Method for producing infected animal model

Abstract

The purpose of the present invention is to provide a method for producing an animal model infected with Enterobacteriaceae bacteria such as pathogenic Escherichia coli. Provided is a method for producing an animal model infected with Enterobacteriaceae bacteria, the method comprising step (1) and step (2) in the following order. (1) A step for administering a β-lactam antibacterial agent to a non-human mammal; (2) a step including the following steps (2-1) and (2-2) in random order; (2-1) a step for administering at least one selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to the non-human mammal; and (2-2) a step for administering Enterobacteriaceae bacteria to the non-human mammal

Description

Method for producing an infected animal model

[0001] The present invention relates to a method for producing an infection model animal.

[0002] Infection with Enterobacteriaceae bacteria causes various intestinal infections. For example, enterohemorrhagic E. coli O157, a pathogenic E. coli, has caused numerous outbreaks in Japan and abroad in the past. Under these circumstances, methods for treating and preventing infectious diseases based on enterohemorrhagic E. coli infections are needed. However, Enterobacteriaceae bacteria are known to have difficulty colonizing the intestinal tract of non-human animals such as mice, and there are no suitable animal models for infection. For example, in Non-Patent Document 1, instead of Enterobacteriaceae bacteria that have difficulty colonizing the intestinal tract of mice, the pathogenic bacterium Citrobacter rodentium is used, which is an intestinal pathogenic bacterium of mice similar to human enteropathogenic / enterohemorrhagic E. coli. The authors then introduced genes containing virulence factors of enteropathogenic / enterohemorrhagic E. coli into this bacterium and expressed them to analyze their effect on pathogenesis. In Non-Patent Document 2, a model using streptomycin-treated mice infected with enterohemorrhagic E. coli showed persistent infection for more than five days compared to untreated mice. However, enterohemorrhagic E. coli was detected in the cecum and lower gastrointestinal tract, with only small numbers of bacteria detected in the feces. Furthermore, the pathological condition could not be confirmed. In Non-Patent Document 3, mice were treated with several antibiotics for three days, then orally administered large amounts of enterohemorrhagic E. coli. The intestines were removed from the mice six hours later, and infection was considered established. In this case, it is unclear whether the infection is adhesion or colonization. Therefore, the research and development environment is insufficient, and the mechanisms of bacterial infection and host defense responses are largely unknown, making it difficult to develop new treatment and prevention methods.

[0003] Mallick, Emily M et al. The Journal of clinical investigation vol. 122, 11 (2012): 4012-24. Etienne-Mesmin, Lucie et al. Applied and environmental microbiology vol. 77, 3 (2011): 1127-31. Wen Yang et al. Cell Reports vol. 42, 6 (2023): 112638.

[0004] An object of the present invention is to provide a method for producing an animal model infected with Enterobacteriaceae bacteria such as pathogenic Escherichia coli, and a method for using the same.

[0005] The present inventors discovered that an animal model of infection in which Enterobacteriaceae bacteria have colonized the intestinal tract can be produced by administering multiple types of antibacterial agents in a specific order, and thus completed the present invention.

[0006] The present invention includes the following aspects [1] to

[14] . [1] A method for producing an animal model infected with Enterobacteriaceae bacteria, comprising the following steps (1) and (2) in this order: (1) administering a β-lactam antibacterial agent to a non-human mammal; (2) comprising the following steps (2-1) and (2-2) in any order: (2-1) administering at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to the non-human mammal; and (2-2) administering Enterobacteriaceae bacteria to the non-human mammal. [2] The method for producing an animal model infected with Enterobacteriaceae bacteria according to [1], wherein the step (2) comprises the steps (2-1) and (2-2) in this order. [3] The method for producing an animal model for Enterobacteriaceae infection according to [1] or [2], wherein the β-lactam antibacterial agent is at least one selected from the group consisting of penicillin antibacterial agents, cephem antibacterial agents, carbapenem antibacterial agents, and monobactam antibacterial agents. [4] The method for producing an animal model for Enterobacteriaceae infection according to any one of [1] to [3], wherein the β-lactam antibacterial agent is at least one selected from the group consisting of ampicillin, penicillin, and cefoperazone. [5] The method for producing an animal model for Enterobacteriaceae infection according to any one of [1] to [4], wherein the lincomycin antibacterial agent is at least one selected from the group consisting of clindamycin and lincomycin. [6] The method for producing an animal model for Enterobacteriaceae infection according to any one of [1] to [4], wherein the macrolide antibacterial agent is at least one selected from the group consisting of tylosin, erythromycin, and clarithromycin. [7] The method for producing an Enterobacteriaceae bacteria-infected model animal according to any one of [1] to [6], wherein the Enterobacteriaceae bacteria are bacteria of the genus Escherichia, Klebsiella, Salmonella, or Citrobacter. [7-1] The method for producing an Enterobacteriaceae bacteria-infected model animal according to any one of [1] to [6], wherein the Enterobacteriaceae bacteria are bacteria of the genus Escherichia, Klebsiella, or Salmonella.[8] The method for producing an Enterobacteriaceae bacteria infection model animal according to any one of [1] to [7-1], wherein the Enterobacteriaceae bacteria is Escherichia coli. [9] The method for producing an Enterobacteriaceae bacteria infection model animal according to [8], wherein the Escherichia coli is pathogenic Escherichia coli or drug-resistant Escherichia coli.

[10] The method for producing an Enterobacteriaceae bacteria infection model animal according to any one of [1] to [9], wherein the non-human mammal is a mouse.

