Methods for the extraction and preservation of microorganisms from a stool sample of a living being

Abstract

Methods for the extraction and preservation of microorganisms from a stool sample of a living being, in particular a human being, comprising the following steps: - Separating a stool sample into several fractions, resulting in a microorganism fraction containing microorganisms from the intestinal microbiome of the organism; - Freezing the microorganism fraction for preservation in such a way that the microorganisms are at least partially preserved in a viable form; - wherein the stool sample is separated into several fractions by means of density gradient centrifugation, wherein, prior to separating the stool sample into several fractions, an auxiliary substance with a preformed density gradient is prepared in a container, and the stool sample is diluted and / or homogenized, in particular with Ringer's solution, so that a stool suspension is formed, wherein, for density gradient centrifugation, the stool suspension together with the auxiliary substance with the preformed density gradient is centrifuged in a container, so that different layered fractions are formed in the container.

Description

[0001] The present invention relates to a method for extracting and preserving microorganisms from a stool sample of a living being, in particular a human.

[0002] Trillions of bacteria live in the human gut. Several hundred different species colonize the large intestine in particular and form what's known as the microbiome. These bacteria are not only important for the human immune system but also responsible for the digestion of fiber. They produce enzymes that break down these otherwise indigestible food components. They also fight off pathogens and produce vitamins.

[0003] It is known that the composition of the microbiome depends on many different factors, including the lifestyle of each individual. Shifts and imbalances in the composition of the intestinal flora can cause allergies, a predisposition to obesity, and other diseases. Against this backdrop, a healthy, balanced intestinal flora is essential for everyone's health.

[0004] In recent years, the intestine and its bacterial flora have received increasing attention, particularly in the context of eliminating chronic intestinal inflammation. When the intestine is affected by bacterial infections, such as inflammation of the intestinal wall caused by the toxins of the bacterium Clostridium difficile, treatment is routinely carried out with antibiotics. One problem, however, is that the antibiotics used do not distinguish between good bacteria in the intestine that are essential for health and those that caused the inflammation being treated. The result is a general bacterial die-off in the patient's intestine, and not infrequently, harmful clostridia spread in the spaces vacated by the dead good bacteria. This is frequently followed by inflammation of the intestine, which can even progress to pseudomembranous colitis, accompanied by persistent diarrhea.After a long-term treatment with antibiotics, the intestinal flora is regularly rebuilt with medication and / or probiotic foods.

[0005] An alternative treatment for symptomatic Clostridium difficile infections that has been used for several years and has also been approved in Germany since mid-2016 is fecal transplantation, in which stool from healthy donors is injected into the intestines of sick patients. This involves transferring the intestinal flora from one person to another, a process referred to by various terms. In English, the term "fecal microbiota transplantation" is often used.

[0006] Such transfer of intestinal flora is often performed after treatment with antibiotics. The approach is based on the fact that damaged intestinal flora strongly favors relapses, as the corresponding bacteria can multiply unhindered due to a lack of competition from physiological intestinal bacteria. However, the biodiversity of stool in antibiotic-associated colitis is significantly reduced compared to normal stool.

[0007] One problem with stool transplantation from an unrelated donor is that a suitable donor must be available. In some cases, it is possible to collect stool from a donor immediately before the transplant and prepare it for transplantation. This is especially true if, for example, the stool comes from a patient's children. However, this is not always possible. The problem is that there is often a long period of time between collection and implantation. However, long-term storage of stool has so far been difficult because the microorganisms it contains can change.

[0008] Furthermore, there are recent efforts to perform autologous stool transplantation. This involves taking stool samples from the patient before antibiotic treatment and then storing them for an extended period of time so that the microorganisms they contain can be introduced into the patient's intestine after antibiotic treatment. Even with this approach, there is a need to be able to store the microorganisms contained in the stool for an extended period.

[0009] A method for detecting intestinal parasites is already known from the publication by Albrecht, E., and Schubert, S.: The MIFC method in various modifications for routine parasitology. 1991. In: Mitt. Österr. Ges. Tropenmed. Parasitol., Vol. 13, pp. 189-190. Furthermore, the preservation of microorganisms with glycerol is known from the online publication https: / / illumina-chemie.de / viewtopic.php?t=2648; by iTim, February 20, 2011.

[0010] Further methods for preserving microorganisms from the stool sample of a living being are known from EP 3 443 967 A1 and EP 3 389 679 B1.

[0011] Against this background, the object of the following invention is to provide a method by which the stool of a donor for transplantation can be stored even for a longer period of time.

[0012] This object is achieved by a method having the features of claim 1.

