Fecal sample evaluation method and fecal sample evaluation system

Abstract

Provided is a method capable of rapidly and simply evaluating the state of intestinal bacterial flora by using a fecal sample. The present invention provides a fecal sample evaluation method including: an immobilization step for immobilizing a fecal sample; a labeling step for labeling the fecal sample that has undergone the immobilization step; and a measurement step for subjecting the fecal sample that has undergone the labeling step to measurement using a flow cytometry device, wherein the labeling of the fecal sample in the labeling step involves labeling individual bacteria constituting a bacterial population contained in the fecal sample using a total bacterial count detection probe for detecting the total bacterial count and a viable bacterial count detection probe for detecting the viable bacterial count, and, in the measurement step, the ratio (B / A) of the viable bacterial count to the total bacterial count for the bacterial population contained in the fecal sample is obtained.

Description

Method for evaluating fecal sample and system for evaluating fecal sample

[0001] The present invention relates to a method for evaluating a fecal sample and a system for evaluating a fecal sample.

[0002] Various types of bacteria exist in the digestive tract, particularly the intestinal tract, of a host such as a human, and these bacteria form what is called the intestinal flora as a bacterial group. The state of the intestinal flora can be regarded as a barometer that indicates the health condition of the host. That is, for example, due to some trigger such as a change in the host's physical condition, unbalanced dietary habits, infection, or use of antibiotics, the total number of intestinal bacteria may significantly decrease, the composition ratio of the bacteria may change, or bacterial species that normally have a low bacterial count may abnormally increase, thereby disrupting the normal bacterial composition.

[0003] In recent years, a state in which the balance of intestinal flora is disrupted, in contrast to when the intestinal flora is in a balanced state, is generally called dysbiosis. It has become clear that as this dysbiosis progresses, the homeostasis of the intestinal environment cannot be normally maintained, which exerts various influences on the health condition of the host. For example, it has been revealed that dysbiosis is closely associated with the progression of various diseases such as inflammatory bowel disease, obesity, and diabetes.

[0004] One method for evaluating dysbiosis includes detecting bacteria contained in the intestinal flora by distinguishing between live bacteria and dead bacteria. This is because compared with live bacteria, dead bacteria cause fragmentation and lysis of bacteria, leakage of bacteria-related molecules, and the like, which cause increased intestinal permeability, induction of necrosis, exacerbation of inflammatory reactions, and the like due to the immune response of the host.

[0005] Conventionally, a technique for distinguishing and detecting live bacteria from dead bacteria in the gut microbiota has been known, which involves applying nucleic acid probes that specifically bind to ribosomal RNA across paneubacteria to fecal samples (Non-Patent Literature 1). Since RNA is rapidly degraded when bacteria die, using nucleic acid probes that specifically bind to ribosomal RNA allows only live bacteria to be detected. On the other hand, the total number of bacteria, including dead bacteria, can be detected by probes intercalated into DNA, allowing for a quantitative evaluation of live bacteria based on the total number of bacteria.

[0006] Hermie JM, et al. (2002) Extensive Set of 16S rRNA-Based Probes for Detection of Bacteria in Human Feces. Applied and Environmental Microbiology 68(6):2982-2990.

[0007] However, the technique described in Non-Patent Document 1 involved directly observing the fluorescence signal from nucleic acid probes labeled with fluorescent substances to detect live bacteria, along with the bacterial morphology, under a microscope. Therefore, it was not possible to evaluate fecal samples quickly and easily.

[0008] The present invention aims to overcome these inconveniences and provide a method for rapidly and easily evaluating the state of the intestinal microbiota, which can be considered a barometer of the host's health status, using fecal samples.

[0009] To achieve this objective, the present invention, in its first aspect, provides a method for evaluating a fecal sample, comprising: an immobilization step of immobilizing a fecal sample; a labeling step of labeling the fecal sample after the immobilization step; and a measurement step of subjecting the fecal sample after the labeling step to measurement using a flow cytometry apparatus, wherein the labeling of the fecal sample in the labeling step involves labeling individual bacteria constituting a bacterial population contained in the fecal sample using a total bacterial count detection probe for detecting the total bacterial count and a viable bacterial count detection probe for detecting the viable bacterial count, and in the measurement step, a measurement amount (A) corresponding to the total bacterial count contained in the bacterial population is detected based on the labeling of individual bacteria constituting the bacterial population using the total bacterial count detection probe, and a measurement amount (B) corresponding to the viable bacterial count contained in the bacterial population is detected based on the labeling of individual bacteria constituting the bacterial population using the viable bacterial count detection probe, thereby obtaining the ratio of viable bacteria to the total bacterial count (B / A) for the bacterial population contained in the fecal sample.

[0010] According to the fecal sample evaluation method provided by the present invention, by immobilizing the fecal sample and labeling it using a total bacterial count detection probe for detecting the total bacterial count and a viable bacterial count detection probe for detecting the viable bacterial count, live bacteria can be distinguished and detected from dead bacteria. Then, after labeling, the sample is subjected to measurement using a flow cytometry device to obtain the ratio of viable bacteria to the total bacterial count of the bacterial community contained in the fecal sample. This makes it possible to quickly and easily evaluate the state of the intestinal microbiota, which can be considered a barometer of the host's health status, using a fecal sample.

[0011] In the above method for evaluating fecal samples, it is preferable that the probe for detecting the total bacterial count is a probe containing a DNA intercalator molecule, and the probe for detecting the viable bacterial count is a probe containing a nucleic acid molecule having a common sequence of 16S rDNA of eubacteria.