[11] The method for producing an Enterobacteriaceae bacteria infection model animal according to any one of [1] to

[10] , wherein the step (2) comprises administering at least one antibiotic selected from the group consisting of lincomycin antibiotics and macrolide antibiotics 5 to 10 days after the start of administration of the β-lactam antibiotic.

[12] The method for producing an Enterobacteriaceae bacteria infection model animal according to any one of [2] to

[11] , wherein the step (2-2) comprises inoculating the Enterobacteriaceae bacteria 3 to 24 hours after the start of administration of at least one antibiotic selected from the group consisting of lincomycin antibiotics and macrolide antibiotics.

[13] A method for screening for a therapeutic agent for an Enterobacteriaceae bacteria infection, comprising the following steps (p1) to (p3) in this order: (p1) a step of preparing an Enterobacteriaceae bacteria-infected model animal by the method described in any one of [1] to

[12] , (p2) a step of administering a test substance to the Enterobacteriaceae bacteria-infected model animal obtained in step (p1), and (p3) a step of selecting the test substance as a candidate substance for a therapeutic agent for an Enterobacteriaceae bacteria-infected model animal, when the symptoms of the infection are improved in the model animal administered with the test substance in step (p2) compared to the control.

[14] A method for evaluating a therapeutic agent for an Enterobacteriaceae bacteria-infected model animal, comprising the following steps (q1) to (q3) in this order: (q1) a step of preparing an Enterobacteriaceae bacteria-infected model animal by the method described in any one of [1] to

[12] , (q2) a step of administering a therapeutic agent to be evaluated to the Enterobacteriaceae bacteria-infected model animal obtained in step (q1), and (q3) a step of evaluating the therapeutic effect of the therapeutic agent to be evaluated on an Enterobacteriaceae bacteria-infected model animal administered with the therapeutic agent to be evaluated in step (q2).

[0007] The present invention provides a method for producing an animal model infected with Enterobacteriaceae bacteria such as pathogenic Escherichia coli, and a method for using the same.

[0008] FIG. 1 is a diagram showing the results of Reference Example 1. FIG. 2 is a diagram showing the results of Reference Example 1. FIG. 3 is a diagram showing the results of Reference Example 1. FIG. 4 is a diagram showing the results of Reference Example 1. FIG. 5 is a diagram showing the steps of Example 1. FIG. 6 is a diagram showing the results of Example 1. FIG. 7 is a diagram showing the results of Example 2. FIG. 8 is a diagram showing the results of Example 2-1. FIG. 9 is a diagram showing the results of Example 3. FIG. 10 is a diagram showing the results of Example 4. FIG. 11 is a diagram showing the results of Example 5. FIG. 12 is a diagram showing the results of Example 6.

[0009] [Method for producing a model animal infected with Enterobacteriaceae bacteria] The method for producing a model animal infected with Enterobacteriaceae bacteria of the present invention comprises the following steps (1) and (2) in this order: (1) a step of administering a β-lactam antibacterial agent to a non-human mammal; (2) a step of comprising the following steps (2-1) and (2-2) in any order: (2-1) a step of administering at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to the non-human mammal; and (2-2) a step of administering Enterobacteriaceae bacteria to the non-human mammal.

[0010] <Infected Animal Model> The infected animal model of the present invention is a disease model animal in which Enterobacteriaceae bacteria colonize in the intestinal tract, and if the infected bacteria are pathogenic to the animal, the majority of the animals will die during rearing, reproducing the symptoms of human infectious disease patients. As used herein, "colonization" means that the infected bacteria can be detected in the feces of the model animal even three days after infection with Enterobacteriaceae bacteria. Typically, in wild-type untreated mice, Enterobacteriaceae bacteria are no longer detected in the feces within about three days after administration.

[0011] The animal species constituting the infection model animal are non-human mammals, including mice, rats, guinea pigs, rabbits, dogs, cows, pigs, monkeys, chimpanzees, etc. In one aspect of the present invention, the non-human mammal is a mouse (Mus musculus) from the viewpoints of ease of acquisition and handling. Genetically controlled infection model animals can be produced by using inbred mouse strains such as C57BL / 6J and BALB / c.

[0012] The rearing environment for non-human mammals is not particularly limited. When the non-human mammal is a mouse, the rearing environment can be, for example, a room set at a temperature of 15°C to 25°C, a humidity of 40% to 60%, and a light-dark cycle of approximately 8 hours to 16 hours light and 8 hours to 16 hours dark.

[0013] <Enterobacteriaceae> The Enterobacteriaceae bacteria are not particularly limited as long as they belong to the Enterobacteriaceae family. Enterobacteriaceae bacteria are typically bacilli that are facultatively anaerobic and gram-negative. Examples of Enterobacteriaceae bacteria in the present invention include bacteria that inhabit the human intestinal tract and cause intestinal infections, such as the genus Escherichia (Escherichia coli), the genus Klebsiella, the genus Salmonella, the genus Citrobacter, and the genus Shigella. Examples of bacteria belonging to the genus Escherichia include Escherichia coli, particularly pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, and drug-resistant Escherichia coli such as carbapenem-resistant Escherichia coli. Examples of bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae. Examples of bacteria of the genus Salmonella include Salmonella enterica, and particularly pathogenic Salmonella such as Salmonella enterica subsp. enterica serovar Typhi, Salmonella enterica subsp. enterica serovar Paratyphi A, and food-toxic Salmonella. Examples of food-toxic Salmonella include Salmonella enterica subsp. enterica serovar Typhimurium. Examples of bacteria of the genus Citrobacter include Citrobacter rodentium. Examples of bacteria belonging to the Shigella genus include Shigella dysenteriae. Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to the genus Escherichia, Klebsiella, Salmonella, and Citrobacter, and more preferably bacteria belonging to the genus Escherichia, Salmonella, and Citrobacter.As the Enterobacteriaceae family bacteria, bacteria of the genus Escherichia, bacteria of the genus Klebsiella, or bacteria of the genus Salmonella are also preferred. Among them, as the Enterobacteriaceae family bacteria, Escherichia coli is more preferred, and pathogenic Escherichia coli or drug-resistant Escherichia coli is even more preferred.

