Composition, food / beverage item, and method for assisting examination and diagnosis of disease caused by pathogenic bacterium or pathogenic fungus

Abstract

The present invention provides: a composition and a food / beverage item which can promote the elongation of pili of an intestinal bacterium and exhibits an anti-bacterial activity; and a method for assisting the examination and diagnosis of a disease caused by a pathogenic bacterium or a pathogenic fungus. The present invention provides a composition containing a bacterium capable of producing 3-phenylpropionic acid (PPA) or 3-(4-hydroxyphenyl)propionic acid (4OHPPA). The present invention also provides a food / beverage item containing PPA or 4OHPPA. The present invention further provides a method for assisting the examination and diagnosis of a disease caused by a pathogenic bacterium or a pathogenic fungus. The method comprises: quantifying the amount of PPA or the amount of 4OHPPA in feces from a subject by using 2-nitrophenylhydrazine; and comparing a value obtained by the quantification of the amount of PPA or the amount of 4OHPPA with a reference value. When the value obtained by the quantification of the amount of PPA or the amount of 4OHPPA is smaller than the reference value, it is determined that the subject is possibly affected by the disease.

Description

Composition, food and drink, and method for assisting in testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi

[0001] The present invention relates to compositions, foods and beverages, and methods for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi. This application claims priority to Japanese Patent Application No. 2023-191108, filed November 8, 2023, the contents of which are incorporated herein by reference.

[0002] Fimbriae are multicomponent proteins produced by both Gram-negative and Gram-positive bacteria. They are proteinaceous polymers with long filamentous structures and are involved in a variety of phenotypes, including interactions and aggregation between multiple bacterial species, biofilm formation, and colonization of host tissues (particularly the intestinal tract). Bifidobacteria are one of the dominant bacterial species that colonize the human intestinal tract and are known to be beneficial to host health (see, for example, Non-Patent Document 1). Bifidobacteria possess sortase-dependent pili and type IV pili (see, for example, Non-Patent Documents 2 and 3), and these pili genes are conserved in the genomes of many Bifidobacteria (see, for example, Non-Patent Document 4). However, no fimbrial structures have been confirmed in Bifidobacteria isolated from humans and cultured under standard conditions.

[0003] On the other hand, 3-phenylpropionic acid (PPA) is known to be a metabolic product of phenylalanine produced by the enterobacterium Clostridium sporogenes (see, for example, Non-Patent Document 5).

[0004] Schroeder BO et al., “Bifidobacteria or fiber protects against diet-induced microbiota-mediated colonic mucus deterioration.”, Cell Host Microbe., Vol. 23, Issue 1, pp. 27-40. e7, 2018.O’Connell Motherwaya M et al., “Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor.”, PNAS, Vol. 108, No. 27, pp. 11217-11222, 2011.Turroni F et al., “Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium-host interactions.”, PNAS, Vol. 110, No. 27, pp. 11151-11156, 2013.Milani C et al., “The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue.”, Applied and Environmental Microbiology, Vol. 83, No. 19, e01295-17, 2017.Elsden SR et al., “The end products of the metabolism of aromatic amino acids by Clostridia.”, Archives of Microbiology, Vol. 107, No. 3, pp. 283-288, 1976.

[0005] The inventors have hypothesized that, because fimbrial structures were confirmed in Bifidobacterium cultured in fecal culture medium, metabolites produced by symbiotic bacteria induce the elongation of Bifidobacterium fimbriae, promoting their colonization in the intestinal tract. However, the details of these metabolites and their effects on the intestinal flora have not been clarified.

[0006] The present invention has been made in consideration of the above circumstances, and provides a composition and food and drink that can promote the extension of enterobacterial pili, as well as a method for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi.

[0007] It is already known from Non-Patent Document 5 and the like that 3-phenylpropionic acid (PPA) is biosynthesized from phenylalanine in Clostridium sporogenes by a group of enzymes consisting of phenyllactate dehydrogenase H (FldH), phenyllactate dehydrogenase B (FldB), phenyllactate dehydrogenase C (FldC), and acyl-CoA dehydrogenase (AcdA). However, as a result of intensive research by the inventors to achieve the above-mentioned object, they discovered that PPA can also be biosynthesized from phenylalanine by a group of enzymes consisting of phenylalanine ammonia-lyase (PAL) and hydroxycinnamate reductase B (HcrB), that PPA upregulates the expression of sortase-dependent (SD) fimbriae genes in Bifidobacterium, Blautia, Ruminococcus, and the like, and induces the elongation of these fimbriae, and that PPA not only promotes colonization of symbiotic bacteria in the intestinal tract, but also exhibits a growth-inhibitory effect on Gram-negative pathogenic bacteria by damaging their cell membranes, thereby completing the present invention.

[0008] That is, the present invention includes the following aspects: (1) A composition for producing 3-phenylpropionic acid (PPA) or 3-(4-hydroxyphenyl)propionic acid (4OHPPA), the composition comprising at least one bacterium, and the PPA or 4OHPPA is produced by the at least one bacterium. (2) The composition according to (1), wherein the at least one bacterium has a nucleotide sequence encoding a protein having the activity of phenylalanine ammonia-lyase (PAL), and a nucleotide sequence encoding a protein having the activity of hydroxycinnamate reductase B (HcrB), the protein having the activity of PAL having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having the activity of HcrB having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 2. (3) The composition according to (1) or (2), wherein the at least one bacterium has a nucleotide sequence encoding an aromatic amino acid lyase domain or a domain having a function equivalent to the aromatic amino acid lyase domain. (4) The composition according to any one of (1) to (3), wherein the at least one bacterium has a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain, or a domain having a function equivalent to the flavin adenine dinucleotide-binding domain and the flavin mononucleotide-binding domain. (5) The composition according to any one of (1) to (4), wherein the at least one bacterium further has a nucleotide sequence encoding an NADPH-dependent flavin mononucleotide reductase domain or a domain having a function equivalent to the NADPH-dependent flavin mononucleotide reductase domain.(6) The composition according to (1), wherein the composition comprises a plurality of bacteria, and the plurality of bacteria are classified into two groups, Group A and Group B, wherein Group A includes bacteria having a nucleotide sequence encoding a protein having phenylalanine ammonia-lyase (PAL) activity, and Group B includes bacteria having a nucleotide sequence encoding a protein having hydroxycinnamate reductase B (HcrB) activity, the protein having PAL activity has an amino acid sequence having 60% or more homology to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having HcrB activity has an amino acid sequence having 60% or more homology to the amino acid sequence set forth in SEQ ID NO: 2. (7) The composition according to (6), wherein Group A also includes bacteria having a nucleotide sequence encoding an aromatic amino acid lyase domain or a domain having a function equivalent to the aromatic amino acid lyase domain. (8) The composition according to (6) or (7), wherein Group B also includes bacteria having a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain, or a domain having a function equivalent to a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain.(9) The composition according to (8), wherein the bacterium classified into Group B and having a nucleotide sequence encoding the flavin adenine dinucleotide-binding domain and the flavin mononucleotide-binding domain, or a domain having a function equivalent to the flavin adenine dinucleotide-binding domain and the flavin mononucleotide-binding domain, further has a nucleotide sequence encoding an NADPH-dependent flavin mononucleotide reductase domain or a domain having a function equivalent to the NADPH-dependent flavin mononucleotide reductase domain. (10) The composition according to any one of (6) to (9), wherein the bacteria classified into Group A are at least one selected from the group consisting of Bacteroides intestinalis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides dorei, Blautia hansenii, and Bifidobacterium longum. (11) The composition according to any one of (6) to (10), wherein the bacterium classified into Group B is at least one selected from the group consisting of Blautia producta, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactiplantibacillus plantarum.(12) The composition according to any one of (1) to (12), further comprising a bacterium having: a nucleotide sequence encoding a protein having the activity of acyl-CoA dehydrogenase (AcdA); a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase B (FldB); a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase C (FldC); and a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 3; the protein having the activity of FldB has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 4; the protein having the activity of FldC has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 5; and the protein having the activity of FldH has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 6. (13) The composition according to (12), wherein the bacteria having the nucleotide sequence encoding a protein having the activity of AcdA, the nucleotide sequence encoding a protein having the activity of FldB, the nucleotide sequence encoding a protein having the activity of FldC, and the nucleotide sequence encoding a protein having the activity of FldH are bacteria selected from the group consisting of Clostridium sporogenes, Clostridium cadaveris, and Peptostreptococcus anaerobius. (14) The composition according to any one of (1) to (13), further comprising an acid or a bacterium producing the acid. (15) The composition according to (14), wherein the acid is at least one selected from the group consisting of short-chain fatty acids and inorganic acids. (16) The composition according to (14), wherein the acid is at least one selected from the group consisting of acetic acid, fumaric acid, butyric acid, propionic acid, lactic acid, and hydrochloric acid.(17) The composition according to any one of (14) to (16), wherein the acid-producing bacterium is at least one selected from the group consisting of bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Bacteroides, bacteria of the genus Blautia, and bacteria of the genus Clostridium. (18) The composition according to any one of (1) to (17), which is a pharmaceutical composition. (19) The composition according to (18), which has an effect of promoting the elongation of pili of enterobacteria. (20) The composition according to (19), wherein the enterobacterium has a sortase-dependent pilus gene. (21) The composition according to (19) or (20), wherein the intestinal bacterium is at least one selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Ruminococcus gnavus, Blautia hansenii, and Lactobacillus rhamnosus. (22) The composition according to any one of (18) to (21), which is for treating a disease caused by a pathogenic bacterium. (23) The composition according to (22), wherein the pathogenic bacterium is a bacterium belonging to the phylum Proteobacteria. (24) The composition according to (23), wherein the bacterium of the phylum Proteobacteria is at least one selected from the group consisting of Salmonella enterica subsp. enterica serovar typhimurium, Escherichia coli, Citrobacter redentium, Klebsiella pneumoniae, and Proteus mirabilis. (25) The composition according to any one of (22) to (24), wherein the disease caused by the pathogenic bacterium is at least one selected from the group consisting of bacterial food poisoning, typhoid fever, gastroenteritis, respiratory tract infection, urinary tract infection, bacterial vaginosis, sepsis, and meningoencephalitis. (26) The composition according to any one of (18) to (25), which is for treating a disease caused by a pathogenic fungus.(27) The composition according to (26), wherein the pathogenic fungus is a fungus of the genus Saccharomycetales or a fungus of the genus Trichosporonaceae. (28) The composition according to (27), wherein the fungus of the genus Saccharomycetales is at least one selected from the group consisting of Candida albicans, Candida auris, Candida famata, and Pichia fermentans. (29) The composition according to (27) or (28), wherein the fungus of the genus Trichosporonaceae is Cryptococcus humicola. (30) The composition according to any one of (26) to (29), wherein the disease caused by the pathogenic fungus is at least one selected from the group consisting of inflammatory bowel disease, oral candidiasis, vaginal candidiasis, pneumonia, and meningitis. (31) The composition according to any one of (1) to (17), which is a food or drink. (32) A composition comprising 3-phenylpropionic acid or 3-(4-hydroxyphenyl)propionic acid. (33) The composition according to (32), which has the effect of promoting the elongation of pili of enterobacteria. (34) The composition according to (33), wherein the enterobacteria have a sortase-dependent pilus gene. (35) The composition according to (33) or (34), wherein the enterobacteria is at least one selected from the group consisting of Ruminococcus gnavus, Blautia hansenii, and Lactobacillus rhamnosus. (36) The composition according to any one of (32) to (35), which is a pharmaceutical product. (37) The composition according to any one of (32) to (35), which is a food or drink product. (38) A composition containing a protein having the activity of phenylalanine ammonia-lyase (PAL) and a protein having the activity of hydroxycinnamate reductase B (HcrB), wherein the protein having the activity of PAL has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having the activity of HcrB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.(39) The composition according to (38), further comprising a protein having the activity of acyl-CoA dehydrogenase (AcdA), a protein having the activity of phenyllactate dehydrogenase B (FldB), a protein having the activity of phenyllactate dehydrogenase C (FldC), and a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 3, the protein having the activity of FldB has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 4, the protein having the activity of FldC has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 5, and the protein having the activity of FldH has an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 6. (40) The composition according to (38) or (39), which is a pharmaceutical product. (41) The composition according to (38) or (39), which is a food or drink. (42) A method for assisting in the examination and diagnosis of a disease caused by pathogenic bacteria or pathogenic fungi, comprising: quantifying the amount of 3-phenylpropionic acid (PPA) or the amount of 3-(4-hydroxyphenyl)propionic acid (4OHPPA) in the stool of a subject using 2-nitrophenylhydrazine; comparing the value obtained by quantifying the amount of PPA or the amount of 4OHPPA with a standard value; and indicating the possibility that the subject is suffering from the disease if the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is less than the standard value. (43) The method according to (42), further comprising quantifying the amount of short-chain fatty acids.(44) A method for assisting in the testing and diagnosis of a disease caused by pathogenic bacteria or pathogenic fungi, comprising: quantifying the expression level of a phenylalanine ammonia-lyase (PAL) gene or the expression level of a hydroxycinnamate reductase B (HcrB) gene in the feces of a subject; comparing the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene with a standard value; and indicating the possibility that the subject is suffering from the disease when the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene is lower than the standard value. (45) The method according to (44), further comprising quantifying the expression level of at least one gene selected from the group consisting of an acyl-CoA dehydrogenase gene, a phenyllactate dehydrogenase B gene, a phenyllactate dehydrogenase C gene, and a phenyllactate dehydrogenase H gene. (46) A method for assisting in the testing and diagnosis of a disease caused by pathogenic bacteria or pathogenic fungi, comprising: quantifying the expression level of at least one gene selected from the group consisting of a gene comprising a nucleotide sequence encoding an aromatic amino acid lyase domain, a gene comprising a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain, and a gene comprising a nucleotide sequence encoding a flavin mononucleotide-binding domain in the feces of a subject; comparing the value obtained by quantifying the expression level of the gene with a reference value; and indicating the possibility that the subject is suffering from the disease when the value obtained by quantifying the expression level of the gene is less than the reference value.

