Biofilm clearing reagent, and method for observing biofilms using the clearing reagent.

Abstract

Provided is a biofilm transparentizing reagent that makes observation of a three-dimensional structure possible by instantly making even a thick biofilm transparent without causing changes in the physiological condition of cells. The present invention has an X-ray contrast agent. The X-ray contrast agent is an iodinated X-ray contrast agent such as iohexol, ioversol, iopamidol, iomeprol, iotrolan, ioxilan, iodixanol, ioxaglic acid, or sodium iotalamate, and is preferably iohexol. The present invention can instantly make a MRSA biofilm transparent.

Description

[Technical Field]

[0001] This invention relates to a biofilm clearing reagent and a method for observing biofilms using the clearing reagent. [Background technology]

[0002] A biofilm is a structure consisting of a matrix of polysaccharides and other substances secreted by bacteria outside the cell, along with an aggregate of bacteria. When bacteria attach to and proliferate, they form a biofilm, becoming covered by the matrix, which makes them resistant to drugs and more likely to evade the body's defense mechanisms, thus making treatment difficult.

[0003] Staphylococcus aureus is a causative agent of surgical site infections, bloodstream infections, respiratory infections, and urinary tract infections, primarily in immunocompromised patients in healthcare settings. Depending on the site of infection, these infections can be difficult to treat and sometimes lead to death. In particular, methicillin-resistant Staphylococcus aureus (MRSA), a causative agent of hospital-acquired infections, is said to enter the body via indwelling blood catheters in 48% of cases and urinary catheters in 14% of cases, posing a problem as it can become difficult to treat and lead to severe illness.

[0004] In the case of Staphylococcus aureus, airborne bacteria attach to and proliferate on surfaces such as catheters, producing an extracellular matrix and maturing into a biofilm. Eventually, airborne bacteria are released from the biofilm, and the infection spreads. In recent years, biofilm-related infections (biofilm infections) have been increasing in connection with treatments using artificial medical materials such as central venous catheters and artificial joints.

[0005] While previous research on biofilms has been conducted at the laboratory level, important basic clinical data, such as the composition of biofilms at infection sites and their relationship to sampling sites and clinical findings, are currently lacking (Non-Patent Literature 1, 2).

[0006] Biofilms differ not only at the bacterial species level but also at the strain level. Accurately evaluating the components that make up the extracellular matrix (ECM) of individual biofilms is considered important for understanding the molecular mechanisms of biofilm formation and for developing flexible biofilm infection control measures tailored to the specific properties of each biofilm (Non-patent documents 3, 4, 5, 6, 7).

[0007] Three-dimensional structure observation of biofilms is generally performed using a confocal laser fluorescence microscope (Non-Patent Literature 8). However, it is difficult to observe the three-dimensional structure of biofilms with a thickness exceeding 20 micrometers using fluorescence microscopy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Yoshimitsu Mizunoe, Shinya Sugimoto, Tadayuki Iwase: Mechanism of Staphylococcus aureus elimination by commensal bacteria. Respiratory Medicine. 26(1):49-52, 2014. [Non-Patent Document 2] Yoshimitsu Mizunoe, Shinya Sugimoto, Kenichi Okuda: Prospects for controlling biofilm infections. Journal of the Japanese Society of Veterinary Infectious Diseases. 5(4): 113-120. 2016. [Non-Patent Document 3] Yoshimitsu Mizunoe, Akio Chiba, Tadayuki Iwase, Shinya Sugimoto: Isolation and analysis of extracellular matrix in biofilms. Field of Chemotherapy. 31(11): 2158-2165, 2015. [Non-Patent Document 4] Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, Mizunoe Y: Broad impact of extracellular DNA on biofilm formation by clinically isolated methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep. 8(1): 2254,

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

[0009] This invention has been made in view of the above problems, and aims to provide a biofilm clearing reagent that enables the observation of a three-dimensional structure by instantly making even thick biofilms transparent. It also aims to provide a method for observing biofilms using this clearing reagent. [Means for solving the problem]

[0010] The biofilm clearing reagent according to the present invention is characterized by containing an X-ray contrast agent.

