Antibacterial composition for drug-resistant bacteria or inflammation-inducing bacteria

Abstract

The present invention is an antibacterial composition containing intestinal bacteria as an effective ingredient for targeting drug-resistant bacteria or inflammation-inducing bacteria, or a pharmaceutical composition for treating, improving or preventing infectious diseases and inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to an antibacterial composition for drug-resistant bacteria or inflammation-inducing bacteria. In addition, the present invention relates to a pharmaceutical composition or method for treating, ameliorating or preventing diseases caused by drug-resistant bacteria or inflammation-inducing bacteria. Background Art

[0002] There are a variety of normal bacteria in the mucous membranes of the digestive tract and oral cavity, which together form a flora. The normal flora plays a great role in maintaining the physiology or health of the host. Abnormal composition of the normal flora is called dysbiosis, which has been gradually clarified as the cause of various diseases. If we further understand the normal flora of the mucosa, it is very likely to contribute to the development of new disease countermeasures or treatments for various diseases, but due to its complexity, its detailed mechanism has not yet been fully clarified.

[0003] People secrete and swallow about 1.5L of saliva every day. Generally speaking, the bacteria contained in saliva (oral bacteria) only pass through the intestines and do not colonize. However, in some cases, oral bacteria sometimes colonize in the intestines. In particular, it has been reported that in Crohn's disease, cirrhosis, and colorectal cancer, intestinal colonization of oral bacteria can be observed from the early stages of the disease. Moreover, it is known that colonized oral bacteria can affect the symptoms of the disease (Non-Patent Literature 1 to 6).

[0004] In addition, the inventors have successfully isolated, cultured, and identified bacteria related to the onset of the disease from oral bacteria of patients with Crohn's disease, etc., by colonizing the intestines and inducing Th1 cells (Patent Document 1). More specifically, the inventors orally administered saliva taken from a Crohn's patient to germ-free mice and found that interferon-γ (IFN-γ)-producing CD4-positive T cells (Th1 cells) increased significantly in the large intestine. Moreover, the Kp2H7 strain, which can be considered to belong to Klebsiella pneumoniae, was successfully isolated and cultured from the intestines of mice in which the increase in Th1 cells can be seen. It is further clarified that the proliferation or activation of Th1 cells induced by colonizing the bacteria taken from the saliva of Crohn's patients in the intestines is also related to the occurrence of enteritis.

[0005] Furthermore, the present inventors found that when the Kp2H7 strain was orally administered to SPF (specific-pathogen-free) mice, unlike the germ-free mice, intestinal colonization of these bacterial strains was not confirmed. Furthermore, it was also clarified that these bacterial strains can sometimes colonize in the intestines of SPF mice by administering antibiotics to the mice. Moreover, based on such results, the present inventors assume that intestinal bacteria that inhibit intestinal colonization of Th1 cell-inducing bacteria such as the Kp2H7 strain exist in the intestines, and that the intestinal bacteria are eliminated from the intestines by administering the antibiotics, thereby colonizing the bacteria in the intestines.

[0006] Therefore, an attempt was made to identify bacteria that inhibit the intestinal colonization of Th1 cell-inducing bacteria in human intestinal bacteria. As a result, 68, 37 and 42 intestinal bacterial strains were successfully isolated and cultured from stool samples taken from three healthy people (#K, #F and #I), respectively, and the 16SrDNA sequence of each strain was determined. Furthermore, it was clarified that the intestinal colonization of Th1 cell-inducing bacteria can be inhibited by administering these bacterial strains (Patent Document 2).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2018 / 084172

[0010] Patent Document 2: International Publication No. 2019 / 017389

[0011] Non-patent literature

[0012] Non-patent literature 1: Y. Chenet et al., Scientific reports 6, 34055 (2016)

[0013] Non-patent document 2: D. Gevers et al., Cell host µbe 15, 382-392 (2014)

[0014] Non-patent document 3: CALozupone et al., Cell host µbe 14, 329-339 (2013)

[0015] Non-patent literature 4: I. Vujkovic-Cvijin et al., Science translational medicine 5, 193ra191 (2013)

[0016] Non-patent document 5: N. Qin et al., Nature 513, 59-64 (2014)

[0017] Non-patent document 6: CL Sears, WS Garrett, Cell host µbe 15, 317-328 (2014) Summary of the invention

[0018] Problems to be solved by the invention

[0019] The present invention aims to discover intestinal bacteria that have antibacterial activity against drug-resistant bacteria or inflammation-inducing bacteria, to provide an antibacterial composition for drug-resistant bacteria or inflammation-inducing bacteria containing the intestinal bacteria as an active ingredient, and to provide a pharmaceutical composition or method for treating, ameliorating or preventing diseases caused by drug-resistant bacteria or inflammation-inducing bacteria.

[0020] Means for solving problems

[0021] The results of multiple studies conducted by the inventors to achieve the above-mentioned purpose have shown that bacteria that inhibit the intestinal colonization of the Th1 cell-inducing bacteria (68 strains of intestinal bacteria obtained from healthy person #K, 37 strains of intestinal bacteria obtained from healthy person #F, and 42 strains of intestinal bacteria obtained from healthy person #I) can inhibit the intestinal colonization of multidrug-resistant bacteria (carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus, Clostridium difficile, Campylobacter jejuni) and inflammation-inducing bacteria (adherent and invasive Escherichia coli).

[0022] Furthermore, regarding such ability to inhibit intestinal bacterial colonization, 18 strains that can exert the same degree of ability as 37 intestinal bacterial strains isolated from healthy person #F were successfully selected from the 37 strains, thereby completing the present invention.

[0023] That is, the present invention provides the following inventions.

[0024] [1] An antibacterial composition for drug-resistant bacteria or inflammation-inducing bacteria, comprising intestinal bacteria as an active ingredient.

[0025] [2] The antibacterial composition according to [1], wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity with the base sequence.

[0026] [3] An antibacterial composition as described in [1], wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence recorded in any one of sequence numbers: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105, or a base sequence that is at least 90% identical to the base sequence.

[0027] [4] The antibacterial composition according to [1], wherein the intestinal bacteria is at least one type of bacteria specifically defined by any one of the deposit numbers NITE BP-03147 to 03164.

[0028] [5] The antibacterial composition as described in [1], wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 1 to 147 or a base sequence that is at least 90% identical to the base sequence.

[0029] [6] The antibacterial composition as described in [1], wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 1 to 68 or a base sequence that is at least 90% identical to the base sequence.

[0030] [7] The antibacterial composition as described in [1], wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 106 to 147 or a base sequence that is at least 90% identical to the base sequence.

[0031] [8] The antibacterial composition according to any one of [1] to [7] is a pharmaceutical composition.

[0032] [9] The antibacterial composition according to any one of [1] to [7] is a pharmaceutical composition for treating, ameliorating or preventing an infectious disease or an inflammatory disease.

[0033] Effects of the Invention

[0034] According to the present invention, by inhibiting the colonization of drug-resistant bacteria or inflammation-inducing bacteria in the intestine, the proliferation or activation of these bacteria can be inhibited, and diseases caused by these bacteria can be treated, improved or prevented. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the time-dependent changes in the number of Klebsiella in feces (CFU) when Klebsiella 2H7 strain (Kp2H7) was administered to germ-free mice and fecal samples (FMT) from healthy individuals were administered to the mice one week later. Klebsiella was significantly reduced in all five fecal samples.

[0036] Figure 2 This is a histogram showing the results of 16S meta-analysis of three types of feces collected from healthy donors F, I, and K. Each square represents a strain, and its size represents the proportion of the bacteria in the total bacterial count. The three types of feces were cultured under an anaerobic environment, and the yellow color of the adjacent histogram (under color display) represents the strain that can be isolated from the culture. The total number of bacteria that can be isolated is shown below.

[0037] Figure 3 This is a graph showing the time-dependent changes in the amount of Kp2H7 in stool when Kp2H7 was colonized in germ-free mice and then mixed with strains isolated from feces. The 37 strains derived from feces (F stool) taken from healthy donor F reduced Klebsiella to the same extent as the fecal sample.

[0038] Figure 4 This is a graph showing the time-dependent changes in the amount of Kp2H7 in the stool when strains isolated from feces were mixed and administered to germ-free mice after Kp2H7 was colonized. Like the 37 strains derived from F stool, the 68 strains isolated from the feces of healthy donor K (K stool) reduced the number of Klebsiella to the same extent as the fecal sample.

[0039] Figure 5 This is a graph showing the time-dependent changes in the number of Kp2H7 bacteria in the feces of germ-free mice, when Kp2H7 was administered to the mice, F37mix (strain 37 derived from F feces) was administered one week later, and ampicillin was administered to the drinking water one month later. The Klebsiella genus increased temporarily due to the administration of ampicillin, but then decreased again.

[0040] Fig. 6A Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F31, F22, F20, F32) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0041] Figure 6B Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F26, F28, F21, F30) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0042] Figure 6C Yes means Figure 5The graph shows the temporal changes in the abundance ratio of each bacterial amount (F24, F23 / F25, F35 / F36, F09) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0043] Fig.6D Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F33, F12, F17 / F19, F18) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0044] Fig. 6E Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F34, F03 / F08, F29, F13) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0045] Fig. 6F Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F04 / F08, F37, F01, F02) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0046] Figure 6G Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F05, F07, F14) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0047] Figure 6H Yes means Figure 5 The graph shows the temporal changes in the abundance ratio of each bacterial amount (F10 / F15, F16, F11 / F27) in the total bacterial amount in the experiment. The number, r (Spearman rank correlation coefficient with Klebsiella) and bacterial name of each bacterial species are shown below the graph.

[0048] Figure 7 The graph arranges bacteria in the order of positive correlation between Kp2H7 and the Spearman rank correlation coefficient of each bacterial population. Most of the Bacteroides species showed no correlation with Kp2H7, and most of the negatively correlated species were Furmicutes.

