Microbial profiling and analysis methods

Abstract

[Problem] To provide a microorganism image analysis method capable of capturing information on the fine morphology of individual microorganisms with the resolution of an electron microscope and evaluating the ratio of microorganisms. [Solution] A microorganism image analysis method according to one aspect of the present invention comprises a first step of acquiring an image of a specimen obtained by staining a sample containing microorganisms using an electron microscope, a second step of acquiring a luminance profile relating to the brightness of the image, a third step of setting a first standard luminance range in the profile that satisfies a first condition relating to luminance as an area in which a first group of microorganisms exists, and setting a second standard luminance range in the profile that satisfies a second condition relating to luminance as an area in which a second group of microorganisms exists, and a fourth step of calculating the ratio of microorganisms existing within each of the first and second standard luminance ranges. [Selected Figure] Figure 1

Description

Technical Field

[0001] The present invention relates to a method for analyzing microbial images.

Background Art

[0002] With the worldwide spread of antimicrobial-resistant bacteria a concern, infectious disease tests such as the identification of the causative microorganisms of infectious diseases and antimicrobial susceptibility testing have become important. Here, as a method for identifying microorganisms and measuring their susceptibility to antimicrobial agents, after incubating the microorganisms in a liquid together with a staining agent, they are recovered by filtration onto a filter, and a viability test is used to count the microorganisms stained with a substance that stains both live and dead cells within the optical microscope field of view, and the microorganisms stained with a substance that stains only dead cells. A drug susceptibility test method is known (see Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] According to Patent Document 1, there is a problem that only a method using an optical microscope is disclosed, and finer forms of microorganisms cannot be captured.

[0005] Therefore, an object of the present invention is to provide a method for analyzing microbial images that can capture information on the fine form of individual microorganisms with the resolution of an electron microscope and evaluate the ratio of microorganisms.

Means for Solving the Problems

[0006] A microbial image analysis method according to one aspect of the present invention comprises: a first step of acquiring an image of a stained specimen containing microorganisms using an electron microscope; a second step of acquiring a brightness profile relating to the brightness of the image; a third step of setting a first standard brightness range in the profile that satisfies a first condition relating to brightness as the region where a first group of microorganisms exists, and a second standard brightness range in the profile that satisfies a second condition relating to brightness as the region where a second group of microorganisms exists; and a fourth step of calculating the ratio of microorganisms present within each of the first and second standard brightness ranges. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a microbial image analysis method that can capture information on the fine morphology of individual microorganisms with the resolution of an electron microscope and evaluate the proportion of microorganisms. [Brief explanation of the drawing]

[0008] [Figure 1] A diagram showing microbial images with different image brightness levels and their respective brightness ranges. [Figure 2] A diagram showing an example of the image brightness range. [Figure 3] A diagram illustrating an example of microbial image classification. [Figure 4] A diagram showing the results of a microbial survival rate test after preservation treatment. [Figure 5] A diagram showing the results of a survival rate test of microorganisms exposed to oxygen. [Figure 6] A diagram showing an electron staining agent. [Figure 7] A figure showing the results of image analysis of microorganisms treated with antibiotics. [Modes for carrying out the invention]

[0009] Before describing the examples, the background of the present invention will be explained.

[0010] From the perspective of background technology, one of the important fundamental technologies is microscopy, which utilizes images of individual microorganisms or colonies. For example, in clinical testing, differences in bacterial cell wall structure can be distinguished using dyes and observed with a light microscope to differentiate between Gram-positive and Gram-negative bacteria, classify bacteria into rods and cocci based on their appearance, and obtain information related to bacterial species such as Staphylococcus and Streptococcus from the general shape of aggregates of isolated and cultured bacteria.

[0011] In addition to identification through such staining, it is expected that information on the fine morphology of individual microorganisms can be obtained. However, since the resolution of optical microscopes commonly used in clinical examinations is only about a few hundred nanometers, it is not easy to distinguish the morphological differences of individual microorganisms, even if the general shape and staining information of the microorganism can be obtained.

[0012] Therefore, electron microscopes are used. Because electron beams have a much shorter wavelength than visible light, high-resolution images can be obtained, allowing observation of even minute morphologies that cannot be captured with optical microscopes. For example, species-specific structures of microorganisms, structures observed during cell division, and abnormal morphologies caused by the effects of drugs, etc.