[0012] According to this, a probe containing a DNA intercalator molecule can detect DNA derived from it regardless of whether the bacteria are alive or dead, and this can be correlated with the total bacterial count. Furthermore, a probe containing a nucleic acid molecule with a common 16S rDNA sequence can detect 16S rRNA contained in the 16S ribosomes of eubacteria when the bacteria are alive. However, when the bacteria are dead, the 16S rRNA released from the 16S ribosome is rapidly degraded by the bacteria's own RNA-degrading enzymes, etc., and is therefore not detected, and this can be correlated with the number of viable bacteria.

[0013] In the above-described method for evaluating fecal samples, it is preferable that in the measurement step, the detection of the measured amount (A) corresponding to the total bacterial count and the measured amount (B) corresponding to the viable bacterial count by the flow cytometry device is based on the number of bacteria detected with a signal strength equal to or greater than a predetermined threshold corresponding to the probe used for each detection.

[0014] According to this method, outliers in measurements can be excluded, allowing for a more accurate evaluation of fecal samples that reflect the proportion of live bacteria.

[0015] In the above method for evaluating fecal samples, it is preferable that the probe for detecting the total bacterial count contains a fluorescent substance, and the probe for detecting the viable bacterial count contains a fluorescent substance other than the aforementioned fluorescent substance.

[0016] This allows for improved measurement sensitivity and selectivity, enabling a more accurate evaluation of fecal samples that reflect the proportion of live bacteria.

[0017] In a second aspect, the present invention provides an evaluation system for evaluating a fecal sample by the evaluation method described above, comprising: a flow cytometry device for obtaining the ratio of viable bacteria to the total number of bacteria in a bacterial community contained in the fecal sample; and a computer device for displaying the health status of the host of the fecal sample, which is pre-associated according to the ratio of viable bacteria.

[0018] The fecal sample evaluation system provided by the present invention includes a flow cytometry device that obtains the ratio of viable bacteria to the total number of bacteria in the bacterial community contained in the fecal sample. Therefore, the state of the intestinal microbiota, which can be considered a barometer of the host's health, can be evaluated quickly and easily using the fecal sample. Furthermore, since it includes a computer device that displays the results of the fecal sample evaluation by referring to a pre-associated host health status according to the obtained ratio of viable bacteria, the results of the fecal sample evaluation can be presented to the user in an easy-to-understand manner.

[0019] In the above method for evaluating fecal samples, the health status is preferably at the level of dysbiosis or ulcerative colitis.

[0020] According to this method, the state of dysbiosis in the host intestine, which is often difficult to express as a disease state or symptom, can be quickly and easily categorized and presented to consumers using fecal samples. Furthermore, the state of ulcerative colitis in the host can be quickly and easily categorized and presented to consumers using fecal samples.

[0021] According to the present invention, the state of the intestinal microbiota, which can be considered a barometer of the host's health status, can be rapidly and easily evaluated using a fecal sample.

[0022] This is a flowchart of the fecal sample evaluation method according to the present invention. This is a schematic diagram of the fecal sample evaluation system according to the present invention. In Test Example 1, this figure shows an example of an observation image by fluorescence microscopy when a fecal sample was evaluated by a conventional method. The left figure shows the observation image based on the DAPI signal, the center figure shows the observation image based on the Cy5-labeled Eub338 signal, and the right figure shows an observation image obtained by merging (combining) the observation images based on those signals. In Test Example 2, this figure shows an example of an analysis plot when a fecal sample was evaluated using a flow cytometry device. The left figure shows the analysis plot based on the Cy5-labeled Non-Eub338 signal, and the right figure shows the analysis plot based on the Cy5-labeled Eub338 signal. In Test Example 3, this figure shows the results of investigating the amount of calprotectin, IgG, complement 3 (C3), and adenosine triphosphate (ATP), which are indicator molecules of intestinal permeability that are thought to be related to the pathogenesis of ulcerative colitis, in the fecal supernatant for each patient group and healthy control group. Figure 7A is a scatter plot showing the correlation between the obtained viable cell count (%LB) and the amount of calprotectin, an indicator molecule for intestinal permeability, in Test Example 3. Figure 7B is a scatter plot showing the correlation between the obtained viable cell count (%LB) and the disease state score of each patient separately evaluated in Test Example 3. This chart shows the results of the investigation into the relationship with intestinal status in Test Example 4. It is a scatter plot showing the correlation between the obtained viable cell count (%LB) value and the amount of calprotectin, an indicator molecule of intestinal permeability.This figure shows the results of an investigation into the relationship between the intestinal status of the elderly group in Test Example 4. It shows the results of determining the Eub338 index (EI), which is the relative abundance of bacterial taxa in the viable cell fraction of the bacterial population contained in fecal samples from the elderly. This figure shows the results of an investigation into the relationship between the intestinal status of the elderly group in Test Example 4. It is a scatter plot showing the correlation between the relative abundance (EI) of bacteria belonging to the Bifidobacteriae family and the abundance of calprotectin, an indicator molecule of intestinal permeability. This figure shows the results of an investigation into the relationship between the intestinal status of the elderly group in Test Example 4. It is a scatter plot showing the correlation between the obtained viable cell rate (%LB) value and the relative resistance value when transepithelial electrical resistance (TEER) was evaluated separately using the T84 cell monolayer test, a model system for the intestinal epithelium. This chart shows the results of an investigation into the relationship between the gut microbiome of healthy individuals in Test Example 4, and shows the results of determining the Eub338 index (EI), which is the relative abundance of bacterial taxa in the viable fraction of the bacterial population contained in fecal samples from healthy individuals. This chart shows the results of an investigation into the relationship between the gut microbiome of healthy individuals in Test Example 4, and is a scatter plot showing the correlation between the obtained viable cell rate (%LB) value and the stool quality score (BS) according to the Bristol Stool Scale. This chart shows the results of an investigation into the relationship between the gut microbiome of healthy individuals in Test Example 4, and is a scatter plot showing the correlation between the obtained viable cell rate (%LB) value and the interval between bowel movements. This chart shows the results of an investigation into the relationship between the gut microbiome of healthy individuals in Test Example 4, and is a scatter plot showing the correlation between the obtained viable cell rate (%LB) value and the frequency of bowel movements. This figure shows the results of an investigation into the relationship between the intestinal status of the healthy control group in Test Example 4, and is a scatter plot showing the correlation between the obtained viable cell count (%LB) value and the abdominal distension tension score during defecation. This is a schematic chart explaining the technical significance brought about by the fecal sample evaluation method or evaluation system according to the present invention.