[0009] The composition and food / drink of the above aspect can promote the growth of pili of enterobacteria.The method of the above aspect can evaluate the possibility of suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0010] 1 is a graph showing the average PPA (3-phenylpropionic acid) concentration (left), average 4OHPPA (3-(4-hydroxyphenyl)propionic acid) concentration (center), and average IPA (3-indolepropionic acid) concentration (right) in the feces of healthy humans (KHC) and Crohn's disease patients (CD) in Experimental Example 1. FIG. 2 is a phylogenetic diagram of 50 types of symbiotic bacteria in Experimental Example 1. FIG. 3 is a graph showing the contents of PPA and 4OHPPA in the culture medium of 50 types of symbiotic bacteria in Experimental Example 1. FIG. 4 is a graph showing the contents of PPA, 4OHPPA, and IPA in the culture medium of screened bacteria in Experimental Example 1.

[0023] Figure 1 shows a diagram (top) illustrating a new metabolic pathway for synthesizing PPA or 4OHPPA from Phe or Tyr in Experimental Example 1, and a diagram (bottom) illustrating the production amounts of PAA, 4OHPAA, and IAA (3-indoleacrylic acid) in Bacteroides thetaiotaomicron wt strain and pal gene-deficient (Δpal) strain, or PPA, 4OHPPA, and IPA in L. plantarum.

[0024] Figure 2 shows a protocol (top) for an in vivo test using germ-free (GF) mice in Experimental Example 1, and a graph (bottom) illustrating the results.

[0025] Figure 3 shows a diagram (top) illustrating an in vivo test using germ-free (GF) mice in Experimental Example 1, in which GF mice were colonized with B. thetaiotaomicron wt strain and L. plantarum (BTwt+LP) or B. 1 is a graph showing the time course of the bacterial counts of B. longum, B. thetaiotaomicron, or L. plantarum in the feces of mice colonized with the thetaiotaomicron Δpal strain and L. plantarum (BTpal+LP).

[0023] FIG. 1 is a graph showing the amount of B. longum colonized in the intestinal lumen or mucin layer of GF mice colonized with the B. thetaiotaomicron wt strain and L. plantarum (BTwt+LP) or mice colonized with the B. thetaiotaomicron Δpal strain and L. plantarum (BTpal+LP) in Experimental Example 1.Fluorescent staining images showing the results of fluorescent in situ hybridization of the intestines of GF mice colonized with the B. thetaiotaomicron wt strain and L. plantarum (BTwt+LP) or mice colonized with the B. thetaiotaomicron Δpal strain and L. plantarum (BTpal+LP) in Experimental Example 1. DAPI indicates the localization of nucleic acids, and WGA indicates the localization of cell membrane sugar chains. In Experimental Example 1, B. thetaiotaomicron was colonized using mice colonized with the B. thetaiotaomicron wt strain and L. plantarum (BTwt+LP) or mice colonized with the B. thetaiotaomicron Δpal strain and L. plantarum (BTpal+LP). 1 is a graph (left: feces, right: mucin layer) showing the results of a colonization test of a mixed bacterium of B. longum wt strain (BL wt) and ΔlacY strain (ΔlacY). It is an image showing the antibacterial effect of a culture medium in which C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS fldC)) and B. longum (BL) were cultured alone or together against Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (S. Typhimurium) in Experimental Example 2. It is a graph (top) showing the protocol of an in vivo test using GF mice in Experimental Example 2 and the results thereof (bottom). It is an image showing the antibacterial effect of a culture medium in which C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS fldC)) and B. longum (BL) were cultured alone or together against Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (S. Typhimurium) in Experimental Example 2. ... a graph (bottom) showing the protocol of an in vivo test using GF mice in Experimental Example 2. It is an image showing the antibacterial effect of a culture medium in which C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS fldC 1 is a graph showing the number of S. Typhimurium bacteria in the feces of GF mice colonized with C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS ko)) and B. longum (BL), either singly or in combination. 2 is a graph showing the concentrations of Phe, Tyr, Trp, and their intermediate metabolites and final metabolites in the feces of GF mice colonized with C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS ko)) and B. longum (BL), either singly or in combination, in Experimental Example 2. 3 is a graph showing the concentrations of Phe, Tyr, Trp, and their intermediate metabolites and final metabolites in the feces of GF mice colonized with C. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS ko)) and B. longum (BL), either singly or in combination, in Experimental Example 2. 1 is a graph showing the concentration of short-chain fatty acids in the feces of GF mice colonized with B. sporogenes (CS) (wild-type strain (CS wt) and fldC-deficient strain (CS ko)) and B. longum (BL), alone or together.1 is a graph showing the number of S. Typhimurium cells in the presence or absence of different concentrations of PPA and acetic acid in Experimental Example 2. The MIC of PPA in the presence or absence of acetic acid against each pathogen in Experimental Example 2. ９０ 1 shows the minimum inhibitory concentration (MIC) that inhibited the growth of 90% of bacterial strains. It shows the protocol (top) of an in vivo test using GF mice in Experimental Example 2, and a graph (bottom) showing the survival rate of GF mice colonized with B. thetaiotaomicron (wt (wild) strain or PAL ko (knockout) strain) and L. plantarum. It shows the number of S. Typhimurium bacteria in the feces of GF mice colonized with B. thetaiotaomicron (wt (wild) strain or PAL ko (knockout) strain) and L. plantarum in Experimental Example 2. It shows the MIC in Experimental Example 2. ９０1 shows images of S. Typhimurium cell division monitored for 120 minutes in the presence of 2.5 mM PPA, which is twice the concentration of PPA, and 10 mM acetic acid. 1 shows graphs showing growth inhibition of S. Typhimurium by the addition of culture media and PPA of various bacteria that produce short-chain fatty acids (SCFAs) in Experimental Example 2. 1 shows graphs showing changes in the pH of the cytoplasm of S. Typhimurium over time in the presence of acetic acid and PPA or Phe in Experimental Example 2. 1 shows SEM (scanning electron microscopy) images of S. Typhimurium in the presence of PPA or PPA and acetic acid in Experimental Example 2. 1 is a graph showing the cell viability by MTT assay of human colon cancer-derived Caco2 cells after 24 hours of culture in the presence of various concentrations of PPA and acetic acid in Experimental Example 2. 2 is a graph showing the pH of the medium when various short-chain fatty acids were added to LB medium at various concentrations in Experimental Example 2. 3 is a graph showing the growth inhibitory effect on S. Typhimurium by the addition of acetic acid, fumaric acid, or propionic acid alone, or by the co-addition of acetic acid, fumaric acid, or propionic acid with PPA in Experimental Example 2. 4 is a graph showing the growth inhibitory effect on S. Typhimurium by the addition of lactic acid or hydrochloric acid alone, or by the co-addition of lactic acid or hydrochloric acid with PPA in Experimental Example 2. 5 is a graph showing the growth inhibitory effect on S. Typhimurium by the administration of PPA or acetic acid alone, by co-administration of PPA and acetic acid, or by the absence of PPA and acetic acid in Experimental Example 2. 1 shows a TEM (Transmission Electron Microscope) image of Typhimurium. 1 shows a TEM image of Escherichia coli ATCC BAA-2777 after administration of PPA or acetic acid alone, after co-administration of PPA and acetic acid, or after no administration of PPA or acetic acid in Experimental Example 2. 1 shows a TEM image of Klebsiella pneumoniae 2H7 after administration of PPA or acetic acid alone, after co-administration of PPA and acetic acid, or after no administration of PPA or acetic acid in Experimental Example 2.

[0011] <Composition> The composition of this embodiment is a composition that produces 3-phenylpropionic acid (hereinafter sometimes abbreviated as "PPA") or 3-(4-hydroxyphenyl)propionic acid (hereinafter sometimes abbreviated as "4OHPPA"). The composition of this embodiment contains at least one bacterium, and the PPA or 4OHPPA is produced by the at least one bacterium. That is, the composition of this embodiment contains a bacterium that produces PPA or a bacterium that produces 4OHPPA. Of the at least one bacterium, the bacterium that produces PPA promotes the extension of enterobacteria's fimbria more than other bacteria, and therefore, it is preferable that the composition of this embodiment contains a bacterium that produces PPA or a bacterium that produces 4OHPPA as an active ingredient. In addition, it is preferable that the bacterium contained in the composition of this embodiment is a live bacterium.

[0012] <Bacteria Producing PPA or Bacteria Producing 4OHPPA> It is already known that PPA or 4OHPPA is biosynthesized from phenylalanine or tyrosine in Clostridium sporogenes by an enzyme group consisting of phenyllactate dehydrogenase H (FldH), phenyllactate dehydrogenase B (FldB), phenyllactate dehydrogenase C (FldC), and acyl-CoA dehydrogenase (AcdA). Meanwhile, as shown in the Examples below, the inventors have discovered a new metabolic pathway in which PPA or 4OHPPA is biosynthesized from phenylalanine or tyrosine by an enzyme group consisting of phenylalanine ammonia-lyase (PAL) and hydroxycinnamate reductase (HcrB).

[0013] In the composition of this embodiment, bacteria having the PAL gene and the HcrB gene may be used, or bacteria having the PAL gene and bacteria having the HcrB gene may be used in combination.

[0014] That is, at least one bacterium contained in the composition of this embodiment preferably has a nucleotide sequence encoding a protein having PAL activity and a nucleotide sequence encoding a protein having HcrB activity, the protein having PAL activity has an amino acid sequence that is 60% or more homologous to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having HcrB activity has an amino acid sequence that is 60% or more homologous to the amino acid sequence set forth in SEQ ID NO: 2.

[0015] Alternatively, the composition preferably comprises a plurality of bacteria, the plurality of bacteria being classified into two groups, Group A and Group B, wherein Group A includes bacteria having a nucleotide sequence encoding a protein having PAL activity, and Group B includes bacteria having a nucleotide sequence encoding a protein having HcrB activity, the protein having PAL activity having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having HcrB activity having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0016] The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of PAL of Bacteroides thetaiotaomicron strain VPI-5482. The amino acid sequence of SEQ ID NO: 2 is the amino acid sequence of HcrB of Lactobacillus plantarum (Lactobacillus plantarum) strain WCFS1.

[0017] In a protein having PAL activity, the homology (synonymous with "sequence identity") between the amino acid sequence set forth in SEQ ID NO: 1 and the protein having PAL activity is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and most preferably 100%.

[0018] As used herein, the homology of an amino acid sequence is a value indicating the percentage of identity between a target amino acid sequence (target amino acid sequence) and a reference amino acid sequence (reference amino acid sequence). The homology of a target amino acid sequence to a reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize homology. Next, the number of identical amino acids in the reference amino acid sequence and the target amino acid sequence is calculated, and the homology can be determined according to the following formula.