[0011] The present invention provides a method for observing biofilms, characterized by making the biofilm transparent by applying an X-ray contrast agent to the biofilm to be observed.

Advantages of the Invention

[0012] According to the present invention, even a thick biofilm can be instantaneously promoted to be transparent, so that it becomes easy to observe the three-dimensional structure of the biofilm. Further, by making it transparent, it becomes possible to observe the biofilm not only after formation but also during the formation process.

Brief Description of the Drawings

[0013] [Figure 1] It is a photographic view showing the transparency of MRSA biofilm when iohexol is used. [Figure 2] It is a photographic view showing the localization of proteins in MRSA biofilm when iohexol is used. Among them, (A) is a photographic view showing the localization of SasG protein, (B) is a photographic view showing the localization of MRSA cells, and (C) is an overlay of (A) and (B). [Figure 3] It is a photographic view showing the localization of sugars in MRSA biofilm when iohexol is used. Among them, (A) is a photographic view showing the localization of glycoconjugates on the cell membrane, (B) is a photographic view showing the localization of MRSA cells, and (C) is an overlay of (A) and (B). [Figure 4] It is a photographic view showing the transparency of MRSA biofilm when iobelsole is used. Among them, (A) is a low magnification image and (B) is a high magnification image. [Figure 5] It is a photographic view showing the transparency of Escherichia coli biofilm when iohexol is used. [Figure 6] It is a photograph showing the transparency of a biofilm of fungus (Candida albicans) when iodixanol is used. Among them, (A) is a plan view taken from the upper part of the culture dish, and (B) is a cross-sectional view by the XZ plane. [Figure 7]These are photographs showing the clearing of Bacillus subtilis biofilms when iohexol is used, with (A) being a low-magnification image and (B) being a high-magnification image. [Figure 8] This is a photograph showing a biofilm of Staphylococcus epidermidis that was made clear while the bacteria remained alive, without the use of a fixative, when using iodixanol. [Modes for carrying out the invention]

[0014] The embodiments of the present invention will be described below with reference to the attached drawings. However, these embodiments are provided to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which those skilled in the art appropriately substitute the configurations of the embodiments below are also included in the scope of the present invention.

[0015] The biofilm clearing reagent according to the present invention contains an X-ray contrast agent. While X-ray contrast agents are used to enhance contrast during X-ray imaging, the inventors have discovered, as a novel finding, that biofilms can be instantly cleared by applying an X-ray contrast agent to them, and have completed the present invention based on this fact.

[0016] The main factor hindering transparency is the scattering of visible light. However, by applying the transparency reagent of the present invention to a biofilm, the scattering of visible light by the biofilm can be suppressed. Specifically, biofilms are thought to be composed of microbial cells, polysaccharides, DNA, and proteins. By applying the transparency reagent of the present invention (refractive index of approximately 1.4), the refractive index of the solution can be matched with that of the microbial cells in the constituent components (approximately 1.4), which are the main cause of scattering. This suppresses the scattering of visible light.

[0017] When using the clearing reagent of the present invention, the transparency of the biofilm can be increased to 75-99%, preferably 80-90%, in terms of transmittance. For example, when iohexol is used as the X-ray contrast agent in the clearing reagent of the present invention, the transmittance of an MRSA biofilm can be instantly increased to 80-90%.

[0018] The X-ray contrast agent is preferably an iodine-containing X-ray contrast agent. The iodine-containing X-ray contrast agent contains a compound having a triiodobenzene ring structure. Depending on the type of group (side chain) that provides water solubility, it is classified into ionic and nonionic types, and further classified into monomer type and dimer type depending on the number of benzene rings in one molecule.

[0019] The iodine-containing X-ray contrast agent is not particularly limited, but examples include iohexol, iobersol, iopamidol, iomeprole, iotrolan, ioxilan, iodixanol, ioxaglucic acid, or sodium iotalamate, and is preferably iohexol.