[0023] The present invention will be described in more detail below with reference to the drawings.

[0024] Figure 1 shows a flowchart of the fecal sample evaluation method according to the present invention.

[0025] As shown in Figure 1, in the fecal sample evaluation method according to the present invention, the fecal sample is immobilized (indicated as S1 in the figure). In this immobilization step, the fecal sample is treated with an immobilizing agent commonly used for staining tissues and cells, thereby immobilizing the individual bacteria contained in the sample and preventing instability such as leakage of biological components from the bacteria. Furthermore, the state of DNA fragmentation, degradation, etc., related to the life and death of the bacteria is preserved, resulting in a sample that retains that state well. Therefore, this makes it possible to perform labeling with a probe, as described later, and subsequent measurement using a flow cytometry device on the bacterial population contained in the fecal sample collected from the host.

[0026] Treatment with an immobilizing agent can be carried out by suspending the fecal sample in an aqueous solution such as a buffer solution containing the immobilizing agent. Examples of immobilizing agents include aldehyde-based immobilizing agents such as paraformaldehyde, formaldehyde, and glutaraldehyde, although these are not limited to those mentioned above. The concentration of the immobilizing agent can be set appropriately, for example, 0.5 to 5.0 v / v%. Typically, for paraformaldehyde, the concentration can be 3.0 to 4.0 v / v%. Treatment with an immobilizing agent can be carried out, for example, at a temperature of 4 to 10°C for 12 to 24 hours. Furthermore, larger contaminants than bacteria can be removed by passing the fecal sample, while suspended in an aqueous solution such as a buffer solution, through a mesh or filter with an appropriate pore size. The removal of impurities using a mesh or filter may be performed before, after, or both before and after treatment with the immobilizing agent. The fecal sample after treatment with the immobilizing agent can be recovered by separating the solid and liquid by centrifugation or the like.

[0027] As shown in Figure 1, in the fecal sample evaluation method according to the present invention, the fecal sample that has undergone an immobilization step is labeled (indicated as S2 in the figure). In this labeling step, the fecal sample treated with an immobilization agent is labeled by treating it with at least two types of probes. One of the at least two types of probes is a total bacterial count detection probe for detecting the total bacterial count, and the other is a viable cell count detection probe for detecting the viable cell count. More specifically, labeling means that the individual bacteria constituting the bacterial population contained in the fecal sample bind to one or both of the at least two types of probes, and produce a specific signal from that probe. Therefore, in the labeling of the fecal sample described here, the individual bacteria constituting the bacterial population contained in the fecal sample are labeled in such a way that they either produce a specific signal from the total bacterial count detection probe, a specific signal from the viable cell count detection probe, or both signals. In some cases, among the bacteria constituting the bacterial population, some may not produce any signal or may produce a weak signal even after this labeling process. However, as long as the viability and viability of the bacteria that make up the majority of the bacterial population can be determined, this does not hinder the objective of the present invention.

[0028] The probe used for detecting the total bacterial count can be any probe capable of producing a signal corresponding to the total bacterial count of the bacterial population contained in a fecal sample; there are no particular restrictions, but probes containing DNA intercalator molecules are preferred examples. Probes containing DNA intercalator molecules can be bound to DNA derived from bacteria, whether alive or dead, by intercalation, thereby allowing them to correspond to the total bacterial count of the bacterial population contained in the fecal sample. Examples of DNA intercalator molecules include "Syto BC" and "Syto 9" manufactured by Thermo Fisher Scientific. These nuclear stains are cell-permeable and diffuse well into bacterial cells, allowing for more efficient labeling of bacteria.

[0029] The probe used for detecting viable cell counts can be any probe capable of generating a signal corresponding to the viable cell count of a bacterial population contained in a fecal sample; there are no particular restrictions. However, a probe containing a nucleic acid molecule having a common sequence of 16S rDNA of eubacteria is a preferred example. A probe containing a nucleic acid molecule having a common sequence of 16S rDNA of eubacteria can broadly match the 16S rDNA sequences of bacteria that constitute the majority of the bacterial population in a fecal sample, bind to them by hybridization, and detect them based on the resulting signal. Furthermore, if the bacteria are dead, the 16S rRNA released from the 16S ribosome is rapidly degraded by the bacteria's own RNA-degrading enzymes, etc., and is therefore not detected. This allows the probe to correspond to the viable cell count of the bacterial population contained in the fecal sample. An example of a probe containing a nucleic acid molecule having a common sequence of 16S rDNA of eubacteria is "Eub338" manufactured by Sigma-Aldrich.

