# Patent

## Title
Production of enteritis evoking causal factor by highly pathogenic oral bacteria and high sensitivity detection method therefor

## Abstract

Disclosed is the construction of a system capable of quickly and easily identifying patients having a risk for worsening inflammatory digestive tract disease by identifying a causal factor for the worsening of the inflammatory digestive tract disease. Specifically disclosed is a method for detecting oral bacteria that cause worsening of inflammatory digestive tract disease and/or a screening that targets high risk for worsening of inflammatory digestive tract disease and/or determining the risk for worsening of the inflammatory digestive tract disease by PA not being detected and/or CBP being detected, inclusive of the detection of PA, which is an oral bacteria protein antigen, and/or CBP, which is collagen binding protein, in samples. Also disclosed are a detection reagent and kit used for this method.

## Background
RELATED APPLICATION
This application is a national stage filing under 35 U.S.C. §371 of international application PCT/JP2011/055688, filed Mar. 10, 2011, the disclosure of which is incorporated by reference herein in its entirety.
TECHNICAL FIELD
The present invention relates to a method of detecting an oral bacterium that aggravates inflammatory digestive tract disorders, in particular inflammatory bowel disease (IBD).
BACKGROUND ART
Inflammatory bower disease (IBD) is a chronic and intractable inflammatory disease of the intestine, and is principally classified into ulcerative colitis and Crohn's disease. In Japan, the number of patients with IBD in 2003 (the number of certified patients, because IBD is designated to be an intractable disease) is approximately 80 thousands patients with ulcerative colitis and approximately 20 thousands patients with Crohn's disease. However, the number of patients is steadily increasing due to Westernized dietary habit; the number of patients in 2003 is 20 times that in 1980 (the number of patients was approximately 5 thousands). In the future, it is expected that the number of young patients increases as well due to further Westernized dietary habit, and there is no doubt about a further increase in the number of patients.
It is indicated that IBD is caused by genetic predisposition, high fat and high protein diet, abnormality in the immune system, and intestinal bacteria, etc., but risk factors of the onset of IBD have not yet been clarified. However, abnormality in the immune system is considered to play an important role in the disease onset.
There is no established therapeutic method for IBD, and diet and rest are applied as general therapy, and steroids and immunosuppressive agents are administered as drug therapy; in addition, antibacterial agents such as Sarazopirin and mesalazine as well as antibiotics may have effects in some cases. Based on these, involvement of intestinal bacteria is indicated, but to date there has been no convincing evidence that a specific intestinal bacterium leads to the onset of disease symptoms.
In recent years, an oral bacterium as a major pathogenic bacterium of caries, i.e., Streptococcus mutans (S. mutans) as a kind of Mutans streptococci, is known to be a causative organism of bacteremia and infective endocarditis. In addition, since bacterial DNA of S. mutans was detected from specimens of heart valve and aortic aneurysm, its association with circulatory diseases has been reported (Non-patent Literature 1). However, there has been no report on the involvement of oral bacteria in inflammation of the digestive tract such as inflammatory bowel disease.
CITATION LIST
Non-Patent Literature


    Non-patent Literature 1: Nakano et al., 2008, Japanese Dental Science Review, 44: 29-37.


SUMMARY OF INVENTION
Problems to be Solved by the Invention
An object of the present invention is to provide a system to identify responsible factors that aggravate inflammatory digestive tract disorders, and to rapidly and readily specify a patient having a risk of aggravation of inflammatory digestive tract disorders.
Means for Solving the Problems
The inventors have already found that hemorrhage is aggravated in a subject who has been infected with a particular strain of Streptococcus mutans (S. mutans), and that the most severe virulence of hemorrhage aggravation is exerted by bacterial strains that do not carry a protein antigen (PA, also known as PAC, SpaP, antigen I/II, antigen B, SR, IF, P1, MSL-1), i.e., a major bacterial surface protein having a molecular weight of about 190 kDa, and that carry a collagen binding protein (CBP, also known as Cnm) having a molecular weight of about 120 kDa; the inventors have filed a patent application of an invention on the basis of these findings (JP A 2009-88239).
Streptococcus mutans is known to have four serotypes (c, e, f and k). The inventors revealed that intravenous administration of some of different S. mutans strains inhibits spontaneous hemostatic action and induces aggravation of hemorrhage, when mild cerebral hemorrhage has been induced by damaging the middle cerebral artery. Of the S. mutans strains, generally c-serotype strains are isolated from the oral cavity, and their standard strain MT8148 (serotype c) (Minami et al., 1990, Oral Microbiol. Immunol., 5: 189-194) does not cause such effects; however, there are strains among k-serotype strains that evoke aggravation of inflammatory digestive tract disorders. In particular, TW295 strain and TW871 strain (Nakano et al., 2004, Journal of Clinical Microbiology, 42(1): 198-202), and SA53 strain (Nakano et al., 2007, J. Clin. Microbiol., 45: 2614-2625) cause a significant hemorrhage aggravation.
The inventors have found that these highly virulent S. mutans strains lack PA, a major bacterial surface protein, and that among PA-deleted strains, the virulence of the strains carrying CBP, another bacterial surface protein, is particularly high. Moreover, the inventors have confirmed that when CBP-encoding gene of TW295 strain has been deleted by genetic engineering, hemorrhage aggravation similar to that caused by TW295 strain is not observed, and that when PA-encoding gene of MT8148 strain has been deleted, hemorrhage aggravation is observed; thus the inventors have confirmed that CBP and PA are involved in hemorrhage aggravation of S. mutans. 
Recently, the inventors have additionally recognized that abnormal reddening occurs in the intestinal tract of a mouse administered with said bacterial strain that causes hemorrhage aggravation, and found a significant association between oral bacteria and inflammatory bowel disease (IBD); after further promotion of the research, the inventors accomplished the present invention.
Namely, the present invention relates to a method of detecting an oral bacterium that aggravates inflammatory digestive tract disorder, comprising detecting PA and/or CBP of oral bacteria in a sample, wherein the presence of the oral bacterium that aggravates inflammatory digestive tract disorder is determined when PA is not detected and/or CBP is detected.
Furthermore, the present invention relates to a method of screening a subject at a high risk of aggravation of inflammatory digestive tract disorder, comprising detecting PA and/or CBP of oral bacteria in a biological sample obtained from a subject, wherein a high risk of aggravation of inflammatory digestive tract disorder is determined when PA is not detected and/or CBP is detected.
In addition, the present invention relates to a method of judging the risk of aggravation of inflammatory digestive tract disorder in a subject, comprising detecting PA and/or CBP of oral bacteria in a biological sample obtained from a subject, wherein a high risk of aggravation of inflammatory digestive tract disorder in the subject is determined when PA is not detected and/or CBP is detected.
Furthermore, the present invention relates to the method wherein the inflammatory digestive tract disorder is inflammatory bowel disease.
In addition, the present invention relates to the method wherein the oral bacterium is Streptococcus mutans. 
Furthermore, the present invention relates to the method wherein the genotype of Streptococcus mutans is cnm(+).
In addition, the present invention relates to the method wherein the serotype of Streptococcus mutans is f-type or k-type.
Furthermore, the present invention relates to the method wherein the PA is selected from the group consisting of: (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23; (2) a polypeptide comprising one or more mutations in the polypeptide of (1), but having an equal function to the polypeptide of (1); (3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 2, 18, 20, 22 or 24 or its complementary sequence or its fragment under stringent conditions, and having an equal function to the polypeptide of (1); (4) a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23, and having an equal function to the polypeptide of (1).
In addition, the present invention relates to the method wherein the PA comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23.
Furthermore, the present invention relates to the method wherein the CBP is selected from the group consisting of: (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9; (2) a polypeptide comprising one or more mutations in the polypeptide of (1), but having an equal function to the polypeptide of (1); (3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 6 or 10 or its complementary sequence or its fragment under stringent conditions, and having an equal function to the polypeptide of (1); (4) a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 5 or 9, and having an equal function to the polypeptide of (1).
In addition, the present invention relates to the method wherein the CBP comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or 9.
Furthermore, the present invention relates to a reagent for detection of an oral bacterium that aggravates inflammatory digestive tract disorder, comprising an oral bacterial PA-detecting agent and/or CBP-detecting agent.
In addition, the present invention relates to an oral bacterial PA-specific antibody for detection of an oral bacterium that aggravates inflammatory digestive tract disorder.
Furthermore, the present invention relates to a kit for detecting an oral bacterium that aggravates inflammatory digestive tract disorder and/or for screening a subject at a high risk of aggravation of inflammatory digestive tract disorder and/or for determining the risk of aggravation of inflammatory digestive tract disorder in a subject, comprising at least a PA-detecting reagent, and a CBP-detecting reagent.
Advantageous Effects of Invention
The present invention enables rapid and simple diagnosis of the risk of causing aggravation of inflammatory digestive tract disorder in an individual. Also, the method of the present invention enables detection of responsible factors of aggravation of inflammatory digestive tract disorder using readily-available biological samples such as saliva and plaque, without employing any special analyzers. As such, the present invention allows to specify a high-risk population of aggravation of inflammatory digestive tract disorder, and to treat the individuals belonging to this population with a regimen such as removing virulent bacteria and advising dental hygiene, thereby effectively preventing aggravation of inflammatory digestive tract disorder.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a flow-chart of a system to detect a S. mutans strain which may cause aggravation of inflammatory digestive tract disorders.
FIG. 2 is a scheme of methods of culturing and detecting S. mutans. 
FIG. 3 is a scheme of methods of detecting PA-deleted S. mutans and CBP-carrying S. mutans. 
FIG. 4 is a graph showing DAI scores of the control group, TW295 administration group, and MT8148 administration group on day 10 following administration.
FIG. 5 shows photographs of typical examples of individual mouse (top) and the large intestine (bottom) in the control group and TW295 administration group on day 11-15 following administration.
FIG. 6 is a graph showing mortality rate of the control group, TW295 administration group, and MT8148 administration group on day 15 following administration.
FIG. 7 is a graph showing mortality rate on day 15 following administration when the amount of TW295 administration was changed.
FIG. 8 is a graph showing mortality rate of the control group, TW295 administration group, MT8148 administration group and CND administration group on day 15 following administration.
FIG. 9 is an electrophoretogram of PCR products showing presence/absence of highly virulent Streptococcus mutans in the small intestine, large intestine, liver and lung.
FIG. 10 shows photographs of typical examples of the results of seeding homogenize of lung and liver onto MSB plate.
FIG. 11 shows photographs of observation of localization in the tissue of a Streptococcus mutans TW295-GFP strain that expresses GFP proteins.
FIG. 12 is a conceptual diagram showing mechanism of aggravating inflammatory digestive tract disorders by highly virulent Streptococcus mutans. 
FIG. 13 shows an example of analytical results determining presence of PA-deleted S. mutans and CBP-carrying S. mutans. 
FIG. 14 is a graph showing results of investigation of optimum conditions for culturing S. mutans (culturing in an aerobic/anaerobic condition, concentration of antibiotics, nutrient concentrations).
FIG. 15 is a graph showing results of investigation of storage period of usable saliva for detecting virulent S. mutans. 
FIG. 16 is a graph showing results of distribution of genotype of Streptococcus mutans (cnm (+) and cnm (−)) in saliva samples.
FIG. 17 is a graph showing results of distribution of serotype of Streptococcus mutans (c-type, e-type, f-type and k-type) in saliva samples.
FIG. 18 is a graph showing changes in phagocytosis action by leucocytes depending on the serotype of Streptococcus mutans. 
FIG. 19 is a graph showing the ratio of strains of Streptococcus mutans in which the genotype is cnm (+) and the serotype is f-type or k-type.