[0014] <β-Lactam Antibacterial Agents> β-Lactam antibacterial agents have a β-lactam structure, specifically inhibit cell wall synthesis enzymes, and exert bacteriostatic or bactericidal effects. Examples of β-lactam antibacterial agents include penicillin-based antibacterial agents such as penicillin, ampicillin, methicillin, and oxacillin; cephem-based antibacterial agents such as cefoperazone; carbapenem-based antibacterial agents such as doripenem; and monobactam-based antibacterial agents such as aztreonam. β-Lactam antibacterial agents also include pharmacologically acceptable salts and solvates thereof. Examples of pharmacologically acceptable salts include base addition salts such as sodium salts, potassium salts, and calcium salts; and acid addition salts such as hydrochlorides. In one aspect of the present invention, ampicillin, penicillin, or cefoperazone is preferred as the β-lactam antibacterial agent from the viewpoint of ease of availability. The β-lactam antibacterial agents can be used singly or in combination of two or more.

[0015] <Lincomycin Antibacterial Agents and Macrolide Antibacterial Agents> Lincomycin antibacterial agents and macrolide antibacterial agents inhibit protein synthesis by acting on the 50S subunit of the ribosome, thereby exerting bacteriostatic activity. Examples of lincomycin antibacterial agents include clindamycin and lincomycin, which are preferably used. Examples of macrolide antibacterial agents include tylosin, erythromycin, clarithromycin, and the like, which are preferably used. Among these, clindamycin is more preferred as at least one selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents. Lincomycin antibacterial agents and macrolide antibacterial agents also include their pharmacologically acceptable salts and solvates. Examples of pharmacologically acceptable salts include base addition salts such as sodium salts, potassium salts, and calcium salts; and acid addition salts such as hydrochlorides. Lincomycin antibacterial agents and macrolide antibacterial agents can be used alone or in combination.

[0016] <Step (1)> Step (1) is a step of administering a β-lactam antibacterial agent to a non-human mammal. The method of administering the β-lactam antibacterial agent is not particularly limited. Examples include oral administration, subcutaneous administration, intravenous administration such as intracaudal administration, and intraperitoneal administration. From the viewpoint of simplicity, a method in which the β-lactam antibacterial agent is added to drinking water and allowed to drink ad libitum is preferred. The dose of the β-lactam antibacterial agent can be appropriately determined by those skilled in the art. For example, when the β-lactam antibacterial agent is added to drinking water and allowed to drink ad libitum in a mouse as a non-human mammal, the content of the β-lactam antibacterial agent in the drinking water is preferably 0.05 g / L or more and 10 g / L or less, more preferably 0.1 g / L or more and 5 g / L or less.

[0017] <Step (2)> Step (2) includes the following steps (2-1) and (2-2) in any order: (2-1) administering at least one antibiotic selected from the group consisting of lincomycin antibiotics and macrolide antibiotics to the non-human mammal; and (2-2) administering Enterobacteriaceae bacteria to the non-human mammal.

[0018] (Step (2-1)) Step (2-1) is a step of administering at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to a non-human mammal. The method of administering at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents is not particularly limited. Examples include oral administration, subcutaneous administration, intravenous administration such as intracaudal administration, and intraperitoneal administration. From the viewpoint of simplicity, a preferred method is to add at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to drinking water and allow the subject to drink the water ad libitum. The dosage of at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents can be appropriately determined by one skilled in the art. For example, when at least one selected from the group consisting of lincomycin-based antibacterial agents and macrolide-based antibacterial agents is contained in drinking water and allowed to be consumed ad libitum, the content of the at least one selected from the group consisting of lincomycin-based antibacterial agents and macrolide-based antibacterial agents in the drinking water is preferably 0.05 g / L or more and 10 g / L or less, more preferably 0.1 g / L or more and 5 g / L or less.

[0019] (Step (2-2)) Step (2-2) is a step of administering Enterobacteriaceae bacteria to a non-human mammal. The method of administering Enterobacteriaceae bacteria is not particularly limited. Examples include oral administration, subcutaneous administration, intravenous administration such as intracaudal administration, and intraperitoneal administration. From the viewpoint of simplicity, oral administration using an appropriate device such as a probe is preferred. The dose of Enterobacteriaceae bacteria can be appropriately determined by those skilled in the art. For example, when the Enterobacteriaceae bacteria is Escherichia coli and is administered to a mouse as the non-human mammal, the dose is preferably 1 x 10 ４ CFU (Colony Forming Units) or more 1×10 ８ CFU or less, more preferably 1 x 10 ５ CFU or more 1 x 10 ７ The Enterobacteriaceae bacteria can be grown by known culture methods to achieve the above-mentioned dosage.

[0020] Step (2) may include the steps (2-1) and (2-2) in any order, and preferably includes the steps (2-1) and (2-2) in this order.