[0020] Iodine-containing X-ray contrast agents can be used in combination, and in such cases, it is preferable to combine iodine-containing X-ray contrast agents with significantly different molecular weights. For example, by combining iopamilon with a molecular weight of 777.1 and iotrolan with a molecular weight of 1626.2, it is possible to penetrate the iodine-containing X-ray contrast agent deep into biofilms with complex constituent components.

[0021] The origin of the biofilm targeted by the clearing reagent of the present invention is not particularly limited, but it is preferably used for biofilms formed by Staphylococcus bacteria, which belong to the genus Staphylococcus, a Gram-positive cocci.

[0022] The clearing reagent of the present invention is preferably used for clearing biofilms derived from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus, among the Staphylococcus species.

[0023] Furthermore, it is preferably used to clear biofilms derived from Staphylococcus aureus, specifically MRSA, MSSA, and vancomycin-resistant Staphylococcus aureus (VRSA).

[0024] Furthermore, the clearing reagent of the present invention is also preferably used for biofilms formed by Gram-negative bacteria.

[0025] The clearing reagent of the present invention is preferably used for clearing biofilms derived from Gram-negative bacteria, particularly Escherichia coli, Enterobacter cloacae, Salmonella enterica, Pseudomonas aeruginosa, Pseudomonas syringae, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0026] Furthermore, the biofilm targeted by the clearing reagent of the present invention is also preferably used for biofilms formed by Candida fungi.

[0027] The clearing reagent of the present invention is used to clear biofilms derived from Candida fungi, preferably Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, or Candida tropicalis.

[0028] Furthermore, the biofilm targeted by the clearing reagent of the present invention is also preferably used for biofilms formed by Bacillus bacteria.

[0029] The clearing reagent of the present invention is used to clear biofilms derived from Bacillus bacteria, preferably Bacillus subtilis, Bacillus cereus, Bacillus megaterium, or Bacillus brevis.

[0030] The thickness of the biofilm in which the clearing reagent of the present invention is used is not particularly limited, but is, for example, 10 μm to 550 μm, preferably 20 μm to 450 μm, and more preferably 30 μm to 350 μm.

[0031] The clearing reagent of the present invention may also contain further components such as pH adjusters, buffers, and osmotic pressure adjusters. Examples of buffers include PBS buffer, HEPES buffer, and Tris buffer.

[0032] The pH of the clearing reagent of the present invention is determined from the viewpoint of rapidly clearing the biofilm and not damaging the structure of the biofilm, and is not particularly limited, but for example, pH 4.5 to 7.0, preferably pH 5.0 to 6.5, and more preferably pH 5.5 to 6.0. Examples of pH adjusting agents include phosphoric acid, citric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, pyrophosphate, sulfuric acid, nitric acid, acetic acid, glycolic acid, boric acid, lactic acid, silicic acid, phosphonic acid, tartaric acid, succinic acid, malic acid, sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, ammonia, and the like.

[0033] The concentration of the X-ray contrast agent in the clearing reagent according to the present invention is not particularly limited, but can be, for example, 100 to 800 mg / mL, preferably 200 to 600 mg / mL.

[0034] The method for using a biofilm according to the present invention includes the step of applying the clearing reagent of the present invention to the target biofilm. Applying the clearing reagent to a biofilm means impregnating the biofilm with the clearing reagent.

[0035] The method for applying the clearing reagent of the present invention to a biofilm is not particularly limited, but one example is to drop the clearing reagent of the present invention onto the biofilm to impregnate the biofilm with the clearing reagent of the present invention.

[0036] Even with a short impregnation time, it is possible to make the biofilm transparent, for example, 1 to 120 minutes, preferably 2 to 60 minutes, and more preferably 3 to 10 minutes. The temperature at which the clearing reagent of the present invention is dropped is not particularly limited, but for example, it is 20°C to 40°C, and preferably 25°C to 30°C.

[0037] It is possible to fix the biofilm to be observed with a fixative solution to make it transparent, or to make the biofilm transparent while it is still alive without using a fixative solution, allowing for observation of the biofilm not only after it has formed but also during the formation process. [Examples]

[0038] (1) Example 1 We attempted to clear MRSA biofilms using iohexol as an X-ray contrast agent. The experiment was conducted according to the following procedure.