[0030] The above-mentioned probe is preferably a fluorescent substance itself or chemically modified with a fluorescent substance. This allows for more accurate detection of the target bacteria by measuring the signal produced by bacteria labeled with the probe as fluorescence emitted by the fluorescent substance. Chemical modification with a fluorescent substance can be carried out according to conventional methods. For example, DNA intercalator molecules or nucleic acid molecules can be chemically modified with a fluorescent substance in a binding manner that does not impair their intercalation or hybridization functions. Without limitation, chemical modification can be carried out by, for example, covalent bonding via a linker molecule.

[0031] Preferably, the probes described above contain different fluorescent substances for labeling the total bacterial count probe and the viable bacterial count probe. More specifically, it is preferable that the detection wavelengths for the fluorescence emitted by these fluorescent substances are different. By using different detection wavelengths, even when a bacterial population is labeled using both probes, the signals from individual bacteria labeled by the total bacterial count probe and the signals from individual bacteria labeled by the viable bacterial count probe can be detected with a single measurement. Examples of fluorescent substances include cyanine-based synthetic dyes belonging to polymethine, such as Cy3 and Cy5, and fluorescein dyes such as FITC.

[0032] The labeling process using the above-described probe can be carried out by suspending the immobilized fecal sample in an aqueous solution such as a buffer solution containing the probe to be used. The fecal sample may be labeled using at least two types of probes, one type at a time, or two or more types may be used together. The labeling process is not limited, but for example, it can be carried out at a temperature of 38 to 42°C for 12 to 16 hours. As mentioned above, the fecal sample can be suspended in an aqueous solution such as a buffer solution and passed through a mesh or filter with an appropriate pore size to remove impurities larger than bacteria. Such removal of impurities using a mesh or filter may be performed before the labeling process, after the process, or both before and after the process. The fecal sample after labeling with the above-described probe can be recovered by solid-liquid separation by centrifugation or the like.

[0033] While not limited to these methods, it is preferable to dehydrate the fecal sample after treatment with an immobilizing agent but before labeling by treating it with alcohol-containing water containing 80 v / v% or more, preferably 85 v / v% or more, more preferably 90 v / v% or more, and most preferably 95 v / v% or more of alcohol. Dehydration removes water, improving the transfer and binding of each probe to nucleic acids, and consequently improving the sensitivity and selectivity of detection. Ethanol is preferred as the alcohol. The dehydration treatment is not limited to these methods, but can be carried out, for example, at a temperature of 15 to 25°C for 5 to 15 minutes. After dehydration with alcohol-containing water, further washing with an aqueous solution such as a buffer solution may be performed.

[0034] (Bacterial count in fecal samples) The bacterial count in fecal samples can be measured separately by methods such as flow cytometry or microscopic observation to standardize the amount of bacteria used in various treatments. For example, although not limited to these, samples can be collected according to the following rules based on the bacterial count per gram of feces and used for the labeling treatment described above.

[0035] Bacterial count (cells / g fecal sample) Number of bacteria to collect Less than 10^10: Volume equivalent to 2 x 10^7 bacteria 10^10 or more but less than 10^11: Volume equivalent to 3 x 10^7 bacteria 10^11 or more: Volume equivalent to 4 x 10^7 bacteria

[0036] As shown in Figure 1, in the fecal sample evaluation method according to the present invention, the fecal sample that has undergone a labeling step is subjected to measurement using a flow cytometry apparatus (indicated as S3 in the figure). In this measurement step, the fecal sample that has undergone labeling treatment is measured using a flow cytometry apparatus. This makes it possible to detect the intensity (luminance) of the signal emitted by each individual bacterium that constitutes the bacterial population contained in the fecal sample, using the principle of a commonly known flow cytometry apparatus. Thus, it is possible to distinguish and detect bacteria labeled with a probe for detecting the total bacterial count and bacteria labeled with a probe for detecting the viable bacterial count, as bacteria that emit signals specific to each probe. More specifically, if the probe is a fluorescent substance itself or a probe that has been chemically modified with a fluorescent substance, the intensity (luminance) of the fluorescent signal emitted by each individual bacterium that constitutes the bacterial population can be detected. Furthermore, in bacteria labeled by both probes, each probe emits a unique signal together. However, by equipping the detection device with an optical filter that matches that unique signal, the unique signals emitted by each probe from a single bacterium can be distinguished and detected.

[0037] Measurement using a flow cytometry apparatus can be performed by suspending a labeled fecal sample in a measurement solution such as PBS and supplying it to the apparatus. In this case, if necessary, the labeled fecal sample may be washed with an aqueous solution such as a buffer solution before being suspended in the measurement solution such as PBS and supplied to the apparatus. The method of supplying to the apparatus can be in accordance with the specifications of the apparatus being used, but it is generally preferable to use a sample in which the number of particles taken up per second falls within a predetermined range. If the number of particles taken up exceeds a predetermined value, the suspended sample can be diluted, or conversely, if it falls below a predetermined value, a sample with a higher suspension concentration can be prepared and remeasured. The number of particles taken up is not limited, but it is preferable to use a sample in which the number of particles taken up is, for example, 800 to 12,000 (events / sec).