[0021] In the method for producing an infection model animal of the present invention, for example, when a β-lactam antibacterial agent is added to drinking water and the animal is allowed to drink ad libitum, it is preferable that in step (2), administration of at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents is initiated 5 to 10 days after the start of administration of the β-lactam antibacterial agent. Furthermore, for example, when at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents is added to drinking water and the animal is allowed to drink ad libitum, it is preferable that in step (2-2), Enterobacteriaceae bacteria are administered 3 to 24 hours after the start of administration of at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents. When at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents is added to drinking water and the animal is allowed to drink ad libitum, administration is preferably continued for at least 3 days, more preferably at least 5 days. It is preferable to continue administration of at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents throughout the use as an Enterobacteriaceae infection model animal.

[0022] Thus, an animal model for infection with Enterobacteriaceae bacteria is produced. Without wishing to be bound by theory, it is believed that in the production method of the present invention, the action of the β-lactam antibacterial agent administered in step (1) largely eradicates the diverse intestinal bacterial flora living in the intestinal tract of the non-human mammal, preferably resulting in a nearly sterile state. It is then believed that the action of at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents administered in step (2) allows the administered Enterobacteriaceae bacteria to grow preferentially over other bacteria ingested from the rearing environment or feed, and establish themselves in the intestinal tract of the non-human mammal.

[0023] The model animal infected with Enterobacteriaceae bacteria produced by the present invention can be suitably used in the development of methods for treating, preventing, and the like infectious diseases caused by Enterobacteriaceae bacteria. For example, there is provided a method for screening for therapeutic agents for Enterobacteriaceae bacteria infection, comprising the following steps (p1) to (p3) in this order: (p1) producing a model animal infected with Enterobacteriaceae bacteria by the method for producing a model animal infected with Enterobacteriaceae bacteria of the present invention; (p2) administering a test substance to the model animal infected with Enterobacteriaceae bacteria obtained in step (p1); and (p3) selecting the test substance as a candidate substance for a therapeutic agent for Enterobacteriaceae bacteria infection when the model animal administered with the test substance in step (p2) shows improved symptoms of infection compared to the control.

[0024] Also provided is a method for evaluating a therapeutic agent for an Enterobacteriaceae infection, comprising the following steps (q1) to (q3) in this order: (q1) preparing an Enterobacteriaceae infection model animal by the method of the present invention for preparing an Enterobacteriaceae infection model animal; (q2) administering a therapeutic agent to be evaluated to the Enterobacteriaceae infection model animal obtained in step (q1); and (q3) evaluating the therapeutic effect of the therapeutic agent to be evaluated on an Enterobacteriaceae infection in the model animal administered with the therapeutic agent obtained in step (q2).

[0025] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0026] [Method] 1-1. Culturing method for pathogenic E. coli The E. coli strains used are as follows: Enterohemorrhagic E. coli O157 clade 8 strain, Enterohemorrhagic E. coli O157 clade 8 Stx2Δ strain, Enterohemorrhagic E. coli O157 Sakai strain, Enterohemorrhagic E. coli O26 WT-1 strain, and Enterohemorrhagic E. coli O26 WT-2 strain. All strains were provided by the National Institute of Infectious Diseases. The O157 clade 8 Stx2Δ strain is a mutant strain of the O157 clade 8 strain in which the gene encoding Shiga toxin is deleted.

[0027] An appropriate amount of 10% glycerol stock of each bacterial strain, which had been frozen and stored at -80°C, was inoculated onto an LB plate and subjected to static culture at 37°C for 24 hours. Next, 3 mL of LB medium was added to a 50 mL conical tube, and a single colony was suspended. After that, shaking culture was performed at 37°C at 150 rpm to obtain a preculture solution. 12 to 15 hours after the start of culture, 2.7 mL of LB medium was added to a new 50 mL conical tube, and 300 μL of the preculture solution was added. Shaking culture was performed at 37°C at 150 rpm for 2 hours. After culture, the turbidity OD600 was measured at 1 (1 x 10 ８ The concentration of the culture medium was adjusted to a final concentration of 1 × 10 CFU / mL. Turbidity was measured at a wavelength of 600 nm using an absorption spectrophotometer. 100 μL of the adjusted culture medium was placed in a 1.5 mL tube and centrifuged at 14,000 rpm for 5 minutes at 4°C. The supernatant was discarded and then suspended in 1 mL of sterile water. The final concentration was 1 × 10 ８ CFU / mL.

[0028] 1-2. Culturing Method of Salmonella Typhimurium Salmonella Typhimurium (Salmonella enterica subsp. enterica serovar Typhimurium, hereinafter also referred to as Salmonella Typhimurium) was a naturally streptomycin-resistant strain selected by culturing a parent strain provided by the ATCC (American Type Culture Collection) on streptomycin-containing LB agar medium. An appropriate amount of a 10% glycerol stock of the bacterial strain, frozen and stored at -80°C, was inoculated onto an LB plate and cultured statically at 37°C for 24 hours. Next, 3 mL of LB medium was added to a 50 mL conical tube, and a single colony was suspended. The suspension was then cultured with shaking at 37°C at 150 rpm to obtain a preculture solution. After 12 to 15 hours from the start of the culture, 2.7 mL of LB medium was added to a new 50 mL conical tube, and 300 μL of the preculture solution was added. The mixture was cultured with shaking at 37°C for 2 hours at a speed of 150 rpm. After the culture, the turbidity (OD600) was 1 (1 × 10 ８The concentration of the culture medium was adjusted to a final concentration of 1 × 10 CFU / mL. Turbidity was measured at a wavelength of 600 nm using an absorption spectrophotometer. 100 μL of the adjusted culture medium was placed in a 1.5 mL tube and centrifuged at 14,000 rpm for 5 minutes at 4°C. The supernatant was discarded and then suspended in 1 mL of sterile water. The final concentration was 1 × 10 ８ CFU / mL.