[0049] Figure 8 After Kp2H7 was administered to germ-free mice to colonize them, the mixture was administered Figure 7The graph shows the time-dependent changes in the amount of Kp2H7 in stool in CFU for 37 strains (F37mix), 8 strains belonging to the Bacteroidetes phylum (F8mix) among the 37 strains, or 29 strains other than these (F29mix). The 29 strains other than Bacteroides also showed a reduction in Klebsiella that was almost the same as that of the 37 strains, and it is believed that Bacteroides is not required for the elimination of Klebsiella.

[0050] Fig. 9 Yes means Figure 8 Phylogenetic tree of the details of F37mix, F8mix and F29mix used in the experiment shown. The phylogenetic tree was created by using MEGA X to analyze the DNA base sequences of the 16SrDNA of the isolated bacteria using the Sanger method by using the neighbor-joining method. Fig.10 and 12 The same is true in Chinese.

[0051] Fig.10 It is a phylogenetic tree showing the details of 18 strains derived from F stool (F18mix).

[0052] Fig.11 After Kp2H7 was colonized in germ-free mice, 37 strains derived from F feces (the aforementioned F37mix) and 18 strains derived from F feces (the aforementioned F37mix) were mixed and administered. Fig.10 The graph shows the time-dependent change of the bacterial count in the stool of Kp2H7 in CFU when the strains were F18mix, or 42 strains derived from the stool of healthy person I (I stool). For F18mix, Klebsiella genus was excluded in the same manner as F37mix.

[0053] Fig.12 This is a phylogenetic tree that divides 18 strains (F18mix) from F stool into 4 groups and shows their details. These 4 groups were extracted from 18 strains (F18mix) to prepare strain groups F15mix (F18mix-other phyla), F12mix (F18mix-Lachnoclostiridum), F14mix (F18mix-Blautia), and F13mix (F18mix-other Firmicutes), and provided to Fig.11 The experiment shown.

[0054] Fig.13This is a graph showing the time-dependent changes in the amount of Kp2H7 in stool in CFU when Kp2H7 was colonized in germ-free mice and then the strains repeated by F37mix and the group without F18mix (F31-18mix), the F15mix, the F12mix, the F14mix, the F13mix, or the F18mix were mixed and administered to each of them. In addition, Fig.13 The data of two experiments are combined in the table. Removed from F18mix Fig.12 In any of the groups shown, the ability to exclude Klebsiella decreased, indicating that any of the groups is important for the exclusion of Klebsiella.

[0055] Fig.14 It means in Fig.13 The graph shows the CFU of Kp2H7 in the stool of each group at the 28th day in the experiment shown. The bacterial count of Klebsiella was statistically significantly lower in the F18mix-administered group than in the other administration groups except for the F37mix-administered group.

[0056] Fig.15 The figure is a scatter plot showing the results of flow cytometric analysis of immune cells in the lamina propria of the mouse colon mucosa in the F37mix administration group, the F18mix administration group, or the Kp2H7 administration group. The numerical values ​​in each gate (quadrilateral) in the figure represent the proportion of CD4+IFNγ+ cells. Compared with the Kp2H7 administration group alone, the induction of CD4+IFNγ+ cells was suppressed in the F37mix administration group and the F18mix administration group.

[0057] Fig.16 This is a graph showing the time-dependent changes in the amount of Kp2H7 in the stool when Kp2H7 was administered to germ-free mice and each strain mix was administered one week later, with CFU representing the change. The "F15mix" in the figure represents the results of mice administered with three strains of E.coli, Fusobacterium, and Bifidobacterium removed from F18mix, while "F18mix-E.coli", "F18mix-Fusobacterium", and "F18mix-Bifidobacterium" represent the results of mice administered with one strain of each of the three strains removed from F18mix. If all three strains are removed from F18mix, their effects are weakened, indicating that each is related to the elimination of Klebsiella.

[0058] Fig.17 Is a sterile Rag2 - / - γc - / - Mouse, MyD88 - / - Triff - / -When Kp2H7 was administered to mice or wild-type mice (WT), and the mixed F37mix was administered one week later, the time-dependent change in the amount of Kp2H7 in the stool was expressed in CFU. All types of mice can eliminate Klebsiella equally. This suggests that the host's main natural immunity and acquired immunity have nothing to do with the elimination of Klebsiella.

[0059] Fig.18 This is a graph showing the time-dependent changes in the amount of CRE in the stool when Klebsiella (Kp-CRE) was administered to germ-free mice and a mix of isolated bacteria (F37mix, K68mix, I42mix) was administered to the mice one week later. F37mix and K68mix can also reduce CRE.

[0060] Fig.19 Yes Fig.18 The following are microscopic photographs of the results of HE staining of the large intestine of mice at the end of the experiment. No signs of inflammation were observed in mice to which any of the isolate mixes were administered.

[0061] Fig. 20 This is a graph showing the time-dependent changes in the amount of VRE in stool in CFU when VRE (vancomycin-resistant enterococci) was administered to germ-free mice and a mix of isolated bacteria (F37mix, K68mix, I42mix) was administered to the mice one week later. K68mix was more effective in reducing the amount of VRE than F37mix.

[0062] Fig.21 Yes Fig. 20 The results of HE staining and analysis of the large intestine of mice at the end of the experiment are shown in the microscopic photographs. No signs of inflammation were observed in mice administered any of the isolate mixes.

[0063] Fig. 22 This is a graph showing the time-dependent changes in the amount of AIEC in feces in CFU when AIEC was administered to germ-free mice and a mix of isolated bacteria (F37mix, K68mix, I42mix) was administered to the mice one week later. F37mix was the most effective in reducing the amount of AIEC.

[0064] Fig.23 This is a graph showing the time-dependent changes in the amount of ESBL-producing Klebsiella in feces in CFU when ESBL-producing Klebsiella was administered to germ-free mice and the isolated bacterial mix (F37mix, K68mix, I42mix) was administered to the mice one week later. F37mix and K68mix can eliminate ESBL-producing Klebsiella in feces as well as feces taken from F.

[0065] Fig.24This is a graph showing the time-dependent changes in the amount of Campylobacter in the stool in CFU when Campylobacter jejuni was administered to germ-free mice and then administered to the mice one week later with a bacterial isolate mix (F37mix, K68mix, I42mix) or a stool sample taken from a healthy person F. Campylobacter jejuni was eliminated to the same extent in all groups administered with the bacterial isolate mix.

[0066] Fig.25 This is a graph showing the time-dependent changes in the number of Campylobacter bacteria in the stool when Campylobacter jejuni was administered to germ-free mice and then, one week later, the mice were administered with an isolated bacterial mix (F37mix, K68mix, I42mix) or a stool sample taken from a healthy person F. The relative value obtained by dividing by the total bacterial count was used. Campylobacter jejuni was eliminated to the same extent in all the groups administered with the isolated bacterial mix.

[0067] Fig.26 This is a graph showing the results of qPCR analysis of the time-dependent changes in the amount of Clostridium difficile in feces when Clostridium difficile was administered to germ-free mice and then, one week later, isolated bacterial mix (F37mix, K68mix, I42mix, K47mix) or fecal samples taken from healthy human F were administered to the mice. K68mix and K47mix were more effective in excreting Clostridium difficile than feces taken from F, but F37mix was less effective. DETAILED DESCRIPTION

[0068] <Intestinal bacteria>

[0069] In the present invention, the intestinal bacteria contained as an active ingredient of the antibacterial composition have an antibacterial effect against drug-resistant bacteria or inflammation-inducing bacteria (hereinafter also referred to as "drug-resistant bacteria, etc.") in the intestine.

[0070] The "antibacterial activity" referred to in the present invention refers to the activity of inhibiting bacterial activity, more specifically, the activity of inhibiting bacterial proliferation or colonization, or the activity of eliminating bacteria, for example, the activity of inhibiting bacterial colonization in the intestine and the activity of eliminating bacteria from the intestine.

[0071] "Intestinal bacteria" refers to bacteria present in the intestines of animals. In addition, as animals where the bacteria exist, humans and non-human animals (mice, rats, monkeys, pigs, cattle, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, etc.) can be cited; among these animals, humans are preferred.

[0072] In the present invention, "intestinal bacteria" may be a single strain of bacteria or a mixture of bacterial strains composed of multiple strains of bacteria. In addition, when composed of multiple strains of bacteria, it is desirable that at least one bacterial strain has antibacterial activity against drug-resistant bacteria and the like. In addition, at this time, the multiple bacterial strains may be bacterial strains that do not have the antibacterial activity, or may include bacterial strains that have the effect of enhancing the activity of bacterial strains with the antibacterial activity, bacterial strains that have the effect of maintaining the proliferation or colonization of bacterial strains with the antibacterial activity, and bacterial strains that have the effect of inhibiting the inhibitory activity of bacteria that inhibit the antibacterial activity.

[0073] In the present invention, the term “intestinal bacteria” includes, for example, at least one bacterium having a DNA consisting of a base sequence described in any one of sequence numbers: 1 to 147 or a base sequence having at least 70% identity with the base sequence; at least one bacterium having a DNA consisting of a base sequence described in any one of sequence numbers: 1 to 68 or a base sequence having at least 70% identity with the base sequence; at least one bacterium having a base sequence described in any one of sequence numbers: 69 to 105 or a base sequence having at least 70% identity with the base sequence; At least one bacterium having a DNA consisting of a base sequence with at least 70% identity to the base sequence (for example, at least one bacterium having a DNA consisting of a base sequence recorded in any one of sequence numbers: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105, or a base sequence with at least 70% identity to the base sequence); or at least one bacterium having a DNA consisting of a base sequence recorded in any one of sequence numbers: 106 to 147, or a base sequence with at least 70% identity to the base sequence.

[0074] The sequences shown in the sequence numbers are the 16SrDNA sequences of K68, F37 and I43 in the attached data. The following Tables 1 to 4 show the correspondence between each bacterium, the sequence number representing each 16SrDNA sequence, and each bacterium inferred from the sequence. In addition, K, F and I represent intestinal bacteria isolated from the feces of three healthy people (Japanese) (see Patent Document 2).