[0013] Conventional electron microscopes required complex mechanisms, such as those for applying high voltage to generate a stable electron beam and for maintaining a vacuum inside the microscope to prevent scattering of the emitted electron beam, making them difficult to use in routine microbiological examinations such as those in clinical settings. However, in recent years, improved desktop electron microscopes have emerged, and their application to microbial samples is expected to improve.

[0014] One challenge with electron microscopy is the limited range of staining methods available. For optical microscopy, advancements in cell biology, histology, and pathology have led to the development of a wide variety of stains for diverse purposes, and numerous staining methods have been established that allow for the differentiation of cell and tissue components and functions through color differences.

[0015] In the case of an electron microscope, for example, by treating a sample with a stain containing heavy metals such as uranium, lead, platinum, and osmium, the structure of the whole microorganism such as the cell membrane and nucleus can be visualized as the contrast of a black-and-white image, or the localization of a specific molecule can be visualized with gold nanoparticle labeling. However, there are few reports of stains that can stain specific components and functions of microorganisms and generate contrast with an electron beam.

[0016] As described above, a method that combines both staining with an optical microscope and high-resolution observation with an electron microscope is desirable, but there is no method that has been put into practical use in daily microorganism tests.

[0017] As a second aspect of the background art, a viability test of microorganisms, which is one of the important test items in microorganism tests, can be mentioned. For example, when developing treatment methods such as chemicals, heat, and gas for removing microorganisms, or when selecting a therapeutic agent for infectious diseases, by measuring the viability of microorganisms in a sample subjected to such treatments, the type of treatment, treatment method, intensity and concentration of the treatment, etc. are determined.

[0018] There are various viability test methods, and the standard method is a culture method such as the colony formation method. In the evaluation by colony formation, a certain amount of a microorganism suspension with a known concentration is thinly spread on an agar medium and cultured for about one day, and the number of colonies of bacteria that have grown within a certain time is regarded as the number of bacteria that survived at the start of the culture. The colony-forming unit (CFU / ml) is calculated from the number of colonies relative to the volume of the seeded bacterial solution and regarded as the viability at the start of the culture.

[0019] The culture method is the most widely used method for the viability test of microorganisms. However, there are problems such as the culture time required for colony formation being one day or more, the need for a trained judge to visually determine the colony-forming ability, and the inability to apply it to microorganisms with unknown culture conditions such as the type of medium. <好きな食べ物は何ですか。

[0020] To solve the problems of such culturing methods, methods using an optical microscope or the like that do not require culturing have been developed. For example, there are methods for detecting functions such as enzyme activity possessed by living cells using a chromogenic substrate, methods for differentiating and detecting based on the difference in the substance permeability of the cell membranes of living cells and dead cells, methods for detecting morphological changes, etc. Since there is no need to wait for the culturing time until the bacteria grow to form visible colonies, they are excellent in terms of speed.

[0021] Here, combining the above first and second viewpoints, with the resolution of an electron microscope, information on the fine morphology of microorganisms, such as species-specific structures of microorganisms, structures observed during cell division, and abnormal morphologies due to the influence of drugs, etc., is captured, and the usefulness of a technique for performing a viability test on the same specimen is considered. For example, by capturing the type and state from the morphological information of microorganisms forming a biofilm, performing a treatment that affects the survival of microorganisms such as an antimicrobial agent or a bactericide on the biofilm, and comparing the viability of the treated specimen and the untreated specimen, the effects of the antimicrobial agent or the bactericide can be evaluated "in situ" (observation in the field). However, in microbial examinations in clinical and environmental settings, a viability test method using an electron microscope has not been put into practical use.

[0022] As a reason why it is difficult to perform a viability test using an electron microscope, in the case of a general electron microscope, there is a pretreatment for observation to preserve the morphology of the microorganisms observed in a vacuum. Since the microorganisms are chemically fixed using a protein cross-linking agent such as glutaraldehyde in advance and then dried, the microorganisms lose their survival functions at the time of fixation. Therefore, it is not possible to perform a viability test that visualizes the survival functions of actually living microorganisms, such as differences in enzyme activity or biomembrane permeability, by staining or the like, as cited in the optical detection examples.

[0023] Therefore, one method for performing both microscopic morphological observation and viability determination on the same sample is to determine morphological features related to microbial viability, such as membrane damage or the breakdown of intracellular ultrastructure, by observing ultrathin sections with a transmission electron microscope. However, this method requires skilled techniques and is time-consuming. Therefore, it is difficult to apply to routine microbiological testing because it requires an enormous amount of work to observe a statistically significant number of microorganisms and calculate their survival rate.