DESCRIPTION OF EMBODIMENTS
The present invention provides a method of detecting an oral bacterium that causes aggravation of inflammatory digestive tract disorder, the method comprising detecting PA and/or CBP of oral bacteria in a sample, wherein the presence of the oral bacterium that causes aggravation of inflammatory digestive tract disorder is determined by nondetection of PA and/or detection of CBP.
The present invention provides, in another embodiment, a method of screening a subject at a high risk of aggravation of inflammatory digestive tract disorder, the method comprising detecting PA and/or CBP of oral bacteria in a biological sample obtained from a subject, wherein a high risk of aggravation of inflammatory digestive tract disorder is determined by nondetection of PA and/or detection of CBP.
The present invention further provides, in another embodiment, a method of determining the risk of aggravation of inflammatory digestive tract disorder in a subject, the method comprising detecting PA and/or CBP of oral bacteria in a biological sample obtained from a subject, wherein a high risk of aggravation of inflammatory digestive tract disorder is determined in the subject by nondetection of PA and/or detection of CBP.
Inflammatory digestive tract disorders referred to in this specification intend to include inflammatory diseases in any digestive tract and digestive gland and their associated organs. More specifically, they include inflammatory disease of the digestive tract such as oral cavity, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, appendix, colon (ascending colon, transverse colon, descending colon, sigmoid colon), rectum) and anus, as well as salivary glands that secrete saliva, pancreas that secretes pancreatic juice, and liver and gallbladder that secrete bile. Their typical example includes inflammatory bowel disease (IBD).
PA (protein antigen) is a surface protein of approximately 190 kDa found in MT8148 strain, a S. mutans wild-type strain, and also known in various other names such as PAc (protein antigen c), SpaP, antigen I/II and antigen B, P1 and MSL-1. PA polypeptide comprises 3 alanine-rich repeat domains (A-region) at N-terminal side and 3 proline-rich repeat domains (P-region) at central part, and has cell wall/membrane-spanning domain at C-terminal. It has been reported that A-regions are involved in the attachment of bacterial cells to the teeth (Matsumoto-Nakano et al., 2008, Oral Microbiology and Immunology, 23:265-270). Also, there have been reports that PA is involved in infective endocarditis by S. mutans (Nakano et al., 2008, Japanese Dental Science Review, 44: 29-37); that an antibody against PA inhibits the attachment of bacterial cells to a hydroxyapatite substrate (Kawato et al., 2008, Oral Microbiology and Immunology, 23:14-20); and that an antiserum against PA is useful as a vaccine for dental caries (Okahashi et al., 1989, Molecular Microbiology, 3(2): 221-228). Although there is a region between A-region and P-region of PA, in which amino acid sequences are highly variable among strains (for example, in MT8148 strain, residues from 679 to 827), the repeat domain and membrane-spanning domain are highly conserved among strains.
Also, it is reported that a high percentage of strains of serotype k, which are often detected in patients with infective endocarditis, lack PA, and that both the hydrophobicity of the bacterial cell and sensitivity to phagocytosis are low in this serotype (Nakano et al., 2008, Journal of Dental Research, 87(10): 964-968).
Known PA includes, for example, PA of serotype c MT8148 (DDBJ Accession No.: X14490, amino acids: SEQ ID NO: 1, nucleic acids: SEQ ID NO: 2), PA of LJ23 strain (DDBJ Accession No.: AB364261, amino acids: SEQ ID NO: 17, nucleic acids: SEQ ID NO: 18), PA of SA98 strain (DDBJ Accession No.: AB364285, amino acids: SEQ ID NO: 19, nucleic acids: SEQ ID NO: 20), as well as spaP gene of antigen I/II (DDBJ Accession No.: X17390, Kelly et al., 1989, FEBS Lett. 258(1), 127-132, amino acids: SEQ ID NO: 21, nucleic acids: SEQ ID NO: 22) and a meningococcus Neisseria meningitidis iron binding protein fbp gene (X53469, Berish et al., 1990, Nucleic Acid Research, 18(15): 4596-4596, amino acids: SEQ ID NO: 23, nucleic acids: SEQ ID NO: 24).
CBP, i.e., another anchor protein of S. mutans (also denoted as Cnm), is a Type I collagen binding protein of approximately 120 kDa molecular weight, and has a collagen binding domain (CBD, residues from 152 to 316), B repeat domain (residues from 328 to 455) and LPXTG motif (residues from 507 to 511) (Sato et al., 2004, Journal of Dental Research, 83(7): 534-539). CBP gene-carrying frequency of S. mutans present in the oral cavity is about 10 to 20%, and CBP-positive strain is predominantly detected in serotypes f and k (Nakano et al., 2007, J. Clin. Microbiol., 45: 2616-2625).
A study by the inventors revealed that, for CBP of serotype k TW295 strain (DDBJ Accession No.: AB102689, amino acids: SEQ ID NO: 3, nucleic acids: SEQ ID NO: 4), CBD (amino acids: SEQ ID NO: 5, nucleic acids: SEQ ID NO: 6) and LPXTG motif are highly conserved among strains, whereas the number of repeats in the B repeat domain varies among strains.
In one embodiment of the present invention, PA is defined as:

    (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23;
    (2) a polypeptide comprising one or more, preferably 1 to 20, 1 to 15, 1 to 10, or one or several mutations in the polypeptide of (1), but having an equal function to the polypeptide of (1);
    (3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 2, 18, 20, 22 or 24 or its complementary sequence or its fragment, and having an equal function to the polypeptide of (1); or
    (4) a polypeptide comprising an amino acid sequence having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology to the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23, and having an equal function to the polypeptide of (1).