[0013] The invention is therefore based on the idea of ​​separating a stool sample into several fractions, forming a fraction containing a concentrated concentration of the relevant microorganisms. This fraction is then frozen, optionally with additional additives, ensuring that the microorganisms are at least partially preserved in a viable form. In particular, the microorganisms should retain an intact cellular status so that they can be introduced into a patient's body and continue to perform their function even after extended storage. In principle, the microorganism fraction can be introduced into the donor's body or into the body of another patient.

[0014] In a further embodiment of the method according to the invention, the stool sample can be separated into several fractions using density gradient centrifugation. This is a physical separation method that utilizes the different densities of different sample components for separation.

[0015] Preferably, an auxiliary substance with a preformed density gradient is prepared in a container before separating the stool sample into several fractions. A preformed density gradient means that the density of the auxiliary substance increases continuously toward the bottom of the container. Specifically, the auxiliary substance can contain a chemically inert agent, in particular cesium chloride, Percoll, sucrose, and / or iodixanol. Such agents are characterized by the fact that, under certain conditions, they can form a density gradient in a container.

[0016] The auxiliary substance preferably contains iodixanol diluted with Ringer's solution. In particular, this can be 1 / 4 Ringer's solution (autoclaved). The use of iodixanol not only has the advantage of being suitable for forming a density gradient, but also acts as a so-called cryoprotectant, meaning that, in conjunction with the microorganisms, it prevents the formation of large ice crystals during subsequent freezing. Such ice crystals could penetrate the cell membranes of the microorganisms being preserved and cause irreparable damage. Thus, the microorganisms survive subsequent freezing and storage largely unscathed, since the intact cell status of the microorganisms is maintained by the presence of iodixanol.

[0017] In a further embodiment, the concentration of iodixanol in the auxiliary substance can be at least 10% by weight, in particular at least 20% by weight, and / or at most 40% by weight, in particular at most 30% by weight, preferably 25% by weight.

[0018] To create a preformed density gradient in the auxiliary substance, the auxiliary substance can be frozen and then thawed. Specifically, the auxiliary substance can be frozen to a temperature of at least -15°C, in particular -20°C, and / or subsequently thawed at an ambient temperature of at least 2°C, in particular 4°C. Freezing and thawing can each take place over a period of approximately 8 to 24 hours. In this way, a preformed density gradient is formed in the auxiliary substance.

[0019] Furthermore, before separating the stool sample into several fractions, the stool sample can be diluted and / or homogenized, particularly with Ringer's solution, to create a stool suspension. Diluting or homogenizing the stool sample renders it more liquid, making it easier to separate into different fractions during subsequent centrifugation with the auxiliary substance.

[0020] Specifically, for density gradient centrifugation, the stool suspension can be centrifuged together with the auxiliary substance in a container using the preformed density gradient, resulting in the formation of different layered fractions in the container. During centrifugation, the different fractions sediment at different speeds until the density of the auxiliary substance is higher than the individual density of the various components. Specifically, an equilibrium is established between the sedimentation and diffusion processes, resulting in the formation of defined fractions. These are found as layers in the container. After density gradient centrifugation, a microorganism fraction is preferably present approximately in the middle of the container, with a supernatant above and a residue of the stool sample below.

[0021] The microorganism fraction can then be separated from the remaining fractions. This can be done by first gradually removing a supernatant from the container using a pipette. The microorganism fraction can then also be removed using a pipette and transferred to a separate container. Microorganisms adhering to the container wall can be mechanically removed and resuspended using a suitable inoculation loop.

[0022] Centrifugation is preferably carried out at a minimum of 5,000 g, in particular at least 10,000 g, preferably at 15,000 g, for a duration of at least 10 minutes, in particular at least 30 minutes, preferably for a duration of 60 minutes. The temperature during centrifugation can be at least 1°C, preferably at least 2°C, and / or at most 10°C, in particular at most 5°C. To prevent mixing of the fractions separated during centrifugation, centrifugation is advantageously completed without braking.

[0023] Before freezing the microorganism fraction, the cell count and / or cell status of the microorganisms in the microorganism fraction can be determined. This design of the procedure is based on the idea that this allows the cells, i.e., the microorganisms, to be counted and analyzed for their molecular and physical properties. This allows the quality of the extracted microorganism fraction to be assessed, which is relevant for its subsequent implantation into a patient's body.

[0024] Specifically, the cell count and / or cell status can be determined using flow cytometry. Individual microorganisms are isolated using hydrodynamic focusing and guided past various lasers. As soon as a laser beam hits the microorganism in question, it scatters the incoming light. A distinction is made between forward scatter (FSC) and side scatter (SSC). Forward scatter provides information about the size of the microorganism, whereas side scatter provides information about its granularity.