[0024] Another method is correlational light and electron microscopy, which links stained images obtained with an optical microscope with morphological images obtained with an electron microscope. In principle, this method allows for determining viability with an optical microscope and then processing the same sample for observation with an electron microscope. However, the sample preparation and image overlay operations are not simple, making it difficult to apply to routine microbiological testing.

[0025] In the following embodiment, as a technique to capture information on the fine morphology of microorganisms with the resolution of an electron microscope and to perform viability tests on the same sample, an aqueous solution of phosphotungstic acid (PTA) is used as a staining agent that strongly adsorbs to microorganisms that have suffered morphological damage related to microbial life and death. By acquiring an image including compositional contrast with backscattered electrons of a scanning electron microscope (SEM), a means is provided to evaluate the ratio of microorganisms corresponding to viability.

[0026] Here, the ratio refers to the ratio obtained by detecting the difference in staining intensity between microorganisms that have not suffered morphological damage related to their survival and those that have suffered morphological damage related to their survival when microorganisms in a suspension are stained with a PTA aqueous solution, and quantifying each microorganism. This ratio is defined as the survival rate according to the present invention. As mentioned above, since observation pre-processing such as fixation is performed, this does not represent the ratio of microorganisms alive at the time of SEM image acquisition, but the examples demonstrate that a ratio equivalent to the survival rate obtained by conventional methods can be obtained.

[0027] This document explains the process that led to the use of PTA as a staining agent in this invention. Generally, when observing biological samples with an electron microscope, staining agents such as uranium acetate aqueous solution, lead salt aqueous solution, and platinum blue are often used. These are excellent staining agents that can visualize intracellular structures such as cell membranes and nuclei by adding image contrast due to differences in affinity with the components that make up cells, but they are not staining methods that distinguish between living and dead microorganisms. Furthermore, uranium is a radioactive material and is subject to restrictions in terms of availability and handling, and along with lead and platinum, they cannot be said to be suitable staining agents for routine microbiological testing due to toxicity, solution stability, and reagent costs.

[0028] Therefore, the inventors considered PTA to be suitable for application to the electron staining of microorganisms in routine microbiological tests due to its relatively low cost, ease of storage, and ease of use as described below. PTA is widely used in the preparation of microscopic samples, such as as a mordant for staining light microscope samples in pathological examinations, and as a negative stain for electron microscopy observation of microorganisms. It is also used for positive staining when observing cells with an electron microscope, and by adjusting the concentration and pH of the solution, it is known to selectively stain basic proteins, glycoproteins, and polysaccharides, and to bind to the carbohydrate portion of glycoproteins. When used for SEM observation of bacteria, it has been reported that the staining intensity differs between Gram-positive and Gram-negative bacteria.

[0029] Here, the inventors treated bacteria with known susceptibility to an antimicrobial agent that exhibits a bactericidal effect that damages the cell membrane and cell wall, prepared PTA-stained specimens, and obtained backscattered electron images using SEM. They found that bacterial individuals with high image brightness were observed in the specimens, and that the proportion of individuals with high image brightness increased with the passage of time since antimicrobial treatment.

[0030] Backscattered electron images have the characteristic of providing contrast not only in surface morphology contrast of the specimen but also in contrast due to the composition of the material. Therefore, it was considered that more PTA bound to bacteria whose cell membranes and cell walls were damaged by the bactericidal action of antibiotics than to live bacteria. This result suggests that morphological damage related to bacterial viability can be identified by PTA staining.

[0031] Therefore, the inventors diligently investigated a sample preparation method including a PTA staining step and backscattered electron observation conditions using a SEM. As an example, microorganisms treated with disinfectants and control microorganisms that were not treated were placed on equipment to prepare specimens, the specimens were fixed with 2.5% glutaraldehyde for 5 minutes, then stained with a 10% PTA aqueous solution by weight for about 2 minutes, and backscattered electron images were acquired using a SEM at an acceleration voltage of 5kV to 10kV.