Preferably, PA comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23. More preferably, PA comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
PA that can be used in the method of the present invention may be a polypeptide comprising one or more amino acid mutations (deletion, substitution, addition), as long as it comprises an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 2, 18, 20, 22 or 24 (nucleic acid sequence that encodes PA protein) or its complementary sequence or its fragment, and has an equal function to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23 (amino acid sequence of PA protein). Mutations may be naturally occurring mutations or mutations generated by any known procedures, e.g., cleavage or insertion of a nucleic acid by restriction enzyme, site-specific mutagenesis, or radiation or ultraviolet irradiation. Moreover, the number of mutated amino acids may be 1 to 20, 1 to 15, 1 to 10, or 1 to several, for example.
Furthermore, in one embodiment of the present invention, CBP is defined as:

    (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9;
    (2) a polypeptide comprising one or more, preferably 1 to 20, 1 to 15, 1 to 10, or one or several mutations in the polypeptide of (1), but having an equal function to the polypeptide of (1);
    (3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 6 or 10 or its complementary sequence or its fragment, and having an equal function to the polypeptide of (1); or
    (4) a polypeptide comprising an amino acid sequence having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology with the amino acid sequence of SEQ ID NO: 5 or 9, and having an equal function to the polypeptide of (1).


CBP polypeptide that can be used in the method of the present invention may be a polypeptide comprising one or more, e.g., 1 to 20, 1 to 15, 1 to 10, or one or several amino acid mutations (deletion, substitution, addition), as long as it comprises an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 6 or 10 (nucleic acid sequence encoding CBD of S. mutans TW295 strain or TW871 strain) or its complementary sequence or its fragment, and has an equal function to a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9 (CBD amino acid sequence of S. mutans TW295 strain or TW871 strain).
For instance, CBP polypeptide may be a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 4 or 8 (a nucleic acid sequence encoding CBP of S. mutans TW295 strain or TW871 strain) or its complementary sequence or its fragment, and has an equal function to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 7 (an amino acid sequence of CBP protein of S. mutans TW295 strain or TW871 strain).
Preferably, CBP comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or 9.
Whether or not a PA mutant or CBP mutant has a function equal to that of PA or CBP may be confirmed using any known means. For instance, the ability of PA mutant making the bacterial cell adhere to a hydroxyapatite substrate may be determined by raising a specific antibody against the mutant peptide by a known method, and assaying the inhibition of adhesion of bacteria to the hydroxyapatite by said antibody in accordance with a method described in Kawato et al., 2008, Oral Microbiology and Immunology, 23:14-20. Alternatively, the binding ability of a CBP mutant to Type I collagen may be determined by collagen binding assay described in Nomura et al., 2009, J. Med. Microbiol., 58(4): 469-475. By such means, the ability of a mutant can be assessed in comparison with an appropriate negative control, or with PA or CBP as a positive control. For instance, a certain mutant is considered as a functional mutant when at least one function described above is better, e.g., 10% or more, 25% or more, 50% or more, 75% or more, or even 100% or more, than that of the negative control, and/or when said function is 1/100 or less, 1/50 or less, 1/25 or less, 1/10 or less, 1/5 or less, or even 1/2 or less, than that of the positive control.
Major bacterial species identified as oral bacteria that cause aggravation of inflammatory digestive tract disorders include mutans streptococci such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, Streptococcus downei; as well as Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, and Streptococcus salivarius. Particularly, S. mutans TW295 strain and TW871 strain would cause severe aggravation of inflammatory digestive tract disorders.
Screening of other bacteria that could induce aggravation of inflammatory digestive tract disorders can be carried out utilizing databases such as NCBI GenBank®, DDBJ (DNA Data Bank of Japan, http://www.ddbj.nig.ac.jp/) and EMBL, and publicly available search tools such as BLAST.
The present invention provides, in one embodiment, a reagent for detection of an oral bacterium that causes aggravation of inflammatory digestive tract disorder, comprising an oral bacterial PA-detecting agent and/or an oral bacterial CBP-detecting agent.
In one embodiment, the PA-detecting agent comprises an oral bacterial PA-specific antibody. Using the PA-specific antibody developed by the inventors, presence or absence of highly virulent S. mutans can be rapidly and easily detected. The PA-specific antibody is preferably an antibody or its fragment induced from a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or its immunogenic fragment. Alternatively, the PA-specific antibody may be an antibody or its fragment induced from a polypeptide having at least 80% homology with the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23, and having an immunogenicity to induce antibody production against a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23. For example, a recombinant PA comprising the above polypeptide (see, e.g., Nakano et al., 2006, Microbes and Infection, 8:114-121) may be used as an antigen to produce a monoclonal or polyclonal antibody.
In one embodiment, the CBP detecting agent comprises a substrate (such as a microplate, test tube or slide glass) coated with Type I collagen. The binding ability of CBP to Type I collagen (Nomura et al., 2009, J. Med. Microbiol., 58 (4): 469-475) can be utilized to allow CBP-expressing bacterial cell to attach the substrate coated with Type I collagen, which can easily be detected.
In another embodiment, the CBP-detecting agent comprises a specific antibody against an oral bacterial CBP. The CBP-specific antibody may be a specific antibody against the collagen binding domain of CBP, preferably, an antibody or its fragment induced from a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9, or its immunogenic fragment. Alternatively, the CBP-specific antibody may be an antibody or its fragment induced from a polypeptide having at least 80% homology with the amino acid sequence of SEQ ID NO: 5 or 9, and having an immunogenicity to induce an antibody production against a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9.
In the present invention, the antibody fragment comprises, for example, without limitation, various functional fragments such as Fab, Fab′, F(ab′)2, scFv, dsFv (disulfide-stabilized V region fragment), and CDR-containing fragment.
The present invention provides, in one embodiment, a kit for detecting an oral bacterium that causes aggravation of inflammatory digestive tract disorder, and/or for screening a subject at a high risk of aggravation of inflammatory digestive tract disorder, and/or for the determination of the risk of aggravation of inflammatory digestive tract disorder in a subject. The kit comprises at least a PA-detecting reagent and a CBP-detecting reagent.
In one embodiment, the kit comprises as a PA-detecting reagent an oral bacterial PA-specific antibody.
In one embodiment, the kit comprises as a CBP-detecting reagent a substrate coated with Type I collagen (such as a microplate, test tube or slide glass).
In another embodiment, the kit comprises as a CBP-detecting reagent a CBP-specific antibody.
The kit of the present invention may further comprise one or more of the followings for culturing S. mutans: 

    
    
        An instrument for collecting saliva such as a spitz for collecting saliva (the material and shape is not particularly limited as long as it is sterilized and suitable for collecting and seeding).
        A collecting instrument such as a dropper capable of collecting saliva of approximately 10 μl.
        S. mutans selection medium (Special Medium A). For example, sterile substrate coated with MSB agar medium (Mitis-salivariusagar medium (e.g., Difco Laboratories) supplemented with an antibiotic (e.g., bacitracin (e.g., SIGMA-ALDRICH)) and sucrose (e.g., Wako Pure Chemical Industries, Ltd.)). The substrate is not particularly limited as long as it is such as a dish or well plate, though typically roughly a 24-well plate (e.g., 24 well with Lid MICROPLATE (IWAKI)) is used. Bacitracin is preferably used at about 100 unit/ml. Sucrose is preferably used at about 15%.
        A sealing and/or deoxygenating instrument for culturing under an anaerobic condition, such as Anaero Pack® or a CO2 chamber.
        A sterile stick for picking up bacterial colonies (such as a toothpick or tip).
        A liquid medium for culturing the picked-up colonies (Special Medium B). For example, sterilized Brain Heart Infusion (BHI) liquid medium (Difco Laboratories) contained in a disposable test tube.
    
    


The kit of the present invention may further comprise one or more of the followings for detecting S. mutans: 

    
    
        A collecting instrument suitable for collecting bacterial solution of approximately 10 μl such as a dropper.
        A special medium for detecting S. mutans (Special Medium C). For example, a medium in which 100 μl of a sucrose (Wako Pure Chemical Industries, Ltd.)-containing BHI solution is added to a substrate. The substrate is not particularly limited as long as it is such as a well plate or test tube, though typically a 96-well plate (e.g., MULTI WELL PLATE for ELISA (SUMIRON)) is used. Sucrose is used at about 1%.
        A wash buffer (Wash Buffer A: PBS solution or sterile water may be used, though preferably PBS solution is used.)
        A Gram-positive bacteria detecting reagent (Buffer 1: for example, a solution in which about 0.5% crystal violet (e.g., Wako Pure Chemical Industries, Ltd.) as a Gram-positive bacteria detecting reagent is added to sterile distilled water.)
        A mordanting reagent (Buffer 2: a suitable mordanting reagent may be selected depending on the bacteria detecting reagent. For example, 7% acetate (e.g., Wako Pure Chemical Industries, Ltd.) solution or sterile water may be used for the crystal violet, though preferably acetate solution is used.)
    