[0019] Homology (%) = number of matching amino acids / number of amino acids in the aligned region of the target amino acid sequence × 100 (%)

[0020] A protein having PAL activity may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 199, more preferably 1 to 149, even more preferably 1 to 99, still more preferably 1 to 49, particularly preferably 1 to 24, and most preferably 1 to 4.

[0021] In a protein having HcrB activity, the amino acid sequence homology with the amino acid sequence set forth in SEQ ID NO: 2 is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and most preferably 100%.

[0022] A protein having HcrB activity may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 324, more preferably 1 to 243, even more preferably 1 to 162, still more preferably 1 to 81, particularly preferably 1 to 40, and most preferably 1 to 8.

[0023] PAL has an aromatic amino acid lyase domain as a functional domain. Therefore, the bacterium contained in the composition of this embodiment preferably has a nucleotide sequence encoding the aromatic amino acid lyase domain (Pfam ID: PF00221, InterPro entry: IPR001106) or a domain having a function equivalent thereto.

[0024] Alternatively, it is preferable that PAL has, as a functional domain, a nucleotide sequence encoding phenylalanine ammonia-lyase (InterPro entry: IPR005922) or a domain having a function equivalent thereto.

[0025] HcrB has, as functional domains, a flavin adenine dinucleotide (FAD)-binding domain (Pfam ID: PF00890, InterPro entry: IPR003953) and a flavin mononucleotide (FMN)-binding domain (Pfam ID: PF04205, InterPro entry: IPR007329). Therefore, the bacterium contained in the composition of this embodiment preferably has a nucleotide sequence encoding the FAD-binding domain and the FMN-binding domain, or domains having equivalent functions thereto.

[0026] Furthermore, HcrB further has an NADPH-dependent flavin mononucleotide reductase domain (Pfam ID: PF03358, InterPro entry: IPR005025) as a functional domain. Therefore, the bacterium contained in the composition of this embodiment preferably further has a nucleotide sequence encoding an NADPH-dependent flavin mononucleotide reductase domain or a domain having a function equivalent thereto.

[0027] The function equivalent to a specific domain is, for example, when the domain is an aromatic amino acid lyase domain, the function is to catalyze the reaction of eliminating ammonia from aromatic amino acids (such as histidine and phenylalanine) and the reaction of transferring the amino group of aromatic amino acids (such as tyrosine and phenylalanine). For example, when the domain is a phenylalanine ammonia-lyase domain, the function is to catalyze the reaction of eliminating ammonia from phenylalanine. For example, when the domain is an FAD-binding domain or an FMN-binding domain, the function is to bind to FAD or FMN. For example, when the domain is an NADPH-dependent flavin mononucleotide reductase domain, the function is to catalyze the following oxidation-reduction reaction:

[0028] FMNH ２ + NADP ＋ ⇔ FMN + NADPH +H ＋

[0029] Furthermore, the amino acid sequence of an enzyme having a domain with a function equivalent to that of a specific domain can be obtained by a homology search using the method described below.

[0030] First, the known amino acid sequences of each enzyme shown below were used as reference sequences, and the NCBI nr database (downloaded on April 22, 2021) was used as a search target, using DIAMOND (version 0.9.30; diamond blastp -- evaluate 0.00001 -- id 10 -- query-cover 50 -- max-target-seqs 1000000 -- more-sensitive) (Reference 1: "B. Buchfink, C. Xie, and D.H. Huson, "Fast and sensitive protein alignment using DIAMOND," Nat. Methods 2014 121, Vol. 12, No. 1, pp. 59-60, Nov. 2014, doi: 10.1038 / nmeth.3176.

[0031] PAL: SEQ ID NO: 1 (bth:BT_2690; KEGG; Bacteroides thetaiotaomicron VPI-5482) HcrB: SEQ ID NO: 2 (CCC78762.1; NCBI; Lactiplantibacillus plantarum WCFS1) AcdA: SEQ ID NO: 3 (EDU39257.1; NCBI; Clostridium sporogenes ATCC 15579) FldB: SEQ ID NO: 4 (EDU39255.1; NCBI; Clostridium sporogenes ATCC 15579) FldC: SEQ ID NO: 5 (EDU39256.1; NCBI; Clostridium sporogenes ATCC 15579) FldH: SEQ ID NO: 6 (EDU39261.1; NCBI; Clostridium sporogenes ATCC 15579)

[0032] An amino acid sequence is obtained as a "hit sequence" if it has the following characteristics a) and b): a) the amino acid homology with the reference sequence is greater than or equal to an e-value of 0.00001 (1 x 10 －５a) The amino acid identity within the alignment region is 10% or more. b) The reference sequence coverage of the alignment region is 50% or more. By calculating the amino acid identity relative to the full length of the reference sequence, it is possible to quantify to what extent each hit sequence conserves the amino acid sequence of the reference sequence.

[0033] Next, the hit sequences and each reference sequence were subjected to HMMER (version 3.3.2; hmmscan -E 0.01) (Reference 3: R.D. Finn, J. Clements, and S.R. Eddy, "HMMER web server: interactive sequence similarity searching," Nucleic Acids Res., vol. 39, no. suppl_2, pp. W29-W37, Jul. 2021) using the Pfam database (downloaded on April 23, 2021) (Reference 2: J. Mistry et al., "Pfam: The protein families database in 2021," Nucleic Acids Res., vol. 49, no. D1, pp. D412-D419, Jan. 2021, doi: 10.1093 / NAR / GKAA913.) Protein functional domain prediction was performed using the "Nuclear Protein Analysis and Enzyme Analysis for the Chromosome 1" method (http: / / www.narc.org / narc. 2011, doi: 10.1093 / NAR / GKR367.). e-value 0.01 (1 x 10 －２ ) or less is set as a threshold to detect the functional domain of each sequence. If an amino acid sequence has the same functional domain as a reference sequence, the amino acid sequence is acquired as a homologue sequence of each enzyme.

[0034] Examples of bacteria having a PAL gene include bacteria of the genus Bacteroides, bacteria of the genus Blautia hansenii, and bacteria of the genus Bifidobacterium. More specifically, examples of bacteria having a PAL gene include Bacteroides intestinalis (NCBI; AB214328.1; 16S rDNA (partial sequence): SEQ ID NO: 7), Bacteroides ovatus (NCBI; L16484.1; 16S rDNA: SEQ ID NO: 8), Bacteroides thetaiotaomicron (NCBI; M58763.2; 16S rDNA (partial sequence): SEQ ID NO: 9), Bacteroides uniformis (NCBI; L16486.1; 16S rDNA: SEQ ID NO: 10), Bacteroides dorei (NCBI; AB242142.1; 16S rDNA (partial sequence): SEQ ID NO: 11), Blautia hansenii (NCBI; M59114.2; 16S rDNA (partial sequence): SEQ ID NO: 12), and Bifidobacterium longum (NCBI; LC071818.1; 16S rDNA (partial sequence): SEQ ID NO: 13).

[0035] Examples of bacteria having the HcrB gene include bacteria of the genus Blautia and bacteria of the genus Lactobacillus, and more specifically, bacteria of the genus Blautia include Blautia producta (NCBI; AB600998.1; 16S rDNA (partial sequence): SEQ ID NO: 14), Lactobacillus crispatus (NCBI; AJ421225.1; 16S rDNA (partial sequence): SEQ ID NO: 15), Lactobacillus gasseri (NCBI; M58820.1; 16S rDNA: SEQ ID NO: 16), Lactobacillus johnsonii (NCBI; AJ002515.1; 16S rDNA: SEQ ID NO: 17), and Lactiplantibacillus plantarum (NCBI; X52653.1; 16S rDNA: SEQ ID NO: 18).

[0036] Furthermore, as the bacterium having each of the above-mentioned genes, a transformant bacterium into which at least one gene selected from the group consisting of the PAL gene, the HcrB gene, and genes containing nucleotide sequences encoding domains having functions equivalent to those of these genes may be used. Such transformant bacteria may be used alone or in combination of two or more types. Examples of bacteria that can serve as hosts include known enterobacteria that do not have each of the above-mentioned genes. Furthermore, known methods such as lipofection and electroporation can be used to introduce the above-mentioned genes into the host. The above-mentioned genes may be introduced in the form of a vector containing the above-mentioned genes, or the above-mentioned genes may be integrated into the genomic DNA of the host.

[0037] Alternatively, as shown in the examples below, the composition of this embodiment contains a novel bacterium that synthesizes PPA from phenylalanine using a group of enzymes consisting of FldH, FldB, FldC, and AcdA.

[0038] That is, in another embodiment of the composition, the composition further contains a bacterium having: a nucleotide sequence encoding a protein having the activity of acyl-CoA dehydrogenase (AcdA); a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase B (FldB); a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase C (FldC); and a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:3; the protein having the activity of FldB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:4; the protein having the activity of FldC has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:5; and the protein having the activity of FldH has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:6.

[0039] The amino acid sequences set forth in SEQ ID NOs: 3 to 6 are the amino acid sequences of AcdA, FldB, FldC, and FldH of Clostridium sporogenes ATCC 15579 strain, respectively.

[0040] In a protein having the activity of AcdA, FldB, FldC, or FldH, the homology of the amino acid sequence of each protein to the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 6 is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and most preferably 100%.

[0041] A protein having AcdA activity may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 3. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 150, more preferably 1 to 113, even more preferably 1 to 75, still more preferably 1 to 37, particularly preferably 1 to 18, and most preferably 1 to 3.

[0042] A protein having the activity of FldB may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 4. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 167, more preferably 1 to 125, even more preferably 1 to 83, still more preferably 1 to 41, particularly preferably 1 to 20, and most preferably 1 to 4.

[0043] A protein having FldC activity may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 5. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 149, more preferably 1 to 112, even more preferably 1 to 74, still more preferably 1 to 37, particularly preferably 1 to 18, and most preferably 1 to 3.

[0044] A protein having the activity of FldH may have one to several amino acids deleted, inserted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 6. The number of amino acids that may be deleted, inserted, substituted, or added is preferably 1 to 132, more preferably 1 to 99, even more preferably 1 to 66, still more preferably 1 to 33, particularly preferably 1 to 16, and most preferably 1 to 3.

[0045] AcdA, FldB, FldC, and FldH each have a functional domain. Therefore, the bacterium contained in the composition of this embodiment preferably has a gene comprising a nucleotide sequence encoding the functional domain of AcdA, FldB, FldC, or FldH, or a domain having a function equivalent thereto.

[0046] The amino acid sequences of enzymes having domains with functions equivalent to those of AcdA, FldB, FldC, and FldH can be obtained using the methods described above for PAL and HcrB.

[0047] Examples of bacteria having the AcdA gene, FldB gene, FldC gene, and FldH gene include Clostridium bacteria and Peptostreptococcus bacteria. More specifically, Clostridium sporogenes (NCBI; AJ579907.1; 16S rDNA (partial sequence): SEQ ID NO: 19), Clostridium cadaveris (NCBI; M59086.1; 16S rDNA: SEQ ID NO: 20), and Peptostreptococcus anaerobius (NCBI; NR_042847.1; 16S rDNA (partial sequence): SEQ ID NO: 21) can be mentioned.

[0048] Furthermore, the bacterium having each of the above-mentioned genes may be a transformant into which at least one gene selected from the group consisting of the AcdA gene, the FldB gene, the FldC gene, the FldH gene, and genes containing nucleotide sequences encoding domains having functions equivalent to those of these genes has been introduced. Such transformant bacteria may be used alone or in combination of two or more. Examples of bacteria that can serve as hosts include known enterobacteria that do not have each of the above-mentioned genes. Furthermore, known methods such as lipofection and electroporation can be used to introduce the above-mentioned genes into the host. The above-mentioned genes may be introduced in the form of a vector containing the above-mentioned genes, or may be integrated into the genomic DNA of the host.

[0049] In the composition of this embodiment, the content of the PPA-producing bacteria or the 4OHPPA-producing bacteria can be preferably 0.01% by mass or more, more preferably 0.1% by mass or more, relative to the total mass of the composition. On the other hand, the upper limit of the content of the PPA-producing bacteria or the 4OHPPA-producing bacteria is not particularly limited, and can be, for example, 100% by mass, preferably 100% by mass or less, more preferably 99% by mass or less, and even more preferably 95% by mass or less, relative to the total mass of the composition.