[0029] 1-3. Citrobacter rodentium Citrobacter rodentium was provided by ATCC. An appropriate amount of 10% glycerol stock of each bacterial strain, frozen and stored at -80°C, was inoculated onto an LB plate and cultured statically at 37°C for 24 hours. Next, 3 mL of LB medium was added to a 50 mL conical tube, and a single colony was suspended. The suspension was then cultured with shaking at 150 rpm at 37°C to obtain a preculture solution. 12 to 15 hours after the start of culture, 2.7 mL of LB medium was added to a new 50 mL conical tube, and 300 μL of the preculture solution was added. The suspension was then cultured with shaking at 150 rpm at 37°C for 2 hours. After culture, the turbidity OD600 was 1 (1 x 10 ８ The concentration of the culture medium was adjusted to a final concentration of 1 × 10 CFU / mL. Turbidity was measured at a wavelength of 600 nm using an absorption spectrophotometer. 100 μL of the adjusted culture medium was placed in a 1.5 mL tube and centrifuged at 14,000 rpm for 5 minutes at 4°C. The supernatant was discarded and then suspended in 1 mL of sterile water. The final concentration was 1 × 10 ８ CFU / mL.

[0030] 2. Mouse rearing method: Mice were reared under a 12-hour light / dark cycle and given filter-sterilized tap water as drinking water and gamma-ray sterilized mouse feed (product name: CL-2, manufactured by CLEA Japan, Inc.). Mice administered pathogenic E. coli were reared in cages placed in an isolator equipped with a HEPA filter. Cages, food, and drinking water were changed once a week in a safety cabinet.

[0031] The 1 g / L ampicillin solution administered to mice was prepared by dissolving ampicillin sodium salt (Tokyo Chemical Industry Co., Ltd.) in tap water to a final concentration of 1 g / L and filter sterilization. The 1 g / L penicillin solution was prepared by dissolving penicillin G sodium (Tokyo Chemical Industry Co., Ltd.) in tap water to a final concentration of 0.5 g / L and filter sterilization. The 0.5 g / L cefoperazone solution was prepared by dissolving cefoperazone sodium (MP Biomedicals) in tap water to a final concentration of 0.5 g / L and filter sterilization. The 0.5 g / L clindamycin solution was prepared by dissolving clindamycin hydrochloride monohydrate (Tokyo Chemical Industry Co., Ltd.) in tap water to a final concentration of 0.5 g / L and filter sterilization. The pathogenic E. coli suspension was administered to mice using an oral tube (manufactured by Fuchigami Kikai Co., Ltd.).

[0032] Fresh feces excreted from the mouse anus in a safety cabinet were directly collected into sterilized 1.5 mL tubes, and the mass of the feces was measured. Sterile water was added to each fecal sample to a concentration of 0.1 g / mL, and the feces were suspended using a homogenizing rod (trade name: Pestles for 1.5 mL Tubes, manufactured by SSI Bio) to obtain a fecal suspension.

[0033] 4. Statistical analysis In the figures, * indicates a significant difference of p<0.05 by t-test, ** indicates a significant difference of p<0.01, *** indicates a significant difference of p<0.001, and NS indicates no significant difference. "Quantitation limit" and "ND" (Not Detected) indicate values ​​below the detection limit.

[0034] Reference Example 1 Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were bred as follows. n=3 per group. i) Ampicillin treatment group: Mice were administered a 1 g / L ampicillin aqueous solution by allowing them to drink water ad libitum for 7 days. ii) Clindamycin treatment group: Mice were administered a 0.5 g / L clindamycin aqueous solution by allowing them to drink water ad libitum for 7 days. iii) Penicillin treatment group: Mice were administered a 1 g / L penicillin aqueous solution by allowing them to drink water ad libitum for 7 days. iv) Cefoperazone treatment group: Mice were administered a 0.5 g / L cefoperazone aqueous solution by allowing them to drink water ad libitum for 7 days.

[0035] Fresh feces were collected from the anus of each mouse in each group. Enterobacterial DNA was extracted from the collected feces, and the intestinal microbiota was analyzed by quantitative PCR to determine the cell number per gram (cell number / g) or relative cell number (relative number) of total bacteria (Total Bacteria), Enterobacteriaceae, Clostridiales, Bacteroidales, and Lactobacillaceae. The relative cell number was calculated by setting the average value of either the control or antibiotic-treated sample as 1, and representing the relative value of the other sample.

[0036] The results of the ampicillin treatment group are shown in FIG. 1, the results of the clindamycin treatment group in FIG. 2, the results of the penicillin treatment group in FIG. 3, and the results of the cefoperazone treatment group in FIG.

[0037] Ampicillin and penicillin treatment eliminated bacteria from the intestines of mice, resulting in mice that appeared almost germ-free. Cefoperazone treatment reduced the total bacterial count to less than 1 / 10,000, and the count of Enterobacteriaceae bacteria was reduced to almost zero. Furthermore, clindamycin treatment caused Enterobacteriaceae bacteria to become the predominant bacteria in the intestines.