[0032] As a result, the microorganisms contained in the disinfectant-treated specimens were mainly classified into two groups: those with high image brightness and those with low image brightness. The image brightness of the latter group of low-brightness microorganisms was equivalent to that of the control microorganism specimens that had not been treated with the disinfectant. In other words, we found that in a microbial population treated with a disinfectant, it is possible to distinguish between bacteria that have been morphologically damaged due to the bactericidal effect and bacteria that have not been damaged, and to determine their ratio through staining.

[0033] Based on the above findings, the present inventors provide a method for capturing the type and morphological characteristics of microorganisms using an electron microscope, and for easily distinguishing between microorganisms that have suffered morphological damage related to their survival and those that have not, based on the image brightness derived from the intensity of electron staining using PTA.

[0034] Detailed examples are described below. Note that the procedures, chemical concentrations, processing times, and image analysis procedures described below are examples only and do not limit the scope of the present invention. [Examples]

[0035] Example 1 demonstrates an example of classifying microbial images based on differences in image brightness of electron-stained microorganisms in electron microscope images. Figure 1 shows a schematic diagram (101) of electron microscope images of specimens in which microorganisms were placed on equipment and electronically stained, illustrating an example where microorganism 1 (102), which has weak staining and low image brightness, and microorganism 2 (103), which has strong staining and high image brightness, are mixed together. In this example, the microbial images are classified into two groups, microorganism 1 and microorganism 2, based on the difference in image brightness.

[0036] Examples of electron microscope images showing microorganism 1 (102) (104) and microorganism 2 (103) (105) were obtained by placing a Pseudomonas aeruginosa strain on a polycarbonate track-etched membrane, fixing it with 2.5% glutaraldehyde, then electron-staining it with a 10% PTA aqueous solution, inserting the specimen with the Pseudomonas aeruginosa on the membrane into the SEM sample chamber, setting the SEM acceleration voltage to 5kV, and keeping the brightness contrast settings constant during image generation. As the SEM image generation conditions were constant, the difference in brightness between microorganism 1 (102) in image (104) and microorganism 2 (103) in image (105) is due to the difference in PTA staining intensity.

[0037] As an example, a plastic track-etched membrane was used for the equipment, but it is not limited to this. Here, from the perspective of subsequent image analysis, it is preferable for the user to select equipment made of a material that exhibits different image brightness in the backscattered electron image from the stained microorganisms 1 and 2 being analyzed.

[0038] The luminance profile (106) for image (104) and the luminance profile (107) for image (105) are difference histograms obtained by calculating the number of pixels for each luminance level for each image in which microorganism 1 or microorganism 2 is placed on the membrane, and then subtracting the number of pixels for each luminance level for the membrane image without the microorganism.

[0039] For example, the images of microorganism 1 and microorganism 2 are classified by characterizing the image region of microorganism 1 with a standard brightness range 1 (108) that includes peak 1 of the brightness profile (106), and the image region of microorganism 2 with a standard brightness range 2 (109) that includes peak 2 of the brightness profile (107).

[0040] As mentioned above, when acquiring images (104), (105), images of other regions of the same specimen, and images of multiple specimens prepared with the same materials and methods, if the brightness contrast of the SEM is adjusted to a constant value, then when performing image analysis, the standard brightness range 1 (108) that characterizes the image region of microorganism 1 and the standard brightness range 2 (109) that characterizes the image region of microorganism 2 can be set to the same value.

[0041] Figure 2 shows an example of setting a standard brightness range (201, 202). 201 and 202 are the same histogram, representing brightness profiles related to the brightness of electron microscope images. As shown in the schematic diagram (101) in Figure 1, they originate from images containing microorganisms with different brightness levels, hence the presence of peak 1 and peak 2.

[0042] Another peak located to the left of peak 1 corresponds to the electron microscope image region of the equipment containing the microorganisms; therefore, excluding this range, we determine the standard brightness range that characterizes the microbial image.

[0043] As shown in histogram 1(201), a standard brightness range 1 containing peak 1 derived from the image of microorganism 1 is determined, and a standard brightness range containing peak 2 derived from the image of microorganism 2 is set in a range that does not overlap with this standard brightness range 1. This allows for the classification of microorganism 1, characterized by standard brightness range 1, and microorganism 2, characterized by standard brightness range 2.

[0044] Alternatively, as shown in histogram 2(202), a standard brightness range 1 can be determined that includes both peak 1 derived from the image of microorganism 1 and peak 2 derived from the image of microorganism 2, and a standard brightness range 2 can be set that does not include peak 1 but includes peak 2. This allows for classification into all microorganisms included in the image, microorganism 2 characterized by standard brightness range 2, and microorganism 1, which is obtained by subtracting microorganism 2 from all microorganisms.