    


The kit of the present invention may further comprise one or more of the followings for detecting PA-deleted S. mutans: 

    
    
        A plate for detecting PA-deleted S. mutans. It is not particularly limited as long as it is a sterile well plate, though typically a 96-well plate (e.g., MICROTEST U-Bottom (BECTON DICKINSON)) is used.
        A wash buffer (Wash Buffer B: a solution in which about 0.05% of a surfactant such as Triton X-100 (e.g., Wako Pure Chemical Industries, Ltd.) is added to PBS solution or sterile water. Preferably PBS solution is used.)
        A buffer (Buffer 3: a mixture of Tris buffered saline (pH 6.8), 100 mM dithiothreitol (e.g., Wako Pure Chemical Industries, Ltd.) and 20% glycerin (e.g., Wako Pure Chemical Industries, Ltd.).)
        A blocking solution (Buffer 4: a PBST solution containing approximately 5% of skimmed milk (e.g., BECTON DICKINSON).)
        A primary antibody (Buffer 5: a PBST solution containing approximately 0.1% of anti-PA antiserum.)
        A secondary antibody (Buffer 6: a PBST solution containing approximately 0.1% of an antibody against immunoglobulin of the primary antibody of the host (e.g., Dakopatts).)
        A color-developing reagent (Buffer 7: AP (100 mM 2-amino-2-hydroxymethyl-1,3-propanediol, 5 mM magnesium chloride, 100 mM sodium chloride) buffer supplemented with NBT solution (Wako Pure Chemical Industries, Ltd.) at final concentration of 0.6% and BCIP solution (Wako Pure Chemical Industries, Ltd.) at final concentration of 0.33%.)
    
    


The kit of the present invention may further comprise one or more of the followings for detecting CBP-carrying S. mutans: 

    
    
        A special medium for detecting CBP-carrying S. mutans (Special Medium D: a mixed solution of 0.6% acetate-containing sterile distilled water and Type I collagen (Sigma) in 9:1 ratio contained in the Special Plate used in Analysis 3.)
        A wash buffer (Wash Buffer A: PBS solution or sterile water may be used, though preferably PBS solution is used.)
        A buffer (Buffer 8: Wash Buffer A containing approximately 5% bovine albumin (Sigma).)
        A wash buffer (Wash Buffer C: PBS solution or sterile water containing a surfactant such as approximately 0.01% Tween 20 (Wako Pure Chemical Industries, Ltd.). Preferably, PBS solution is used.)
        A fixative solution (Buffer 9: for example, sterile distilled water containing approximately 25% formaldehyde (Wako Pure Chemical Industries, Ltd.).)
        A Gram-positive bacteria detecting reagent (e.g., above Buffer 1: a solution in which approximately 0.5% crystal violet (Wako Pure Chemical Industries, Ltd.) as a Gram-positive bacteria detecting reagent is added to sterile distilled water.)
        A mordanting reagent (e.g., above Buffer 2: 7% acetate (e.g., Wako Pure Chemical Industries, Ltd.) solution or sterile water may be used, though preferably acetate solution is used.)
    
    


A skilled person in the art may appropriately adjust the concentration of the above-mentioned components, e.g., antiserum, secondary antibody, formaldehyde or crystal violet, to be optimum depending on experimental conditions.
The method of the present invention for detecting an oral bacterium that causes aggravation of inflammatory digestive tract disorder is carried out, specifically, in a scheme comprising the following four steps, for example as shown in FIGS. 1 to 3:
Analysis 1. Culturing of S. mutans 
Analysis 2. Detection of S. mutans 
Analysis 3. Detection of PA-deleted S. mutans 
Analysis 4. Detection of CBP-carrying S. mutans 
In Analysis 1, culturing of bacteria is carried out by the following procedures using for example instruments and reagents in the aforementioned kit for culturing mutans streptococci.
The saliva of a subject is collected in a small amount using a spitz for collecting saliva. 10 μl of the saliva is taken from the spitz using a dropper, plated on a S. mutans selection agar medium (e.g., the above-mentioned Special Medium A), and cultured at 37° C. for 48 hours, preferably under an anaerobic condition. After culturing, the presence of bacterial colonies is grossly confirmed, the colonies are picked up and added to a liquid medium (e.g., the above-mentioned Special Medium B) and cultured at 37° C. for 18 hours, then used for the following Analyses 2, 3 and 4. Preferably, rough colonies are picked up, since S. mutans forms rough colonies, whereas S. sobrinus forms smooth colonies.
In Analysis 2, detection of S. mutans is carried out by the following procedures using for example instruments and reagents in the aforementioned kit for detecting S. mutans. 
10 μl of the bacterial solution cultured from the method of Analysis 1 is added to a medium (e.g., the above-mentioned Special Medium C), and incubated at 37° C. for 3 hours. The medium is washed with a wash buffer (e.g., the above Wash Buffer A) for three times, then left still about 15 minutes with the last wash buffer. The wash buffer is removed, and again the medium is washed with Wash Buffer A for once, then a buffer containing a Gram-positive bacteria staining reagent (e.g., above Buffer 1) is added and left still for 1 minute. It is washed with the wash buffer for three times, and a buffer containing a mordanting agent (e.g., above Buffer 2) is added. If the color of the medium changes, it is determined to be S. mutans-positive, if the color of the medium does not change, it is determined to be S. mutans-negative. A reagent in which a staining reagent and a mordanting agent are already combined may also be used.
In Analysis 3, detection of PA-deleted S. mutans is carried out by the following procedures using for example instruments and reagents in the aforementioned kit for detecting PA-deleted S. mutans. 
(1) Sample Preparation
To the bacterial solution cultured by the method of Analysis 1 above a suitable buffer (e.g., the above-mentioned Buffer 3) is added, which is then immersed in boiling water for 10 minutes, and frozen if it is to be stored.
(2) Detection of PA-Deleted S. mutans 


    1) The sample produced from (1) above is added to a plate, left still overnight at 4° C.
    2) The plate is washed three times with a wash buffer (e.g., above Wash Buffer B), then skimmed milk (e.g., above Buffer 4) is added thereto, and left still at room temperature for 1 hour.
    3) The plate is washed three times with the wash buffer, then a primary antibody (e.g., above Buffer 5) is added, and reacted at room temperature for 1 hour.
    4) The plate is washed three times with the wash buffer, then a labeled secondary antibody (e.g., above Buffer 6) is added, and reacted at room temperature for 1 hour.
    5) The plate is washed three times with the wash buffer, then a color-developing reagent (e.g., above Buffer 7) is added, and after appropriate time period, changes in the color of the solution are observed. When the color of the solution changes, it is determined to be PA-positive, and when the color of the solution does not change, it is determined to be PA-negative.


In Analysis 4, detection of CBP-carrying S. mutans is carried out by the following procedures using for example instruments and reagents in the aforementioned kit for detecting CBP-carrying S. mutans. 

    (1) The medium (e.g., above Special Medium D) is washed three times with a wash buffer (e.g., above Wash Buffer A), then albumin-containing buffer (e.g., above Buffer 8) is added, and left still at 37° C. for 1 hour.
    (2) After washing three times with a wash buffer containing a surfactant (e.g., above Wash Buffer C), bacterial solution cultured by the method of Analysis 1 above is added, and incubated at 37° C. for 2 hours.
    (3) After washing three times with the wash buffer (e.g., above Wash Buffer A), a fixative solution (e.g., above Buffer 9) is added and left still at room temperature for 30 minutes.
    (4) After washing three times with the wash buffer, a Gram-positive bacteria staining reagent (e.g., above Buffer 1) is added and left still for 1 minute.
    (5) After washing three times with Wash Buffer A, a mordanting agent (e.g., above Buffer 2) is added.


