[0034] Silkworms have a simple caterpillar-like form specialized for nutritional supplementation, and have clearly defined instars that are determined by several moltings. Therefore, by aligning the instars, it is possible to precisely align the growth conditions of individuals used in tests, and it is also possible to select the most appropriate instar depending on the purpose. In addition, silkworms have many excellent characteristics as test animals, such as being easy to raise because they move slowly, and being easy to inject drugs into.

[0035] There are no particular limitations on the size and age of the silkworms, but from the standpoint of stable growth conditions and ease of operation such as injection of drugs and insertion and removal of medical device materials, silkworms of 3rd instar or higher are preferred, and 5th instar silkworms are more preferred.

[0036] <Medical equipment materials> The medical device material used in this embodiment may be a material used for a medical device that is placed in a living body. Examples of the medical device include a catheter and an artificial joint. Polyurethane may be used as a material for the catheter. Other materials that may be used for the medical device include silicon, polyethylene, polypropylene, and polystyrene.

[0037] In addition, when performing an experiment assuming a catheter as a medical device, a catheter needs to be inserted into the blood vessels of a mouse or rat, etc., so a hollow tubular catheter of a size that can be inserted into the blood vessels of a mouse or rat, etc. is required. According to this embodiment, by using an invertebrate, the medical device material can be simply inserted into the body fluid of the invertebrate, and there is no need to make it hollow tubular to pass blood, so the preparation of the medical device material can be simplified. In addition, in the evaluation of biofilm formation described below, the biofilm formed on the surface of the medical device material can be evaluated by dissolving it with a chemical. In the case of a hollow tubular catheter, it is difficult to extract the liquid inside the hollow tube due to capillary action, so it may be difficult to analyze the biofilm formed inside the hollow tube. By using an invertebrate, the biofilm formed on the surface of the medical device material can be quantitatively evaluated.

[0038] The thickness of the medical device material is preferably 1 / 5 or less of the thickness of the silkworm's body. By setting the thickness of the medical device material within this range, the medical device material can be placed at a predetermined position inside the silkworm's body.

[0039] [Step of removing medical equipment materials] (Step S20) After a predetermined time has elapsed, the medical device material 30 is taken out from the silkworm 10. The method for taking out the medical device material 30 is not particularly limited, and it can be done by pulling out the medical device material 30 from the silkworm 10.

[0040] [Step of evaluating biofilm formation] (Step S30) The step of evaluating biofilm formation is a process of analyzing the biofilm formed on the surface of the medical device material 30 extracted from the silkworm 10 and evaluating the biofilm formation. The biofilm formation can be evaluated by staining the biofilm using a staining method and observing it under a microscope. In addition, the stained biofilm can be quantified by dissolving it and measuring its absorbance.

[0041] As described above, unlike rats or mice, the medical device material 30 can be inserted into the silkworm 10 between the subcutaneous layer and the intestinal tract, not into the bloodstream. Therefore, it is not necessary to form the medical device material 30 into a hollow cylinder, and it is sufficient to analyze the biofilm formed on the surface of the medical device material 30, so that the amount of the formed biofilm can be accurately quantified by measuring the absorbance. Crystal violet can be used as the staining solution used in the staining method, and the absorbance of the solution obtained by eluting the biofilm stained with this crystal violet with an aqueous acetic acid solution is measured. A wavelength of 590 nm can be used to measure the absorbance.

[0042] Other Embodiments Fig. 4 is a flowchart showing a method for evaluating biofilm formation according to another embodiment. The method for evaluating biofilm formation according to the embodiment shown in Fig. 4 differs from the method for evaluating biofilm formation shown in Fig. 1 in that it includes a step of infecting a fungus (step S12).

[0043] [Step of infecting with fungus] (Step S12) The step of infecting with a fungus is a process of inoculating the silkworms 10 with a fungus after the step of inserting the medical device material 30 (step S10) and infecting the silkworms 10 with the fungus. By inoculating the silkworms 10 with a fungus after inserting the medical device material 30, it is possible to promote biofilm formation on the medical device material 30. It is also possible to evaluate whether the inoculated fungus forms a biofilm.

[0044] As the fungus, for example, C. albicans, Staphylococcus aureus, Candida glabrata, etc. can be inoculated. In addition, a fungus to be evaluated for the formation of a biofilm can be inoculated.

[0045] The method for inoculating the fungus is not particularly limited, but a suspension of the fungus in physiological saline can be administered using a syringe into the body fluids of the silkworm 10. The concentration of the fungus to be inoculated can be appropriately changed depending on the purpose and method of the experiment, but it is preferable that the concentration does not kill the silkworm during the experiment.