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079] Table 3

[0080]

[0081] Table 4

[0082]

[0083] Tables 1 to 4 show the species names and RefSeq accessions (as of January 8, 2020) of the sequences recorded in each sequence number after BLAST search against the RefSeq 16sDNA sequence database. In addition, generally speaking, if the % identity is >97%, it can be identified to the species; if it is >94%, it can be identified to the genus. Therefore, strains with a % identity of >94% should be understood as bacteria that can be specifically specified at the genus level.

[0084] The so-called "at least 70% identity" in the intestinal bacteria of the present invention means that the identity with respect to each base sequence is preferably 80% or more, more preferably 85% or more, further preferably 90% or more (for example, 91% or more, 92% or more, 93% or more, 94% or more), more preferably 94% or more (for example, 95% or more, 96% or more, 97% or more, 98% or more), and particularly preferably 99% or more.

[0085] In addition, the homology or identity of sequences (amino acid sequences or nucleotide (base) sequences) can be determined using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing the homology or identity between sequences by BLAST, for example, the BLAST of the National Center for Biological Information (NCBI) of the United States can be used (for example, using default values, i.e., initially set parameters).

[0086] In the present invention, the "intestinal bacteria" having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 147 or a base sequence having at least 70% identity with the base sequence is preferably at least 15 bacteria among these intestinal bacteria groups, more preferably at least 30 bacteria among the intestinal bacteria groups, further preferably at least 75 bacteria among the intestinal bacteria groups, more preferably at least 120 bacteria among the intestinal bacteria groups, further preferably at least 135 bacteria among the intestinal bacteria groups, more preferably at least 140 bacteria among the intestinal bacteria groups, further preferably 147 intestinal bacteria each having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 147 or a base sequence having at least 70% identity with the base sequence, and particularly preferably 147 bacteria each having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 147.

[0087] In the present invention, the "intestinal bacteria" having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 68 or a base sequence having at least 70% identity with the base sequence is preferably at least 7 bacteria among these intestinal bacteria groups, more preferably at least 15 bacteria among the intestinal bacteria groups, further preferably at least 35 bacteria among the intestinal bacteria groups, more preferably at least 60 bacteria among the intestinal bacteria groups, further preferably at least 65 bacteria among the intestinal bacteria groups, more preferably 68 intestinal bacteria each having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 68 or a base sequence having at least 70% identity with the base sequence, and particularly preferably 68 bacteria each having a DNA consisting of a base sequence recorded in any one of sequence numbers: 1 to 68. In addition, as the "intestinal bacteria" having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 1 to 68 or a base sequence having at least 70% identity with the base sequence, it is desired that the bacteria have resistance to ampicillin. In addition, 46 types of bacteria each having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 1 to 46 or a base sequence having at least 70% identity with the base sequence are also suitable for use in the present invention.

[0088] In the present invention, the "intestinal bacteria" having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity with the base sequence is preferably at least 4 bacteria among these intestinal bacteria groups, more preferably at least 8 bacteria among the intestinal bacteria groups, further preferably at least 18 bacteria among the intestinal bacteria groups, more preferably at least 29 bacteria among the intestinal bacteria groups, further preferably at least 33 bacteria among the intestinal bacteria groups, more preferably at least 35 bacteria among the intestinal bacteria groups, further preferably 37 intestinal bacteria each having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity with the base sequence, and particularly preferably 37 bacteria each having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 69 to 105. Furthermore, the "intestinal bacteria" having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity with the base sequence is desired to be sensitive to ampicillin.

[0089] In the present invention, the “intestinal bacteria” having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105, or a base sequence having at least 70% identity with the base sequence, is preferably at least 2 bacteria among these intestinal bacteria groups, more preferably at least 5 bacteria among the intestinal bacteria groups, further preferably at least 10 bacteria among the intestinal bacteria groups, more preferably at least 14 bacteria among the intestinal bacteria groups, further preferably at least 15 bacteria among the intestinal bacteria groups, more preferably at least 16 bacteria among the intestinal bacteria groups, and further preferably at least 17 bacteria among the intestinal bacteria groups. The at least 17 bacteria in the intestinal bacterial group are more preferably 18 intestinal bacteria each having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69, 80, 85-92, 94, 96, 98-101, 103 and 105 or a base sequence having at least 70% identity with the base sequence, and particularly preferably 18 bacteria each having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69, 80, 85-92, 94, 96, 98-101, 103 and 105 (the bacteria having a DNA consisting of a base sequence described in SEQ ID NO: 69, the bacteria having a DNA consisting of a base sequence described in SEQ ID NO: 80, the bacteria having a DNA consisting of a base sequence described in SEQ ID NO: 91, the bacteria having a DNA consisting of a base sequence described in SEQ ID NO: 93). Bacteria having a DNA consisting of the base sequence recorded in sequence number: 85, bacteria having a DNA consisting of the base sequence recorded in sequence number: 86, bacteria having a DNA consisting of the base sequence recorded in sequence number: 87, bacteria having a DNA consisting of the base sequence recorded in sequence number: 88, bacteria having a DNA consisting of the base sequence recorded in sequence number: 89, bacteria having a DNA consisting of the base sequence recorded in sequence number: 90, bacteria having a DNA consisting of the base sequence recorded in sequence number: 91, bacteria having a DNA consisting of the base sequence recorded in sequence number: 92 Bacteria, bacteria having a DNA consisting of the base sequence recorded in sequence number: 94, bacteria having a DNA consisting of the base sequence recorded in sequence number: 96, bacteria having a DNA consisting of the base sequence recorded in sequence number: 98, bacteria having a DNA consisting of the base sequence recorded in sequence number: 99, bacteria having a DNA consisting of the base sequence recorded in sequence number: 100, bacteria having a DNA consisting of the base sequence recorded in sequence number: 101, bacteria having a DNA consisting of the base sequence recorded in sequence number: 103, and bacteria having a DNA consisting of the base sequence recorded in sequence number: 105).

[0090] In addition, typical examples of 18 bacteria each having a DNA consisting of a base sequence described in any one of sequence numbers: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105 are the deposited strains shown in the following Table 5. All bacterial strains were entrusted to the National Institute for Technology Evaluation (NITE, Room 122, Kazusakamatari 2-5-8, Kisarazu City, Chiba Prefecture, zip code 292-0818) for deposit on March 2, 2020.

[0091] Table 5

[0092]

[0093] In addition, bacteria (derivative strains, induced strains, etc.) cultivated from these bacteria by mutation treatment, gene recombination, genome editing, selection of natural mutant strains, etc. are also included in the intestinal bacteria of the present invention, as long as the antibacterial effect against drug-resistant bacteria or inflammation-inducing bacteria is not impaired.

[0094] In the present invention, the "intestinal bacteria" having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity with the base sequence is preferably at least 4 bacteria among these intestinal bacteria groups, more preferably at least 9 bacteria among the intestinal bacteria groups, further preferably at least 22 bacteria among the intestinal bacteria groups, more preferably at least 34 bacteria among the intestinal bacteria groups, further preferably at least 39 bacteria among the intestinal bacteria groups, more preferably at least 41 bacteria among the intestinal bacteria groups, further preferably 42 intestinal bacteria each having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity with the base sequence, and particularly preferably 42 bacteria each having a DNA consisting of a base sequence recorded in any one of SEQ ID NOs: 106 to 147. Furthermore, the "intestinal bacteria" having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity with the base sequence is desired to be sensitive to ampicillin.

[0095] In addition, in the present invention, as one embodiment of the "intestinal bacteria", there can be mentioned intestinal bacteria that show resistance to at least one compound selected from the group consisting of spectinomycin and / or show sensitivity to at least one compound selected from the group consisting of ampicillin, tylosin and chloroform. In addition, as another embodiment, there can be mentioned intestinal bacteria that show resistance to metronidazole and / or show sensitivity to at least one compound selected from the group consisting of vancomycin and tylosin.

[0096] Furthermore, as shown in the Examples described below, the above-mentioned intestinal bacteria were isolated by the present inventors and are useful for exerting antibacterial action against drug-resistant bacteria, inflammation-inducing bacteria, etc. Therefore, the present invention can also provide the following inventions.

[0097] (1) At least one bacterium having a DNA consisting of a base sequence as set forth in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity with the base sequence.

[0098] (2) At least one bacterium having a DNA consisting of a base sequence described in any one of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105, or a base sequence having at least 90% identity with the base sequence.

[0099] (3) At least one type of bacteria specified by any one of the accession numbers NITE BP-03147 to 03164.

[0100] (4) At least one bacterium having a DNA consisting of a base sequence as set forth in any one of SEQ ID NOs: 1 to 147 or a base sequence having at least 90% identity with the base sequence.

[0101] (5) At least one bacterium having a DNA consisting of a base sequence as set forth in any one of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity with the base sequence.

[0102] (6) At least one bacterium having a DNA consisting of a base sequence as set forth in any one of SEQ ID NOs: 106 to 147 or a base sequence having at least 90% identity with the base sequence.

[0103] (7) The bacterium according to any one of (1) to (6), which has an antibacterial effect against drug-resistant bacteria, inflammation-inducing bacteria, or bacteria that induce proliferation or activation of Th1 cells in the intestine.

[0104] <Antibacterial Composition and Pharmaceutical Composition>

[0105] The composition of the present invention can be any composition containing the intestinal bacteria, which may be live bacteria or dead bacteria. In addition, the composition can be used in combination, and in the case of being ingested or absorbed as a result of combined use (the case of combined use of the composition), the intestinal bacteria may also exist separately in two or more compositions.

[0106] The composition of the present invention may be in the form of a pharmaceutical composition, a quasi-drug composition, a food or beverage (including animal feed), or a reagent for research purposes (eg, in vitro or in vivo experiments).

[0107] Since the composition of the present invention exhibits antibacterial activity against drug-resistant bacteria and the like, it is suitable as a pharmaceutical composition, a quasi-drug composition, or a food or drink for treating, preventing, or ameliorating diseases caused by the bacteria.