When the color of the solution changes, it is determined to be CBP-positive, and when the color of the solution does not change, it is determined to be CBP-negative.
In any of the detecting methods described above, detection is possible when bacterial concentration is 1 CFU or more.
Moreover, a culture of e.g., S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius may be used as a control to confirm in Analysis 1 that any bacterium other than S. mutans and S. sobrinus does not grow; in Analysis 3 that any bacterium other than PA-carrying S. mutans does not show a positive reaction; and in Analysis 4 that any bacterium other than CBP-carrying S. mutans does not show a positive reaction, respectively.
A skilled person in the art may appropriately modify the method of the present invention according to its object. For example, for detecting PA-deleted S. mutans, a substrate to which a specific antibody against PA or CBP is attached may be contacted with a bacterial solution, washed to remove the bacteria that do not attach to the substrate, then only the bacteria that attached to the substrate can be detected by the Gram-positive bacteria staining reagent. Alternatively, primers or probes for a PA or CBP-coding nucleic acid may be used to detect whether the cultured bacterium has the gene of PA or CBP.
In preferred embodiments of the present invention, S. mutans MT8148 strain may be used as a positive control for detection of a PA-deleted oral bacterium, and/or as a negative control for detection of a CBP-carrying oral bacterium. As a positive control for detection of a PA-deleted oral bacterium, depending on the detection method, an isolated PA protein, a nucleic acid or a vector comprising a DNA encoding PA or its fragment, a cell transformed with said vector may also be used. As a negative control for detection of a CBP-carrying oral bacterium, CND strain, which is a TW295 strain in which CBP-encoding gene has been knocked out, and a Gram-positive bacterium that does not express CBP may also be used.
The term “subject” in the present invention means any living organism, preferably an animal, more preferably a mammal, still more preferably a human individual.
Hereinafter, the present invention is explained in detail using working examples; however, the present invention should not be limited to these examples.
EXAMPLES
Example 1
Examination of Virulence of Streptococcus mutans in Mouse Model of Dextran Sulfate Sodium (DSS)-Induced Enteritis
Effects of each strain of S. mutans on inflammatory bowel disease and their causative factors were examined using a mouse model of dextran sulfate sodium (DSS)-induced enteritis.
Materials and Methods


    1. S. mutans Bacterial Strain


ATW295 strain (serotype k) isolated from the blood of a patient with bacteremia after tooth extraction was used (provided from Tokyo Women's Medical University). As a standard strain, a MT8148 strain (serotype c) isolated from the oral cavity of a Japanese infant was used as a tested bacterium.

    2. Mouse Model of Dextran Sulfate Sodium (DSS)-Induced Enteritis


In order to induce a mild inflammation in a BALB/c mouse (8-week old male, body weight from 20 to 30 g), the concentration of DDS used was set to be 2.5% (DDS was dissolved in tap water before administration). Under this condition, usually inflammatory symptoms develop from day 4 after DSS administration, and approximately 40% of the mice are dead on day 15. Disease activity index (DAI) which includes this mortality rate on day 15 time-course change in body weight, and bleeding in the digestive tract (bloody stool) was employed as a major index of the analysis.
To mimic transient bacteremia by oral bacteria, 1×107 colony forming unit (CFU) of the tested bacteria were bolus-administered via the cervical vein.
For detection of the bacteria, PCR method using the following S. mutans-specific primer (Hoshino et al. (2004) Dian Microbiol Infect Dis 48: 195-199, 2004) was carried out.
S. mutans-Specific Primer


    Forward: 5′-GGC ACC ACA ACA TTG GGA AGC TCA GTT-3′ (SEQ ID NO: 11)
    Reverse: 5′-GGA ATG GCC GCT AAG TCA ACA GGA T-3′ (SEQ ID NO: 12)

3. Results



When oral bacteria were administered simultaneously with the start of DSS administration, no significant changes were observed in both the TW295 administration group and MT8148 administration group compared to the control group without administration of oral bacteria.
When oral bacteria were administered at 4 days after the start of DSS administration, at which inflammation begins to occur, the DAI score showed a high value (namely, aggravation of enteritis) in the TW295 administration group on day 7, and the value significantly increased on day 10. In contrast, no significant increase in the value was observed in the MT8148 group compared to the control (FIG. 4).
Regarding the change in body weight, a decreasing tendency was observed in the TW295 administration group, compared to other groups. A significant decrease in the body weight was observed in the TW295 group compared to the control on days 11-15 (top photos in FIG. 5), and observation of the large intestine by dissection revealed aggravation of enteritis (bottom photos in FIG. 5).
Mortality rate on day 15 also showed a remarkably high value in the TW295 group compared to the control; however, no difference was observed between the MT8148 strain as a standard strain and the control (FIG. 6).
By way of precaution, effects of oral administration of TW295 at 1×108 CFU on the disease state were also examined; as a result, no worsening of the disease state was observed in the oral administration group. These results suggested a possibility that highly virulent TW295 invades into the blood via the transient bacteremia and worsens enteritis not from the mucosal side of the intestinal tract, but from the inner side of the blood vessel, thereby increasing mortality rate.
Example 2
Examination of Amount of Administration of Streptococcus mutans and Mortality Rate
For the DSS-induced enteritis mouse model used in Example 1, the amount of highly virulent TW295 strain was changed between 1×103 CFU and 1×107 CFU, and their effect on mortality rate was examined. Here, the tested bacteria and their administration method, etc. were in accordance with Example 1.
As a result, no difference in the mortality rate between the administration group and the control up to the amount of administration of 1×104 CFU, but an increase in the mortality rate was observed from 1×105 CFU, and a statistically significant increase in the mortality rate was observed from 1×106 CFU (FIG. 7). From this, it is suggested that the highly virulent TW295 strain aggravates enteritis when 1×105 CFU or more of the bacteria invade in the blood, and as a result the strain exhibits a risk of increasing the mortality rate.
Example 3
Examination of Mechanism of Aggravation of Enteritis by Streptococcus mutans TW 295
The reason why the standard strain MY8148 does not aggravate enteritis while TW295 aggravates enteritis and increases mortality was examined with focusing on collagen binding protein possessed by TW295.
Production of CBP Gene Deletion Strain (CND Strain):
A CND strain, which is a TW295 strain from which a collagen binding protein (CBP)-coding gene is knocked out, was produced. Namely, using the following primer that was designed based on the cnm gene total sequence (SEQ ID NO: 4; DDBJ accession No. AB469913) that encodes CBP of TW295 strain, cnm gene fragments of the TW295 strain were amplified.
Cnm Amplification Primer:









(SEQ ID NO: 13)



cnm1F 5′-GAC AAA GAA ATG AAA GAT GT-3′



 



(SEQ ID NO: 14)



cnm1R 5′-GCA AAG ACT CTT GTC CCT GC-3′






The amplified fragment was integrated into pGEM-T Easy vector (Promega, Madison, Wis., USA) to produce a plasmid pTN11. pTN11 was treated by a restriction enzyme Bsml, and around the center of the open reading frame of cnm was digested to produce a plasmid pTN12 in which erythromycin-resistant gene fragment taken out from the plasmid pKN100 was integrated. After pTN12 was made into a single strand using restriction enzyme Pstl, a homologous recombination of the TW295 strain was performed by a chemical method using equine serum. Screening of the strain having erythromycin-resistant gene at the center of the cnm gene (CND strain) was carried out using a S. mutans selection medium comprising erythromycin. The produced strain was confirmed by Southern hybridization and measurement of collagen binding ability.
Aggravation of enteritis was examined using the produced S. mutans CND strain. Here, other tested bacteria and their administration method, etc. were in accordance with Example 1.
As a result, DAI score of the CND strain on day 10 was significantly lower than that of TW295, and was almost equal to that of the control. Furthermore, the mortality rate of the CND strain on day 15 was remarkably low, which was significantly lower than not only the parent TW295 strain, but also the MT8148 and the control (FIG. 8). These results indicate that the collagen binding protein (CBP) plays an important role in the mechanisms of aggravation of enteritis and of increase in mortality rate of the highly virulent TW295.
Example 4
Examination of Presence/Absence of Localization of Streptococcus mutans in Various Organs
In order to investigate bacteremia caused by a highly virulent TW295 strain, namely, to investigate the mechanism through which a TW295 strain that invades into the blood aggravates enteritis and, in the first place, whether the administered strain actually reaches the intestinal tract, each organ was extracted immediately after intravenous administration of the bacteria, and presence of the bacteria was confirmed by PCR method. Here, tested bacteria and their administration method, etc. were in accordance with Example 1.
As a result, no band was observed in the intestinal tract such as the large intestine and small intestine, and presence of the bacteria was not confirmed (FIG. 9). On the other hand, a band presumed to be derived from the bacteria was observed in the liver and lung (FIG. 9). These results suggest a possibility that the administered bacteria accumulate in these organs.
For the purpose of further confirmation, a homogenize of each organ 3 hours after administration of the bacteria was plated on a MSB plate, a selection medium of S. mutans, and proliferation of the bacteria was determined. As a result, almost no bacterial colony was observed in the lung, and many bacterial colonies were observed only in the liver (FIG. 10). These results suggest a possibility that the TW295 after administration accumulates in the liver, thereby aggravating enteritis. Each colony was confirmed to be the administered TW295 by means of PCR using a specific primer. Interestingly, in the liver of a mouse in which enteritis was induced by administration of DSS, with an estimated number of 1.80-2.00 million of TW295 accumulated at the time point 3 hours after the administration. This number is actually 20% of the total number of bacteria administered. In contrast, in a mouse without administration of DSS, namely, without induction of enteritis, only an estimated number of 0.02 million of TW295 accumulated. These results suggest a possibility that highly virulent TW295 specifically accumulate in the liver with induced enteritis.
Example 5
Observation of Localization in Tissue of Streptococcus mutans TW295-GFP Strain (TW295 with Integration of GFP Genes)
A TW295 strain genetically modified to express a green fluorescent protein (GFP), a fluorescent label, was produced in accordance with a routine method (TW295-GFP strain).
The produced TW295-GFP strain was administered to a mouse treated with DSS, and localization of the bacteria in the tissue was observed. As a result, it was clarified that the bacteria after administration were not taken up by the cells of the immune system such as vascular endothelium and Kupffer cells, instead they were taken up by the hepatocytes (FIG. 11). This is a new finding because to date, there has been no report describing that a large amount of bacteria was taken up by hepatocytes.
Example 6
Analysis Using DNA Microarray
Since accumulation of highly virulent TW295 in hepatocytes of the liver is considered to be a mechanism for aggravating enteritis, an exhaustive analysis of changed genes was carried out using the liver tissue after bacterial administration. The analysis was performed using DNA microarray.
As a result, it was recognized that expression of a number of interesting genes increased. Some of such genes are those coding for inflammation-related proteins that are generated by the hepatocytes, and that are known to be secreted in the blood. As a typical example of such inflammation-related proteins includes orosomucoid; and the following mechanism of aggravation of enteritis is suggested: (1) invasion of highly virulent bacteria into the blood vessel, (2) accumulation of said highly virulent bacteria in the liver, (3) production of orosomucoid by the hepatocytes due to stimulation by the highly virulent bacteria, and (4) aggravation of inflammation and the inflammation becoming chronic by the orosomucoid (FIG. 12).
Reference Example
Production of PA Gene-Deleted Strain (PD Strain)
In accordance with a method by Nakno et al., Microbes, Infect. 2006 8 (1): 114-21, a primer on the basis of pac gene total sequence (SEQ ID NO: 2, DDBJ accession number X14490) that encodes PA of MT8148 strain was used to produce a PD strain, by means of a method similar to that for the above CND strain.
Primer for pac Amplification