[0025] To perform flow cytometry, a sample of the microorganism fraction can be diluted to 10 -5diluted, with Ringer's solution being preferred. After dilution, a working solution containing fluorescent dyes can be added to the sample. By using appropriate fluorescent dyes, additional information about the microorganism in question can be obtained during flow cytometry.

[0026] Specifically, the working solution can contain the fluorescent dyes SYBR Green I and propidium iodide. This design of the method is based on the idea that SYBR can be incorporated into the DNA of microorganisms with intact, but also permeable, cell membranes, whereas propidium iodide cannot penetrate intact cell membranes and can therefore only be incorporated into the DNA of microorganisms with damaged or destroyed cell membranes. Depending on the intensity of the two fluorescent dyes, the cells can be differentiated according to the state of their cell membrane. The cells are generally classified as "living," "dead," or "damaged." In cells with a living cell status, the scattering of the SYBR dye is highly intense, whereas the scattering of propidium iodide is very low.When the cell status is "dead," both fluorescent dyes exhibit high intensity; when the cell status is damaged, both dyes exhibit medium intensity. This allows individual microorganisms to be analyzed based on their cell status.

[0027] In general, flow cytometry can detect the forward scatter and side scatter of a laser beam incident on the sample to determine the cell count and cell status of the microorganisms in the sample.

[0028] The microorganism fraction can be frozen to a temperature below -50°C, in particular below -60°C, preferably to a temperature of -80°C. The cooling rate can be at least 0.1°C per minute, in particular at least -0.5°C per minute, and / or at most -5°C per minute, in particular at most -2.5°C per minute, preferably -1°C per minute. Such cooling enables long-term preservation, with the microorganisms predominantly surviving freezing and storage undamaged. Particularly when iodixanol is contained in the auxiliary substance and thus comes into contact with the stool sample or stool suspension during centrifugation, it acts as a cryoprotectant and prevents damage to the microorganisms during freezing.

[0029] This allows stool samples to be taken from a patient using the procedure described above, whereby the microorganisms of the intestinal microbiome are extracted and stored before being reinserted into the same or another patient.

[0030] For the further development of the method according to the invention, reference is made to the subclaims and to the following description of an embodiment.

[0031] To carry out the method according to the invention for the extraction and preservation of microorganisms from a stool sample, in this embodiment, an iodixanol auxiliary substance with a concentration of 25% by weight is first prepared. For this purpose, the OptiPrep™ solution from PROGEN Biotechnik GmbH, which contains 60% by weight iodixanol, is diluted with 1 / 4 Ringer's solution (autoclaved). Subsequently, 75 ml of this iodixanol auxiliary substance is placed in a 225 ml centrifuge tube and frozen at -20°C overnight, i.e., for approximately 10 to 12 hours. Subsequent thawing for a similar period at 4°C in a refrigerator leads to the formation of a continuous density gradient in the centrifuge tube.

[0032] Furthermore, for preparation, 50 g of stool is weighed to the nearest 0.1 g and placed in a filter bag. After a further 150 ml of Ringer's solution (autoclaved) is added to the filter bag, the filter bag is closed 2 cm below the opening with a suitable clip. The sample can then be homogenized in a mixer for approximately 5 minutes with a paddle spacing of 3 mm. The resulting stool suspension is removed from the pocket of the filter bag using a 100 ml serological pipette, the volume is determined simultaneously, and transferred to a 225 ml centrifuge tube. If the volume is less than 150 ml, the appropriate amount can be made up to 150 ml by adding ¼ Ringer's solution (autoclaved).

[0033] The stool suspension is then added to the preformed density gradient using a 50 ml serological pipette while tilting the 225 ml centrifuge tube. The filled centrifuge tube is then centrifuged at 15,000 g for 60 minutes at a temperature of 4°C. The centrifuge is accelerated to its highest speed, while the centrifugation is stopped without stopping to prevent mixing of the different fractions.

[0034] Through this density gradient centrifugation, several fractions are formed in the centrifuge vessel, with a microorganism fraction forming approximately in the middle of the vessel, which contains microorganisms of the intestinal microbiome of the organism in concentrated form.

[0035] Next, the supernatant, which is located above the microorganism fraction in the centrifuge tube, is removed. This can be done using a 50 ml serological pipette or a 5 ml piston pipette.

[0036] The microorganism fraction is then transferred to a separate tube using a 5 ml piston pipette while gently rotating the centrifuge tube along its longitudinal axis. The microorganisms adhering to the tube wall are mechanically detached using a 10 µl inoculation loop and resuspended in the microorganism fraction. During the collection of the microorganism fraction, the centrifuge tube can be tilted to prevent accidental aspiration of residual components contained beneath the microorganism fraction in the centrifuge tube. The volume of the collected microorganism fraction can be determined using appropriate pipettes to later determine the exact count based on samples.