[0108] Composition of the present invention can be prepared by known pharmaceutics method.For example, can be made into capsule, lozenge, pill, liquid, powder, granule, fine granule, film-coated agent, granule, tablet, sublingual agent, chewable agent, buccal agent, paste, syrup, suspension, elixir, emulsion, coating agent, ointment, plaster, patch, percutaneous absorption type preparation, lotion, inhalant, aerosol, injection, suppository etc., and can be used for via oral, parenteral (for example intestinal, intramuscular, intravenous, intratracheal, intranasal, percutaneous, intradermal, subcutaneous, intraocular, vagina, intraperitoneal, rectal or suction) or the administration of the path comprising these multiple combined approaches.

[0109] In these formulations, it is possible to suitably combine with a carrier that is pharmacologically or permissible as a food and beverage, and the carrier is specifically sterile water, physiological saline, buffer, culture medium, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, aromatic agent, excipient, vehicle, preservative, adhesive, diluent, isotonic agent, analgesic, extender, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetener, thickener, flavoring agent, solubilizer or other additives etc.

[0110] In addition, in these formulations, based on the viewpoint of more efficiently exerting antibacterial activity against drug-resistant bacteria in the intestine, especially in formulations for the purpose of oral administration, a composition that can effectively deliver the composition of the present invention to the intestine can also be combined. There is no particular limitation on such a composition that can be delivered to the intestine, and a known composition can be appropriately used, for example, a pH-sensitive composition, a composition that can inhibit release to the intestine (cellulose polymers, acrylic acid polymers and copolymers, ethylene acid polymers and copolymers, etc.), a bioadhesive composition that can specifically adhere to the intestinal mucosa (for example, a polymer described in the specification of U.S. Patent No. 6.368.586), a composition containing a protease inhibitor, and a composition that can be specifically decomposed by an enzyme in the intestine).

[0111] In addition, when the antibacterial composition of the present invention is used as a pharmaceutical composition, it may further contain known substances (such as other antibacterial agents, anti-inflammatory agents, immunosuppressants) for the treatment, prevention or improvement of diseases caused by drug-resistant bacteria, etc., and may also be used in combination with the above substances.

[0112] When the composition of the present invention is used as a food and drink, the food and drink can be, for example, a health food, a functional food, a specific health food, a nutritional functional food, a functional labeled food, a nutritional supplement, a patient's food or an animal feed. As a specific example of food and drink, liquid foods such as fermented beverages, oil-containing products, soups, milk beverages, refreshing drinks, tea beverages, alcoholic beverages, beverages, jelly beverages, carbohydrate-containing foods, livestock processed foods, and aquatic processed foods can be cited; vegetable processed foods, semi-solid foods, fermented foods, cakes, pre-packaged products, microwave oven-specific foods, etc. Furthermore, healthy and regular food and drinks modulated into powders, granules, tablets, capsules, liquids, pastes or jelly can also be cited. In addition, the manufacture of the food and drink in the present invention can be implemented by manufacturing techniques known in the art. In the food and drink, ingredients (such as nutrients, etc.) that effectively improve or prevent diseases caused by drug-resistant bacteria, etc. can also be added. Furthermore, multifunctional food and drinks can also be made by combining with other ingredients or other functional foods that can play functions other than the improvement, etc.

[0113] The product (drug, quasi-drug, food and drink, reagent) or the instructions of the composition of the present invention may be attached with an indication that the product exerts antibacterial activity against drug-resistant bacteria, etc., or is used to treat, improve or prevent diseases caused by drug-resistant bacteria, etc. In addition, with regard to food and drink, in order to distinguish it from general food in terms of form and object of use, the indication of health function may be attached to the product of the composition of the present invention as a health functional food (specific health food, nutritional functional food, functional labeled food). The term "additional labeling to the product or instructions" herein refers to additional labeling to the product itself, container, packaging, etc., or additional labeling to the instructions, additional documents, promotional materials, other printed materials, etc. that disclose product information. In addition, the composition of the present invention may also be in the form of a kit.

[0114] In addition, as described above, the intestinal bacteria of the present invention can be used to produce pharmaceutical compositions by known pharmaceutical formulation techniques. Thus, the present invention also provides a use of the intestinal bacteria of the present invention for producing a pharmaceutical composition for treating, ameliorating or preventing diseases caused by drug-resistant bacteria.

[0115] <Treatment methods, etc.>

[0116] The present invention also provides a method for treating, improving or preventing diseases caused by drug-resistant bacteria, etc. in a subject, characterized in that the subject is allowed to ingest the above-mentioned antibacterial composition or pharmaceutical composition, or the above-mentioned intestinal bacteria as their active ingredients (hereinafter collectively referred to as "the pharmaceutical composition of the present invention, etc. or their active ingredients, etc.").

[0117] The "drug-resistant bacteria" referred to in the present invention refers to bacteria that are resistant to antimicrobial agents (antibiotics, etc.) and are ineffective or difficult to be effective against. In addition, the agent may be one agent or multiple agents. That is, the drug-resistant bacteria of the present invention also include multidrug-resistant bacteria. The bacteria are not particularly limited, and examples thereof include carbapenem-resistant Enterobacteriaceae (CRE, Klebsiella pneumoniae producing KPC-2, etc.), vancomycin-resistant Enterococci (VRE, bacteria with a vancomycin resistance gene (vanA), etc.), Clostridium difficile, and Campylobacter jejuni. More specifically, the strains include Klebsiella pneumoniae (ATCC BAA-1705), Enterococcus faecium (Orla-Jensen) Schleifer and Kilpper-Balz (ATCC 700221), Clostridium difficile (Prevot) Lawson et al. (ATCC 43255, strain designation: VPI 10463), Clostridium difficile (Prevot) Lawson et al. (ATCC BAA-1382, strain designation: 630), and Campylobacter jejuni 81-176 (ATCC BAA2151).

[0118] Examples of "diseases caused by drug-resistant bacteria" include infections caused by drug-resistant bacteria. In addition, diseases caused by or related to the infections are also included. Examples of the diseases include respiratory infections such as sepsis, peritonitis, meningitis, gastroenteritis, pneumonia, urinary tract infections, surgical site infections, soft tissue infections, and medical device-related infections (medical device-related bloodstream infections, etc.).

[0119] The "inflammation-inducing bacteria" referred to in the present invention refers to bacteria that induce inflammation in the intestine, and examples thereof include adherent invasive Escherichia coli (AIEC) and more specifically AIEC LF82.

[0120] Examples of "diseases caused by inflammation-inducing bacteria" include diseases caused by inflammation induced by the bacteria or diseases related to the inflammation. Examples of such diseases include inflammatory bowel diseases (chronic inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, and inflammatory bowel diseases).

[0121] The pharmaceutical composition of the present invention or its active ingredients can be used for animals including humans. Animals other than humans are not particularly limited, and various livestock, poultry, pets, experimental animals, etc. can be used as targets.

[0122] In addition, as the ingestion subjects of the intestinal bacteria of the present invention, animals that have drug-resistant bacteria, etc., regardless of whether or not they develop diseases caused by drug-resistant bacteria, etc. In addition, from the perspective of prevention, animals that do not have or may not have the bacteria may also be made to ingest the pharmaceutical compositions of the present invention, etc., or their active ingredients, etc.

[0123] The method for taking the pharmaceutical composition of the present invention or their active ingredients is not particularly limited, and may be oral administration or parenteral administration (e.g., administration into the intestine). If it is administered orally, from the viewpoint of further enhancing the effects of the pharmaceutical composition of the present invention or their active ingredients, the subject of ingestion of the pharmaceutical composition of the present invention or their active ingredients is preferably to reduce gastric acid production in advance by taking a proton pump inhibitor (PPI) or the like.

[0124] In addition, when taking the pharmaceutical composition of the present invention or their active ingredients, the intake amount can be appropriately selected by those skilled in the art according to the age, weight, symptoms of the disease, health status, type of composition (drugs, food, etc.), intake method, etc. of the subject.

[0125] Preferred embodiments of the antibacterial composition, pharmaceutical composition, and therapeutic method of the present invention have been described above, but the present invention is not limited to the above-described embodiments.

[0126] As shown in the examples described below, 18 strains of Klebsiella 2H7 strain (Kp2H7), which is a Th1 cell-inducing bacterium, were successfully screened to have the same degree of intestinal colonization inhibition as 37 strains of intestinal bacteria isolated from healthy person #F. Thus, the present invention relates to antibacterial compositions and pharmaceutical compositions, as well as treatment methods, and can also provide the following schemes.

[0127] <1> An antibacterial composition for Th1 cell-inducing bacteria, comprising intestinal bacteria as an active ingredient, wherein the intestinal bacteria is at least one type of bacteria having a DNA consisting of a base sequence recorded in any one of sequence numbers: 69, 80, 85 to 92, 94, 96, 98 to 101, 103 and 105, or a base sequence having at least 90% identity with the base sequence.

[0128] <2> like <1> The antibacterial composition is a pharmaceutical composition.

[0129] <3> like <1> or <2> The antibacterial composition is a pharmaceutical composition used for treating, improving or preventing diseases caused by Th1 cells.

[0130] <4> A method for inhibiting the proliferation or activation of Th1 cells in a subject, a method for inhibiting immunity in the subject, or a method for treating, ameliorating or preventing a disease caused by Th1 cells in the subject, characterized in that the subject is allowed to ingest <1> ~ <3> The antibacterial composition of any one of the items, or at least one bacterium having a DNA consisting of a base sequence recorded in any one of sequence numbers: 69, 80, 85-92, 94, 96, 98-101, 103 and 105, or a base sequence that is at least 90% identical to the base sequence.

[0131] The "Th1 cell-inducing bacteria" of the present invention are bacteria that are usually present in the human oral cavity, but induce the proliferation or activation of Th1 cells by colonizing in the intestine. Preferably, it is a bacterium belonging to the genus Klebsiella, more preferably a bacterium belonging to Klebsiella pneumoniae or Klebsiella aeromobilis, and inducing the proliferation or activation of Th1 cells in the intestine. In addition, the "Th1 cell-inducing bacteria" are preferably bacteria that are easy to colonize in an intestinal environment where the diversity has changed compared to the normal state due to the administration of antibacterial agents. In addition, it can also be bacteria that are easy to colonize in an intestinal environment where the diversity has changed compared to the normal state due to colitis, etc.