[0046] After the step of infecting with a fungus (step S12), a step of removing the medical device material (step S20) and a step of evaluating biofilm formation (step S30) are performed to evaluate biofilm formation, similar to the method of evaluating biofilm formation shown in Figure 1.

[0047] Fig. 5 is a flowchart showing a method for evaluating biofilm formation according to yet another embodiment. The method for evaluating biofilm formation according to the embodiment shown in Fig. 5 differs from the method for evaluating biofilm formation shown in Fig. 4 in that it includes a step of administering an antibacterial agent (step S14).

[0048] [Step of administering antibiotics] (Step S14) The step of administering an antibacterial agent is a process of administering an antibacterial agent to the silkworms 10 after the step of infecting the silkworms with a fungus (step S12). By administering the antibacterial agent, the effect of the antibacterial agent can be confirmed.

[0049] The method of administering the antibacterial agent is not particularly limited, but it can be administered by administering an antibacterial agent prepared to a predetermined concentration into a body fluid. The concentration and the dosage can be appropriately changed depending on the purpose and method of the experiment.

[0050] The timing of administration of the antibacterial agent can also be changed as appropriate depending on the purpose and method of the experiment. For example, by administering the antibacterial agent immediately after administration of the fungus, the effect of the antibacterial agent before the formation of a biofilm on the medical device material can be confirmed. Furthermore, by administering the antibacterial agent after a predetermined time has elapsed (after biofilm formation) after inoculation of the fungus, the effect of the antibacterial agent after the formation of the biofilm can be confirmed. This makes it possible to identify antibiofilm compounds that have a different mechanism of action for destroying biofilms after their formation, unlike conventional antibacterial agents. For example, by administering the antibacterial agent 18 hours after inoculation of the fungus, the effect of the antibacterial agent after the formation of the biofilm can be confirmed.

[0051] Antifungal drugs include amphotericin B, fluconazole, and micafungin.

[0052] After performing the step of administering an antibiotic (step S14), similar to the method of evaluating biofilm formation shown in Figure 1, a step of removing the medical device material (step S20) and a step of evaluating biofilm formation (step S30) are performed to evaluate biofilm formation.

[0053] As described above, according to the present embodiment, it is possible to use invertebrates to evaluate biofilm formation on medical device materials in vivo. In addition, invertebrates can be used to evaluate biofilm formation on medical device materials in vivo. EXAMPLES

[0054] The present invention will be described in more detail below with reference to examples. Note that the materials, amounts, ratios, processing contents, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0055] (C. albicans culture) C. albicans (strain SC5314) was spread onto YPD agar medium (peptone 20 g / L, bacto yeast extract 10 g / L, glucose 20 g / L, agar 15 g / L) and cultured at 37°C to allow colonies to grow.

[0056] (Silkworm breeding) Silkworms were reared according to the method described in Kaito C, et al., Silkworm larvae as an animal model of bacterial infection pathogenic to humans. Microb Pathog 2002; 32: 183-190. Silkworm eggs were purchased from Ehime Sanshu Co., Ltd. Silkworm eggs were hatched at 25°C and fed Silkmate 2S, an artificial diet purchased from Ehime Sanshu Co., Ltd. Five-instar silkworms were used in the test.

[0057] [Test Example 1] (In vitro biofilm formation on polyurethane fiber surface) First, to confirm whether biofilms could form on the surface of polyurethane fiber, the material of the catheter, biofilm formation was performed on the surface of polyurethane fiber in vitro. The biofilm formation test was performed using a modified method described in Kurakado S, et al. Association of the hypha-related protein Pra1 and zinc transporter Zrt1 with biofilm formation by the pathogenic yeast Candida albicans. Microbiol Immunol 2018; 62: 405-410.

[0058] Polyurethane fibers (0.5 mm thick, rubber line F046, No. H3) were cut to a length of 2 cm, treated with 70% EtOH (ethanol) for 15 minutes, and dried under UV irradiation for 30 minutes. C. albicans (SC5314 strain) grown on YPD agar medium was scraped off with a platinum loop and 7 × 106 The cells were inoculated into RPMII640 buffered with 165mM MOPS at 1.5x10 cells / ml. Polyurethane fibers were added to the medium containing C. albicans and incubated at 37°C for 2 days. The control was RPMI medium with polyurethane fibers. Six samples were used for each test.