[0045] Figure 3 shows an example of classifying microbial images based on differences in brightness in electron microscope images using image analysis software, and creating image masks for each classification. The electron microscope image of the microbial specimen (301) contains a mixture of microorganisms 1 (102) with weak staining and low image brightness, and microorganisms 2 (103) with strong staining and high image brightness, as shown in the schematic diagram (101) in Figure 1.

[0046] Mask (302), as shown in histogram 2(202) in Figure 2, is a mask image created by binarizing the image by setting a threshold from a standard brightness range 1 that includes both peak 1 derived from the image of microorganism 1 and peak 2 derived from the image of microorganism 2, excluding the brightness range of the equipment, and then removing particles significantly smaller than microorganism 1 and microorganism 2 using a particle analysis algorithm. Mask (303), as shown in histogram 2(202) in Figure 2, is a mask image created by binarizing the image by setting a threshold from a standard brightness range 2 that does not include peak 1 but includes peak 2, and then removing particles significantly smaller than microorganisms using a particle analysis algorithm. Using image analysis software, it is possible to identify individual microorganisms classified as mask 1 and mask 2, and further determine the number of microorganisms, the microorganism image area, and the number of pixels, and calculate the ratio.

[0047] In this way, by electronically staining a microbial specimen to generate a backscattered electron image, obtaining brightness profiles for microorganism 1 (104), which has weak staining and low image brightness, and microorganism 2 (105), which has strong staining and high image brightness, and classifying the microbial images according to their image brightness range, the ratio of microorganism 1 to microorganism 2 in the specimen can be easily calculated.

[0048] In Example 1, two types of differences in microbial staining intensity were set, and two types of standard brightness ranges were determined in the brightness profile related to image brightness. However, there may be more than two types of standard brightness ranges. [Examples]

[0049] <Microbial survival rate test> Example 2 describes an example in which the ratio of microorganisms obtained by the method of the present invention is compared with the survival rate of microorganisms obtained by the colony formation method, which is a conventional method for survival rate testing, and the flow cytometry method, which is a non-culture method.

[0050] As an example, after isolating the bacterium (Akkermansia muciniphila) by anaerobic culture on agar medium for 48 hours, the bacterial concentration was increased to approximately 10% in a preservation medium containing an antioxidant. 10 The solution was suspended in CFU / mL, frozen at -80°C for 24 hours, and then lyophilized.

[0051] After freezing and lyophilization, bacteria were stained with 10% PTA for 5 minutes at 37°C. The bacterial suspension was then centrifuged and spread onto glass slides. After drying at room temperature, the slides were observed using SEM at an accelerating voltage of 10kV. Images of 500 bacterial cells were obtained, classified into two groups based on staining intensity, and the ratio was determined.

[0052] In the colony formation method, bacteria after freezing and freeze-drying were diluted in 10 steps under anaerobic conditions using anaerobic PBS, seeded on Colombian blood agar plates, and cultured at 37°C for 48 to 72 hours. Bacterial viability was calculated as the ratio of the number of bacteria that formed colonies during culture to the number of bacteria at the start of culture.

[0053] Figure 4 shows the results of measuring the survival rate of bacteria after freezing and freeze-drying using the method of the present invention (scanning electromicroscopy), flow cytometry, or colony forming unit. The comparison revealed no statistically significant difference in the survival rates obtained by the three methods after freezing or freeze-drying.

[0054] As another example, bacteria (Akkermansia muciniphila) were suspended in Mueller Hinton medium (MHB) or a medium containing antioxidants and left at room temperature for 1 hour under anaerobic or aerobic conditions.

[0055] Figure 5 shows the bacterial ratios obtained by the method of the present invention and the survival rates obtained by the colony formation method, similar to the example described above. The comparison revealed no statistically significant difference in the survival rates obtained by the two methods under any of the conditions.

[0056] As shown in these examples, a correlation was observed between the survival rate obtained by conventional microbial survival rate testing methods and the ratio of microorganisms obtained by the method of the present invention. Furthermore, the method of the present invention allows for the observation of the morphology of individual microorganisms at the resolution of an electron microscope, enabling analysis of the presence or absence of antioxidants in the culture medium and abnormal morphologies caused by oxygen exposure. In other words, this embodiment demonstrates that it is possible to capture the type and morphological characteristics of microorganisms using an electron microscope and to evaluate their survival rate. [Examples]

[0057] Example 3 describes an example of quantifying the effect of a treatment that has a bactericidal effect on microorganisms by analyzing electron microscope images of PTA-stained microorganisms.