[0132] For examples of "Th1 cell-inducing bacteria", see Patent Document 1, and typically include Kp2H7, Ka11E12, 34E1, BAA-1705, 700603, and 40B3 belonging to the genus Klebsiella. Among these, Kp2H7 or Ka11E12 is more preferred, and Kp2H7 is particularly preferred. For details of these bacteria, see Table 6.

[0133]

[0134] In addition, examples of the “Th1 cell-inducing bacteria” of the present invention include bacteria containing a DNA consisting of a nucleotide sequence having 90% or more (91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with a nucleotide sequence encoding 16S rRNA of Kp2H7 strain, Ka11E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain or 40B3 strain; In addition, bacteria that contain DNA consisting of a nucleotide sequence that has 70% or more (preferably 80% or more, more preferably 85% or more, further preferably 90% or more, more preferably 94% or more (for example, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more)) homology or identity with a specific nucleotide sequence of Kp2H7 strain, Ka11E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain or 40B3 strain can also be mentioned.

[0135] In the present invention, "Th1 cells" are a subpopulation of CD4-positive helper T cells (Th cells), and refer to cells that enhance cellular immunity. In addition, "Th1 cell activity" includes the production of Th1 cytokines (IFN-γ, etc.) by the cells, the activation of cells such as macrophages and cytotoxic T cells (CTL) by the cytokines, and the enhancement of cellular immunity by the activation. Furthermore, "induction of proliferation or activation of Th1 cells" also includes the induction of differentiation from naive T cells to Th1 cells that achieves proliferation or activation of Th1 cells.

[0136] The effect of inducing proliferation or activation of Th1 cells in the intestine can be evaluated by quantitatively detecting Th1 cell-specific markers (e.g., CD4 and IFN-γ). The quantitative detection can be performed by known methods, such as flow cytometry, image cytometry, ELISA, radioimmunoassay, immunohistochemical staining, immunoprecipitation, immunoblotting, antibody array analysis, etc., which use antibodies for detection (immunological methods).

[0137] Whether any bacteria or the like has the effect of inducing the proliferation or activation of Th1 cells in the intestine can be determined by, for example, detecting CD4 + TCRβ + IFN-γ in T cells +When the cell ratio is 10% or more, it can be determined that the bacteria, etc. have the effect of inducing the proliferation or activation of Th1 cells in the intestine (preferably when it is 25% or more, it is determined that the bacteria, etc. have the effect of inducing the proliferation or activation of Th1 cells in the intestine; more preferably when it is 30% or more, it is determined that the bacteria, substances, etc. have the effect of inducing the proliferation or activation of Th1 cells in the intestine).

[0138] The "diseases caused by Th1 cells" referred to in the present invention refer to diseases induced by the proliferation or activation of Th1 cells, and examples thereof include inflammatory bowel diseases (chronic inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, inflammatory bowel diseases, etc.), type 1 diabetes, rheumatoid arthritis, experimental autoimmune encephalitis (EAE), multiple sclerosis, autoimmune diseases such as systemic lupus erythematosus, and chronic inflammatory diseases. In addition, the "immunity" suppressed in the present invention includes not only mucosal immunity (intestinal immunity, etc.), but also systemic immunity. Moreover, it includes not only cellular immunity but also humoral immunity.

[0139] When the antibacterial composition of the present invention is used as a pharmaceutical composition, it may further contain known substances for treating, preventing or improving diseases caused by Th1 cells (eg, anti-inflammatory agents, immunosuppressants), and may be used in combination with such substances.

[0140] In addition, regarding the above <1> ~ <4> For other statements in the <Antibacterial compositions and pharmaceutical compositions> and <Therapeutic methods, etc.>, please refer to the above-mentioned <Antibacterial compositions and pharmaceutical compositions> and <Therapeutic methods, etc.> as appropriate.

[0141] <Composition for testing diseases caused by drug-resistant bacteria, etc.>

[0142] In the present invention, it has been demonstrated that the presence of intestinal bacteria such as drug-resistant bacteria that colonize the intestine etc. can be suppressed. Therefore, by detecting the presence of such intestinal bacteria, diseases caused by drug-resistant bacteria etc. can be examined.

[0143] Therefore, the present invention provides the following composition for examining diseases caused by drug-resistant bacteria and the like.

[0144] The composition for examining a disease caused by drug-resistant bacteria or the like comprises an antibody that specifically recognizes the intestinal bacteria or the like of the present invention.

[0145] A composition for examining a disease caused by drug-resistant bacteria or the like comprises a polynucleotide for detecting a specific nucleotide sequence of the drug-resistant bacteria or the like of the present invention.

[0146] In the present invention, "antibodies that specifically recognize the intestinal bacteria of the present invention, etc." may be polyclonal antibodies or monoclonal antibodies, as long as they can specifically recognize the bacteria, or functional fragments of antibodies (e.g., Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide bond Fv, single-chain Fv (scFv), sc(Fv)2, bispecific antibodies, multispecific antibodies, or polymers thereof). If the antibody of the present invention is a polyclonal antibody, it can be obtained by immunizing an immunized animal with an antigen (polypeptide, polynucleotide, sugar chain, lipid, etc. derived from the intestinal bacteria of the present invention, etc.), and purifying the antiserum thereof by existing means (e.g., salting out, centrifugal separation, dialysis, chromatography, etc.). In addition, monoclonal antibodies can be produced by the hybridoma method or the recombinant DNA method.

[0147] In addition, as the antibody used in the inspection of the present invention, an antibody combined with a marker can be used. By detecting the marker, the amount of the antibody combined with the intestinal bacteria of the present invention or the substance derived from the bacteria can be directly measured. As the marker, as long as it can be combined with the antibody and can be detected by chemical or optical methods, there is no particular limitation, and for example, fluorescent pigments (GFP, etc.), enzymes (HRP, etc.), and radioactive substances can be cited.

[0148] In the inspection composition of the present invention, in addition to the antibody component, other components that are permissible as the composition may also be included. As such other components, for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological salts, markers, and secondary antibodies can be cited. In addition, in addition to the above-mentioned inspection composition, substrates required for the detection of markers, buffers for dilution or washing of positive controls, negative controls or samples, tubes or plates for the reaction of samples with antibodies of the present invention, etc., can also be combined, and a test kit for diseases caused by drug-resistant bacteria, etc. can also be made. In addition, when an unlabeled antibody is used as an antibody standard, a substance obtained by labeling a substance (such as a secondary antibody, protein G, protein A, etc.) that is bound to the antibody can be combined. Furthermore, the inspection kit for diseases caused by the drug-resistant bacteria, etc. may include the instructions for use of the kit.

[0149] Furthermore, the test composition of the present invention may be combined with a device for detecting the antibody of the present invention. Examples of such a device include a flow cytometer and an ELISA reader.

[0150] In the present invention, the "polynucleotide for detecting a specific nucleotide sequence of intestinal bacteria, etc. of the present invention" is not particularly limited as long as it can detect the specific sequence of the bacteria, and examples thereof include a polynucleotide having a chain length of at least 15 nucleotides and any of the polynucleotides described in (a) to (b) below.

[0151] (a) a polynucleotide as a pair of primers designed to sandwich the specific nucleotide sequence

[0152] (b) A polynucleotide serving as a primer or a probe that hybridizes with a nucleotide sequence comprising the specific nucleotide sequence.

[0153] The polynucleotides of the present invention have base sequences complementary to the nucleotide sequences of the intestinal bacteria of the present invention. "Complementary" as used herein may be incompletely complementary as long as hybridization is possible. These polynucleotides generally have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% with respect to the nucleotide sequences.

[0154] The "chain length" of the polynucleotide of the present invention is usually 15 to 100 nucleotides, preferably 17 to 30 nucleotides, and more preferably 20 to 25 nucleotides when used as a primer. In addition, when used as a probe, it is usually 15 to 1000 nucleotides, and preferably 20 to 100 nucleotides.

[0155] The polynucleotide of the present invention may be DNA or RNA, and nucleotides may be replaced in part or all of it by artificial nucleic acids such as LNA (registered trademark, cross-linked nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acid), TNA (threose nucleic acid), and PNA (peptide nucleic acid).

[0156] In addition, the polynucleotide of the present invention can be synthesized chemically using a commercially available nucleotide automatic synthesizer or the like. In addition, as the polynucleotide used for the inspection of the present invention, a polynucleotide combined with a label can be used. As a label, there is no particular restriction as long as it can be combined with a polynucleotide and can be detected by a chemical or optical method, for example, fluorescent pigments (DEAC, FITC, R6G, TexRed, Cy5, etc.), and pigments (chromogens, chromogens) such as DAB, enzymes, and radioactive substances can be cited in addition to fluorescent pigments.

[0157] The test composition of the present invention may contain, in addition to the polynucleotide, other pharmacologically acceptable components, such as buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological salts, and the like.

[0158] In addition to the above-mentioned test composition, a substrate required for detection of a marker added to a polynucleotide, a positive control, a negative control, or a standard such as a buffer for dilution or washing of a sample, or a tube or plate for reaction of a sample with the polynucleotide of the present invention may be combined, and a test kit for diseases caused by drug-resistant bacteria, etc., etc. may be prepared. Furthermore, the test kit for diseases caused by drug-resistant bacteria, etc., may include instructions for use of the kit.

[0159] Furthermore, the test composition of the present invention may be combined with a device for detecting a specific nucleotide sequence of the intestinal bacteria of the present invention, etc. Examples of the device include a thermal cycler, a sequencer, and a microarray.

[0160] Furthermore, the present invention also provides a method for testing a disease caused by drug-resistant bacteria, etc., wherein the antibody, polynucleotide, or test composition described above is used.