[0050] <Acid or Acid-Producing Bacteria> The composition of the present embodiment preferably further contains an acid or a bacterium that produces the acid.

[0051] As shown in the examples described below, the composition of this embodiment preferably contains bacteria that produce PPA, bacteria that produce 4OHPPA, or bacteria that produce PPA and 4OHPPA, or a mixture thereof, and further contains an acid or bacteria that produce an acid. By having such a configuration, the composition of this embodiment can damage the cell membrane of pathogenic bacteria or pathogenic fungi, and further suppress the growth of pathogenic bacteria or pathogenic fungi.

[0052] The acid may be an organic acid or an inorganic acid. Specific examples include, but are not limited to, acetic acid, fumaric acid, butyric acid, propionic acid, lactic acid, and hydrochloric acid. These acids may be used alone or in combination of two or more. Among these, the acid is preferably a short-chain fatty acid or an inorganic acid, more preferably a short-chain fatty acid such as acetic acid, fumaric acid, propionic acid, hydrochloric acid, or lactic acid, and even more preferably acetic acid or lactic acid.

[0053] Examples of the bacteria that produce the above-mentioned acids include bacteria of the genus Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Bacteroides, Blautia, and Clostridium. More specifically, Bifidobacterium longum (16S rDNA: SEQ ID NO: 13), Lactobacillus gasseri (16S rDNA: SEQ ID NO: 16), Lactobacillus johnsonii (16S rDNA: SEQ ID NO: 17), Lacticaseibacillus rhamnosus (16S rDNA (partial sequence): SEQ ID NO: 22), Lactiplantibacillus plantarum (16S rDNA (partial sequence): SEQ ID NO: 23), Blautia hansenii (16S rDNA (partial sequence): SEQ ID NO: 24), sequence): SEQ ID NO: 12), Clostridium butyricum (16S rDNA (partial sequence): SEQ ID NO: 24), Bacteroides fragilis (16S rDNA: SEQ ID NO: 25), and Bacteroides thetaiotaomicron (16S rDNA: SEQ ID NO: 1).

[0054] In the composition of this embodiment, the content of the acid or acid-producing bacteria can be preferably 0.01% by mass or more, more preferably 0.1% by mass or more, relative to the total mass of the composition. On the other hand, the upper limit of the content of the acid or acid-producing bacteria is not particularly limited, but can be, for example, preferably 95% by mass or less, relative to the total mass of the composition.

[0055] Pharmaceutical Composition The composition of the present embodiment is preferably used as a pharmaceutical product. That is, one embodiment of the composition of the present embodiment is a pharmaceutical composition.

[0056] The pharmaceutical composition of this embodiment preferably has the effect of promoting the extension of pili of enterobacteria.

[0057] Enterobacteria possess sortase (SD)-dependent pili and type IV pili, etc. Among enterobacteria, enterobacteria having SD-dependent pili genes are preferred as targets of the pharmaceutical composition of this embodiment.

[0058] In the fimbriae of enterobacteria having SD-dependent pilus genes, the FimA protein is polymerized on the bacterial cell surface by the SD (SrtC or SrtA), and the FimB protein binds to the tip of the fimbria to form giant fibers. As shown in the Examples below, in the presence of PPA or 4OHPPA, the expression of genes involved in pilus formation is enhanced, resulting in pilus formation. Specifically, the expression of genes containing nucleotide sequences encoding at least the pilus structural proteins (FimA and FimB) and the gene containing a nucleotide sequence encoding an enzyme that polymerizes the structural proteins (SrtC) is enhanced, resulting in pilus formation. Methods for confirming the formation of fimbriae include observing the bacterial cell surface of enterobacteria using a scanning electron microscope and using well-known methods (e.g., Western blotting analysis) using antibodies that recognize proteins that constitute fimbriae (e.g., anti-FimA antibodies).

[0059] Examples of enterobacteria having SD-dependent pilus genes include, but are not limited to, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Ruminococcus gnavus, Blautia hansenii, and Lactobacillus rhamnosus.

[0060] The pharmaceutical composition of this embodiment is preferably used for treating or preventing a disease caused by a pathogenic bacterium.

[0061] Target pathogenic bacteria include bacteria of the phylum Proteobacteria, and more specifically, include, but are not limited to, Salmonella typhimurium, Escherichia coli, Citrobacter redentium, Klebsiella pneumoniae, and Proteus mirabilis.

[0062] Diseases caused by these pathogenic bacteria include, but are not limited to, bacterial food poisoning, typhoid fever, gastroenteritis, respiratory infections, urinary tract infections, bacterial vaginosis, sepsis, and meningoencephalitis.

[0063] Alternatively, in another embodiment, the pharmaceutical composition is preferably used for treating or preventing a disease caused by a pathogenic fungus.

[0064] Target pathogenic fungi include fungi of the genus Saccharomycetales and fungi of the genus Trichosporonaceae.

[0065] Examples of fungi of the genus Saccharomycetales include, but are not limited to, Candida albicans, Candida auris, Candida famata, and Pichia fermentans.

[0066] Examples of fungi of the genus Trichosporonaceae include, but are not limited to, Cryptococcus humicola.

[0067] Diseases caused by these pathogenic fungi include, but are not limited to, inflammatory bowel disease, oral candidiasis, vaginal candidiasis, pneumonia, and meningitis.

[0068] Alternatively, the pharmaceutical composition of another embodiment may be used for the treatment or prevention of diseases other than those mentioned above, specifically, inflammatory diseases such as ulcerative colitis, functional gastrointestinal disorders such as functional constipation and functional diarrhea, intestinal cancer, metabolic syndrome, and neurological diseases.

[0069] Intestinal cancers include duodenal cancer, small intestinal cancer, and large intestinal cancer, etc. Large intestinal cancers include cecum cancer, colon cancer, and rectal cancer, etc.

[0070] Examples of metabolic syndrome include obesity (particularly visceral fat obesity), hypertension, dyslipidemia, and diabetes.

[0071] Neurological disorders include anxiety disorders, autism, and depression.

[0072] The subject to which the pharmaceutical composition of the present embodiment is administered (ingested) is not particularly limited as long as it is an animal, but is preferably a mammal, more preferably a human, and may be any of adults, children, infants, and newborns (including low birth weight infants).

[0073] The intake (administration) amount of the pharmaceutical composition of this embodiment is appropriately selected depending on the age, sex, condition, and other conditions of the subject. The total intake of PPA and 4OHPPA produced by the bacteria is preferably in the range of 100 mg / day to 1000 mg / day, more preferably 100 mg / day to 500 mg / day, and even more preferably 100 mg / day to 300 mg / day. Regardless of the amount or period of administration, the pharmaceutical composition can be administered once a day or in multiple divided doses.

[0074] The lower limit of the content of the PPA-producing bacteria or 4OHPPA-producing bacteria contained in the pharmaceutical composition of this embodiment is not particularly limited and may be selected as appropriate, but may be, for example, 10% by mass or more relative to the total mass of the pharmaceutical composition. Preferably, it can be 20% by mass or more, more preferably 30% by mass or more, even more preferably 40% by mass or more, particularly preferably 50% by mass or more, and most preferably 97% by mass or more. Meanwhile, the upper limit of the content of the PPA-producing bacteria or 4OHPPA-producing bacteria contained in the pharmaceutical composition of this embodiment is not particularly limited, but may be, for example, 100% by mass or less relative to the total mass of the pharmaceutical composition, preferably 100% by mass or less, more preferably 99% by mass or less, and even more preferably 95% by mass or less.

[0075] The pharmaceutical composition of this embodiment may be taken (administered) either orally or parenterally, with oral administration being preferred. Parenteral administration includes transdermal, intravenous, rectal, vaginal, nasal, and inhalation routes. It is desirable that effective amounts or more of PPA and 4OHPPA are retained in the intestines after the pharmaceutical composition of this embodiment is taken (administered).

[0076] The timing of administration of the pharmaceutical composition of this embodiment is not particularly limited and may be any timing, such as before meals, after meals, between meals, or before going to bed.

[0077] When the composition of this embodiment is used as a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment can be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. In addition, in the case of parenteral administration, it can be formulated into suppositories, ointments, injections, etc. Since it is desirable that an effective amount or more of PPA or 4OHPPA is retained in the intestine after administration of the pharmaceutical composition of this embodiment, when the pharmaceutical composition of this embodiment is in the form of an oral preparation, enteric capsules, acid-resistant sugar-coated tablets, etc. are preferred.

[0078] In the formulation, in addition to the PPA-producing bacteria or the 4-OHPPA-producing bacteria, ingredients such as excipients, pH adjusters, colorants, and flavoring agents that are commonly used in formulations can be used. It is also possible to use other medicinal ingredients, ingredients known to have an intestinal flora-improving effect, and ingredients that will be discovered in the future to have an intestinal flora-improving effect. In addition, in the formulation, a known method can be used as appropriate depending on the desired dosage form, and a pharmaceutical carrier may be added to the formulation.

[0079] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, starch, and dextrin carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0080] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0081] Disintegrants include the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

[0082] Lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gaelt; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, etc.

[0083] Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid; and the like.

[0084] Flavoring agents include sweeteners, acidulants, and fragrances.

[0085] When the composition is formulated as a liquid for oral administration, examples of the pharmaceutical carrier to be added include solvents such as water.

[0086] <Food and Drink> The composition of the present embodiment is preferably used as a food or drink. Examples of the food and drink include functional foods and foods for specified health uses.

[0087] Alternatively, in another embodiment, the food or beverage contains PPA or 4OHPPA.

[0088] Alternatively, in another embodiment, the food or beverage contains a protein having the activity of phenylalanine ammonia-lyase (PAL) and a protein having the activity of hydroxycinnamate reductase B (HcrB), wherein the protein having the activity of PAL has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, and the protein having the activity of HcrB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0089] Alternatively, in another embodiment, the food or drink product may contain a protein having an aromatic amino acid lyase domain (Pfam ID: PF00221, InterPro entry: IPR001106) or phenylalanine ammonia-lyase (InterPro entry: IPR005922), or a domain having a function equivalent thereto, and a flavin adenine dinucleotide (FAD)-binding domain (Pfam ID: PF00890, InterPro entry: IPR003953), and a flavin mononucleotide (FMN)-binding domain (Pfam ID: PF00890, InterPro entry: IPR003953). ID: PF04205, InterPro entry: IPR007329), or a protein having a domain having a function equivalent thereto.

[0090] Furthermore, the food and drink of the above embodiment, i.e., the food and drink containing the PAL protein and HcrB protein, or proteins having the activity of these enzymes, may further contain a protein having the activity of acyl-CoA dehydrogenase (AcdA), a protein having the activity of phenyllactate dehydrogenase B (FldB), a protein having the activity of phenyllactate dehydrogenase C (FldC), and a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:3, the protein having the activity of FldB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:4, the protein having the activity of FldC has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:5, and the protein having the activity of FldH has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:6.

[0091] The form and properties of the food and drink product are not particularly limited as long as they do not impair the effects of the PPA-producing bacteria or the 4-OHPPA-producing bacteria and can be taken orally, and they can be produced by a conventional method using raw materials that are normally used in food and drink products, except that they contain the PPA-producing bacteria or the 4-OHPPA-producing bacteria. When the composition of the present embodiment is used as a food or drink product, it can contain an acceptable carrier.

[0092] The food and drink may be in any form, such as liquid, paste, gel, solid, or powder. Specific examples of the food and drink include tablets; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, and breadcrumbs; instant noodles, cup noodles, retort-packed foods, canned foods, microwave-safe foods, instant soups, stews, instant miso soup, instant clear soups, canned soups, freeze-dried foods, and other instant foods; canned agricultural products, canned fruit, jams and marmalades, pickles, Agricultural processed products such as boiled beans, dried agricultural products, and cereals (processed grain products); processed seafood products such as canned seafood, fish ham and sausage, fish paste products, seafood delicacies, and tsukudani (simmered fish paste); processed livestock products such as canned livestock products and pastes, and livestock ham and sausage; milk and dairy products such as processed milk, milk drinks, yogurt, fermented milk, lactic acid bacteria drinks, cheese, ice cream, infant formula, cream, and other dairy products; butter, margarine, and vegetable oil complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredient foods, semi-prepared frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice crackers, bean snacks, dessert sweets, jelly, and other sweets; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit drinks with fruit pieces, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, sprinkles, and ochazuke nori seaweed; infant formula; and enteral nutritional foods.