    pac-F 5′-GCG CGC ATG CTT TAT TCA GAT TTG GAG GAT-3′ (SEQ ID NO: 15)
    pac-R 5′-GCG AAA GCG CAT GCT GTG ATT TAT CGC TTC-3′ (SEQ ID NO: 16)


Detection Example 1
Detection of Streptococcus mutans that Aggravates Inflammatory Digestive Tract Disorders
Materials and Methods


    Tested bacteria: Following bacteria were used in the establishment of the detection system.


















S. mutans
MT8148 strain (PA+/CBP−)/TW295 strain




(PA−/CBP+)



S. sobrinus
B13 strain/6715 strain



S. sanguinisA
TCC10556 strain



S. oralis
ATCC10557 strain



S. gordonii
ATCC10558 strain



S. salivarius
HHT strain










Analysis 1. Method for Culturing S. MUTANS (Mutans Streptococci) (Operation Time: about 5 Minutes, Waiting Time (Such as During Culturing of a Bacterium): 2 Days)

Culturing of S. mutans employs the following things:

    
    
        spitz for collecting saliva (not particularly limited as long as it is sterilized and suitable for collecting and seeding)
        a special dropper capable of collecting saliva of 10 μl
        Special Medium A (agar medium) (24-well plate (it is not particularly limited as long as it is a plate of about 24-well, e.g., 24 well with Lid MICROPLATE (IWAKI)) coated with MSB agar medium e.g., Mitis-salivarius agar medium (Difco Laboratories) supplemented with bacitracin (100 unit/ml; SIGMA-ALDRICH) and 15% sucrose (Wako Pure Chemical Industries, Ltd.). It is preferred to be provided with Anaero Pack®.)
        a sterilized toothpick and the like for picking up bacterial colonies
        Special Medium B (liquid medium) (sterilized Brain Heart Infusion (BHI) liquid medium (Difco Laboratories) contained in a disposal test tube).
    
    


Culturing of S. mutans is carried out as follows.
The saliva of the subject is collected in a small amount using a spitz for collecting saliva. 10 μl of the saliva is taken from the spitz using a special dropper, plated onto Special Medium A, then cultured at 37° C. for 48 hours, preferably in an anaerobic condition. After culturing, presence of bacterial colonies is confirmed with the naked eye, and the colonies (rough colonies are desirable) are picked up and added into Special Medium B, cultured at 37° C. for 18 hours, and used in the following Analyses 2, 3 and 4. Cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius are used as controls to confirm that no bacterium other than S. mutans and S. sobrinus grows in Analysis 1.
Analysis 2. Method for Detecting S. mutans (Mutans Streptococci) (Operation Time: about 15 Minutes, Waiting Time (Such as During Culturing of Bacteria): about 3 Hours)
Although the method of culturing mutans streptococci of the above Analysis 1 is provided with conditions in which the mutans streptococci group (S. mutans/S. sobrinus) can preferably grow, a bacterium having bacitracin-resistance other than mutans streptococci may grow. Therefore, confirmation is done in this step.
Detection employs the following things:

    
    
        a special dropper capable of collecting bacterial solution of 10 μl
        Special Medium C (96-well plate (e.g., MULTI WELL PLATE for ELISA (SUMIRON)) containing 100 μl of BHI solution containing 1% sucrose (Wako Pure Chemical Industries, Ltd.))
        Wash Buffer A (PBS solution)
        Buffer 1 (a solution in which 0.5% crystal violet (Wako Pure Chemical Industries, Ltd.) is added to sterile distilled water)
        Buffer 2 (7% acetate (Wako Pure Chemical Industries, Ltd.) solution)
    
    


Detection is carried out as follows:
10 μl of the bacterial solution cultured according to the method of Analysis 1 is added to Special Medium C, incubated at 37° C. for 3 hours. The Special Medium C is washed three times with Wash Buffer A, then left still for approximately 15 minutes after the last Wash Buffer A is added. This Wash Buffer A is removed, and the Special Medium C is washed once again with Wash Buffer A, then 100 μl of Buffer 1 is added to the Special Medium C and left still for 1 minute. This is washed three times with Wash Buffer A, and 200 μl of Buffer 2 is added thereto.
It is determined to be S. mutans-positive when the color of the medium changes, and S. mutans-negative when the color of the medium does not change.
Analysis 3. Method for Detecting PA-Deleted S. mutans (Operation Time: about 30 Minutes, Waiting Time (Such as During Culturing of Bacteria): about 11 Hours and 30 Minutes)
Detection of PA-deleted S. mutans employs the following things:

    
    
        Special Plate (96-well plate; MICROTEST U-Bottom (BECTON DICKINSON))
        Wash Buffer B (a PBST solution in which 0.05% of Triton X-100 (Wako Pure Chemical Industries, Ltd.) is added to Wash Buffer A used in Analysis 2)
        Buffer 3 (a mixture of Tris buffered saline, pH6.8, 100 mM dithiothreitol (Wako Pure Chemical Industries, Ltd.) and 20% glycerin (Wako Pure Chemical Industries, Ltd.))
        Buffer 4 (a PBST solution supplemented with 5% skimmed milk (BECTON DICKINSON))
        Buffer 5 (a PBST solution supplemented with 0.1% rabbit anti-PA antiserum (stored in our laboratory))
        Buffer 6 (a PBST solution supplemented with 0.1% porcine anti-rabbit immunoglobulin antibody (Dakopatts))
        Buffer 7 (a solution in which AP buffer (100 mM 2-amino-2-hydroxymethyl-1,3-propanediol, 5 mM magnesium chloride, 100 mM sodium chloride) is supplemented with NBT solution (Wako Pure Chemical Industries, Ltd.) at 0.6% final concentration and BCIP solution (Wako Pure Chemical Industries, Ltd.) at 0.33% final concentration.)
    
    


Detection of PA-deleted S. mutans is carried out as follows:

    (1) Sample Preparation


To 100 it of the bacterial solution cultured according to the method of Analysis 1 above, Buffer 3 is added, and immersed in boiling water for 10 minutes, and frozen if it is to be stored.