[0039] The methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate MR23 was inoculated into 2 mL of Brain Heart Infusion (BHI) medium (BD) and incubated overnight with shaking at 37°C.

[0040] 2 μL of the pre-culture medium was added to 2 mL of 1% glucose-containing BHI (BHIG) medium.

[0041] All bacterial solutions were transferred to glass-bottomed culture dishes (manufactured by Matsunami Glass Co., Ltd.) and incubated at 37°C for 24 hours.

[0042] After removing the culture medium, fixative (1% glutaraldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0043] The fixative was removed, and the specimens were washed three times with PBS buffer.

[0044] FilmTracer diluted in PBS buffer to a final concentration of 5 μg / mL TM FM TM 1-43 Green Biofilm Cell Stain (Life Technology) was added to the biofilm and stained at room temperature for 30 minutes.

[0045] After removing the staining solution, iohexol solutions at concentrations of 0 wt% (i.e., no iohexol added), 28 wt%, 35 wt%, 56 wt%, and 70 wt% were added.

[0046] After adding iohexol solutions of various concentrations, the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880 (Carl Zeiss) 5 minutes later. The lens of the confocal laser microscope was placed against the bottom of the culture dish, and observation was attempted from the bottom of the culture dish towards the aperture.

[0047] Figure 1 is a photograph showing the clearing of an MRSA biofilm using iohexol. The upper part of Figure 1 is a horizontal view taken from above the culture dish, and the lower part of Figure 1 shows a cross-sectional view of the biofilm in the XZ plane.

[0048] As shown in Figure 1, in the absence of iohexol, the MRSA biofilm has opaque parts, so the bottom of the culture dish is visible, but the top of the culture dish (i.e., the part facing the opening of the culture dish) is not visible.

[0049] However, at iohexol concentrations of 28 wt%, 35 wt%, 56 wt%, and 70 wt%, the MRSA biofilm showed almost no opacity, and transparency was promoted from the bottom of the culture dish to the opening.

[0050] (2) Example 2 We used iohexol as an X-ray contrast agent to clear MRSA biofilms, and then attempted to visualize the localization of SasG, a protein essential for promoting biofilm formation, within the cleared MRSA biofilms. The experiment was conducted according to the following procedure.

[0051] A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), MR23, was inoculated into 2 mL of BHI medium and incubated overnight with shaking at 37°C.

[0052] The pre-culture medium was added to 2 mL of BHIG medium in 2 μL.

[0053] All bacterial solutions were transferred to glass-bottomed culture dishes and incubated at 37°C for 24 hours.

[0054] After removing the culture medium, pre-fixation solution (4% paraformaldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0055] The fixative was removed, and the specimens were washed three times with PBS buffer.

[0056] The biofilm was immersed in 1 mL of blocking buffer (3% BSA, 0.05% Triton X100, 1% goat serum, PBS) and incubated at room temperature for 30 minutes.

[0057] The blocking buffer was removed.

[0058] One mL of SasG antiserum (primary antibody solution), diluted 200-fold with blocking buffer, was added, and the mixture was allowed to react at room temperature for 2 hours.

[0059] After removing the primary antibody solution, the biofilm was washed three times with 1 mL of blocking buffer.

[0060] One mL of Alexa 647-labeled anti-rabbit IgG secondary antibody (Life Technology), diluted 200-fold with blocking buffer (secondary antibody solution), was added and the mixture was reacted at room temperature for 2 hours.

[0061] After removing the secondary antibody solution, the biofilm was washed three times with 1 mL of PBS.

[0062] Post-fixation solution (1% glutaraldehyde, 4% paraformaldehyde, PBS) was added, and the specimens were fixed at room temperature for 10 minutes.

[0063] It was washed three times with PBS.

[0064] FilmTracer diluted in PBS buffer to a final concentration of 5 μg / mL TM FM TM 1-43 Green Biofilm Cell Stain was added to the biofilm and stained at room temperature for 30 minutes.

[0065] After removing the staining solution, a 56 wt% iohexol solution was added, and the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880.