[0093] Furthermore, one aspect of the food and drink product may be feed, such as pet food, livestock feed, and fish feed.

[0094] The form of the feed is not particularly limited, and may contain, in addition to the PPA-producing bacteria or the 4-OHPPA-producing bacteria, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake; bran such as wheat bran, wheat bran, rice bran, and defatted rice bran; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, and tallow; yeasts such as torula yeast and brewer's yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; oils and fats; simple amino acids; sugars, etc.

[0095] The amount of PPA-producing bacteria or 4OHPPA-producing bacteria contained in the food or drink of this embodiment is not particularly limited and may be selected appropriately, but may be, for example, preferably 0.01% by mass or more, more preferably 0.1% by mass or more, relative to the total mass of the food or drink. On the other hand, the upper limit of the content of PPA-producing bacteria or 4OHPPA-producing bacteria is not particularly limited, but may be, for example, preferably 70% by mass or less, more preferably 40% by mass or less, and even more preferably 5% by mass or less, relative to the total mass of the composition.

[0096] The subjects, timing of intake, and intake amount of the food and drink of this embodiment are as exemplified for the pharmaceutical composition above.

[0097] <<Method for assisting in testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi>> The method of this embodiment is a method for assisting in testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi, and includes the steps of quantifying the amount of PPA or the amount of 4OHPPA in the stool of a subject using 2-nitrophenylhydrazine (2-NPH) (hereinafter, this may be referred to as a "first quantification step"), comparing the value obtained by quantifying the amount of PPA or the amount of 4OHPPA with a standard value (hereinafter, this may be referred to as a "first comparison step"), and indicating the possibility that the subject is suffering from the disease when the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is less than the standard value.

[0098] As shown in the examples described below, the concentrations of PPA or 4OHPPA in the feces of patients with diseases caused by pathogenic bacteria or pathogenic fungi are significantly lower than the concentrations of PPA or 4OHPPA in the feces of healthy individuals, and a clear correlation was observed between diseases caused by pathogenic bacteria or pathogenic fungi and the concentrations of PPA or 4OHPPA in the feces. Therefore, the method of this embodiment can evaluate the possibility that a subject is suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0099] Each step of the method of this embodiment will be described in detail below.

[0100] <First Quantification Step> In the first quantification step, the amount of PPA or the amount of 4OHPPA in the subject's feces is quantified using 2-NPH.

[0101] HPLC analysis of underivatized PPA and underivatized 4OHPPA has low sensitivity, and the peaks of underivatized PPA and underivatized 4OHPPA are not well separated. 2-NPH reacts with the carboxyl group of PPA or 4OHPPA in the presence of a condensing agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) to form a 2-nitrophenylhydrazide derivative. Converting PPA and 4OHPPA to their 2-nitrophenylhydrazide derivatives changes the hydrophobicity of PPA and 4OHPPA, allowing the PPA derivative and the 4OHPPA derivative to be detected as different peaks. Furthermore, such conversion enables the PPA derivative and the 4OHPPA derivative to be detected with high sensitivity. In other words, by detecting the 2-nitrophenylhydrazide derivatives of PPA and 4OHPPA, rather than underivatized PPA and underivatized 4OHPPA, the amount of PPA or 4OHPPA in a subject's feces can be quantified.

[0102] In the first quantification step, it is preferable to further quantify the amount of short-chain fatty acids. As shown in the examples below, the combination of PPA or 4OHPPA and short-chain fatty acids damages the cell membrane of pathogenic bacteria or pathogenic fungi in the intestine, and significantly suppresses the growth of pathogenic bacteria or pathogenic fungi. Therefore, by quantifying the amount of short-chain fatty acids in addition to the amount of PPA or 4OHPPA in the subject's feces, the intestinal environment of the subject can be evaluated in more detail.

[0103] Short-chain fatty acids can also be quantified using 2-NPH. Specifically, since short-chain fatty acids also have a carboxy group, they react with 2-NPH in the presence of a condensing agent and are converted to 2-nitrophenylhydrazide derivatives. 2-nitrophenylhydrazide derivatives derived from each compound can be detected by analyzing each compound using a known method such as HPLC. As a result, the amount of PPA or 4OHPPA, and the amount of short-chain fatty acids in the subject's feces can each be quantified.

[0104] In the first quantification step, the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) may be quantified in addition to the subject fecal sample to be quantified. Examples of control samples include a negative control sample that does not contain PPA, 4OHPPA, and short-chain fatty acids, and a positive control sample that contains PPA, 4OHPPA, and short-chain fatty acids. In this case, the presence or absence of PPA or 4OHPPA (and, if necessary, short-chain fatty acids) in the subject fecal sample can be determined by comparing the results obtained from the subject fecal sample with the results obtained from the negative control sample that does not contain PPA, 4OHPPA, and short-chain fatty acids, and the results obtained from the positive control sample that contains PPA, 4OHPPA, and short-chain fatty acids. Alternatively, a series of control samples with gradually varying concentrations can be prepared, and the quantitative results for each control sample can be obtained as numerical values ​​to create a standard curve. Based on the numerical values ​​of the subject fecal sample, the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) in the subject fecal sample can be quantified based on the standard curve.

[0105] Furthermore, an internal standard reagent can be added to all of the samples (standards) for preparing the standard curve and the analytical samples, so that handling errors can be corrected when calculating the concentration. The internal standard reagent is not particularly limited as long as it is a compound that is not synthesized in vivo, and examples thereof include benzoic acid-α- １３ Examples include C.

[0106] <First Comparison Step> In the first comparison step, the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is compared with a reference value.

[0107] In the first comparison step, if the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is less than the standard value, the method of this embodiment indicates that the subject may be suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0108] On the other hand, if the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is equal to or greater than the standard value, the method of this embodiment indicates that the subject is unlikely or not likely to be suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0109] The reference value is a reference value for distinguishing between a group of subjects suffering from a disease caused by pathogenic bacteria or pathogenic fungi and a group of subjects not suffering from the disease.

[0110] The reference value is experimentally determined as a threshold value capable of distinguishing between a patient group suffering from a disease caused by pathogenic bacteria or pathogenic fungi and a patient group not suffering from the disease, by quantifying the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) in the feces of the patient group. In this embodiment, the method for determining the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) is not particularly limited, and may be, for example, a determination method using a general statistical method.

[0111] Examples of methods for determining the reference value include quantifying the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) in feces collected before diagnosis (such as at the time of hospitalization) from patients who have been diagnosed with a disease caused by the target pathogenic bacteria or fungus by methods other than the method of this embodiment, such as commonly performed clinical diagnosis based on symptoms and patient background, and definitive diagnosis by either or both of a stool test and a blood test. After quantifying the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) in the feces of multiple patients, the amount of PPA or 4OHPPA (and, if necessary, the amount of short-chain fatty acids) in the patient's feces can be calculated from the average or median, and the numerical range containing the calculated value can be used as the reference value.

[0112] In addition, as a method for determining the reference value, the amount of PPA or the amount of 4OHPPA (and the amount of short-chain fatty acids, if necessary) in the feces of patients suffering from diseases caused by multiple pathogenic bacteria or pathogenic fungi and patients not suffering from the diseases are quantified before diagnosis (at the time of hospitalization, etc.), and the amount of PPA or the amount of 4OHPPA (and the amount of short-chain fatty acids, if necessary) in the feces of a group of patients suffering from the above-mentioned diseases and a group of patients not suffering from the above-mentioned diseases and their variation are calculated from the average value or median value, etc., and then a threshold value is determined that allows the two values ​​to be distinguished taking into account the variation, and this threshold value can be used as the reference value.

[0113] Alternatively, in another embodiment, the method is a method for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi, comprising: quantifying the expression level of the PAL gene or the expression level of the HcrB gene in the stool of a subject (hereinafter, this may be referred to as a "second quantification step"); comparing the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene with a standard value (hereinafter, this may be referred to as a "second comparison step"); and indicating the possibility that the subject is suffering from the disease if the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene is less than the standard value.

[0114] The enzymes encoded by the PAL gene and the HcrB gene biosynthesize PPA or 4OHPPA from phenylalanine or tyrosine. The PPA or 4OHPPA damages the cell membrane of pathogenic bacteria or fungi, significantly suppressing their growth. Therefore, quantifying the expression levels of genes containing nucleotide sequences encoding these enzymes can be used to assess the likelihood that a subject is suffering from a disease caused by pathogenic bacteria or fungi.

[0115] <Second Quantification Step> In the second quantification step, the expression level of the PAL gene or the expression level of the HcrB gene in the stool of the subject is quantified.

[0116] The expression level of the PAL gene or the HcrB gene can be quantified by known quantification methods such as real-time PCR and digital PCR. Primers for detecting the PAL gene or the HcrB gene can be appropriately designed by those skilled in the art from the nucleotide sequence of the gene using known methods.

[0117] In the second quantification step, it is preferable to further quantify the expression level of at least one gene selected from the group consisting of the AcdA gene, the FldB gene, the FldC gene, and the FldH gene.

[0118] The enzymes encoded by the AcdA gene, FldB gene, FldC gene, and FldH gene biosynthesize PPA or 4OHPPA from phenylalanine or tyrosine, respectively. Therefore, quantifying the expression levels of these genes in addition to the PAL gene or HcrB gene allows for a more accurate assessment of the possibility of suffering from a disease caused by pathogenic bacteria or fungi. The expression levels of the AcdA gene, FldB gene, FldC gene, and FldH gene can be quantified using the same method as that for quantifying the expression levels of the PAL gene and HcrB gene.

[0119] In the second quantification step, in addition to the subject fecal sample for which the expression level of the above-mentioned gene is to be quantified, a control sample may also be set and quantified. Examples of control samples include a negative control sample that does not contain the above-mentioned gene and a positive control sample that contains the above-mentioned gene. In this case, the presence or absence of expression of the above-mentioned gene in the subject fecal sample can be determined by comparing the results obtained from the subject fecal sample with the results obtained from the negative control sample that does not contain the above-mentioned gene and the results obtained from the positive control sample that contains the above-mentioned gene. Alternatively, a series of control samples with gradually varying concentrations can be prepared, and the quantification results for each control sample can be obtained as numerical values ​​to create a standard curve, which can then be used to quantitate the expression level of the above-mentioned gene in the subject fecal sample based on the numerical values ​​of the subject fecal sample.

[0120] <Second Comparison Step> In the second comparison step, the value obtained by quantifying the expression level of the PAL gene or the HcrB gene is compared with a reference value.

[0121] If the value obtained by quantifying the expression level of the PAL gene or the HcrB gene is less than the reference value, the method of this embodiment indicates that the subject is likely suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0122] On the other hand, if the value obtained by quantifying the expression level of the PAL gene or the HcrB gene is equal to or greater than the reference value, the method of this embodiment indicates that the subject is unlikely or unlikely to be suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0123] The reference value is a reference value for distinguishing between a group of subjects suffering from a disease caused by pathogenic bacteria or pathogenic fungi and a group of subjects not suffering from the disease.

[0124] The reference value can be experimentally determined as a threshold value capable of distinguishing between a patient group suffering from a disease caused by pathogenic bacteria or pathogenic fungi and a patient group not suffering from the disease, for example, by measuring the expression levels of the above-mentioned genes in the feces of the two groups. In this embodiment, the method for determining the amount of PPA or the amount of 4-OHPPA (and, if necessary, the amount of short-chain fatty acids) is not particularly limited, and may be, for example, a determination method using a general statistical method.

[0125] Examples of methods for determining the reference value include measuring the expression level of the above-mentioned gene in feces collected before diagnosis (such as at the time of hospitalization) from patients who have been diagnosed with a disease caused by the target pathogenic bacterium or pathogenic fungus by a method other than the method of this embodiment, such as a commonly performed clinical diagnosis based on symptoms and patient background, and a definitive diagnosis by either or both of a stool test and a blood test. After measuring multiple patients, the expression level of the above-mentioned gene in the patient's feces can be calculated from the average or median, and a numerical value including the calculated value can be used as the reference value.