[0058] As an example, Figure 6 shows an example of a backscattered electron image obtained by SEM observation at an accelerating voltage of 5kV. The sample was prepared by culturing a Pseudomonas aeruginosa strain in a control group (untreated with antibiotics) and a test group (treated with the antibiotic colistin) at 37°C for 30 minutes, then collecting the bacteria on a track-etched membrane, fixing with 2.5% glutaraldehyde, and then electron-staining with a 10% PTA aqueous solution (by weight) and, for comparison, with a platinum blue aqueous solution for 2 minutes.

[0059] In specimens stained with PTA aqueous solution, images of bacteria with low image brightness were observed in the control group, while images of bacteria with high image brightness were observed in the test group. On the other hand, in specimens stained with platinum blue aqueous solution, the image brightness of bacteria in both the control and test groups was high, and no significant difference was observed between the control and test groups.

[0060] In addition to the staining solutions shown in the figure, when specimens were stained with a PTA aqueous solution adjusted to a neutral pH, the brightness of bacterial images in the test group was low, similar to the control group. On the other hand, when stained with sodium tungstate aqueous solution or ammonium molybdate aqueous solution, no positive staining images were observed in specimens from either the control or test group. No significant differences were observed between the control and test groups with these three types of staining solutions.

[0061] Thus, by selecting a 10% PTA aqueous solution by weight as the staining agent, it is possible to differentiate between bacteria in the control group (which was not treated with an antibiotic) and bacteria in the test group (which was treated with an antibiotic) based on the difference in image brightness.

[0062] Figure 6 shows the effect of colistin on Pseudomonas aeruginosa by PTA staining, but this example is not limited to this combination. As another example, when Escherichia coli and Pseudomonas aeruginosa were treated with the bactericidal β-lactam antibiotic imipenem, the number of bacteria with high image intensity increased by PTA staining.

[0063] Thus, by selecting an electron staining agent that increases the staining intensity of microorganisms that have experienced bactericidal activity, it is possible to classify images of bacteria damaged by bactericidal activity from those that have not been damaged by the bactericidal activity based on the difference in image brightness during electron microscopy. In particular, strong PTA staining in colistin-treated bacteria is thought to be related to survival due to damage to the membrane and cell wall. That is, by classifying and quantifying PTA-stained bacteria, the bactericidal effect of the antibacterial agent colistin can be analyzed and evaluated.

[0064] In this example, and in any case where a treatment affecting microorganisms is performed, by utilizing the fact that the intensity of electron staining changes depending on the state of the microorganisms, it is possible to obtain images of the control group and the test group, determine the microbial image brightness range that characterizes the microbial image of the control group and the microbial image brightness range that characterizes the microbial image of the test group, which has a different brightness from the control specimen, and then classify the microbial images of the control group and the test group and calculate the ratio, thereby analyzing and evaluating the effect of the treatment affecting the microorganisms.

[0065] For example, many antibacterial drugs have been developed that work by affecting the cell wall of bacteria, thereby inhibiting bacterial growth or exhibiting bactericidal activity. Since human cells lack a cell wall, compounds that selectively act on bacterial cell walls are expected to have fewer side effects on humans. The method described in this example can be used to evaluate the effects of such compounds and for screening new compounds based on their mechanism of action.

[0066] Furthermore, for infectious disease treatment and hospital infection control, it is crucial to quickly determine whether the causative bacteria are resistant or not, and drug susceptibility testing is performed on each bacterium isolated from a patient. Conventional methods determine resistance from the minimum inhibitory concentration (MIC) value that inhibits bacterial growth, but determining the MIC requires about one day of culturing. In contrast, this embodiment allows for rapid determination of the susceptibility of causative bacteria by analyzing whether or not damage has occurred due to the effect of the antibiotic.

[0067] Another application is the evaluation of bacteriophages that infect bacteria and induce lytic activity. Phages kill bacteria by destroying their membranes and peptidoglycans. Phage therapy is being re-evaluated as a treatment for multidrug-resistant bacteria, and there is a need for a rapid method to select a combination of phages specific to the causative bacteria from a library of phages found in the environment or artificially modified phages. In this example, the lytic activity of phages can be evaluated and used for phage screening.