[0066] Figure 2(A) is a photograph showing the localization of the SasG protein. SasG promotes biofilm formation by anchoring to the cell wall, and further contributes to biofilm stabilization by binding to extracellular DNA. Figure 2(B) is a photograph showing the localization of MRSA cells. Figure 2(C) is a superposition of (A) and (B).

[0067] The photographic images in Figure 2(A),(B), and(C) clearly show that the localization of SasG, a protein essential for promoting biofilm formation, is appropriately visualized in MRSA biofilms.

[0068] (3) Example 3 We used iohexol as an X-ray contrast agent to clear MRSA biofilms, and then attempted to visualize the localization of carbohydrates on the cell surface within the cleared MRSA biofilms. The experiment was conducted according to the following procedure.

[0069] A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), MR10, was inoculated into 2 mL of BHI medium and incubated overnight at 37°C.

[0070] The pre-culture medium was added to 2 mL of BHIG medium in 2 μL.

[0071] All bacterial solutions were transferred to glass-bottomed culture dishes and incubated at 37°C for 24 hours.

[0072] After removing the culture medium, fixative (1% glutaraldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0073] The fixative was removed, and the specimens were washed three times with PBS buffer.

[0074] Alexa 647-labeled wheat germ agglutinin (Alexa 647-WGA) (Life Technology), diluted in PBS buffer to a final concentration of 5 μg / mL, was added to the biofilm and stained at room temperature for at least 16 hours.

[0075] The staining solution was removed.

[0076] FilmTracer diluted in PBS buffer to a final concentration of 5 μg / mL TM FM TM 1-43 Green Biofilm Cell Stain was added to the biofilm and stained at room temperature for 30 minutes.

[0077] After removing the staining solution, a 56 wt% iohexol solution was added, and the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880.

[0078] Figure 3(A) is a photograph showing the localization of complex carbohydrates (i.e., N-acetylglucosamine and N-acetylneuroamine) on the cell surface. Wheat germ agglutinin (WGA) is a type of lectin, and when conjugated with Alexa Fluor® dye, wheat germ agglutinin detects complex carbohydrates on the cell membrane. Figure 3(B) is a photograph showing the localization of MRSA cells. Figure 3(C) is a superposition of (A) and (B).

[0079] The photographic images in Figures 3(A), 3(B), and 3(C) reveal that the localization of complex carbohydrates on the cell membrane is appropriately visualized in MRSA biofilms.

[0080] (4) Example 4 In Example 1, iohexol was used as an X-ray contrast agent to attempt to clear the MRSA biofilm, while in Example 4, iobelsol was used to attempt to clear the MRSA biofilm. The experiment was carried out according to the following procedure.

[0081] A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), MR23, was inoculated into 2 mL of BHI medium and incubated overnight with shaking at 37°C.

[0082] The pre-culture medium was added to 2 mL of BHIG medium in 2 μL.

[0083] All bacterial solutions were transferred to glass-bottomed culture dishes and incubated at 37°C for 24 hours.

[0084] After removing the culture medium, fixative (1% glutaraldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0085] The fixative was removed, and the specimens were washed three times with PBS buffer.

[0086] FilmTracer diluted in PBS buffer to a final concentration of 5 μg / mL TM FM TM1-43 Green Biofilm Cell Stain was added to the biofilm and stained at room temperature for at least 30 minutes.

[0087] After removing the staining solution, a 74 wt% Optiray (generic name Ioversol) solution (Optiley 350, manufactured by Guerbet Japan) was added, and the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880.

[0088] Figure 4 shows photographs illustrating the clearing of an MRSA biofilm using Ioversol, with (A) being a low-magnification image and (B) being a high-magnification image. As shown in Figures 4(A) and (B), even when using Ioversol, the MRSA biofilm was almost completely free of opacity, and its clearing was promoted. While examples for iohexol and Ioversol are given in this specification, biofilm clearing is also possible with other X-ray contrast agents such as iopamidol, iomeprole, iotrolan, ioxiran, iodixanol, ioxaglucate, or sodium iotalamate.