[0059] (Quantification of biofilm) After cultivation, the biofilm formed on the surface of the polyurethane fiber was stained with crystal violet, and the amount of biofilm was quantified. To stain the biofilm, the polyurethane fiber was transferred to a 1.5 ml tube and washed twice with 1 ml of distilled water. 500 μl of 0.1% crystal violet aqueous solution was added and left at room temperature for 20-60 minutes. The staining solution was discarded, and the fiber was washed three times with 1 ml of distilled water. It was washed twice with 1 ml of ethanol and then air-dried. The polyurethane fiber was observed and photographed under a microscope (×10, CH30, OLYMPUS). 500 μl of 33% acetic acid solution was added and left at room temperature for more than 2 hours. 500 μl of distilled water was added to bring the total volume to 1 ml, and the absorbance (OD 590 ) was measured.

[0060] The results of forming a biofilm on a polyurethane surface in vitro are shown in Figures 6 and 7. Figure 6(a) is a photograph of the polyurethane fiber after staining, and Figure 6(b) is a magnified photograph taken under a microscope. Figure 7 shows the absorbance of each sample. A t-test was performed on the absorbance values ​​of the samples, and a P value of 0.05 or less was determined to be a statistically significant difference.

[0061] As shown in Figure 6(a), the polyurethane fiber was stained blue when C. albicans was added compared to the control where no C. albicans was added. Also, as shown in Figure 6(b), the formation of hyphae on the polyurethane fiber surface was observed when C. albicans was added. Also, the absorbance results shown in Figure 7 show that the amount of dye attached to the polyurethane fiber was significantly greater with the addition of C. albicans.

[0062] These results confirmed that C. albicans forms a biofilm on the surface of polyurethane fibers and that polyurethane fibers can be used for in vivo animal tests of biofilm formation.

[0063] [Test Example 2] (Infection experiment of silkworms with polyurethane fibers inserted) Next, polyurethane fibers were inserted into the bodies of silkworms to examine whether an infection experiment with C. albicans could be performed. A hole was made near the crescent crest on the back of the silkworm with a 27-gauge injection needle, and 2 cm of polyurethane fiber was inserted through the hole. The polyurethane fiber used was polyurethane fiber that had been treated and dried in the same manner as in Test Example 1. The silkworms into which the polyurethane fiber had been inserted were administered physiological saline and a suspension of C. albicans SC5314 strain (2 x 10 7 Silkworms were inoculated with 50μl of C. albicans (cells / ml) to confirm the effect of inoculation with C. albicans. Silkworms were also prepared without polyurethane fibers inserted, administered with saline, or inoculated with C. albicans to confirm the effect of inserting polyurethane fibers. Each test was performed using six samples.

[0064] The results are shown in Figure 8. Silkworms administered saline did not die even after 48 hours had passed, even when polyurethane fibers were inserted. Furthermore, all silkworms inoculated with C. albicans died within 48 hours, regardless of whether polyurethane fibers were inserted or not. From these results, it was confirmed that silkworms can survive for more than 48 hours even when polyurethane fibers are inserted, and that infection experiments with C. albicans can be carried out under these conditions.

[0065] [Test Example 3] (In vivo silkworm infection experiment) Next, we investigated whether biofilms of C. albicans could be formed on the surface of polyurethane fibers inside the silkworm body. Silkworms with polyurethane fibers inserted were inoculated with saline or a suspension of C. albicans SC5314 (7 × 10 7 The silkworms were inoculated with 50 μl of 10 ...

[0066] The extracted polyurethane fibers were stained with crystal violet. The results are shown in Figures 9 and 10. As shown in Figure 9(a), the polyurethane fibers were stained bluer when silkworms were inoculated with C. albicans than when they were inoculated with saline. Also, as shown in Figure 9(b), microscopic observation confirmed the presence of C. albicans on the polyurethane fiber surface. No such image was observed when silkworms were inoculated with saline. Figure 10 is a graph showing the absorption at 590 nm measured after dissolving the dye on the polyurethane fiber with a 33% aqueous acetic acid solution. The amount of dye attached to the polyurethane fiber was significantly increased by inoculation with C. albicans compared to when silkworms were inoculated with saline.

[0067] These results confirmed that C. albicans forms a biofilm on the surface of polyurethane fibers inside the silkworm body.