[0068] As another example, when microorganisms, including not only bacteria but also fungi and archaea, are exposed to heat and oxygen, evaluating the decrease in their survival rate due to damage to the microorganisms can be used to select storage conditions or sterilization methods.

[0069] Using Figure 7, we will explain the results of analyzing the time course of the effect of the antibiotic colistin on Pseudomonas aeruginosa, as a concrete example of analyzing treatments that affect microorganisms, by treating Pseudomonas aeruginosa with the antibiotic colistin and measuring the brightness of bacterial images.

[0070] Based on MIC values ​​measured by conventional methods, we used Pseudomonas aeruginosa strains known to be susceptible to colistin and strains known to be resistant to colistin. Bacterial suspensions were initially placed in a culture medium at a concentration of 10⁶ / ml, treated with 2 mg / L of colistin (the concentration used to distinguish between susceptible and resistant strains by conventional methods), and cultured at 37°C. A fixed amount of the bacterial suspension was sampled at various time points.

[0071] A polycarbonate track-etched membrane with a pore diameter of 0.2 μm was used as specimen preparation material for SEM observation of bacteria, and bacteria were uniformly collected on a fixed area of ​​the membrane. The membrane surface was pre-treated with platinum-palladium to impart conductivity to the specimen.

[0072] After washing the culture medium components from the bacterial collection surface with physiological saline, the bacteria were fixed for 5 minutes with a 2.5% glutaraldehyde fixative, which has protein cross-linking properties, to prevent changes in bacterial morphology. After washing off excess fixative with water, the bacteria were stained with a 10% PTA aqueous solution by weight for 2 minutes. After washing off excess staining solution with water, the bacteria were dried on a track-etched membrane to prepare specimens for SEM observation.

[0073] The SEM observation conditions were set to an acceleration voltage of 5kV, backscattered electron detection, and a magnification of 7000x. The reason for using an acceleration voltage of 5kV is to prevent the image of the Pseudomonas aeruginosa from overlapping with the image of the holes in the tracked membrane caused by the electron beam that has passed through the bacterial cells. The reason for using a magnification of 7000x is to observe the morphology of the Pseudomonas aeruginosa, but these are not the only reasons.

[0074] As part of the observation process, before observing the bacterial specimens, the brightness contrast adjustment of the SEM was set to a constant level using a brightness standard sample, ensuring that the SEM brightness contrast adjustment remained consistent throughout the image acquisition process for all specimens. This facilitates subsequent image analysis.

[0075] The backscattered electron image series (701) in Figure 7 is an example of an image obtained by SEM observation using a colistin-sensitive Pseudomonas aeruginosa strain. Backscattered electron image (702) is an example of an image obtained by SEM observation of microorganism 1 found in a sample at 0 minutes of culture, which is Pseudomonas aeruginosa unaffected by colistin and equivalent to an untreated control sample. Backscattered electron image (703) is an example of an image obtained by SEM observation of microorganism 2 found after 8 minutes of culture, which is Pseudomonas aeruginosa with damaged membrane and cell wall due to the effect of colistin and stained with PTA.

[0076] A brightness profile was obtained for the image of microorganism 1 (702) to determine the image brightness range 1 (108) as exemplified in Figure 1. Similarly, a brightness profile was obtained for the image of microorganism 2 (703) to determine the image brightness range 2 (107) as exemplified in Figure 1.

[0077] Next, each specimen from which a backscattered electron image series (701) was acquired, as well as a control specimen prepared at the same time, were observed using SEM, acquiring 25 images and 300-500 images of bacterial cells per specimen. Note that the number of images and the number of bacterial cells imaged are not limited to these, as long as the number ensures the reproducibility of quantitative analysis.

[0078] Microbial image regions were extracted from all images taken of each specimen and classified into microorganism 1, characterized by image brightness range 1 (10⁸), and microorganism 2, characterized by image brightness range 2 (10⁷). This allowed identification of whether a microbial individual was unaffected by colistin or whether its membrane and cell wall were damaged by colistin. Furthermore, the classified individuals were counted, and the total number of microorganisms (microorganism 1 and microorganism 2) was taken as the total number of microorganisms, and the proportion of microorganism 1 was calculated. Since microorganism 1 is considered unaffected by colistin and is considered to be alive, a change in the proportion of microorganism 1 corresponds to a change in survival rate.