[0161] The present invention provides a method for inspecting diseases caused by drug-resistant bacteria, etc., comprising: a step of bringing the antibody, polynucleotide or inspection composition into contact with a sample separated from a subject; and a step of detecting the presence or absence of the intestinal bacteria of the present invention in the intestine through the contact.

[0162] The subject is not particularly limited, and examples include animals such as humans suspected of suffering from diseases caused by drug-resistant bacteria, etc. In addition, the sample separated from the subject is not particularly limited, and a stool sample of the subject, a culture thereof, or polypeptides, polynucleotides, sugar chains, lipids, etc. extracted therefrom are suitable for use in the method of the present invention.

[0163] As a method for detecting the presence or absence of the intestinal bacteria of the present invention by bringing the antibody of the present invention or a test composition containing the antibody into contact with the sample, for example, there can be mentioned methods (immunological techniques) using antibodies for detection, such as ELISA, immunoblotting, antibody array analysis, immunohistochemical staining, flow cytometry, image cytometry, radioimmunoassay, and immunoprecipitation.

[0164] In addition, as a method for detecting the presence or absence of the intestinal bacteria of the present invention by contacting the polynucleotide of the present invention or a test composition containing the polynucleotide with the sample, for example, PCR (RT-PCR, real-time PCR, quantitative PCR), DNA microarray analysis, Northern blotting, 16srRNA sequencing, next-generation sequencing (sequencing-by-synthesis, for example, sequencing by Solexa genome analyzer or Hiseq (registered trademark) 2000 manufactured by Illumina), pyrosequencing (for example, sequencing by sequencer GSLX or FLX manufactured by Roche Diagnostics (454) (so-called 454 sequencing)), ligase reaction sequencing (for example, sequencing by Solid (registered trademark) or 5500xl manufactured by Life Technology), beads array method, in situ hybridization method, dot blotting method, RNase protection assay method, mass spectrometry, genomic PCR method, Southern blotting method can be used.

[0165] The "inspection" of diseases caused by drug-resistant bacteria, etc. referred to in the present invention includes not only checking whether the disease has occurred, but also checking the risk of developing the disease; as long as the presence of the intestinal bacteria, etc. of the present invention is detected in the intestine through the method, it can be determined that the disease caused by drug-resistant bacteria, etc. has not occurred or the risk of developing the disease is low.

[0166] The diagnosis of diseases caused by drug-resistant bacteria in a subject is usually performed by a physician (including a person instructed by the physician); the data obtained according to the method of the present invention is helpful for the physician's diagnosis. Therefore, the method of the present invention can also be expressed as a method of collecting data that is helpful for the physician's diagnosis and presenting it.

[0167] In addition, the present invention can also provide a companion diagnostic method and a pharmaceutical agent using the above-mentioned test method. That is, the present invention provides the following aspects.

[0168] A method for determining the effectiveness of the pharmaceutical composition of the present invention, etc. or their active ingredients, etc. in the treatment, improvement or prevention of diseases caused by drug-resistant bacteria, etc., the method comprising: a step of contacting the antibody, polynucleotide or inspection composition with a sample separated from a subject; a step of detecting the presence or absence of the intestinal bacteria, etc. through the contact; when the presence of the bacteria is not detected in the step, determining that the pharmaceutical composition of the present invention, etc. or their active ingredients, etc. in the subject are more effective in the treatment, improvement or prevention of the disease.

[0169] A method for treating, ameliorating or preventing a disease caused by drug-resistant bacteria, etc., the method comprising: causing a patient who is determined by the determination method to have a high effectiveness of the pharmaceutical composition, etc. of the present invention or their active ingredients, etc. to take the pharmaceutical composition, etc., etc. or their active ingredients, etc.

[0170] A composition for treating, ameliorating or preventing a disease caused by drug-resistant bacteria or the like, comprising the intestinal bacteria or the like of the present invention as an effective ingredient, the composition being ingested by a subject determined to be highly effective by the determination method.

[0171] <Method for screening intestinal bacteria having antibacterial activity against drug-resistant bacteria in the intestine>

[0172] The present inventors have first demonstrated that intestinal bacteria include bacteria that inhibit colonization of drug-resistant bacteria, etc. in the intestine. Therefore, the present invention provides a method for screening intestinal bacteria having antibacterial activity against drug-resistant bacteria, etc., comprising the following steps.

[0173] A process of causing non-human germ-free animals to ingest drug-resistant bacteria and the like in their intestinal tracts and test intestinal bacteria;

[0174] A step of detecting the drug-resistant bacteria in the intestinal tract of the non-human sterile animal;

[0175] A step of determining that the subject intestinal bacteria are intestinal bacteria having antibacterial activity against drug-resistant bacteria or the like when the number of bacteria detected in the step is reduced compared to a case where the subject intestinal bacteria are not ingested.

[0176] "Drug-resistant bacteria, etc." are as described above. "Non-human germ-free animals" refer to animals other than humans that are born and raised under sterile conditions. Examples of animals other than humans include, but are not limited to, mice, rats, monkeys, pigs, cattle, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, and hamsters. In addition, among these animals, mice are preferably used.

[0177] The intestinal bacteria to be ingested by the non-human sterile animal may be any bacteria present in the intestine of the animal, and examples of the animal include humans and non-human animals (mice, rats, monkeys, pigs, cattle, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, etc.). In addition, the intestinal bacteria to be ingested by the non-human sterile animal may be isolated intestinal bacteria, but examples may also include samples containing intestinal bacteria (e.g., fecal samples of the animal, or cultures thereof).

[0178] In addition, the method for causing the non-human animal to "ingest" the test intestinal bacteria and the drug-resistant bacteria, etc. is not particularly limited, and is generally performed by oral administration, and may also be non-oral administration (e.g., administration into the intestine). In addition, the ingestion of the test intestinal bacteria and the drug-resistant bacteria, etc. may be simultaneous, or the non-human animal may ingest the test intestinal bacteria and then ingest the drug-resistant bacteria, etc., or the non-human animal may ingest the drug-resistant bacteria, etc. and then ingest the test intestinal bacteria.

[0179] "Detection" of drug-resistant bacteria in the intestine can be performed by detecting the specific nucleotide sequence of the drug-resistant bacteria. Examples of the detection method include PCR (RT-PCR, real-time PCR, quantitative PCR), DNA microarray analysis, Northern blotting, 16srRNA sequencing, next-generation sequencing (sequencing-by-synthesis, such as sequencing by Solexa genome analyzer or Hiseq (registered trademark) 2000 manufactured by Illumina), pyrophosphate sequencing (such as sequencing by GSLX or FLX sequencer manufactured by Roche Diagnostics (454) (so-called 454 sequencing)), ligase reaction sequencing (such as sequencing by Solid (registered trademark) or 5500xl manufactured by Life Technology), beads array, in situ hybridization, dot blotting, RNase protection assay, mass spectrometry, genomic PCR, and Southern blotting.

[0180] In addition, the "detection" of drug-resistant bacteria in the intestine can be performed, for example, by detecting the specific amino acid sequence of the drug-resistant bacteria. As the detection method, ELISA, immunoblotting, antibody array analysis, immunohistochemical staining, flow cytometry, image cytometry, radioimmunoassay, immunoprecipitation and other methods (immunological methods) using antibodies for detection can be cited. The time point of detection is not particularly limited, and can be appropriately adjusted according to the type of animal used by those skilled in the art.

[0181] In addition, in the screening method of the present invention, when it is impossible to screen out intestinal bacteria having antibacterial activity against drug-resistant bacteria after implementing it once, the intestinal sample containing the bacteria can be used as the next test intestinal bacteria, and a new non-human sterile animal can be ingested and the screening can be carried out multiple times to isolate intestinal bacteria having the antibacterial activity.

[0182] Example

[0183] (Example 1)

[0184] like Figure 1As shown in the upper part of the figure, Klebsiella 2H7 strain (Kp2H7) was administered to germ-free mice, and fecal samples of healthy volunteers were administered one week later.

[0185] Specifically, regarding germ-free mice, 4 to 8-week-old C57BL / 6N (CLEA Co., Ltd., Japan) were raised in a plastic isolator (sterile isolator) (manufactured by ICM Co., Ltd.; ICM-1B) for feeding with free drinking water for more than 1 week, and used after environmental acclimation. The age at the start of the experiment was 8 to 14 weeks old. The same applies to other embodiments in this specification.

[0186] The bacterial solution of Klebsiella was placed in LB liquid culture medium and cultured at 37°C overnight, the OD value was adjusted to 1.2 (equivalent to 1*10^9 CFU / mL), and 200 μL / mouse (equivalent to 2*10^8 CFU / mouse) of the bacterial solution was administered into the stomach of the mouse using a probe.

[0187] For fecal samples, feces provided by healthy Japanese volunteers (#A, #F, #I, #J, #K) were diluted to 5 times their weight with a glycerol PBS solution (final concentration of glycerol: 20% by volume), filtered with a 100 μm filter to prepare a stock solution and stored at -80°C. When administering feces, the stock solution was diluted 10 times with PBS in an anaerobic chamber and administered to the stomach of mice at 200 μL / mouse using a probe.

[0188] The fecal sample of the mouse was dissolved in a solution of glycerol (final concentration 20%) and EDTA (final concentration 10 mM) mixed in PBS at a ratio of 50 mg feces / mL. The fecal solution was appropriately diluted and inoculated into DHL medium containing 50 mg / L ampicillin and 50 mg / L spectinomycin, and the number of colonies was counted after incubation at 37°C overnight to calculate the number of CFU per 1 g of feces.

[0189] In other Examples in the present specification, the bacterial solution and feces of the genus Klebsiella were administered in the same manner, and the CFU were counted in the same manner.

[0190] The results are as follows Figure 1 As shown in the lower part (curve graph), although 5 types of feces were used, it can be seen that the amount of Kp2H7 bacteria was significantly reduced in any sample.