[0038] In this invention, by detecting a measured quantity (A) corresponding to the total bacterial count using a probe for detecting the total bacterial count from measurements using the flow cytometry apparatus, and detecting a measured quantity (B) corresponding to the viable bacterial count using a probe for detecting the viable bacterial count, the ratio of viable bacteria to the total bacterial count (B / A) of the bacterial population contained in the fecal sample is obtained. Here, "measured quantity (A) corresponding to the total bacterial count" may be the cumulative number of bacteria for which a signal originating from the probe for detecting the total bacterial count was detected by measurement using the flow cytometry apparatus. Alternatively, it may be a weighted average of the signal strength from the probe for detecting the total bacterial count for individual bacteria, and may be a weighted average over time. Furthermore, "measured quantity (B) corresponding to the viable bacterial count" may be the cumulative number of bacteria for which a signal originating from the probe for detecting the viable bacterial count was detected by measurement using the flow cytometry apparatus. Alternatively, it may be a weighted average of the signal strength from the probe for detecting the viable bacterial count for individual bacteria, and may be a weighted average over time. The ratio of viable bacteria (B / A) may also be multiplied by 100 and expressed as a percentage, which is called the viability rate (unit: %).

[0039] In any non-limiting embodiment of the present invention, in the measurement using the flow cytometry apparatus described above, the detection of the measured quantity (A) corresponding to the total bacterial count and the measured quantity (B) corresponding to the viable bacterial count may be based on the number of bacteria detected with a signal strength above a predetermined threshold corresponding to the probe used for each detection. More specifically, for example, if the signal strength from a certain bacterium is below a predetermined value, that bacterium is not counted in the cumulative count, and is only counted if the signal strength from the bacterium is above a predetermined value. Setting such thresholds and measuring the cumulative count while omitting weak signals based on them is a function that is usually provided in flow cytometry apparatuses. By adopting such an embodiment, abnormal values ​​in the measurement can be eliminated, and the evaluation of fecal samples that reflect the proportion of live bacteria can be performed more accurately.

[0040] Figure 2 shows a schematic diagram of the fecal sample evaluation system according to the present invention.

[0041] As shown in Figure 2, the fecal sample evaluation system according to the present invention comprises a flow cytometry device 1 for carrying out the aforementioned fecal sample evaluation method. The system also comprises a computer device 2 that receives data transmitted from the flow cytometry device 1. In the computer device 2, an evaluation result can be displayed on a display unit 3 thereof based on the data from the flow cytometry device 1. Here, the evaluation result is the ratio of the number of viable bacteria to the total number of bacteria (B / A) for the bacterial population contained in the fecal sample obtained by the aforementioned evaluation method. The system is configured to be capable of referencing and displaying the health status of the host of the fecal sample that is pre-associated in accordance with the ratio of the number of viable bacteria. More specifically, although the embodiment is not limited thereto, for example, the association between data of the ratio of the number of viable bacteria (B / A) and the level of intestinal dysbiosis as the health status of a subject can be established as described below, and the associated level can be displayed on the display unit 3 based on the result of each fecal sample. It goes without saying that the data of the ratio of the number of viable bacteria (B / A) can also be handled as data of the viable bacteria rate (%) which is grasped as a percentage thereof.

[0042] (Association) Ratio of viable bacteria (B / A) more than 70: Level 1 Ratio of viable bacteria (B / A) more than 55 and 70 or less: Level 2 Ratio of viable bacteria (B / A) more than 40 and 55 or less: Level 3 Ratio of viable bacteria (B / A) more than 20 and 40 or less: Level 4 Ratio of viable bacteria (B / A) 0 or more and 20 or less: Level 5

[0043] By adopting such an aspect, the result of evaluation of a fecal sample can be presented to consumers in an easily understandable manner. In addition, the state of dysbiosis in the host intestine, which is less likely to appear as a pathological condition or symptom, can be quickly and simply leveled using a fecal sample and presented to consumers.

[0044] Furthermore, the subject's health condition can be set arbitrarily. For example, in another embodiment, the state of ulcerative colitis can be associated with its state level stepwise in the same manner as described above with the data of the ratio of the number of viable bacteria (B / A). Then, based on the results of each fecal sample, the associated state level can be displayed on the display unit 3. This makes it possible to rapidly and easily level the state of the host's ulcerative colitis using a fecal sample and present it to the demander.

[0045] The present invention will be described more specifically with reference to examples below. However, these examples do not limit the scope of the present invention.

[0046] [Test Example 1] Fecal samples were evaluated by the conventional FISH method. Specifically, the fecal samples were labeled with a fluorescent probe and observed under a microscope as follows.

[0047] (Fluorescent Probe) ・Probe for detecting total bacterial count: DAPI (manufactured by Vector Laboratories) ・Probe for detecting viable bacterial count: "Eub338 Cy5" (Cy5-labeled at the 5' end of HPLC-purified synthetic oligonucleotide (5'-GCTGCCTCCCGTAGGAGT-3')) (hereinafter, may be simply referred to as "Eub338", "Eub338 Cy5", or "Cy5-labeled Eub338") ・Control probe consisting of a complementary strand of the probe for detecting viable bacterial count: "NON Eub338 Cy5" (Cy5-labeled at the 5' end of HPLC-purified synthetic oligonucleotide (5'-ACTCCTACGGGAGGCAGC-3')) (hereinafter, may be simply referred to as "Non Eub338", "Non Eub338 Cy5", or "Cy5-labeled Non Eub338")

[0048] (Immobilization) A fecal specimen was provided by one volunteer, which was diluted 10-fold with a PBS solution to prepare a suspension of the fecal sample. Three volumes of 4 (v / v)% paraformaldehyde (PFA)-PBS solution was added to this suspension (1 / 40 dilution of the fecal sample), and the mixture was allowed to stand overnight at 4°C.