[0126] Furthermore, a method for determining the reference value includes measuring the amount of the above-mentioned genes in feces collected before diagnosis (such as at the time of hospitalization) from patients suffering from diseases caused by multiple pathogenic bacteria or fungi and patients not suffering from the diseases. The expression levels and variability of the above-mentioned genes in the feces of a group of patients suffering from the disease and a group of patients not suffering from the disease are calculated from the average value, median value, etc., and then a threshold value that allows the two values ​​to be distinguished taking variability into consideration can be determined, and the threshold value can be used as the reference value.

[0127] Alternatively, a method of another embodiment is a method for assisting in the testing and diagnosis of a disease caused by pathogenic bacteria or pathogenic fungi, comprising: quantifying the expression level of at least one gene selected from the group consisting of a gene comprising a nucleotide sequence encoding an aromatic amino acid lyase domain, a gene comprising a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain, and a gene comprising a nucleotide sequence encoding a flavin mononucleotide-binding domain in the stool of a subject (hereinafter, this may be referred to as a "third quantification step"); comparing the value obtained by quantifying the expression level of the gene with a reference value (hereinafter, this may be referred to as a "third comparison step"); and, if the value obtained by quantifying the expression level of the gene is less than the reference value, indicating the possibility that the subject is suffering from the disease.

[0128] A group of enzymes having an aromatic amino acid lyase domain, a flavin adenine dinucleotide-binding domain, and a flavin mononucleotide-binding domain biosynthesizes PPA or 4OHPPA from phenylalanine or tyrosine, and the PPA or 4OHPPA damages the cell membrane of pathogenic bacteria or fungi, significantly suppressing their growth. Therefore, by quantifying the expression levels of genes containing nucleotide sequences encoding these domains, the possibility of suffering from a disease caused by pathogenic bacteria or fungi can be evaluated.

[0129] <Third quantification step> In the third quantification step, the expression level of at least one gene selected from the group consisting of a gene comprising a nucleotide sequence encoding an aromatic amino acid lyase domain, a gene comprising a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain, and a gene comprising a nucleotide sequence encoding a flavin mononucleotide-binding domain is quantified in the feces of a subject.

[0130] In the third quantification step, the method for quantifying the expression level of a gene containing a nucleotide sequence encoding the domain can be the method described in the second quantification step.

[0131] In the third quantification step, it is preferable to further quantify the expression level of a gene comprising a nucleotide sequence encoding a functional domain of at least one enzyme selected from the group consisting of AcdA, FldB, FldC, and FldH.

[0132] The enzyme group consisting of AcdA, FldB, FldC, and FldH biosynthesizes PPA and 4OHPPA from phenylalanine and tyrosine, respectively. Therefore, by quantifying the expression levels of a gene containing a nucleotide sequence encoding an aromatic amino acid lyase domain, a gene containing a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain, and a gene containing a nucleotide sequence encoding a flavin mononucleotide-binding domain, as well as a gene containing a nucleotide sequence encoding a functional domain of at least one enzyme selected from the group consisting of AcdA, FldB, FldC, and FldH, the possibility that a subject is suffering from a disease caused by pathogenic bacteria or pathogenic fungi can be more accurately assessed. The method described in the second quantification step above can be used as a method for quantifying a gene containing a nucleotide sequence encoding a functional domain of at least one enzyme selected from the group consisting of AcdA, FldB, FldC, and FldH.

[0133] <Third Comparison Step> In the third comparison step, the value obtained by quantifying the expression level of the gene is compared with a reference value.

[0134] When the value obtained by quantifying the expression level of the gene is less than the reference value, the method of this embodiment indicates the possibility that the subject is suffering from the disease.

[0135] On the other hand, if the value obtained by quantifying the expression level of the gene is equal to or greater than the reference value, the method of this embodiment indicates that there is a low or no possibility of the subject suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0136] The reference value is a reference value for distinguishing between a group of subjects suffering from a disease caused by a pathogenic bacterium or a pathogenic fungus and a group of subjects not suffering from the disease. The method for determining the reference value can be the same as the method for determining the reference value in the second comparison step.

[0137] <Treatment or Prevention Method> If the results obtained from the above-mentioned method for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or pathogenic fungi indicate that a subject may be suffering from a disease caused by pathogenic bacteria or pathogenic fungi, the subject can be administered a pharmaceutical composition containing an effective amount of bacteria that produce PPA or bacteria that produce 4-OHPPA, thereby treating or preventing the disease caused by pathogenic bacteria or pathogenic fungi.

[0138] That is, in one embodiment, the present invention provides a method for treating or preventing a disease caused by pathogenic bacteria or pathogenic fungi, comprising administering to a subject a pharmaceutical composition containing an effective amount of bacteria that produce PPA or bacteria that produce 4OHPPA, when the results obtained from the above-mentioned method for assisting in the testing and diagnosis of a disease caused by pathogenic bacteria or pathogenic fungi indicate that the subject may be suffering from a disease caused by pathogenic bacteria or pathogenic fungi.

[0139] Alternatively, in another embodiment, the present invention provides a method for treating or preventing a disease caused by pathogenic bacteria or pathogenic fungi, comprising administering to a subject a pharmaceutical composition containing an effective amount of a PPA-producing bacterium or a 4OHPPA-producing bacterium.

[0140] In the treatment or prevention methods of each embodiment, diseases caused by pathogenic bacteria or pathogenic fungi include those exemplified in the above "pharmaceutical composition."

[0141] In the treatment or prevention methods of each embodiment, the pharmaceutical composition to be administered may further contain an acid or an acid-producing bacterium exemplified in the above "Composition."

[0142] Other Embodiments In one embodiment, the present invention provides use of a PPA-producing bacterium or a 4OHPPA-producing bacterium for promoting pilus elongation of enterobacteria.

[0143] Alternatively, in another embodiment, the present invention provides the use of PPA-producing bacteria or 4OHPPA-producing bacteria, and acid or acid-producing bacteria, to promote pilus elongation of enterobacteria.

[0144] Alternatively, in another embodiment, the present invention provides a method for promoting the extension of enterobacterial pili, comprising administering to an animal a bacterium that produces PPA or a bacterium that produces 4OHPPA.

[0145] Alternatively, in another embodiment, the present invention provides a method for promoting pilus elongation of intestinal bacteria, comprising administering to an animal a PPA-producing bacterium or a 4OHPPA-producing bacterium, and an acid or an acid-producing bacterium.

[0146] Alternatively, in another embodiment, there is provided the use of a PPA-producing bacterium or a 4OHPPA-producing bacterium in the manufacture of a composition for promoting pilus elongation of enterobacteria.

[0147] Alternatively, in another embodiment, there is provided the use of a PPA-producing bacterium or a 4OHPPA-producing bacterium, and an acid or an acid-producing bacterium in the manufacture of a composition for promoting pilus elongation of enterobacteria.

[0148] In each embodiment, the enterobacteria, PPA-producing bacteria or 4OHPPA-producing bacteria, and acid or acid-producing bacteria may be the same as those exemplified in the "Composition" above.

[0149] Alternatively, in another embodiment, the present invention provides the use of a PPA-producing bacterium or a 4OHPPA-producing bacterium in the manufacture of a pharmaceutical composition for the treatment or prevention of a disease caused by a pathogenic bacterium or a pathogenic fungus.

[0150] Alternatively, in another embodiment, the present invention provides the use of PPA-producing bacteria or 4OHPPA-producing bacteria, and acid or acid-producing bacteria in the manufacture of a pharmaceutical composition for the treatment or prevention of a disease caused by pathogenic bacteria or pathogenic fungi.

[0151] In each embodiment, diseases caused by pathogenic bacteria or pathogenic fungi include those exemplified in the above "pharmaceutical composition."

[0152] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0153] Experimental Example 1 (Identification of PPA-producing bacteria and the role of PPA on B. longum) The average PPA concentration in healthy human feces quantified using LC-MS / MS was 0.03 μmol / g wet stools (Figure 1; in Figure 1, "KHC" represents healthy human feces, and "CD" represents feces from Crohn's disease (CD) patients). Meanwhile, the average PPA concentration in CD patient feces was significantly lower than that in healthy human feces. These results suggest that PPA-producing bacteria may be present in large quantities in samples from healthy individuals. PPA can induce the elongation of SD (sortase-dependent) fimbriae of B. longum even at concentrations of several μM, and can induce pilus elongation in a concentration range similar to that of acylhomoserine lactone, a substance that induces bacterial quorum sensing. Therefore, it is suggested that the metabolite PPA produced by bacteria acts as a novel signal molecule to change the physiological state of other bacteria.

[0154] Clostridia produce PPA and 4-OHPPA via the aromatic amino acid (AAA) metabolic pathway mediated by the enzymes FldB, FldC, and AcdA. However, no bacteria possessing FldB, FldC, and AcdA were detected in the culture medium of feces in which PPA was identified as a factor inducing pilus elongation. Therefore, the inventors hypothesized that bacteria possessing a different PPA production pathway exist in the human intestinal bacteria.

[0155] To identify bacteria with the above-mentioned alternative PPA production pathways, 50 types of enterobacteria (Figure 2A) were cultured in GAM (Gifu anaerobic medium). Figures 2B and 2C show the production amounts of PPA, 4OHPPA, and IPA (3-indolepropionic acid) in the 50 types of symbiotic bacteria. Testing of the 50 symbiotic bacteria revealed that C. sporogenes, P. anaerobious, Clostridioides difficile, and Paeniclostridium sordellii, which possess FldB, FldC, and AcdA, produced PPA, 4OHPPA, and IPA. Interestingly, PPA and 4OHPPA production was also confirmed in the co-culture of 46 strains, excluding the above four strains. These 46 strains were classified into four bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria, or Proteobacteria), but PPA and 4OHPPA were not detected in the monocultures of each strain. Furthermore, these 46 strains were phylogenetically classified into seven types and co-cultured with bacteria of the genus Bacteroidetes. The results suggested that PPA and 4OHPPA were produced by a combination of Bacteroidetes and Firmicutes (mainly Lactobacilli), and that the two species cooperated to contribute to the production of PPA and 4OHPPA. Phenylacrylic acid (PAA) and 4-hydroxycinnamic acid (4OH-phenylacrylic acid; 4OHPAA) were also detected in Bacteroidetes monocultures. PAL is an enzyme that converts Phe and Tyr to PAA and 4OHPAA, respectively. Furthermore, HcrB has been identified in Lactobacillus species as an enzyme that converts PAA and 4OHPAA to PPA and 4OHPPA, respectively.

[0156] The inventors prepared a recombinant enzyme, His6-PAL, derived from Bacteroides thetaiotaomicron K-22 strain, and a recombinant enzyme, His6-HcrB, derived from Lactobacillus plantarum JCM1149T. Enzyme activity tests confirmed that His6-PAL converts Phe and Tyr to PAA and 4OHPAA, respectively, and that His6-HcrB converts PAA and 4OHPAA to PPA and 4OHPPA, respectively. Furthermore, the inventors transformed Lactococcus lactis subsp. 1403, expressing the PAL gene and HcrB gene. It was confirmed by enzyme activity tests that the L. lactis IL 1403 pal / hcrB co-expressing strain also produced PPA and 4OHPPA.

[0157] These results demonstrate that when B. thetaiotaomicron and L. plantarum are cultured in combination, they cooperate to biosynthesize PPA and 4OHPPA. In other words, B. thetaiotaomicron biosynthesizes PAA and 4OHPAA using the enzyme PAL, while L. plantarum converts PAA and 4OHPAA to PPA and 4OHPPA, respectively, using HcrB (Figure 2D). Similar results were obtained when B. thetaiotaomicron and L. plantarum were cultured in combination in minimal medium supplemented with L-Phe or L-Tyr. Furthermore, PPA and 4OHPPA were not biosynthesized when the B. thetaiotaomicron Δpal strain (a pal gene-deficient strain) was combined with L. plantarum (Figure 2D). Therefore, it was confirmed that PAL-carrying bacteria and HcrB-carrying bacteria cooperate to contribute to the reaction of producing PPA and 4OHPPA, thereby producing PPA and 4OHPPA in the intestinal tract.