[0037] To determine the cell count and the cell status of the microorganisms in the microorganism fraction, a sample can be taken from it and tested up to level 10 -5 This can be done in 1.5 ml reaction tubes, using a ¼ Ringer's solution (autoclaved, sterile filtered 0.2 µm) for dilution.

[0038] Transfer 195 µl of the diluted solution to a 5 ml sample tube before adding 2.5 µl each of SYBR Green I working solution and propium iodide working solution. These are fluorescent dyes.

[0039] After incubation for 10 minutes at a temperature of 37°C in the dark, the samples can be measured on a flow cytometer with appropriate settings.

[0040] In this case, a laser with a wavelength of 488 nm can be used for this purpose. This allows the cell count and cell status of the sample to be determined, from which the total cell count of the entire microorganism fraction can be deduced.

[0041] Finally, after completion of the flow cytometric measurements, the collected microorganism fraction can be deep-frozen to a temperature of -80°C at a cooling rate of -1°C per minute. Since the auxiliary substance contains iodixanol with a preformed density gradient, freezing occurs without damaging the microorganisms, as iodixanol also acts as a cryoprotectant.

[0042] The method described above makes it possible to extract a microbial fraction from a patient's stool sample and then store it for an extended period of time until it is reintroduced to the same patient or to another patient, particularly after treatment with antibiotics has taken place in the meantime.

Claims

[1] A method for extracting and preserving microorganisms from a stool sample of a living being, in particular a human, comprising the following steps: - Separating a stool sample into several fractions, forming a microorganism fraction that contains microorganisms from the intestinal microbiome of the organism; - freezing the microorganism fraction for preservation in such a way that the microorganisms are at least partially preserved in a viable form; - wherein the separation of the stool sample into several fractions is carried out by means of density gradient centrifugation, wherein before the separation of the stool sample into several fractions, an auxiliary substance with a preformed density gradient is prepared in a container and the stool sample is diluted and / or homogenized, in particular with Ringer's solution, so that a stool suspension is formed, wherein for the density gradient centrifugation, the stool suspension is centrifuged together with the auxiliary substance with the preformed density gradient in a container so that various layered fractions are formed in the container. [2] Method according to claim 1, characterized by that the excipient contains a chemically inert agent, in particular caesium chloride, Percoll, sucrose and / or iodixanol. [3] Method according to claim 2, characterized by that the excipient contains iodixanol, which is diluted with Ringer's solution. [4] Method according to claim 2 or 3, characterized by that the concentration of iodixanol in the auxiliary substance is at least 10% by weight, in particular at least 20% by weight, and / or at most 40% by weight, in particular at most 30% by weight, preferably 25% by weight. [5] Method according to one of claims 2 to 4, characterized by that the auxiliary substance is frozen and then thawed to create a preformed density gradient in the auxiliary substance. [6] Method according to claim 5, characterized by that the auxiliary substance is frozen to a temperature of at least -15°C, in particular -20°C and / or is subsequently thawed at an ambient temperature of at least 2°C, in particular 4°C. [7] Method according to one of the preceding claims, characterized by that after centrifugation the microorganism fraction is separated from the other fractions. [8] Method according to one of the preceding claims, characterized by that the microorganism fraction is frozen to a temperature below -50°C, in particular below -60°C, preferably -80°C. [9] Method according to claim 8, characterized by that the freezing takes place at a cooling rate of at least -0.1°C per minute, in particular of at least -0.5°C per minute, and / or of at most -5°C per minute, in particular of at most -2.5°C per minute, preferably of -1°C per minute. [10] Method according to one of the preceding claims, characterized by that the cell count and / or cell status of the microorganisms in the microorganism fraction is determined before freezing the microorganism fraction. [11] Method according to one of the preceding claims, characterized by that the determination of cell number and / or cell status is carried out by flow cytometry. [12] Method according to claim 11, characterized bythat to perform flow cytometry, a sample of the microorganism fraction up to dilution level 10 -5 is diluted, in particular with Ringer's solution. [13] Method according to claim 12, characterized by that after dilution, a working solution containing fluorescent dyes is added to the sample. [14] Method according to claim 13, characterized by that the working solution contains the fluorescent dyes SYBR Green I and propidium iodide. [15] Method according to one of claims 12 to 14, characterized by that in flow cytometry, the forward scatter and side scatter of a laser beam incident on the sample are recorded in order to determine the cell number and cell status of the microorganisms in the sample.