[0049] (Labeling) The immobilized sample (10 μL) was diluted 100 to 400 times with cold PBS and measured on a 1 cm MAS-coated glass slide (Matsunami Glass Industry Co., Ltd.). ２ The smears were spread evenly on a frame. After dehydrating and drying by immersion in 96 v / v% ethanol for 10 minutes, 100 μL of hybridization solution (750 mM NaCl, 100 mM Tris-HCl [pH 7.8], 5 mM EDTA, 0.01 w / v% BSA, 0.2 w / v% poly-A, 10 w / v% dextran sulfate) containing 450 ng of Cy5-labeled Eub338 or Cy5-labeled Non-Eub338 was dropped onto the cell smears. The smears were covered with coverslips, and the slides were incubated overnight at 45°C in a dark box humidified with SET solution (750 mM NaCl, 100 mM Tris-HCl, 5 mM EDTA). After the hybridization reaction was complete, the glass slides were incubated in a washing solution (50 mM NaCl, 4 mM Tris-HCl, 0.02 mM EDTA) at 50°C for 20 minutes. Then, the glass slides were washed again with distilled water, air-dried, and the bacteria were embedded in a mounting medium containing the nuclear staining reagent DAPI.

[0050] (Microscopic observation) The slides prepared as described above were observed using a fluorescence microscope (product name "Leica Q550FW", manufactured by Leica), and images were taken.

[0051] Figure 3 shows an example of the observed images. In the observation image on the right in Figure 3, some bacteria, as indicated by the black arrow, produced signals from both probes, while others, as indicated by the white arrow in the same figure, produced a signal only from the DAPI probe, which is used to detect the total bacterial count. Specifically, of the seven bacteria indicated by both black and white arrows, six produced a signal from Eub338, and one did not. Limiting the bacterial population to these seven, the proportion of bacteria considered to be viable was calculated to be "6 / 7 (0.857142...)", which, when converted to a percentage, was "85.7142...%". However, it was clear that increasing the bacterial population to the necessary number for proper evaluation of fecal samples would be too cumbersome if done by microscopic observation.

[0052] [Test Example 2] (Fluorescent Probe) ・Probe for detecting total bacterial count: Product name "FITC-labeled SYTO BC" (manufactured by Thermo Fisher) (Hereinafter, it may simply be referred to as "SYTO BC" or "FITC-labeled SYTO BC".) ・Probe for detecting viable cell count: "Eub338 Cy5" (a synthetic oligonucleotide (5'-GCTGCTCCCGTAGGAGT-3') purified by HPLC with Cy5 labeled at the 5' end) (Hereinafter, it may simply be referred to as "Eub338" or "Eub338 Cy5" or "Cy5-labeled Eub338".) ・Control probe consisting of the complementary strand of the viable cell count detection probe: "NON Eub338 "Cy5" (a synthetic oligonucleotide (5'-ACTCCTACGGGGAGGCAGC-3') purified by HPLC with Cy5 labeled at its 5' end) (Hereafter, it may simply be referred to as "Non-Eub338," "Non-Eub338 Cy5," or "Cy5-labeled Non-Eub338.")

[0053] (Immobilization) A fecal sample was provided by one volunteer, and this was diluted 10-fold with PBS solution to prepare a suspension of the fecal sample. Three times the volume of 4 (v / v)% paraformaldehyde (PFA)-PBS solution was added to this suspension (a 1 / 40 dilution of the fecal sample), and it was left standing overnight at 4°C.

[0054] (Labeled) Immobilized sample (12 μL) (Bacterial count: 3 × 10⁶) ７ 500 μL of PBS was added to the equivalent of 1 cell and filtered through a 200-mesh nylon filter. The precipitate was collected by centrifugation and washed with 500 μL of PBS. 500 μL of 96 v / v% ethanol was added to the centrifuged precipitate and dehydrated at 20°C for 10 minutes. The precipitate was collected by centrifugation and washed with 500 μL of PBS. It was then suspended in 100 μL of a hybridization solution (750 mM NaCl, 100 mM Tris-HCl [pH 7.8], 5 mM EDTA, 0.01 w / v% BSA, 0.2 w / v% poly-A, 10 w / v% dextran sulfate) containing 450 ng of Cy5-labeled Eub338 or Cy5-labeled Non-Eub338, and incubated overnight at 45°C. After the hybridization reaction was complete, a washing solution (50 mM NaCl, 4 mM Tris-HCl [pH 7.8], 0.02 mM EDTA) that had been preheated to 40°C was added, and the mixture was centrifuged at room temperature. Then, 1 mL of this solution was added and the mixture was incubated at 40°C for 20 minutes. The precipitate was collected by centrifugation, washed with PBS (500 μL), and then suspended in 350 μL of FITC-labeled SytoBC / PBS (4000-fold dilution) solution.

[0055] (Measurement using a flow cytometry instrument) The suspension prepared as described above was subjected to a flow cytometry instrument (product name "CytoFLEX", manufactured by Beckman Coulter), and the acquired data was analyzed using the accompanying software "CytoExpert v2.4" (manufactured by Beckman Coulter). For this measurement, the fecal sample was loaded into the instrument to achieve a bacterial count of approximately 800 to 12,000 bacteria per second.