[0158] Furthermore, we colonized germ-free (GF) mice with B. thetaiotaomicron (wt or Δpal) and L. plantarum (Fig. 2E). Two weeks after colonization, PPA and 4OHPPA were detected at 65 nmol / g feces and 29 nmol / g feces, respectively, in the feces of mice colonized with B. thetaiotaomicron wt and L. plantarum (BTwt+LP). In contrast, neither PPA nor 4OHPPA was detected in the feces of mice colonized with B. thetaiotaomicron Δpal and L. plantarum (BTpal+LP) nor in the feces of mice colonized with B. thetaiotaomicron wt alone (Fig. 2E). Gene homologs of pal and hcrB are highly conserved in the Bacteroitedes phylum and Lactobacillus species, respectively. In fact, bacteria carrying gene homologs of pal or hcrB are frequently detected in human fecal cultures and human bacterial flora, suggesting that this pathway is a true PPA / 4OHPPA production pathway in the host body.

[0159] Furthermore, the amounts of PPA and 4OHPPA produced by the combination of commensal bacteria were sufficient to induce the elongation of SD fimbriae of B. longum in the mouse intestine. Scanning electron microscopy (SEM) also confirmed the presence of numerous large fimbrial structures from bacteria colonizing the mucin layer in the gastrointestinal tracts of mice colonized with B. thetaiotaomicron wt and L. plantarum. The colonization levels of B. longum were similar in the feces of mice colonized with B. thetaiotaomicron wt and L. plantarum and those of mice colonized with B. thetaiotaomicron Δpal and L. plantarum (BTpal + LP) (Figure 2F). Furthermore, the rumen of mice colonized with B. thetaiotaomicron Δpal and L. plantarum (BTpal+LP) contained similar numbers of B. longum as those colonized with B. thetaiotaomicron wt and L. plantarum (BTwt+LP) (Fig. 2G). Meanwhile, the level of B. longum colonization in the mucin layer was significantly higher in mice colonized with B. thetaiotaomicron wt and L. plantarum (Fig. 2G). This was also confirmed by fluorescence in situ hybridization (Fig. 2H).

[0160] These results indicated that PPA and 4OHPPA are involved in colonization of the intestinal tract by B. longum. This conclusion was also supported by experiments using GF mice colonized with a 1:1 mixture (bacterial population ratio) of B. longum wt and B. longum ΔlacY (PPA transporter-deficient strain) (Figure 2I). The B. longum ΔlacY strain was eliminated by the B. longum wt strain between days 7 and 14 after colonization in the intestines of mice colonized with B. thetaiotaomicron wt and L. plantarum. On the other hand, the B. longum ΔlacY strain was eliminated by the B. thetaiotaomicron Δpal strain in the intestines of mice colonized with B. thetaiotaomicron Δpal and L. plantarum. However, the B. longum ΔlacY strain was not eliminated (Fig. 2I). Therefore, these results indicate that B. longum uses PPA as a signal molecule that induces the elongation of SD pili as a survival strategy in the intestinal tract.

[0161] Experimental Example 2 (Action of PPA on Bacteria) PPA isolated from the eubacterial species Streptomyces and its structurally similar compounds exhibited MICs of several bacteria. 90 It is known that PPA exhibits antibacterial activity with a minimum inhibitory concentration (MIN) of several mM or less (the concentration that inhibits the growth of 90% of bacterial strains). Therefore, we hypothesized that the PPA-producing bacteria analyzed in this study could suppress pathogenic bacterial infection in mice.

[0162] The antibacterial activity of PPA was confirmed by agar gel diffusion assay. C. sporogenes (CS) and B. longum (BL) were cultured alone or together in GAM medium, and the culture was added to Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (hereinafter sometimes abbreviated as "S. Typhimurium"). As a result, an inhibition zone was formed in the culture containing the C. sporogenes wild-type strain (CS wt), while no inhibition zone was formed in the culture containing the C. sporogenes ΔfldC strain (CS ΔfldC) (Figure 3A).

[0163] Next, we evaluated whether C. sporogenes and B. longum inhibit colonization of S. Typhimurium. First, GF mice were administered C. sporogenes (C. sporogenes wt strain (CS wt) or C. sporogenes ΔfldC strain (CS ko)) and B. longum (5 × 10 ７ cfu / mouse). Then, 14 days after administration, GF mice were challenged with S. Typhimurium (10 ４ cfu / mouse). The progress up to 10 days after S. Typhimurium administration was then evaluated (Figure 3B). In the control GF mice that had not been administered C. sporogenes or B. longum beforehand, all individuals died within 5 days of infection with S. Typhimurium. In the GF mice colonized with the C. sporogenes wt strain (CS wt) alone and the GF mice colonized with the C. sporogenes ΔfldC strain (CS ko) alone or together with B. longum (BL), all individuals died within 8 days of infection with S. Typhimurium. On the other hand, in the GF mice colonized with both the C. sporogenes wt strain and B. longum, all individuals died within 8 days of infection with S. Typhimurium. On day 10 after infection with S. Typhimurium, half of the mice survived, and the amount of S. Typhimurium (cfu) in feces on days 1 and 2 after infection was significantly lower under the control of the control than under the other conditions (Fig. 3C). These results suggest that the C. sporogenes wt strain alone does not sufficiently inhibit colonization by S. Typhimurium, but that the coexistence of the C. sporogenes wt strain and B. longum sufficiently inhibits colonization by S. Typhimurium.

[0164] In addition, the amounts of aromatic amino acids (Phe, Tyr, or Trp) and their metabolites, as well as short-chain fatty acids (hereinafter also referred to as "SCFAs"), were measured using gas chromatography in the feces of mice immediately before S. Typhimurium administration (day 14) in the S. Typhimurium infection experiment. The amount of PPA in the feces was similarly high in the feces of mice co-colonized with C. sporogenes wt strain (CS wt) and B. longum (BL) and in the feces of mice co-colonized with C. sporogenes wt strain alone (Figure 3D). On the other hand, the amounts of SCFAs, particularly acetic acid and lactic acid, in the feces of mice co-colonized with C. sporogenes wt strain and B. longum (BL) were significantly higher than those of mice co-colonized with C. sporogenes wt strain alone. The feces of mice co-colonized with M. longum were overwhelmingly higher (Fig. 3E).

[0165] Based on this, S. Typhimurium was cultured in GAM or LB medium with different concentrations of PPA and 0 or 10 mM acetic acid, and the growth of S. Typhimurium was monitored (Fig. 3F, left: 0 mM acetic acid added, right: 10 mM acetic acid added). As a result, PPA inhibited the growth of S. Typhimurium, but the MIC ９０ The PPA concentration was 10 mM. This concentration was higher than the PPA concentration (approximately 150 nmol / g feces) detected in the feces of mice colonized with the wild-type C. sporogenes strain (Fig. 3D and Fig. 3F). Interestingly, even PPA at a concentration of 0.5 mM or less exerted an antibacterial effect against S. Typhimurium under the condition of 10 mM acetic acid addition (MIC ９０= 1.25 mM) (Fig. 3F and Fig. 3G). These results strongly suggested that the preventive effect of C. sporogenes wt strain and B. longum against lethal S. Typhimurium infection (lethality caused by S. Typhimurium infection) was due to the synergistic effect of PPA and SCFAs. This synergistic effect also strongly inhibited the growth of pathogenic bacteria belonging to the Proteobacteria phylum, such as Salmonella, Escherichia, Citrobacter, Klebsiella, and Proteus (Fig. 3G). On the other hand, Actinobacteria showed resistance to the combination of PPA and acetic acid (MIC ９０ ≥ 10 mM) (Fig. 3G).

[0166] Furthermore, a S. Typhimurium infection experiment similar to that described above was performed, except that B. thetaiotaomicron and L. plantarum were used instead of C. sporogenes and B. longum. First, GF mice were administered B. thetaiotaomicron (wt strain or PAL ko strain) and L. plantarum (10 ７ -10 ８ cfu / mouse). Then, 14 days after administration, B. longum was administered (10 ７ -10 ８ cfu / mouse). Then, 24 days after the administration of B. thetaiotaomicron and L. plantarum, S. Typhimurium was administered (10 4cfu / mouse). As a result, under both conditions using the B. thetaiotaomicron wt strain and the PAL ko strain, survival of all individuals was confirmed at least until day 2 after S. Typhimurium administration (Figure 3H). No significant difference was observed in the survival curves under these conditions. On the other hand, when the S. Typhimurium colonization load was evaluated one day after S. Typhimurium administration, it was suggested that the S. Typhimurium colonization load was significantly reduced when the wt strain, which is a PPA-producing condition, was administered (Figure 3I). These results suggest that PPA production by the relay between B. thetaiotaomicron and L. plantarum, can also have an inhibitory effect on S. Typhimurium infection.

[0167] Furthermore, the inventors attempted to analyze the mechanism of action of the bacteriostatic effect of the combination of PPA and acetic acid on S. Typhimurium. ９０ When cell division of S. Typhimurium was monitored for 120 minutes under conditions in which 2.5 mM PPA and 10 mM acetic acid, twice the normal concentration, were added (Fig. 3J, "PPA + acetic acid"), almost no cell division was observed, and the doubling time was six times longer than under conditions in which PPA and acetic acid were not added (Fig. 3J, "broth") (Fig. 3J).

[0168] These results demonstrate that when physiological concentrations of PPA produced by the wt strain of C. sporogenes coexist with acetic acid, the combination of PPA and acetic acid exhibits bacteriostatic activity against S. Typhimurium.

[0169] In addition, the culture supernatants of the SCFA-producing strains and the late logarithmic growth phase S. Typhimurium cultured cells (OD ６００The SCFAs were mixed with the SCFAs-producing strains (SEQ ID NO: 1) at a volume ratio of 4:1 and cultured anaerobically at 37°C for 24 hours. Table 1 shows the SCFA-producing strains and the SCFA concentrations in the culture supernatants of the strains. Figure 3K shows the OD of the reaction solution with or without PPA added at a concentration of 2.5 mM ("PPA(+)" in Figure 3K) or without PPA added ("PPA(-)" in Figure 3K). ６００ The addition of PPA was carried out after the culture supernatant of the strain producing the SCFAs was mixed with the cultured S. Typhimurium cells and before the initiation of the anaerobic culture.

[0170]

[0171] When the culture supernatant of a strain producing any of the SCFAs was added or when the culture supernatant was not added ("group without culture supernatant added" in Figure 3K), the OD ６００ The activity was low, and an inhibitory effect on the growth of S. Typhimurium was observed (Figure 3K). Furthermore, when the culture supernatant of a strain producing any of the SCFAs was added, it was suggested that the addition of any of the culture supernatants enhanced the bacteriostatic effect of PPA on S. Typhimurium compared to when the culture supernatant of a strain producing any of the SCFAs was not added. In other words, bacteriostatic activity against S. Typhimurium was confirmed even when bacteria producing SCFAs other than Bifidobacterium were used.

[0172] SCFAs have been reported to inhibit the growth of pathogenic bacteria by acidifying the intracellular environment. Therefore, we investigated the intracellular pH using a pH-sensitive GFP-expressing strain of S. Typhimurium. S. Typhimurium was cultured in the presence of 10 mM acetic acid and PPA or Phe, and the PPA concentration-dependent intracellular pH was evaluated (Figure 3L). As a result, in the absence of PPA, S. Typhimurium was able to neutralize the intracellular environment to a neutral pH. On the other hand, the pH buffering capacity of S. Typhimurium was reduced in the presence of 5 mM PPA.

[0173] These results suggest that PPA exerts its bacteriostatic effect by creating pores in the cell membrane and then lowering the intracellular pH through SCFAs. To support this idea, SEM observation of S. Typhimurium cell membranes in the presence of 5 mM PPA and 10 mM acetic acid revealed membrane collapse (Figure 3M). Meanwhile, human colon carcinoma-derived Caco2 cells were cultured for 24 hours in the presence of various concentrations of PPA (0, 2.5, or 5 mM) and acetic acid (5, 10, 20, or 30 mM), followed by an MTT assay. Cell viability (%) was calculated from the assay results. Figure 3N shows a graph of cell viability (%) for Caco2 cells cultured under each culture condition. In Figure 3N, "untreated" indicates the results for cells cultured in the absence of PPA and acetic acid. In Figure 3N, "1% triton" indicates the results for cells cultured in the absence of both PPA and acetic acid, with a triton concentration of 1%. These results confirmed that the addition of 5 mM PPA and 5 mM acetic acid resulted in significantly low cytotoxicity to human cells (Caco-2 cells). This suggests that the use of PPA and SCFAs may be applicable to the treatment or prevention of diseases caused by pathogenic bacteria or fungi.