[0038] Example 1: Preparation of an infection model mouse (1) Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were bred as follows. n=3 per group. 1-1) WT group: Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. 1-2) Amp group: 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 12 days. 1-3) Amp / Clind group: 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 7 days. Subsequently, 0.5 g / L clindamycin aqueous solution was allowed to be drunk ad libitum for 5 days. 1-4) Clade 8 group: 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 7 days. Subsequently, 0.5 g / L clindamycin aqueous solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin solution, each mouse was given 100 μL (1 × 10 ６ CFU) was administered to the mice using an oral tube. 1-5) Sakai group: The mice were allowed to freely drink 1 g / L of ampicillin solution for 7 days. Then, the mice were allowed to freely drink 0.5 g / L of clindamycin solution for 5 days. Four hours after the start of drinking the clindamycin solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O157 Sakai strain was administered to each mouse. ６ The process of this example is shown schematically in Figure 5.

[0039] Fresh feces were collected from the anus of each mouse in each group. Analysis of the intestinal flora contained in the collected feces was outsourced to Seibutsu Giken Co., Ltd. The analysis results are shown in Table 1. The numbers shown in Table 1 represent the copy number of 16S rRNA for each detected bacterial class. In the table, "Actinobacteria" refers to bacteria of the Actinobacteria class other than Bifidobacteriale, Micrococcales, Propionibacteriale, Corynebacteriale, Pseudonocardiales, Streptomycetales, and Frankiales, and "Bacteria" refers to bacteria of a class other than those listed in the table. The analysis results were subjected to cluster analysis, and the results are shown in Figure 6.

[0040]

[0041] The WT, Amp, Amp / Clind, clade 8, and Sakai groups each had different bacteria present in their intestines. In particular, in the clade 8 and Sakai groups administered with enterohemorrhagic E. coli O157, the majority of the intestinal bacteria were Enterobacteriaceae, which is thought to be the administered enterohemorrhagic E. coli O157.

[0042] Example 2-1: Preparation of an infected mouse model (2) Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were bred as follows: 2a-1), 2a-2), and 2a-3); 2b-1) and 2b-2); and 2c-1), 2c-2), and 2c-3). n=5 per group. 2a-1) Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. On the seventh day, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O157 clade 8 strain was administered per mouse. ６ CFU) was administered to mice using an oral tube. This is indicated as "Control" in the figure. 2a-2) 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 7 days. Next, 0.5 g / L clindamycin aqueous solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin aqueous solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O157 clade 8 strain was administered per mouse. ６ CFU) was administered to mice using an oral tube. This is indicated as "O157 clade 8" in the figure. 2a-3) 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 7 days. Next, 0.5 g / L clindamycin aqueous solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin aqueous solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic E. coli O157 clade 8 Stx2Δ strain was administered per mouse. ６ CFU) was administered to mice using an oral gavage. In the figure, this is indicated as "O157Stx2Δ."

[0043] 2b-1) Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. This is indicated as "Control" in the figure. 2b-2) A 1 g / L ampicillin solution was allowed to be drunk ad libitum for 7 days. Next, a 0.5 g / L clindamycin solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O157 Sakai strain was administered per mouse. ６ CFU) was administered to mice using an oral gavage. In the figure, this is indicated as "O157 Sakai."

[0044] 2c-1) Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. This is indicated as "Control" in the figure. 2c-2) A 1 g / L ampicillin solution was allowed to be drunk ad libitum for 7 days. Next, a 0.5 g / L clindamycin solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O26 WT-1 strain was administered per mouse. ６ CFU) was administered to mice using an oral tube. In the figure, this is shown as "O26 WT-1." 2c-3) 1 g / L ampicillin aqueous solution was allowed to be drunk ad libitum for 7 days. Next, 0.5 g / L clindamycin aqueous solution was allowed to be drunk ad libitum for 5 days. Four hours after the start of drinking the clindamycin aqueous solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic E. coli O26 WT-2 strain was administered per mouse. ６ CFU) was administered to mice using an oral gavage. In the figure, this is indicated as "O26 WT-2."

[0045] One week after oral administration of pathogenic E. coli, feces were collected and a fecal suspension was prepared. After allowing the fecal suspension to stand, a dilution series was prepared by serial dilution of the supernatant, and each dilution was applied to CT-SMAC agar medium (manufactured by Kyokuto Pharmaceutical Industries Co., Ltd.) and allowed to stand at 37°C for 18 to 24 hours. The number of colorless and transparent colonies formed was counted, and the number of bacteria in the feces was calculated. On CT-SMAC agar medium, normal E. coli forms red colonies, while enterohemorrhagic E. coli forms colorless and transparent colonies.

[0046] The mice were continued to be bred, and the survival rate was determined. As a result, the number of bacteria in the feces of the mice in each group and the survival curve are shown in FIG.

[0047] All mice administered with the wild-type enterohemorrhagic E. coli O157 clade 8 strain died within 9 days after administration. On the other hand, all mice administered with the Shiga toxin-deficient enterohemorrhagic E. coli O157 clade 8 Stx2Δ strain survived. This suggests that the mice died due to the Shiga toxin produced by E. coli O157. All mice administered with the enterohemorrhagic E. coli O157 Sakai strain died within 41 days after administration. All mice administered with the enterohemorrhagic E. coli O26 WT-1 strain died within 9 days after administration. All mice administered with the enterohemorrhagic E. coli O26 WT-2 strain died within 48 days after administration.

[0048] The trends, including other trials not shown, are as follows: All mice administered with the wild-type enterohemorrhagic E. coli O157 Clade 8 strain died within 8 to 9 days after administration. All mice administered with the enterohemorrhagic E. coli O157 Sakai strain died within approximately 40 days after administration. All mice administered with the enterohemorrhagic E. coli O26 WT-1 strain died within 7 to 10 days after administration. All mice administered with the enterohemorrhagic E. coli O26 WT-2 strain died within 35 to 50 days after administration. These results suggest that pathogenicity to mice differs between pathogenic E. coli strains.