[0191] (Example 2) Isolation of bacteria from feces of healthy volunteers

[0192] Each frozen stool sample prepared in Example 1, derived from the stools of healthy individuals F, K and I (F stool, K stool and I stool), was thawed at room temperature, diluted with PBS, and cultured in EG medium, modified GAM agar medium (Nissui Pharmaceutical Co., Ltd.; 05426), REINFORCED CLOSTRIDIAL AGAR (RCM AGAR) (Thermo Fisher Scientific Inc; CM0151) or Schaedler blood medium (Wako Co., Ltd.; 517-45805) in an anaerobic environment at 37°C and 10% CO2, and the formed colonies were isolated. 37 strains were isolated from F stool, 42 strains were isolated from I stool, and 47 strains were isolated from K stool. Subsequently, the K stool was isolated again, and finally 68 strains were isolated from K stool.

[0193] The isolated bacteria were sequenced and identified by 16S rDNA analysis using the Sanger method. Sequencing analysis was performed using a 3130 DNA Analyzer manufactured by Thermo Fisher Scientific and the following primer set.

[0194] 27Forward-mod: 5'-AGRGTTTGATYMTGGCTCAG-3' (SEQ ID NO: 148)

[0195] 1492Reverse: 5'-GGYTACCTTGTTACGACTT-3' (SEQ ID NO: 149)

[0196] Among them, R: A or G; Y: C or T; M: A or C.

[0197] And then, for 37 bacterial strains derived from F, use a new generation sequencer to determine the genome sequence.That is, use Illumina company's MiSeq and Pacific Biosciences company's Sequel, carry out genome sequencing respectively, and by using the hybrid assembly device (Hybrid assembly) of Unicycler, obtain the full genome sequence respectively.To this each genome sequence, RNAmmer is used to extract 16SrRNA sequence, thus obtain the sequence of the higher accuracy comprising two terminal sequences that cannot be determined in the 16S rDNA sequence determined with the Sanger method.

[0198] The results of the above analysis are shown in Tables 1 to 4. In addition, the results of 16S meta-analysis of three types of feces collected from donors F, I, and K are shown in Figure 2 .

[0199] (Example 3)

[0200] like Figure 3 As shown in the upper part of the figure, Kp2H7 was administered to germ-free mice, and one week later, mixed isolates (37 strains derived from F feces (F37mix), 42 strains derived from I feces (I42mix), 47 strains derived from K feces (K47mix)) or I feces were administered.

[0201] The isolated bacteria were cultured in an anaerobic chamber at 37°C for 24 to 48 hours using mGAM liquid medium, EG medium or CM0149 medium and mixed. The mixed solution was concentrated 5 times, and 200 μL / mouse (total bacterial count equivalent to 1*10^9 CFU / mouse) of the bacterial solution was administered into the stomach using a probe. The administration of the mixed isolated strains in the following examples was also performed in the same manner.

[0202] The results are as follows Figure 3 As shown in the lower part (curve graph), the 37 strains derived from F stool are related to the ability to eliminate Klebsiella from the mouse intestine and have the same activity as I stool.

[0203] (Example 4)

[0204] like Figure 4 As shown in the upper part of the figure, Kp2H7 was administered to germ-free mice, and mixed isolated bacteria (37 strains from F feces and 68 strains from K feces) were administered one week later. Figure 4 As shown in the lower part (graph), the 37 strains derived from F feces and the 68 strains derived from K feces eliminated Klebsiella from the intestines of mice equally.

[0205] (Example 5)

[0206] like Figure 5 As shown in the upper part of , Kp2H7 was administered to germ-free mice, and mixed isolated bacteria (F37mix) were administered one week later. Ampicillin 200 mg / L was administered in drinking water after the administration of the isolated bacteria. In order to investigate the changes in the bacterial amount of each administered bacterium, PCR was performed using specific primers for each bacterium. The primers used for analysis are shown in Table 7.

[0207] Table 7

[0208]

[0209] The results are as follows Figure 5 As shown in the lower part of , the bacterial count of the genus Klebsiella temporarily increased due to the administration of ampicillin, but then decreased again.

[0210] In addition, the presence ratio of each isolated bacteria in the total bacterial count was analyzed together with the presence ratio of Kp2H7 and its change over time. The results are shown in Figures 6A to 6H. Then, the Spearman rank correlation coefficient between the bacterial count of the genus Klebsiella and each bacterium was calculated and arranged in descending order according to the positive correlation. The obtained results are shown in Figure 7 .

[0211] like Figure 7 As shown, it was found that strains belonging to the phylum Bacteroidetes changed independently of the dynamics of the genus Klebsiella. On the other hand, many strains that were negatively correlated belonged to the genus Furmicutes.

[0212] (Example 6)

[0213] like Fig. 9 As shown, 37 strains from F feces were divided into 8 strains belonging to the Bacteroidetes (F8mix) and 29 strains (F29mix) and mixed separately, and Kp2H7 was colonized in germ-free mice, and then the mixed isolates were administered. Fig. 9 , 10 The phylogenetic trees shown in Figures 1 and 12 were prepared by using MEGA X using the neighbor-joining method to analyze the DNA base sequences of the isolated bacteria using the Sanger method.

[0214] The results are as follows Figure 8 As shown, F37mix and F29mix eliminated Klebsiella from the intestinal tract of mice at the same level. On the other hand, in the group administered with F8mix, which belongs to the phylum Bacteroidetes, the amount of Klebsiella remained unchanged, suggesting that F8mix was not involved in the elimination of Klebsiella.

[0215] (Example 7)

[0216] From the 37 strains, 18 strains were selected (refer to Fig.10 ).

[0217] Then, if Fig.11 As shown in the upper part of the figure, the Kp2H7 strain was administered to germ-free mice, and one week later, mixed isolated bacteria (37 strains derived from F feces (F37mix), 18 strains (F18mix), and 42 strains derived from I feces (I42mix)) were administered.

[0218] The results are as follows Fig.11 As shown in the lower part (curve graph), it shows Fig.10 The 18 strains shown were also able to demonstrate the ability to exclude Klebsiella strains comparable to that of the 37 strains.

[0219] (Example 8)

[0220] Based on the phylogenetic tree Fig.10 The 18 strains shown were divided into 4 groups (Blautia, Lachonoclostridum, otherFirmicutes, otherPhyla) (ref. Fig.12 ). Then, these four groups were extracted from the 18 strains (F18mix) to make the strain groups F15mix (F18mix-other phyla), F12mix (F18mix-Lachnoclostiridum), F14mix (F18mix-Blautia), and F13mix (F18mix-other Firmicutes). In addition, a strain group was also made by excluding duplicate strains from 37 strains and further excluding the above 18 strains from the 31 strains (F13mix (F31-18mix)). Then, as Fig.13 As shown in the upper part of the figure, Kp2H7 was administered to germ-free mice, and each isolated bacteria mixed as described above was administered one week later.

[0221] The results are as follows Fig.13 As shown in the lower part (curve graph), F18mix is ​​the best at excluding Klebsiella. However, when any group is excluded, the amount of Klebsiella increases significantly. Fig.14 As shown, Fig.13 In the experiment shown above, the CFU of Klebsiella in the stool of each administration group at the 28th day was statistically significantly reduced in F18mix compared with the other groups except F37mix.

[0222] From the above inspiration, Fig.10 All of the four groups shown were related to the elimination of Klebsiella genus, and the elimination of Klebsiella genus was performed as bacterial groups.

[0223] In addition, Fig.13 In the experiments shown, lymphocytes from the lamina propria of the large intestine mucosa were extracted from mice of the F37mix, F18mix, and F31-18mix groups and subjected to analysis by flow cytometry.

[0224] The results are as follows Fig.15 As shown, the proportion of CD4+IFNγ+ cells was higher in the F13mix (F31-18mix) group, indicating that F37mix and F18mix inhibited the induction of Th1 cells.

[0225] (Example 9)

[0226] As shown in Example 8, in the experiment of excluding bacteria from each group by F18mix, F15mix, which excluded 3 strains of otherPhyla, had the lowest ability to exclude Klebsiella. Therefore, focusing on this group, a group was prepared by excluding these 3 strains (E.coli, Bifidobacterium, Fusobacterium) one by one from the 18 strains derived from F. Then, as Fig.16 As shown in the upper part of the figure, Kp2H7 strain was administered to germ-free mice, and one week later, the mixed isolates, F18mix or F15mix as described above were administered.

[0227] The results are as follows Fig.16 As shown in the lower part (graph), by excluding any of the three strains, it can be seen that the amount of Klebsiella increases by about 1 log, suggesting that any of the three strains is related to the exclusion of Klebsiella.

[0228] (Example 10)

[0229] In order to explore the mechanism by which F37mix excludes Klebsiella, we focused on whether the host's immunity is related to this mechanism. Fig.17 As shown in the upper part, the sterile Rag2 - / - γc - / - Mouse, MyD88 - / - Triff - / - Mice or wild-type mice were administered with Klebsiella 2H7 strain, and one week later, the mixed F37mix was administered.

[0230] The results are as follows Fig.17 As shown in the lower part (graph), the same degree of elimination of Klebsiella 2H7 strain by F37mix was observed in all types of mice. This suggests that the host's main natural immunity and acquired immunity are not related to the elimination of Klebsiella.

[0231] Next, the effects of the isolated strains on eliminating pathogenic bacteria other than the Kp2H7 strain and drug-resistant bacteria were evaluated. In addition, the primers shown in Table 8 were used as specific primers for each bacterium in the analysis.

[0232] Table 8

[0233]

[0234] (Example 11)

[0235] First, if Fig.18 As shown in the upper part of the figure, carbapenem-resistant Klebsiella (CRE) was administered to germ-free mice, and mixed F37mix, K68mix or I42mix was administered one week later. The obtained results are shown in Fig.18 The lower part.