[0068] [Test Example 4] (Suppressive effect of antifungal drugs on biofilm formation) Next, the effect of antifungal drugs was confirmed. Figure 11 shows the flow of the infection experiment. Silkworms with polyurethane fibers inserted were inoculated with physiological saline or a suspension of C. albicans SC5314 strain (3 × 10 7 Immediately after inoculation with C. albicans, 50μl of amphotericin B (AMPH-B) 50μg / ml, fluconazole (FLCZ) 1.6mg / ml, and micafungin (MCFG) 1mg / ml were inoculated into the body fluids of the silkworms. After 24 hours, polyurethane fibers were extracted from the silkworms' bodies in the same manner as in Test Example 3, stained with crystal violet, and used for evaluation. Seven samples were used for each test.

[0069] The results of the measured absorbance are shown in Figure 12. When antifungal drugs were administered to silkworms inoculated with C. albicans, biofilm formation on polyurethane fibers inside the bodies of the silkworms was significantly suppressed for all antifungal drugs compared to when C. albicans-inoculated silkworms were inoculated with physiological saline.

[0070] [Test Example 5] (Inhibitory effect of antifungal drugs on biofilm formation) FIG. 13 shows the flow of the infection experiment in Test Example 5. In Test Example 5, first, physiological saline or C. albicans was inoculated in the same manner as in Test Example 4. After inoculation with C. albicans, amphotericin B (AMPH-B), fluconazole (FLCZ), or micafungin (MCFG) was administered as an antifungal drug 12 hours later. The polyurethane fibers were extracted 24 hours after inoculation with C. albicans. The extracted polyurethane fibers were stained with crystal violet and used for evaluation. Six samples were used for each test.

[0071] The results of the measured absorbance are shown in Figure 14. When the antifungal drug was administered 12 hours after the silkworms were inoculated with C. albicans, biofilm formation on polyurethane fibers inside the silkworms' bodies was significantly suppressed in the group administered amphotericin B compared to when the silkworms were inoculated with saline 12 hours after the silkworms were inoculated with C. albicans.

[0072] [Test Example 6] (Suppressive effect of antifungal drugs on biofilm formation) FIG. 15 shows the infection experiment flow of Test Example 6. In Test Example 6, physiological saline or C. albicans was inoculated as in Test Example 4. After 18 hours from the inoculation with C. albicans, amphotericin B (AMPH-B), fluconazole (FLCZ), or micafungin (MCFG) was administered as an antifungal drug. The polyurethane fibers were extracted 24 hours after the inoculation with C. albicans. The extracted polyurethane fibers were stained with crystal violet and used for evaluation. Seven samples were used for each test.

[0073] The results of the measured absorbance are shown in Figure 16. When the antifungal agent was administered 18 hours after the silkworms were inoculated with C. albicans, the formation of a biofilm on polyurethane fiber inside the silkworms was not suppressed compared to when the silkworms were inoculated with saline 18 hours after the silkworms were inoculated with C. albicans.

[0074] The results of Test Examples 4 to 6 suggest that antifungal drugs have an inhibitory effect on the early stage of biofilm formation by C. albicans on the surface of polyurethane fibers, but no inhibitory effect is observed in the later stage. [Explanation of symbols]

[0075] 10 Silkworm 12 Head 14 Chest 16 Abdomen 18 Intestinal Tract 20 half moon crest 30 Medical equipment materials

Claims

1. A method for evaluating biofilm formation on a medical device material using an invertebrate animal, which is a larva of the Bombycidae family, comprising: A step of inserting the medical device material from the vicinity of the crescent crest of the second segment from the thorax of the abdomen of the invertebrate along the intestinal tract of the invertebrate between the subcutaneous tissue and the intestinal tract; infecting the invertebrate with a fungus into which the medical device material has been inserted; administering an antimicrobial agent to the invertebrate; Removing the medical device material from the invertebrate animal; evaluating the surface of the medical device material for biofilm formation, the evaluation including evaluating the antimicrobial agent; A method for assessing biofilm formation comprising:

2. 2. The method of claim 1, further comprising the step of inserting the medical device material into the invertebrate and then leaving the medical device material within the invertebrate for a predetermined period of time.

3. The method for evaluating biofilm formation according to claim 1 or 2, wherein the medical device material is a catheter.

4. The method for evaluating biofilm formation according to any one of claims 1 to 3, wherein the invertebrate is a 5th instar larva of the Bombycidae family.

5. The method for evaluating biofilm formation according to any one of claims 1 to 4, wherein the thickness of the medical device material is 1 / 5 or less of the thickness of the body of the invertebrate.

6. The method for evaluating biofilm formation according to any one of claims 1 to 5, wherein the step of evaluating biofilm formation is carried out by a staining method.

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