[0056] Figure 4 shows an example of the analysis plot obtained from the analysis. As shown in the left panel of Figure 4, when a control probe (Cy5-labeled Non Eub338) consisting of the complementary strand of the viable cell count detection probe was used, the proportion of event dots (corresponding to the number of bacteria) with an intensity exceeding a predetermined threshold (brightness of 4500 or more) accounted for only 0.1% of the event dots resulting from the signal of the total bacterial count detection probe (FITC-labeled SYTO BC). In contrast, as shown in the right panel of Figure 4, when the viable cell count detection probe (Cy5-labeled Eub338) was used, the proportion of event dots (corresponding to the number of bacteria) with an intensity exceeding a predetermined threshold (brightness of 4500 or more) accounted for 54.6% of the event dots resulting from the signal of the total bacterial count detection probe (FITC-labeled SYTO BC). This value is based on a signal specific to the viable cell count detection probe, and was therefore considered to represent the proportion of viable cells to the total bacterial population in the fecal sample. Furthermore, for the signal from the total bacterial count detection probe (FITC-labeled SYTO BC), only event dots (corresponding to bacterial counts) with an intensity exceeding a predetermined threshold (brightness of 4500 or higher) were included; all others were excluded to prevent errors in the evaluation.

[0057] From the above, it has become clear that the proportion of viable bacteria among the total number of bacteria in a bacterial community contained in a fecal sample can be rapidly and easily evaluated using a flow cytometry device.

[0058] [Test Example 3] The fecal sample evaluation method according to the present invention was applied to fecal samples provided by patients with ulcerative colitis or healthy individuals. Fecal samples were provided from 17 patients with active ulcerative colitis (aUC), 18 patients with inactive ulcerative colitis (inUC), and 25 healthy individuals (HA).

[0059] First, the characteristics of fecal samples from both the patient and healthy control groups were investigated. Specifically, the amounts of calprotectin, IgG, complement 3 (C3), and ATP—indicator molecules of intestinal permeability thought to be related to the pathogenesis of ulcerative colitis—were examined. The amounts were measured by quantifying the amount of each molecule present in the supernatant of the PBS suspension of the fecal samples using conventional methods, and the results are shown converted to per gram of feces.

[0060] As a result, as shown in Figure 5, patients with active ulcerative colitis (aUC) and inactive ulcerative colitis (inUC) showed elevated levels of calprotectin, IgG, complement 3 (C3), and ATP in the fecal supernatant compared to healthy individuals (HA), and these levels correlated well with the pathogenesis of ulcerative colitis, as previously reported.

[0061] Next, the fecal sample evaluation method according to the present invention was applied to the same fecal sample. Specifically, the fecal sample was immobilized, labeled, and measured using a flow cytometry apparatus following the same procedure as in Test Example 2. The results were expressed as the viability rate (%LB), which is the ratio of viable bacteria to the total number of bacteria as a percentage.

[0062] As a result, as shown in Figure 6, patients with active ulcerative colitis (aUC) and inactive ulcerative colitis (inUC) had lower viable cell counts (%LB) compared to healthy individuals (HA). Furthermore, as shown in Figure 7, a negative correlation was observed between the obtained viable cell count (%LB) values ​​and the amount of calprotectin, an indicator molecule of intestinal permeability, present in the fecal supernatant (Figure 7A) and the disease state scores of each patient evaluated separately (Figure 7B).

[0063] From the above, it has become clear that, according to the fecal sample evaluation method of the present invention, the properties of fecal samples that correlate well with the pathology of ulcerative colitis can be rapidly and easily evaluated using a flow cytometry device.

[0064] [Test Example 4] The fecal sample evaluation method according to the present invention was applied to fecal samples provided by elderly or healthy individuals. Fecal samples were provided from 109 elderly individuals aged 71 years or older (Elderly) and 93 other healthy individuals (HA).

[0065] Specifically, the fecal samples were immobilized, labeled, and measured using a flow cytometry apparatus, following the same procedure as in Test Example 2. The results were expressed as the viability rate (%LB), which is the percentage of viable bacteria relative to the total bacterial count.

[0066] As a result, as shown in Figure 8, the percentage of viable bacteria (%LB) was lower in elderly individuals (Elderly) compared to healthy individuals (HA). Furthermore, as shown in Figure 9, a negative correlation was observed between the obtained percentage of viable bacteria (%LB) and the amount of calprotectin, an indicator molecule of intestinal permeability, present in the fecal supernatant.

[0067] (Elderly) For fecal samples from elderly individuals, bacteria showing a signal of a certain intensity or higher using a viable bacteria detection probe were collected using the sorting function of a flow cytometry instrument (product name "FACSAria Fusion," manufactured by Beckton Dickinson). These samples were then subjected to next-generation sequencing for bacterial flora analysis. The bacterial flora analysis was performed according to standard procedures. This allowed us to determine the Eub338 index (EI), which represents the relative abundance of bacterial taxa in the viable bacterial fraction of the bacterial population contained in the fecal samples from elderly individuals.

[0068] On the other hand, regarding calprotectin, an indicator molecule for intestinal permeability, its amount in the supernatant of the PBS suspension of the fecal sample was quantified in the same manner as in Test Example 3, and this value was converted to a value per gram of feces.

[0069] The results were evaluated by classifying the samples into two groups, a high %LB group (%LB_high) and a low %LB group (%LB_low), based on the median viability (%LB = 48.3% in this study) measured by the flow cytometry apparatus described above.

[0070] As a result, as shown in Figure 10, the relative abundance (EI) of bacteria belonging to the families Bifidobacteriaceae, Lachnospiraceae, and Butyricococcaeaceae was reduced in the low %LB group (%LB_low) compared to the high %LB group (%LB_high).