[0236] In addition, the bacterial solution of CRE was placed in LB liquid medium and cultured at 37°C overnight, the OD value was adjusted to 1.2 (equivalent to 1*10^9 CFU / mL), and 200 μL / mouse (equivalent to 2*10^8 CFU / mouse) of the bacterial solution was administered into the stomach of the mouse using a probe. The CFU count of CRE was performed using DHL medium containing 30 mg / L ampicillin and 30 mg / L spectinomycin as a selective medium, and cultured at 37°C under aerobic conditions overnight.

[0237] In addition, mice were dissected one month after the mixed isolates were administered, and the large intestine was fixed with 4% PFA and embedded in paraffin, and thin sections were prepared. The sections were stained with hematoxylin and eosin to observe the inflammatory images of the tissues. The results are shown in Fig.19 .

[0238] like Fig.18 As shown in the lower part (curve graph), the results show that F37mix and K68mix have the same CRE removal ability, and I42mix is ​​slightly worse than them. Fig.19 As shown, no signs of inflammation such as ulcer formation and infiltration of inflammatory cells were observed in any of the mice. This indicates that administration of the mixed group of each isolated bacteria can suppress the induction of inflammation in the large intestine.

[0239] (Example 12)

[0240] like Fig. 20 As shown in the upper part of the figure, vancomycin-resistant Enterococcus faecium (VRE) was administered to germ-free mice, and one week later, the mixed F37mix, K68mix or I42mix was administered.

[0241] In addition, the bacterial solution of VRE was placed in LB liquid medium and cultured overnight at 37°C to adjust the OD value to 1.2 (equivalent to 1*10^9 CFU / mL), and 200 μL / mouse (equivalent to 2*10^8 CFU / mouse) of the bacterial solution was administered into the stomach of the mice using a probe. The CFU count of VRE was performed using VRE medium (Becton, Japan) and cultured overnight at 37°C under aerobic conditions.

[0242] The results are as follows Fig. 20 As shown in the lower part (curve graph), K68mix has the highest removal capacity for VRE. Fig.21 As shown, no signs of inflammation such as ulcer formation and infiltration of inflammatory cells were observed in any of the mice. This indicates that administration of the mixed group of each isolated bacteria can suppress the induction of inflammation in the large intestine.

[0243] (Example 13)

[0244] like Fig. 22 As shown in the upper part of the figure, adhesion-invasive E. coli (AIEC LF82) was administered to germ-free mice, and one week later, mixed F37mix, K68mix, and I42mix were administered.

[0245] In addition, the bacterial solution of AIEC LF82 was placed in LB liquid medium and cultured overnight at 37°C to adjust to OD 1.2 (equivalent to 1*10^9 CFU / mL), and 200 μL / mouse (equivalent to 2*10^8 CFU / mouse) of the bacterial solution was administered to the stomach of mice using a probe. The CFU count of AIEC LF82 was calculated by using MacConkey medium containing 1 mg / L cefotaxime as the selective medium and culturing overnight at 37°C under aerobic conditions.

[0246] The results are as follows Fig. 22 As shown in the lower part (curve graph), F37mix has the highest exclusion ability for AIEC LF82.

[0247] (Example 14)

[0248] like Fig.23 As shown in the upper part of the figure, ESBL-producing Klebsiella (Kp-ESBL) (ATCC700721) was administered to germ-free mice, and one week later, the mixed F37mix, K68mix, I42mix or F was administered.

[0249] In addition, the bacterial solution of Kp-ESBL was placed in LB liquid medium and cultured overnight at 37°C to adjust to OD 1.2 (equivalent to 1*10^9 CFU / mL), and 200 μL / mouse (equivalent to 2*10^8 CFU / mouse) of the bacterial solution was administered to the stomach of the mouse using a probe. The CFU count of Kp-ESBL was calculated by using DHL medium containing 30 mg / L of ampicillin and 30 mg / L of spectinomycin as a selective medium and culturing overnight at 37°C under aerobic conditions.

[0250] The results are as follows Fig.23 As shown in the lower part (graph), F37mix and K68mix showed Kp-ESBL exclusion ability equivalent to that of F.

[0251] (Example 15)

[0252] like Fig.24 and 25As shown in the upper part of the figure, Campylobacter jejuni 81-176 (ATCC BAA2151) was administered to germ-free mice, and one week later, the mixed F37mix, K68mix, I42mix or F was administered.

[0253] The bacterial liquid of Campylobacter jejuni was placed in TS liquid culture medium and placed in an anaerobic bottle together with the microaerobic AnaeroPack, and cultured at 42°C for 48 hours. The bacterial liquid was then administered into the stomach of mice using a probe.

[0254] For Campylobacter jejuni, CFU and qPCR were used to show the bacterial count.

[0255] CFU counts were performed using CHROMagar Campylobacter, which was placed in an anaerobic bottle together with AnaeroPack and cultured at 42°C for 48 hours. The results are shown in Fig.24 .

[0256] The qPCR assay was performed according to the following steps.

[0257] Amplification was performed using LightCycler (registered trademark) 480II (Roche; 05015243001) and Thunderbird (registered trademark) SYBR (registered trademark) qPCR Mix (TOYOBO; QPS-201X5) with primers specific to the Campylobacter jejuni genome and universal bacterial primers, and the calculated DNA concentration ratio was used as the presence ratio of Campylobacter jejuni. The results are shown in Fig.25 .

[0258] In addition, as primers specific to the Campylobacter jejuni genome for qPCR, the primer set described in SEQ ID NOs: 220 and 221 was used, and as universal bacterial primers, the primer set described in SEQ ID NOs: 222 and 223 was used.

[0259] In addition, bacterial genome extraction was performed through the following steps.

[0260] To 10 mg of mouse feces, 5 times the weight of a PBS solution containing EDTA and glycerol (final concentration of EDTA: 10 mM, final concentration of glycerol: 20% by volume) was added, and the mixture was shaken vigorously to break and suspend. To 100 μL of the sample solution, 800 μL of 10 mM Tris / 10 mM EDTA buffer (pH 8.0, hereinafter also referred to as "TE10") containing 15 mg of lysozyme (Lysozyme from chicken egg white; L4919, manufactured by Sigma-Aldrich) and 5 μL of RNase (PureLink RNase A (20 mg / mL, manufactured by Thermo Fisher Scientific; 12091-021) was added, and the mixture was shaken at 37°C for 1 hour. Next, 2,000 U of Achromopeptidase (registered trademark) (Wako; 015-09951) was added, and the mixture was shaken at 37°C for 30 minutes to dissolve the bacteria. Then, 50 μL of 20% SDS TE10 solution and 50 μL of TE10 solution containing proteinase K (Roche, Proteinase K, recombinant, PCR Grade; 03115852001) at a final concentration of 20 mg / ml were added, and the mixture was shaken at 55° C. for 60 minutes. Next, DNA was obtained from 400 μL of the solution using Maxwell (registered trademark) RSC Cultured Cells DNA Kit (Promega).

[0261] like Fig.24 and 25 As shown, for Campylobacter jejuni, any mixed bacteria had the same good bacteria elimination ability as F stool.

[0262] (Example 16)

[0263] like Fig.26 As shown in the upper part of the figure, Clostridum difficile (St.630) was administered to germ-free mice, and mixed F37mix, K68mix, I42mix, K47mix or F feces were administered one week later. In addition, K47mix is ​​47 strains isolated from the feces sample taken from #K, and except for one strain, it overlaps with the above 68 strains (K1 to K46 described in Tables 1 and 2).

[0264] The bacterial liquid of C. difficle was sporulated, adjusted to about 1x10^5 cells, and administered into the stomach of mice using a probe. The sporulated cells were cultured in Clospore medium for 8 days and cultured in an anaerobic chamber at 37°C. The culture medium was washed with PBS, ultrasonicated, and Lysoizyme and trypsin were added. The cells were treated at 45°C for 6 hours and then at 70°C for 10 minutes.

[0265] qPCR was used to quantify the bacterial count of C. difficle. The primer set described in SEQ ID NOs: 224 and 225 was used for qPCR.

[0266] The results are as follows Fig.26 As shown in the lower part, K68mix and K47mix both have high exclusion capabilities for C.difficile.

[0267] Industrial Availability

[0268] As described above, according to the present invention, by inhibiting the colonization of drug-resistant bacteria and inflammation-inducing bacteria in the intestine, diseases caused by these bacteria can be treated, improved or prevented. Therefore, the present invention is extremely useful for the development, treatment, improvement and prevention of drugs related to infections caused by drug-resistant bacteria or inflammation-inducing bacteria.

Claims

1. An antibacterial composition for drug-resistant bacteria or inflammation-inducing bacteria, comprising intestinal bacteria as an active ingredient, wherein: The intestinal bacteria are (1) intestinal bacteria specifically defined by accession number NITE BP-03147, (2) intestinal bacteria specifically defined by accession number NITE BP-03148, (3) intestinal bacteria specifically defined by accession number NITE BP-03149, (4) intestinal bacteria specifically defined by accession number NITE BP-03150, (5) intestinal bacteria specifically defined by accession number NITE BP-03151, (6) intestinal bacteria specifically defined by accession number NITE BP-03152, (7) intestinal bacteria specifically defined by accession number NITE BP-03153, (8) intestinal bacteria specifically defined by accession number NITE BP-03154, (9) intestinal bacteria specifically defined by accession number NITE BP-03155, (10) intestinal bacteria specifically defined by accession number NITE BP-03156, (11) intestinal bacteria specifically defined by accession number NITE BP-03157, (12) intestinal bacteria specifically defined by accession number NITE BP-03158, (13) intestinal bacteria specifically defined by accession number NITE BP-03159, (14) intestinal bacteria specifically defined by accession number NITE BP-03160, (15) intestinal bacteria specifically defined by accession number NITE BP-03161, (16) intestinal bacteria specifically defined by accession number NITE BP-03162, (17) intestinal bacteria specifically defined by accession number NITE BP-03163, and (18) Intestinal bacteria specifically defined by the deposit number NITE BP-03164.

2. The antibacterial composition according to claim 1, which is a pharmaceutical composition.

3. The antibacterial composition according to claim 1 is a pharmaceutical composition for treating, improving or preventing infectious diseases or inflammatory diseases.

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