    (2) Detection of PA-Deleted S. mutans 
    1) 100 it of the sample prepared as above (1) is added to the Special Plate, left still overnight at 4° C.
    2) The Special Plate was washed three times in Wash Buffer B, then 100 μl of Buffer 4 is added thereto, left still at room temperature for 1 hour.
    3) The Special Plate was washed three times in Wash Buffer B, then 100 μl of Buffer 5 is added thereto, reacted at room temperature for 1 hour.
    4) The Special Plate was washed three times in Wash Buffer B, then 100 μl of Buffer 6 is added thereto, reacted at room temperature for 1 hour.
    5) The Special Plate was washed three times in Wash Buffer B, then 100 μl of Buffer 7 is added thereto, and after 15 minutes changes in the color of the solution are observed. It is determined to be PA-positive when the color of the solution changes, PA-negative when the color of the solution does not change. Cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius are used as controls to confirm that no bacterium other than PA-carrying S. mutans shows a positive reaction in Analysis 3.

Analysis 4. Detection Method of CBP-Carrying S. mutans (Operation Time: about 30 Minutes, Waiting Time (Such as During Culturing of a Bacterium): about 3 Hours and 30 Minutes)



Detection of CBP-carrying S. mutans employs the followings:

    
    
        Special Medium D (the Special Plate used in Analysis 3, to which a mixed solution of sterile distilled water supplemented with 0.6% acetate and Type I collagen (Sigma) in 9:1 ration was added.)
        Wash Buffer A (the same buffer as that used in above Analysis 2 (detection method of S. mutans))
        Buffer 8 (Wash Buffer A supplemented with 5% bovine albumin (Sigma))
        Wash Buffer C (Wash Buffer A which is a PBST solution supplemented with 0.01% Tween 20 (Wako Pure Chemical Industries, Ltd.))
        Buffer 9 (sterile distilled water supplemented with 25% formaldehyde (Wako Pure Chemical Industries, Ltd.))
        Buffer 1 (the same buffer as that used in above Analysis 2)
        Buffer 2 (the same buffer as that used in above Analysis 2)
        Detection of CBP-carrying S. mutans is carried out as follows:
    
    
    (1) Special Medium D is washed three times with Wash Buffer A, then 200 μl of Buffer 8 is added thereto, and left still at 37° C. for 1 hour.
    (2) Washed three times with Wash Buffer C, then 200 μl of the bacterial solution cultured according to the method of 1 described above is added thereto, and incubated at 37° C. for 2 hours.
    (3) Washed three times with Wash Buffer A, then 200 μl of Buffer 9 is added thereto, and left still at room temperature for 30 minutes.
    (4) Washed three times with Wash Buffer A, then 200 μl of Buffer 1 is added to the 96-well plate, and left still for 1 minute.
    (5) Washed three times with Wash Buffer A, then 200 μl of Buffer 2 is added thereto.


It is determined to be CBP-positive when the color of the solution changes, CBP-negative when the color of the solution does not change. Cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius are used as controls to confirm that no bacterium other than CBP-carrying S. mutans shows a positive reaction in Analysis 4.
Analysis Example 1
FIG. 13 is an example of the result of an analysis on whether the S. mutans in saliva samples (A, B and C) collected from 3 subjects are PA and/or CBP-carrying strains. As results of culturing saliva samples in Special Medium A (bacitracin-selection agar medium) in steps in Analysis 1, colony formation was confirmed in all of A, B and C. Formed colonies are picked up and cultured in Special Medium B at 37° C. for 18 hours. In addition, cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius were cultured similarly as controls, and it was confirmed that no bacterium other than S. mutans and S. sobrinus grew in Analysis 1.
Subsequently, in steps in Analysis 2, the bacterial solution cultured in Analysis 1 was added to Special Medium C, incubated at 37° C. for 3 hours, washed with Wash Buffer A, then stained with Buffer 1 containing crystal violet. Since the color of the buffer changed to blue-violet in the medium in which samples A and B has been cultured, the presence of S. mutans was determined. As the buffer remained transparent in the medium in which sample C has been cultured, absence of S. mutans was determined.
In steps in Analysis 3, Buffer 3 was added to each of the bacterial solutions of the samples A and B cultured in Analysis 1 and boiled for 10 minutes, and stored frozen. This was added to Special Plate (96-well plate: MICROTEST U-Bottom (BECTON DICKINSON)), left still overnight at 4° C. After washing with Wash Buffer B, Buffer 4 was added and blocked at room temperature for 1 hour, then Buffer 5 containing rabbit anti-PA antiserum was added and reacted at room temperature for 1 hour. After washing with Wash Buffer B, Buffer 6 containing porcine anti-rabbit immunoglobulin antibody was added and reacted at room temperature for 1 hour. After washing with Wash Buffer B, Buffer 7 which contained an alkaline phosphatase reaction-detecting reagent was added, and after 15 minutes changes in the color of the solution were observed. Since the color of the solution changed to pink in the plate of the sample A, presence of PA-carrying S. mutans was determined. As the color of the solution remained transparent for the sample B, absence of PA-carrying S. mutans was determined. Similar analysis was performed using cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius as controls, confirming that no bacterium other than the PA-carrying S. mutans showed a positive reaction.
In steps in Analysis 4, Buffer 8 containing 5% bovine albumin was added to the Special Medium D coated with Type I collagen (Sigma), and left still at 37° C. for 1 hour. After washing with Wash Buffer C, bacterial solution cultured in Analysis 1 was added and incubated at 37° C. for 2 hours. After washing with Wash Buffer A, Buffer 9 containing 25% formaldehyde was added, left still at room temperature for 30 minutes. After washing with Wash Buffer A, Buffer 1 was added and left still for 1 minute. After washing with Wash Buffer A, Buffer B was added and changes in the color of the solution were observed. Since the color of the solution remained transparent in the plate containing the sample A, absence of CBP-carrying S. mutans was determined. As the color of the solution changed to blue-violet in the plate containing the sample B, presence of CBP-carrying S. mutans was determined. Similar analysis was performed using cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius as controls, confirming that no bacterium other than the CBP-carrying S. mutans showed a positive reaction.
Example 7
Optimal Conditions for Culturing S. mutans 
In order to obtain a determination with higher accuracy in Analyses 2 to 4 above, it is considered to be important to culture the largest possible number of S. mutans in Analysis 1 and to minimize the contamination of bacteria other than S. mutans. As conditions for culturing, (1) culturing in an aerobic condition/anaerobic condition, (2) antibiotics (bacitracin) concentration, and (3) nutrient (sucrose) concentration were investigated. FIG. 14 is a graph showing the percentage of S. mutans to total bacteria isolated when bacitracin was added to the MSB medium at (a) 1 eq. or (b) 5 eq. (assuming the amount of bacitracin in a conventional MSB medium is 1 eq.) and sucrose was added to the MSB medium at 1 to 4 eq. (assuming the amount of sucrose in a conventional MSB medium is 1 eq.). It was shown that S. mutans could be isolated at the highest concentration in an anaerobic condition, when 1 eq. of bacitracin and 1 eq. of sucrose were used. Accordingly, it was shown that in order to obtain a determination with higher accuracy, it is necessary to culture in a sealable container in an anaerobic condition (e.g., in a sealed pack to which Anaero Pack® is attached) in a medium supplemented with bacitracin and sucrose at the same concentration (approx. 100 unit/ml and 15%, respectively) contained in a conventional MSB medium.
Example 8
Storage Period of Samples
In the case when analysis is performed using a saliva sample collected some time ago, in order to detect virulent S. mutans under the optimal condition obtained in Example 4, we investigated allowable storage period of saliva samples.
FIG. 15 is a graph showing isolation rate of S. mutans from saliva samples collected 0-6 months before by Analysis 1, relative to the isolation rate of S. mutans from a saliva sample collected and diluted with sterile saline by serial dilution on the same date and plated on a MSB agar medium as 100%. The saliva samples were frozen-stored at −20° C. after collection. The number of samples: N=8; however, the number of samples stored for 1-2 months was N=6. As a result, it is considered to be desirable that saliva samples are used preferably within 3 months, preferably within 2 months, and most preferably within 1 month after collection.
Example 9
Distribution of Genotype and Serotype of Streptococcus mutans in Saliva Sample
With respect to Streptococcus mutans in saliva samples obtained from 528 healthy subjects and 24 patients with IBD, the genotype of collagen binding protein (CBP) was identified in accordance with a method by Nomura et al., 2009, Journal of Medical Microbiology, 58: 469-475, and the serotype was identified in accordance with a method by Shibata et al., 2003. Journal of Clinical Microbiology, 41, 4107-4112 and Nakano et al., 2004. Journal of Clinical Microbiology, 42, 4925-4930. Results are shown in FIG. 16 (genotype) and FIG. 17 (serotype).
As shown in FIG. 16, the percentage of cnm(+) in the Streptococcus mutans obtained from the saliva samples of healthy subjects was 10%, that in the Streptococcus mutans obtained from the saliva samples of IBD patients was 25%, which was significantly high (odds ratio 2.99).
In addition, as shown in FIG. 17, regarding the serotype of Streptococcus mutans obtained from the saliva samples of healthy subjects, the percentages of c-type (74.6%) and e-type (19.7%) are large, and those of f-type (3.4%) and k-type (2.3%) are extremely small. In contrast, regarding the serotype of Streptococcus mutans obtained from the saliva samples of IBD patients, the percentages of c-type (70.8%) and e-type (4.2%) are smaller than those in the healthy subjects, and those of f-type (12.5%) and k-type (12.5%) are larger than those in the healthy subjects.
In other words, in the case of healthy subjects, the percentage of (c-type+e-type) is 94.3%, and that of (f-type+k-type) is 5.7%; whereas in the case of IBD patients, the percentage of (c-type+e-type) is 75%, and that of (f-type+k-type) is 25%; thus, indicating a large ratio of specific types, i.e., (f-type+k-type) (odds ratio: 5.53).
Example 10
Serotype of Streptococcus mutans and Phagocytosis by Leucocytes
With respect to each of the Streptococcus mutans MT8148 strain (serotype c), MT8148GD strain (serotype k), and TW295 strain (serotype k), its phagocytosis rate by human leucocytes was investigated. Here, MT8148GD strain is a variant of MT8148 in which gluA gene, which encodes the enzyme that catalyzes the production of the immediate precursor of the glucose side chain donor, has been inactivated (WO 2005/063992).
Each bacterial strain was cultured in Brain Heart Infusion broth (Difco) at 37° C. for 18 hour, and the bacterial cells after culturing were washed, then the concentration of the cells was adjusted with PBS to be 1.0×108 CFU/ml. Human peripheral blood (500 μl) collected from a healthy volunteer was incubated with 500 μl of bacteria (5.0×107 CFU/ml) at 37° C. for 10 minutes. The sample was giemsa-stained (Wako Pure Chemical Industries, Osaka, Japan), and the ratio of polymorphonuclear leucocytes (PMN) exhibiting phagocytosis was obtained using an optical microscope (magnification, ×100; Olympus Industries, Tokyo, Japan). The ratio was expressed by an average number of phagocytic PNM per 100 PMN (500 PMN were tested).
It was shown that the k-type MT8148GD strain and TW 295 strain, rather than the c-type MT8148 strain, are hardly phagocytized by leucocytes (FIG. 18). Consequently, k-type and f-type detected at high concentrations in saliva samples of IBD patients are hardly phagocytized by leucocytes, resulting in their long life in the blood, and leading to a tendency to induce bacteremia, etc.
Furthermore, of the Streptococcus mutans in the saliva samples of IBD patients, the ratio of those with f- or k-serotype and genotype cnm(+) is remarkably higher than that in the samples of healthy subjects (FIG. 19). From this, we consider as follows: it is important to have f- or k-serotype in order to persist in the blood for a long period, and it is necessary to have CBP (cnm(+)) in order to invade into the liver as a target cell. Ultimately, it is considered that bacterial strains having both of these characteristics have higher possibility of aggravating disease conditions.
Sequences of protein, polypeptides and nucleic acids used in the present specification are described in the attached sequence list below.
[Table 1]