[0079] Graph (704) shows the time course of the proportion of microorganism 1 in bacteria sampled from the control group, and graph (705) shows the time course of the proportion of microorganism 1 in bacteria sampled from the colistin-treated test group. In the control group, the proportion of microorganism 1 hardly changed, but in the test group, the proportion of microorganism 1 decreased to 80% after 8 minutes, 60% after 30 minutes, and 0% after 60 minutes. This result indicates that the Pseudomonas aeruginosa strain analyzed is susceptible to colistin.

[0080] On the other hand, when colistin-resistant Pseudomonas aeruginosa strains were treated with colistin in the same way as susceptible strains, the proportion of microorganism 1 in the control group (corresponding to graph (704)) and the proportion of microorganism 1 in the test group (corresponding to graph (705)) hardly decreased, remaining at approximately 95% even after 60 minutes. This result indicates that colistin does not affect the Pseudomonas aeruginosa strains being analyzed.

[0081] As described above, quantitative analysis and comparison of the effects of colistin treatment on Pseudomonas aeruginosa makes it possible to show the difference between susceptible and resistant strains, and the results of the susceptibility / resistance determination were shown to be consistent with the determination based on the conventional method of MIC values.

[0082] According to this embodiment, while determining the MIC using conventional methods required approximately one day of culturing, it is possible to rapidly determine the susceptibility / resistance of the causative bacteria by analyzing the presence or absence of morphological damage caused by the effect of the antimicrobial agent.

[0083] Here, we will describe the characteristics of the specimens used in this embodiment. While other viability tests using microscopic observation techniques have been disclosed, viability determination reagents for optical microscopes are based on the enzyme activity of living cells and the selective permeability of the cell membrane, and the prerequisite for staining is that the cells are alive at the time of observation. In contrast, since the target of PTA staining is morphological damage during the process leading to bacterial death, staining and observation are possible even after fixation and preservation with glutaraldehyde, etc., improving the convenience of viability tests. Furthermore, with the recent availability of benchtop SEMs, there is potential for application to routine microbiological tests such as infectious disease testing, environmental monitoring, and food safety testing targeting microorganisms.

Claims

1. The first step involves obtaining an image of a stained specimen containing microorganisms using an electron microscope, A second step involves obtaining a luminance profile relating to the luminance distribution range of the aforementioned image, A third step involves setting a first standard luminance range in the luminance profile that satisfies a first condition related to luminance as the region where the first microbial group exists, and setting a second standard luminance range in the luminance profile that satisfies a second condition related to luminance as the region where the second microbial group exists. The fourth step includes identifying microorganisms present within each of the first and second standard brightness ranges, and / or calculating the ratio of microorganisms present within the first standard brightness range to those present within the second standard brightness range, The first condition is that the luminance profile includes a first peak and does not include a second peak that is different from the first peak. The second condition is that the first peak is not included, and the second peak is included. The fourth step is a microbial image analysis method, which involves identifying the viability and / or morphologically damaged microbial individuals present in each of the first and second standard brightness ranges, and / or calculating the survival rate of microorganisms and / or the ratio of morphologically damaged microorganisms based on the number of microorganisms and / or the microbial image area and the number of image pixels.

2. The microbial image analysis method according to claim 1, wherein the specimen is a smear specimen prepared by using a liquid containing microorganisms in such a way that the microorganisms and solid matter do not generally overlap with each other.

3. The microbial image analysis method according to claim 1, wherein the first step includes chemically fixing the microorganism using at least one of glutaraldehyde, formalin, or alcohol in order to stop and preserve the morphological changes of the microorganism.

4. The microbial image analysis method according to claim 1, wherein the first step is to stain the sample with a phosphotungstic acid solution.

5. The microbial image analysis method according to claim 4, wherein the phosphotungstic acid solution is an acidic solution with a pH of 0.0 to 7.

0.

6. The microbial image analysis method according to claim 4, wherein the phosphotungstic acid solution is an acidic solution with a pH of 0.0 to 3.

0.

7. The microbial image analysis method according to claim 4, wherein the phosphotungstic acid solution is a solution with a concentration of 0.1 to 20%.

8. The microbial image analysis method according to claim 4, wherein the phosphotungstic acid solution is a solution with a concentration of 2 to 10%.

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