[0174] Furthermore, the inventors investigated the bacteriostatic effect of the co-addition of PPA and various SCFAs on S. Typhimurium. First, LB medium was prepared by adding acetic acid, fumaric acid, propionic acid, lactic acid, or hydrochloric acid at 0, 2.5, 5, 10, 20, or 30 mM, respectively, and the pH of the medium was measured (Figure 3O). S. Typhimurium was cultured in each of these media for 25 hours, and the OD ６００ In addition, S. Typhimurium was cultured for 25 hours under the conditions where 2.5 mM PPA was additionally added to the above medium, and the OD ６００ were obtained (right side of Figure 3P and right side of Figure 3Q).

[0175] As shown in Figures 3P and 3Q, acetic acid, fumaric acid, propionic acid, and hydrochloric acid increased the MIC by co-addition with PPA. ９０These results suggest that PPA enhances the ability of SCFAs to inhibit the growth of S. Typhimurium.

[0176] Furthermore, the inventors observed changes in intracellular structures due to co-administration of PPA and acetic acid. Specifically, the intracellular structures of three representative strains of the Enterobacteriaceae family, S. Typhimurium, Escherichia coli ATCC BAA-2777™, and Klebsiella pneumoniae 2H7, were observed using a transmission electron microscope (TEM). More specifically, the following conditions were prepared: LB medium containing 5 mM PPA and 10 mM acetic acid ("5 mM PPA + 10 mM acetic acid" in Figures 3R-3T); LB medium containing 5 mM PPA alone ("5 mM PPA" in Figures 3R-3T); LB medium containing 10 mM acetic acid alone ("10 mM acetic acid" in Figures 3R-3T); and a control condition in which neither PPA nor acetic acid was added to LB medium ("Broth" in Figures 3R-3T). Each bacterium was cultured under each condition for 4 hours at 37°C with agitation. After culture, the microbial cells were gently washed and fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS). After fixation, the samples were treated with a 2% osmium tetroxide solution. The samples were then embedded in 1.5% (wt / vol) agarose gel and dehydrated with ethanol. After dehydration, the samples were embedded in epoxy resin and sectioned at 60 nm thickness (Leica Ultracut UCT ultramicrotome). The sectioned samples were observed using a Hitachi H-7650 TEM (Hitachi, Ltd., Tokyo, Japan).

[0177] As shown in Figures 3R to 3T, the cell membranes of these three bacteria belonging to the Enterobacteriaceae family were damaged by co-administration of PPA and acetic acid, and specifically, peeling of the inner and outer membranes was observed.

[0178] The composition and food / drink of this embodiment can promote the extension of enterobacterial pili, and the method of this embodiment can evaluate the possibility of contracting a disease caused by pathogenic bacteria or pathogenic fungi.

Claims

1. A composition for producing 3-phenylpropionic acid (PPA) or 3-(4-hydroxyphenyl)propionic acid (4OHPPA), characterized in that the composition comprises at least one bacterium, and the PPA or 4OHPPA is produced by the at least one bacterium.

2. The composition of claim 1, wherein the at least one bacterium has a nucleotide sequence encoding a protein having phenylalanine ammonia lyase (PAL) activity and a nucleotide sequence encoding a protein having hydroxycinnamate reductase B (HcrB) activity, wherein the protein having PAL activity has an amino acid sequence having 60% or more homology to the amino acid sequence set forth in SEQ ID NO:1, and the protein having HcrB activity has an amino acid sequence having 60% or more homology to the amino acid sequence set forth in SEQ ID NO:

2.

3. The composition according to claim 1 or 2, wherein the at least one bacterium has a nucleotide sequence encoding an aromatic amino acid lyase domain or a domain having a function equivalent to an aromatic amino acid lyase domain.

4. The composition of claim 1 or 2, wherein the at least one bacterium has a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain, or domains having a function equivalent to the flavin adenine dinucleotide-binding domain and the flavin mononucleotide-binding domain.

5. The composition of claim 4, wherein the at least one bacterium further comprises a nucleotide sequence encoding an NADPH-dependent flavin mononucleotide reductase domain or a domain having a function equivalent to an NADPH-dependent flavin mononucleotide reductase domain.

6. The composition of claim 1, wherein the composition comprises a plurality of bacteria, the plurality of bacteria being classified into two groups, group A and group B, wherein group A includes bacteria having a nucleotide sequence encoding a protein having phenylalanine ammonia lyase (PAL) activity, and group B includes bacteria having a nucleotide sequence encoding a protein having hydroxycinnamate reductase B (HcrB) activity, the protein having PAL activity having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:1, and the protein having HcrB activity having an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:

2.

7. The composition according to claim 6, wherein group A also includes bacteria having a nucleotide sequence encoding an aromatic amino acid lyase domain or a domain having a function equivalent to an aromatic amino acid lyase domain.

8. The composition according to claim 6, wherein group B also includes bacteria having a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain, or a domain having a function equivalent to a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain.

9. The composition according to claim 8, wherein the bacterium classified into group B and having a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain and a flavin mononucleotide-binding domain, or a domain having a function equivalent to the flavin adenine dinucleotide-binding domain and the flavin mononucleotide-binding domain, further has a nucleotide sequence encoding an NADPH-dependent flavin mononucleotide reductase domain, or a domain having a function equivalent to the NADPH-dependent flavin mononucleotide reductase domain.

10. The composition according to claim 7, wherein the bacteria classified into group A is at least one selected from the group consisting of Bacteroides intestinalis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides dorei, Blautia hansenii, and Bifidobacterium longum.

11. The composition according to claim 8, wherein the bacteria classified into group B is at least one selected from the group consisting of Blautia producta, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactiplantibacillus plantarum.

12. The composition of claim 1, further comprising a bacterium having a nucleotide sequence encoding a protein having the activity of acyl-CoA dehydrogenase (AcdA), a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase B (FldB), a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase C (FldC), and a nucleotide sequence encoding a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:3, the protein having the activity of FldB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:4, the protein having the activity of FldC has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:5, and the protein having the activity of FldH has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:

6.

13. The composition according to claim 12, wherein the bacterium having a nucleotide sequence encoding a protein having an activity of AcdA, a nucleotide sequence encoding a protein having an activity of FldB, a nucleotide sequence encoding a protein having an activity of FldC, and a nucleotide sequence encoding a protein having an activity of FldH is a bacterium selected from the group consisting of Clostridium sporogenes, Clostridium cadaveris, and Peptostreptococcus anaerobius.

14. The composition of any one of claims 1, 2, 6 and 12, further comprising an acid or a bacterium that produces said acid.

15. The composition according to claim 14, wherein the acid is at least one selected from the group consisting of short chain fatty acids and inorganic acids.

16. The composition according to claim 14, wherein the acid is at least one selected from the group consisting of acetic acid, fumaric acid, butyric acid, propionic acid, lactic acid and hydrochloric acid.

17. The composition according to claim 14, wherein the acid-producing bacteria is at least one selected from the group consisting of bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Bacteroides, bacteria of the genus Blautia, and bacteria of the genus Clostridium.

18. The composition according to any one of claims 1, 2, 6 and 12, which is a pharmaceutical composition.

19. The composition according to claim 18, which has the effect of promoting the extension of pili of enterobacteria.

20. The composition according to claim 19, wherein the enterobacteria have a sortase-dependent pilus gene.

21. The composition according to claim 19, wherein the intestinal bacteria is at least one selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Ruminococcus gnavus, Blautia hansenii, and Lactobacillus rhamnosus.

22. The composition according to claim 18, for use in treating a disease caused by a pathogenic bacterium.

23. The composition according to claim 22, wherein the pathogenic bacterium is a bacterium of the phylum Proteobacteria.

24. The composition according to claim 23, wherein the Proteobacteria bacterium is at least one selected from the group consisting of Salmonella enterica subsp. enterica serovar typhimurium, Escherichia coli, Citrobacter redentium, Klebsiella pneumoniae, and Proteus mirabilis.

25. The composition according to claim 22, wherein the disease caused by the pathogenic bacteria is at least one selected from the group consisting of bacterial food poisoning, typhoid fever, gastroenteritis, respiratory infections, urinary tract infections, bacterial vaginosis, sepsis, and meningoencephalitis.

26. The composition according to claim 18, for use in treating a disease caused by a pathogenic fungus.

27. The composition according to claim 26, wherein the pathogenic fungus is a fungus of the genus Saccharomycetales or Trichosporonaceae.

28. The composition according to claim 27, wherein the fungus of the genus Saccharomycetales is at least one selected from the group consisting of Candida albicans, Candida auris, Candida famata, and Pichia fermentans.

29. The composition according to claim 27, wherein the fungus of the genus Trichosporonaceae is Cryptococcus humicola.

30. The composition according to claim 26, wherein the disease caused by the pathogenic fungus is at least one selected from the group consisting of inflammatory bowel disease, oral candidiasis, vaginal candidiasis, pneumonia, and meningitis.

31. The composition according to claims 1, 2, 6 and 12, which is a food or beverage.

32. A composition containing 3-phenylpropionic acid or 3-(4-hydroxyphenyl)propionic acid.

33. The composition according to claim 32, which has the effect of promoting the extension of pili of enterobacteria.

34. The composition of claim 33, wherein the enterobacterium has a sortase-dependent pilus gene.

35. The composition of claim 33, wherein the intestinal bacteria is at least one selected from the group consisting of Ruminococcus gnavus, Blautia hansenii, and Lactobacillus rhamnosus.

36. The composition of claim 33, 34, or 35, which is a pharmaceutical product.

37. The composition according to claim 33, 34 or 35, which is a food or drink product.

38. A composition comprising a protein having phenylalanine ammonia-lyase (PAL) activity and a protein having hydroxycinnamate reductase B (HcrB) activity, wherein the protein having PAL activity has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:1, and the protein having HcrB activity has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:

2.

39. The composition of claim 38, further comprising a protein having the activity of acyl-CoA dehydrogenase (AcdA), a protein having the activity of phenyllactate dehydrogenase B (FldB), a protein having the activity of phenyllactate dehydrogenase C (FldC), and a protein having the activity of phenyllactate dehydrogenase H (FldH), wherein the protein having the activity of AcdA has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 3, the protein having the activity of FldB has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 4, the protein having the activity of FldC has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 5, and the protein having the activity of FldH has an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO:

6.

40. The composition according to claim 38 or 39, which is a pharmaceutical product.

41. The composition according to claim 38 or 39, which is a food or drink product.

42. A method for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or fungi, comprising: quantifying the amount of 3-phenylpropionic acid (PPA) or the amount of 3-(4-hydroxyphenyl)propionic acid (4OHPPA) in the stool of a subject using 2-nitrophenylhydrazine; comparing the value obtained by quantifying the amount of PPA or the amount of 4OHPPA with a standard value; and indicating the possibility that the subject is suffering from the disease if the value obtained by quantifying the amount of PPA or the amount of 4OHPPA is less than the standard value.

43. The method of claim 42, further comprising quantifying the amount of short chain fatty acids.

44. A method for assisting in the testing and diagnosis of diseases caused by pathogenic bacteria or fungi, comprising: quantifying the expression level of the phenylalanine ammonia-lyase (PAL) gene or the expression level of the hydroxycinnamate reductase B (HcrB) gene in the stool of a subject; comparing the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene with a standard value; and indicating the possibility that the subject is suffering from the disease if the value obtained by quantifying the expression level of the PAL gene or the expression level of the HcrB gene is less than the standard value.

45. The method of claim 44, further comprising quantifying the expression level of at least one gene selected from the group consisting of an acyl-CoA dehydrogenase gene, a phenyllactate dehydrogenase B gene, a phenyllactate dehydrogenase C gene, and a phenyllactate dehydrogenase H gene.

46. ​​A method for assisting in the testing and diagnosis of a disease caused by pathogenic bacteria or fungi, comprising: quantifying the expression level of at least one gene selected from the group consisting of a gene comprising a nucleotide sequence encoding an aromatic amino acid lyase domain, a gene comprising a nucleotide sequence encoding a flavin adenine dinucleotide-binding domain, and a gene comprising a nucleotide sequence encoding a flavin mononucleotide-binding domain in the feces of a subject; comparing the value obtained by quantifying the expression level of the gene with a standard value; and indicating the possibility that the subject is suffering from the disease when the value obtained by quantifying the expression level of the gene is less than the standard value.