[0049] Example 2-2 Mice were raised in the same manner as in 2a-1) and 2a-2) of Example 2-1 above, except that the 0.5 g / L aqueous clindamycin solution was replaced with a 0.01 g / L aqueous erythromycin solution or a 0.5 g / L aqueous lincomycin solution, and a survival curve was determined. The results are shown in Figure 8.

[0050] Example 3: Phenotype of infected mouse model (1) As in Example 2, section 2a-2), a suspension of enterohemorrhagic E. coli O157 clade 8 strain was orally administered to mice. Seven days after administration of enterohemorrhagic E. coli O157 clade 8 strain, mice were dissected, their kidneys were removed, and the tissues were fixed in 4% paraformaldehyde. The obtained tissues were entrusted to the Sapporo General Pathology Research Institute, Inc., where they were embedded in paraffin, thinly sectioned, and TUNEL stained. As a control, tissue from uninfected mice not administered enterohemorrhagic E. coli O157 clade 8 strain was also similarly embedded in paraffin and stained with TUNEL. The results of TUNEL staining of kidney cortical cells are shown in Figure 9A. In the figure, arrowheads indicate TUNEL-positive cells. TUNEL-stained positive cells are cells undergoing apoptosis.

[0051] In addition, blood was collected from the fundus of mice 7 days after administration of enterohemorrhagic E. coli O157 clade 8 strain and collected in BD Vacutainer Heparin Tubes (Becton, Dickinson and Company) to obtain plasma components. The concentrations of sodium (Na), potassium (K), inorganic phosphorus (IP), creatinine (CRE), urea nitrogen (BUN), and amylase (AMY) contained in the plasma were analyzed by Oriental Yeast Co., Ltd. As a control, the concentrations of components contained in the plasma were also analyzed in the same manner for tissues derived from uninfected mice that were not administered enterohemorrhagic E. coli O157 clade 8 strain. The analysis results are shown in Figure 9B.

[0052] Compared with control mice, mice treated with E. coli O157 clade 8 showed a higher number of apoptotic cells in the kidney cortex. Furthermore, plasma indices in mice treated with E. coli O157 clade 8 showed values ​​similar to those of renal failure or hemolytic uremic syndrome.

[0053] Example 4: Method of using infected mouse model Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were allowed to drink 1 g / L ampicillin solution ad libitum for 7 days. Then, they were allowed to drink 0.5 g / L clindamycin solution ad libitum for 5 days. Four hours after the start of drinking the clindamycin solution, 100 μL (1 × 10) of a suspension of enterohemorrhagic Escherichia coli O157 clade 8 strain was administered per mouse. ６ CFU) was administered to mice using an oral probe. The day administration of the clindamycin aqueous solution began was designated day 0. The mice were then divided into the following four groups and continued to be bred. n=5 mice per group. 4-1) On day 3, the clindamycin aqueous solution was replaced with an ampicillin aqueous solution. 4-2) On day 4, the clindamycin aqueous solution was replaced with an ampicillin aqueous solution. 4-3) On day 3, 500 mL of fecal suspension supernatant prepared from fecal samples collected from 7-week-old C57BL / 6J female mice was administered to each mouse using an oral probe. 4-4) On day 4, 500 mL of fecal suspension supernatant prepared from fecal samples collected from 7-week-old C57BL / 6J female mice was administered to each mouse using an oral probe.

[0054] Groups 4-3) and 4-4) were treated with fecal microbiota transplantation (FMT).

[0055] The survival curves for mice in each group are shown in Figure 10. When ampicillin was administered on day 3, all mice survived, whereas when ampicillin was administered on day 4, 4 out of 5 mice died. Furthermore, all mice administered fecal extract died. These results demonstrate that it is important to treat O157 within 3 days of infection, and that antibiotic administration is more effective than fecal transplantation.

[0056] Example 5: Preparation of infected mouse model (3) Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were bred as described in 5-1) and 5-2) below. Each group consisted of six mice. 5-1) Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. On the seventh day, 100 μL (1 × 10) of a suspension of Salmonella Typhimurium was administered to each mouse. ６CFU) was administered to the mice using an oral tube. This is indicated as "Control" in the figure. 5-2) 1 g / L ampicillin aqueous solution was allowed to be drunk freely for 7 days. Next, 0.5 g / L clindamycin aqueous solution was allowed to be drunk freely for 5 days. Four hours after the start of drinking the clindamycin aqueous solution, 100 μL (1 × 10) of a suspension of Salmonella Typhimurium was administered. ６ CFU) was administered to mice using an oral gavage. This is indicated as "Amp / Clnd" in the figure.

[0057] Five days after oral administration of Salmonella typhimurium, feces were collected and a fecal suspension was prepared. After allowing the fecal suspension to stand, a dilution series was prepared from the supernatant by serial dilution, and each dilution was applied to a streptomycin-containing LB agar medium and incubated at 37°C for 18 to 24 hours. The number of colonies formed was counted, and the number of bacteria in the feces was calculated.

[0058] The mice were continued to be bred, and the survival rate was determined. As a result of the steps and the results of this example, the number of bacteria in the feces of the mice in each group and the survival curve are shown in Figure 11.

[0059] When Salmonella typhimurium was orally administered to antibiotic-treated mice, more Salmonella typhimurium was detected in the feces than in control mice not administered antibiotics, and all of the mice died 6 to 7 days after administration. On the other hand, when Salmonella typhimurium was orally administered to control mice, 100% of the mice survived 7 days after administration, and approximately 30% of the mice survived even 17 days after administration. This suggests that enterobacteria inhibit intestinal colonization and infection by Salmonella typhimurium, and that the present invention is also useful as a Salmonella infection model.