[0089] (5) Example 5 In Example 1, iohexol was used as an X-ray contrast agent to attempt to clear an MRSA biofilm, while in Example 5, iohexol was used to attempt to clear an E. coli biofilm. The experiment was carried out according to the following procedure.

[0090] E. coli strain K-12 was spread onto 2 mL of LB medium and incubated overnight with shaking at 37°C.

[0091] The pre-culture medium was added to 2 mL of YESCA medium (1% casamino acids, 0.1% yeast extract) in a total volume of 2 μL.

[0092] All bacterial solutions were transferred to glass-bottomed culture dishes and incubated at 25°C for 7 days.

[0093] Except for the culture solution, a fixing solution (1% glutaraldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0094] The fixing solution was removed and washed three times with PBS buffer.

[0095] FilmTracer was diluted in PBS buffer to a final concentration of 5 μg / mL. TM FM TM 1-43 Green Biofilm Cell Stain was added to the biofilm and stained at room temperature for 30 minutes or more.

[0096] Except for the staining solution, a 56 wt% iodixanol solution was added, and the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880.

[0097] Figure 5 is a photographic illustration showing the clearing of Escherichia coli biofilm using iodixanol. As shown in Figure 5, clearing was also promoted for Escherichia coli biofilm. Although examples of MRSA biofilm and Escherichia coli biofilm are shown in this specification, clearing is also possible for biofilms derived from other Gram-positive bacteria and Gram-negative bacteria.

[0098] (6) Example 6 Using iodixanol, an X-ray contrast agent, an attempt was made to clear the biofilm of Candida albicans. The experiment was carried out according to the following procedure.

[0099] The Candida albicans standard strain (SC5314) was spread on 2 mL of YPD plate medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar) and cultured at 25°C for 3 days.

[0100] Colonies grown on YPD agar plates were transferred to 5 mL of RPMI-MOPS medium [10.4 mg / mL RPMI-1640 powder (glutamine+, phenol red+, bicarbonate-), 165 mM MOPS (pH 7.0)] in a 1 × 10⁶ solution. 6 The suspension was adjusted to a concentration of CFU / mL.

[0101] 2 mL of bacterial solution was transferred to a glass-bottomed culture dish (manufactured by Matsunami Glass Co., Ltd.) and incubated at 37°C for 24 hours.

[0102] After removing the culture medium, the biofilm formed on the glass surface was fixed with a fixative (1% glutaraldehyde, 4% paraformaldehyde, PBS) and fixed at room temperature for 30 minutes.

[0103] The fixative was removed, and the surface was washed three times with sterile water.

[0104] FilmTracer diluted in sterile water to a final concentration of 5 μg / mL TM FM TM 1-43 Green Biofilm Cell Stain (Life Technology) was added to the biofilm and stained at room temperature for 30 minutes.

[0105] After removing the staining solution, iodixanol solutions at concentrations of 0 wt% (i.e., no iodixanol added), 30 wt%, 45 wt%, and 60 wt% were added.

[0106] After adding iodixanol solutions of various concentrations, the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880 (Carl Zeiss) 5 minutes later. The lens of the confocal laser microscope was placed against the bottom of the culture dish, and observation was attempted from the bottom of the culture dish towards the aperture.

[0107] Figure 6 is a photograph showing the clearing of a Candida albicans biofilm using iodixanol. Figure 6A is a horizontal view taken from the top of the culture dish, and Figure 6B shows a cross-sectional view of the biofilm in the XZ plane.

[0108] As shown in Figure 6, without iodixanol, the Candida albicans biofilm has opaque parts, so the bottom of the culture dish is visible, but the top of the culture dish (i.e., the part facing the opening of the culture dish) is not visible.

[0109] However, when iodixanol was present at 45 wt%, the Candida albicans biofilm was almost completely opaque, and transparency was promoted from the bottom of the culture dish to the opening.

[0110] (7) Example 7 We attempted to clear a Bacillus subtilis biofilm using the X-ray contrast agent iohexol. The experiment was conducted according to the following procedure.

[0111] Bacillus subtilis strain JKBS01 was inoculated into 2 mL of Tryptic Soy Broth (TSB) medium and incubated overnight with shaking at 37°C.