[0071] Furthermore, as shown in Figure 11, a negative correlation was observed between the relative abundance (EI) of bacteria belonging to the Bifidobacteriaceae family and the amount of calprotectin, an indicator molecule of intestinal permeability, in the fecal supernatant.

[0072] Furthermore, in a separate T84 cell monolayer test, a model system for the intestinal epithelium, the transepithelial electrical resistance (TEER) of fecal supernatant from elderly individuals was evaluated according to a standard method, and a positive correlation was observed between the value of viable bacteria (%LB) and the T84 cell monolayer test, as shown in Figure 12.

[0073] From the above, it has become clear that, according to the fecal sample evaluation method of the present invention, the properties of fecal samples that correlate well with the intestinal condition of elderly people can be evaluated quickly and easily using a flow cytometry device.

[0074] (Healthy individuals) Fecal samples from healthy individuals were collected using the sorting function of a flow cytometry instrument, and bacteria showing a signal of a viable bacteria detection probe at or above a predetermined intensity were collected and then subjected to next-generation sequencing for microbial community analysis.

[0075] The results were evaluated by classifying the cells into two groups, a high %LB group (%LB_high) and a low %LB group (%LB_low), using the median viability rate (%LB) measured by the flow cytometry apparatus described above (in this example, %LB = 59.0%).

[0076] As a result, as shown in Figure 13, the relative abundance (EI) of bacteria belonging to the families Lachnospiraceae, Selenomonadaceae, and Oscillospiraceae was reduced in the low %LB group (%LB_low) compared to the high %LB group (%LB_high).

[0077] Furthermore, as shown in Figure 14, the viability rate (%LB), which is the percentage of live bacteria in the bacterial population contained in fecal samples from healthy individuals, showed a positive correlation with the Bristol Stool Scale score (BS) (Figure 14A) and defecation frequency (Figure 14C). In addition, a negative correlation was observed between the defecation interval (Figure 14B) and the abdominal distension tension score during defecation (Figure 14D).

[0078] From the above, it has become clear that, according to the fecal sample evaluation method of the present invention, the properties of fecal samples that correlate well with the defecation status of healthy individuals can be evaluated quickly and easily using a flow cytometry device.

[0079] Figure 15 shows a schematic chart illustrating the technical significance of the fecal sample evaluation method or evaluation system according to the present invention. According to the present invention, the ratio of live bacteria (B / A) based on the total number of bacteria in the bacterial population contained in the fecal sample is obtained, so the state of dysbiosis in the host intestine, which conventionally does not easily manifest as pathological conditions or symptoms, can be evaluated quickly and easily using a fecal sample.

[0080] (Sequence description) Sequence ID 1: A nucleic acid sequence contained in Eub338, a probe for detecting viable cell counts, represented as 5'-GCTGCTCCCGTAGGAGT-3'. Sequence ID 2: A nucleic acid sequence contained in NonEub338, a control probe consisting of the complementary strand of the viable cell count detection probe, represented as 5'-ACTCCTAACGGGAGGCAGC-3'.

[0081] 1...Free cytometry device, 2...Computer device, 3...Display unit

Claims

1. A method for evaluating a fecal sample, comprising: an immobilization step of immobilizing a fecal sample; a labeling step of labeling the fecal sample after the immobilization step; and a measurement step of subjecting the fecal sample after the labeling step to measurement using a flow cytometry apparatus, wherein the labeling of the fecal sample in the labeling step involves labeling individual bacteria constituting a bacterial population contained in the fecal sample using a total bacterial count detection probe for detecting the total bacterial count and a viable bacterial count detection probe for detecting the viable bacterial count, and in the measurement step, a measurement amount (A) corresponding to the total bacterial count contained in the bacterial population is detected based on the labeling of individual bacteria constituting the bacterial population using the total bacterial count detection probe, and a measurement amount (B) corresponding to the viable bacterial count contained in the bacterial population is detected based on the labeling of individual bacteria constituting the bacterial population using the viable bacterial count detection probe, thereby obtaining the ratio of viable bacteria to the total bacterial count (B / A) for the bacterial population contained in the fecal sample.

2. The method for evaluating a fecal sample according to claim 1, wherein the probe for detecting the total bacterial count is a probe containing a DNA intercalator molecule, and the probe for detecting the viable bacterial count is a probe containing a nucleic acid molecule having a common sequence of 16S rDNA of eubacteria.

3. The method for evaluating a fecal sample according to claim 1, wherein in the measurement step, the detection of the measured amount (A) corresponding to the total bacterial count and the measured amount (B) corresponding to the viable bacterial count by the flow cytometry apparatus is based on the number of bacteria detected with a signal strength above a predetermined threshold corresponding to the probe used for each detection.

4. The method for evaluating a fecal sample according to claim 1, wherein the probe for detecting the total bacterial count contains a fluorescent substance, and the probe for detecting the viable bacterial count contains a fluorescent substance other than the fluorescent substance.

5. An evaluation system for evaluating a fecal sample by an evaluation method according to any one of claims 1 to 4, comprising: a flow cytometry device for obtaining the ratio of viable bacteria to the total number of bacteria in a bacterial community contained in the fecal sample; and a computer device for displaying the health status of the host of the fecal sample, which is pre-associated according to the ratio of viable bacteria.

6. The fecal sample evaluation system according to claim 5, wherein the health status is at the level of dysbiosis or ulcerative colitis.

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