TABLE 1







Table of sequences









SEQ ID No.
Species or strain
content of the sequence












1
S. mutans MT8148
PA-amino acid


2
S. mutans MT8148
PA-DNA


3
TW295
CBP-amino acid


4
TW295
CBP-DNA-ORF


5
S. mutans TW295
CBD-amino acid


6
S. mutans TW295
CBD-DNA


7
S. mutans TW871
CBP-amino acid


8
S. mutans TW871
CBP-DNA-ORF


9
S. mutans TW871
CBD-amino acid


10
S. mutans TW871
CBD-DNA


11
Artificial
S. mutans-primer F


12
Artificial
S. mutans-primer R


13
Artificial
S. mutans-CBD-Primer F (cnm1F)


14
Artificial
S. mutans-CBP-primer R (cnm1R)


15
Artificial
S. mutans-PAC-primer F (pac-F)


16
Artificial
S. mutans-PAC-primer R (pac-R)


17
S. mutans LJ23
PA-amino acid


18
S. mutans LJ23
PA-DNA


19
S. mutans SA98
PA-amino acid


20
S. mutans SA98
PA-DNA


21
S. mutans
antigenI/II-amino acid


22
S. mutans
antigenI/II gene (spa)-DNA


23
Neisseria meningitidis
iron-binding protein-amino acid


24
Neisseria meningitidis
iron-binding protein gene (fbp)




DNA

## Claims

1. A method of detecting an oral bacterium for diagnosing whether inflammatory bowel disease is aggravated in a subject, comprising
contacting saliva or plaque obtained from the subject with a collagen binding protein (CBP)-detecting reagent,
performing an assay to detect CBP of oral bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when the CBP is detected, and
diagnosing that inflammatory bowel disease is aggravated in the subject when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

2. The method according to claim 1, further comprising contacting the saliva or plaque obtained from the subject with a PA-detecting reagent, performing an assay to detect protein antigen (PA) of oral bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when PA is not detected and CBP is detected, and
diagnosing that inflammatory bowel disease is aggravated in the subject when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

3. A method of detecting an oral bacterium for diagnosing whether a subject is at a high risk of aggravation of inflammatory bowel disease, comprising
contacting saliva or plaque obtained from the subject with a collagen. binding protein. (CBP)-detecting reagent,
performing an assay to detect CBP of oral bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when the CBI is detected, and
diagnosing that the subject is at a high risk of aggravation of inflammatory bowel disease when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

4. The method according to claim 3, further comprising contacting the saliva or plaque obtained from the subject with a PA-detecting reagent, performing an assay to detect protein antigen (PA) of oral. bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when PA is not detected and CBP is detected, and
diagnosing that the subject is at a high risk of aggravation of inflammatory bowel disease when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

5. A method of judging risk of aggravation of inflammatory bowel disease in a subject, comprising
contacting saliva or plaque obtained from the subject with a collagen binding protein (CBP)-detecting reagent,
performing an assay to detect CBP of oral bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when the CBP is detected, and
judging that the subject is at a high risk of aggravation of inflammatory bowel disease when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

6. The method according to claim 5, further comprising contacting the saliva or plaque obtained from the subject with a PA-detecting reagent, performing an assay to detect protein antigen (PA) of oral bacteria in the saliva or plaque obtained from the subject,
confirming the presence in the saliva or plaque obtained from the subject of an oral bacterium that aggravates inflammatory bowel disease when PA is not detected and CBP is detected, and
judging that the subject is at a high risk of aggravation of inflammatory bowel disease when the oral bacterium that aggravates inflammatory bowel disease is present in the saliva or plaque obtained from the subject.

7. The method according to claim 1, wherein the oral bacterium is Streptococcus mutans.

8. The method according to claim 7. Wherein the genotype of Streptococcus mutans is cnm(+).

9. The method according to claim 7, wherein the serotype of Streptococcus mutans is f-type or k-type.

10. The method according to claim 2, wherein the PA is selected from the group consisting of:
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23;
(2) a polypeptide comprising one or more mutations in the polypeptide of (1), but having an equal function to the polypeptide of (1);
(3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 2, 18, 20, 22 or 24 or its complementary sequence or its fragment under stringent conditions, and having an equal function to the polypeptide of (1);
(4) a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23, and having an equal function to the polypeptide of (1).

11. The method according to claim 10, wherein the PA comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 17, 19, 21 or 23.

12. The method according to claim 1, wherein the CBP is selected from the group consisting of:
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 9;
(2) a polypeptide comprising one or more mutations in the polypeptide of 1), but having an equal function to the polypeptide of (1);
(3) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 6 or 10 or its complementary sequence or its fragment under stringent conditions, and having an equal function to the polypeptide of (1);
(4) a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 5 or 9, and having an equal function to the polypeptide of (1).

13. The method according to claim 12, wherein the CBP comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or 9.

14. The method of claim 1, wherein the CBP-detecting reagent is a medium for detecting CBP-carrying, bacteria or an antibody that binds CBP.

15. The method of claim 3, wherein the CBP-detecting reagent is a medium for detecting CBP-carrying bacteria or an antibody that binds CBP.

16. The method of claim 5, wherein the CBP-detecting reagent is a medium for detecting CBP-carrying bacteria or an antibody that binds CBP.

17. The method of claim 2, wherein the PA-detecting reagent is an antibody that binds PA.

18. The method of claim 4, wherein the PA-detecting reagent is an antibody that binds PA.

19. The method of claim 6, wherein the PA-detecting reagent is an antibody that binds PA.