[0060] Example 6: Preparation of infected mouse model (4) Seven-week-old C57BL / 6J female mice purchased from CLEA Japan, Inc. were bred as described in 6-1) and 6-2) below. Each group consisted of n=5. 6-1) Filter-sterilized tap water was allowed to be drunk ad libitum for 12 days. On the seventh day, 100 μL (1 × 10) of a suspension of Citrobacter rodentium was administered per mouse. ６CFU) was administered to the mice using an oral tube. This is indicated as "Control" in the figure. 6-2) 1 g / L ampicillin aqueous solution was allowed to be drunk freely for 7 days. Next, 0.5 g / L clindamycin aqueous solution was allowed to be drunk freely for 5 days. Four hours after the start of drinking the clindamycin aqueous solution, 100 μL (1 × 10) of a suspension of Citrobacter rodentium was administered. ６ CFU) was administered to mice using an oral gavage. This is indicated as "Amp / Clnd" in the figure.

[0061] Five days after oral administration of Citrobacter rodentium, feces were collected and a fecal suspension was prepared. A dilution series was prepared from the supernatant after allowing the fecal suspension to stand by serial dilution, and each dilution was applied to Chromocult Coliform agar medium (Merck, Millipore brand) and allowed to stand at 37°C for 18 to 24 hours. The number of reddish-purple colonies formed was counted, and the number of bacteria in the feces was calculated. On Chromocult Coliform agar medium, E. coli formed dark purple colonies, while coliform bacteria other than E. coli formed reddish-purple colonies.

[0062] Furthermore, when the mice were continued to be bred, no deaths occurred in either group 6-1) or 6-2). The steps of this example and the number of bacteria in the feces of mice in each group are shown in Figure 12.

[0063] When Citrobacter rodentium was orally administered to antibiotic-treated and control mice, more Citrobacter rodentium was detected in the feces of the antibiotic-treated mice than the control mice, and there was little individual difference between the mice. This indicates that enterobacteria inhibit the colonization of Citrobacter rodentium in the intestinal tract, and that the present invention is also useful as a model for Citrobacter genus bacterial infection.

Claims

1. A method for producing an animal model infected with Enterobacteriaceae bacteria, comprising the following steps (1) and (2) in this order: (1) administering a β-lactam antibacterial agent to a non-human mammal; (2) comprising the following steps (2-1) and (2-2) in any order: (2-1) administering at least one antibacterial agent selected from the group consisting of lincomycin antibacterial agents and macrolide antibacterial agents to the non-human mammal; and (2-2) administering Enterobacteriaceae bacteria to the non-human mammal.

2. The method for producing an Enterobacteriaceae bacteria-infected model animal according to claim 1, wherein step (2) comprises steps (2-1) and (2-2) in this order.

3. A method for producing an Enterobacteriaceae bacteria infection model animal as described in claim 1, wherein the β-lactam antibiotic is at least one selected from the group consisting of penicillin antibiotics, cephalosporin antibiotics, carbapenem antibiotics, and monobactam antibiotics.

4. The method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 1, wherein the β-lactam antibacterial drug is at least one selected from the group consisting of ampicillin, penicillin, and cefoperazone.

5. A method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 1, wherein the lincomycin-based antibacterial drug is at least one selected from the group consisting of clindamycin and lincomycin.

6. A method for producing an animal model infected with Enterobacteriaceae bacteria as described in claim 1, wherein the macrolide antibacterial drug is at least one selected from the group consisting of tylosin, erythromycin and clarithromycin.

7. A method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 1, wherein the Enterobacteriaceae bacteria are bacteria of the genus Escherichia, Klebsiella, Salmonella or Citrobacter.

8. The method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 1, wherein the Enterobacteriaceae bacteria is Escherichia coli.

9. The method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 8, wherein the E. coli is pathogenic E. coli or drug-resistant E. coli.

10. A method for producing an animal model infected with Enterobacteriaceae bacteria according to claim 1, wherein the non-human mammal is a mouse.

11. The method for producing an Enterobacteriaceae bacteria infection model animal according to claim 1, wherein step (2) comprises administering at least one antibiotic selected from the group consisting of lincomycin antibiotics and macrolide antibiotics 5 to 10 days after the start of administration of the β-lactam antibiotic.

12. A method for producing an Enterobacteriaceae bacteria infection model animal according to claim 2, wherein step (2-2) comprises administering Enterobacteriaceae bacteria 3 to 24 hours after the start of administration of at least one antibiotic selected from the group consisting of lincomycin antibiotics and macrolide antibiotics.

13. A method for screening for a therapeutic agent for Enterobacteriaceae infections, comprising the following steps (p1) to (p3) in this order: (p1) preparing an animal model for Enterobacteriaceae infection by the method of claim 1; (p2) administering a test substance to the animal model for Enterobacteriaceae infection obtained in step (p1); and (p3) selecting the test substance as a candidate for a therapeutic agent for Enterobacteriaceae infections when the model animal administered the test substance in step (p2) shows an improvement in the symptoms of infection compared to the control.

14. A method for evaluating a therapeutic agent for Enterobacteriaceae infection, comprising the following steps (q1) to (q3) in this order: (q1) preparing an Enterobacteriaceae infection model animal by the method of claim 1; (q2) administering a therapeutic agent to be evaluated to the Enterobacteriaceae infection model animal obtained in step (q1); and (q3) evaluating the therapeutic effect of the Enterobacteriaceae infection model animal administered the therapeutic agent to be evaluated in step (q2).

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