[0112] 2 μL of the pre-culture solution was added to 2 mL of TSB medium.

[0113] All bacterial solutions were transferred to glass-bottomed culture dishes and incubated at 37°C for 24 hours.

[0114] After removing the culture medium, fixative (1% glutaraldehyde, PBS) was added to the biofilm formed on the glass surface and fixed at room temperature for 30 minutes.

[0115] The fixative was removed, and the specimens were washed three times with PBS buffer.

[0116] FilmTracer diluted in PBS buffer to a final concentration of 5 μg / mL TM FM TM 1-43 Green Biofilm Cell Stain was added to the biofilm and stained at room temperature for at least 30 minutes.

[0117] After removing the staining solution, a 56 wt% iohexol solution was added, and the three-dimensional structure of the biofilm was observed using a confocal laser microscope LSM880.

[0118] Figure 7 shows photographs of Bacillus subtilis biofilms cleared using iohexol, with (A) being a low-magnification image and (B) being a high-magnification image.

[0119] As shown in Figures 7(A) and 7(B), a Bacillus subtilis biofilm with a thickness of approximately 90 μm was observed.

[0120] (8) Example 8 We used the X-ray contrast agent iodixanol to clear a biofilm of Staphylococcus epidermidis while keeping it alive, and performed live-cell imaging. The experiment was carried out according to the following procedure.

[0121] Staphylococcus epidermidis strain SE21 was inoculated into 2 mL of BHI medium and incubated overnight at 37°C.

[0122] The pre-culture medium (3 μL) was added to BHIG medium (3 mL) containing 30 wt% iodixanol solution and 10 μM thioflavin T.

[0123] All the bacterial solution was transferred to a glass-bottomed culture dish.

[0124] The three-dimensional structure of the biofilm was observed every 30 minutes using a confocal laser microscope LSM880 (Carl Zeiss) while the biofilm was incubated at 37°C for 18 hours. The lens of the confocal laser microscope was placed against the bottom of the culture dish, and observation was attempted from the bottom of the dish towards the aperture.

[0125] Figure 8 shows photographs observing the formation process of a Staphylococcus epidermidis biofilm using iodixanol. The time of day when each image was taken is indicated.

[0126] As shown in Figure 8, we were able to observe the formation process of a Staphylococcus epidermidis biofilm while the bacteria were still alive. First, microcolonies, indicated by the arrows, are formed in the initial stages of biofilm formation. Subsequently, bacteria attach to and proliferate around the periphery, causing the biofilm to build up and eventually form a thicker biofilm. [Industrial applicability]

[0127] It can be used in the development of methods for preventing, treating, and diagnosing biofilm infections.

Claims

1. A reagent comprising an iodine-containing X-ray contrast agent, used to clear a biofilm when observing the biofilm with an optical microscope, The biofilm is a biofilm derived from Staphylococcus aureus, Candida fungi, Staphylococcus epidermidis, Bacillus subtilis, or Escherichia coli. A biofilm clearing reagent characterized in that the iodine-containing X-ray contrast agent is iohexol, iobersol, iopamidol, iomeprole, iotrolan, ioxilan, iodixanol, ioxaglucic acid, or sodium iotalamate.

2. The biofilm clearing reagent according to claim 1, characterized in that the thickness of the biofilm is 10 μm to 550 μm.

3. The biofilm clearing reagent according to claim 1, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), or vancomycin-resistant Staphylococcus aureus (VRSA).

4. A method for observing biofilms, comprising: impregnating a biofilm clearing reagent consisting of an iodine-containing X-ray contrast agent into a biofilm derived from Staphylococcus aureus, Candida fungi, Staphylococcus epidermidis, Bacillus subtilis, or Escherichia coli, thereby instantly clearing the biofilm and observing it with an optical microscope; A method for observing a biofilm, characterized in that the iodine-containing X-ray contrast agent is iohexol, iobersol, iopamidol, iomeprole, iotorolan, ioxilan, iodixanol, ioxaglucate, or sodium iotalamate.

5. The method for observing a biofilm according to claim 4, wherein the biofilm is observed without being fixed with a fixative.

Images