JP4324464B2 - Method for determining bacterial coaggregation reaction - Google Patents

Description

  The present invention relates to a method for directly determining a bacterial coaggregation reaction, and more particularly to a method for determining a bacterial coaggregation reaction by measuring the presence or absence of skatole production in saliva or the like.

  Bacteria in the oral cavity are involved in oral diseases such as caries, periodontal disease, and bad breath. Streptococcus mutans (hereinafter referred to as “Sm”) and Streptococcus sobrinus (hereinafter referred to as “Ss”) are known as caries-related bacteria, and Actinobacillus actinomycetemcomitans as a causative agent of periodontal disease. (Actinobacillus actinomycetemcomitans (hereinafter referred to as Aa))), Porphyromonas gingivalis (hereinafter referred to as Pg)), Prevotella intermedia (hereinafter referred to as Pi)), and Fusobacterium nucleatum (Fusobacterium nucleatum) (Hereinafter referred to as Fn bacteria)).

  Most oral diseases and infections have a coaggregation reaction in which pathogenic bacteria aggregate and form a network structure as plaque, and epithelium of the oral mucosa such as hard tissues such as host teeth, tongue, and periodontal pockets. It has been reported that it starts by attaching to cells (Non-patent Document 1).

  As a method for confirming the coaggregation reaction of bacteria, a bacterial agglutination assay as described in Non-Patent Document 1 is common. In other words, bacteria collected from the oral cavity are separated, and each separated bacteria is added to a test tube, etc. and reacted (left) for 30 minutes at 4 ° C., or visually confirmed, or the absorbance of the bacterial solution is measured. In this way, the aggregation rate between bacteria is estimated.

  Further, as a method for examining periodontal diseases, Patent Document 1 discloses that the degree of activity of γ-glutamyl transpeptidase (hereinafter referred to as γ-GTP) in the oral cavity is expressed as γ-glutamyl-p-nitroanilide, γ-glutamyl-α. -After reacting naphthylamine, γ-glutamyl-pN-ethyl-N-hydroxyethylaminoanilide, γ-glutamyl-3,5-dibromo-4-hydroxyanilide, etc. with a synthetic substrate, p-dimethylaminocinnamaldehyde A method of visualizing using a coloring reagent such as (p-dimethylaminocinnamic aldehyde), 3-methyl-2-benzoazolinone hydrazone, 1-naphthol-2-sulfonate, etc. is disclosed.

  However, in the method described in Patent Document 1, it is not easy to handle and obtain a substrate to be reacted with γ-GTP, and further, when a coloring reagent is added to develop a color of the substrate reacted with γ-GTP. It is difficult to control test conditions, such as the need to use enzyme activators.

  Furthermore, Patent Document 2 discloses an oral preliminary screening system and oral preliminary screening method that can easily determine the degree of progression of periodontal disease and bad breath. Patent Document 2 discloses that attribute data received from a user's mobile phone via the Internet and expiration data collected from the user are analyzed, and a periodontal disease is calculated from a correlation value indicating a correlation with the degree of progression of periodontal disease. A method of determining the degree of progress is disclosed. Further, in Patent Document 2, since the gas produced by periodontal disease-causing bacteria is detected by the measurement and analysis of exhalation in the periodontal disease preliminary examination, it is possible to perform a bad breath check indirectly. It is disclosed.

Patent Document 2 states that “the semiconductor gas sensors PA2, T50 / 3, P10 / 9, P40 / 1 built in the breath measuring device 2 can measure and recognize all substances other than skatole. Is disclosed.
P. E. Kolenbrander, Methods Enzymol, 1995, 253, 385-397 Japanese Unexamined Patent Publication No. 4-229198 Japanese Patent Laid-Open No. 15-159262

  The present invention provides a coaggregation reaction determination method capable of directly confirming bacterial coaggregation reactions occurring in the oral cavity.

  Plaque is formed when a coaggregation reaction occurs between Fn bacteria and various oral pathogenic bacteria (for example, Ss bacteria, Aa bacteria, Pi bacteria, etc.). Plaque deposits and accumulates in the oral cavity, deteriorating the oral hygiene environment and causing oral disease. The present inventors have found that there is a high correlation between the coaggregation reaction and the amount of skatole, which is one of the bad breath components. Based on the above findings, the present inventors have made it possible to directly confirm the coaggregation reaction between Fn bacteria and various oral pathogenic bacteria by detecting skatole.

  That is, the present invention is a method for determining a bacterial coaggregation reaction using skatole in an oral sample as an index. The bacterium may be an oral bacterium, and the oral bacterium is selected from the group consisting of Streptococcus mutans, Streptococcus sobrinus, Actinobacillus actinomycetemcomitans, Porphynomonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum. 1 type or 2 types or more may be sufficient.

  Furthermore, according to the present invention, the presence or absence of a coaggregation reaction of oral bacteria can be detected by measuring the amount of skatole in the oral sample. In addition, the degree of oral bacteria aggregation can be determined based on the amount of skatole. As a result, it is possible to grasp the hygiene environment in the oral cavity, periodontal disease and / or the progress or improvement status of dental caries with a simple method, and to early detection, prevention and prevention of bad breath of caries and periodontal disease. Can contribute.

  The present invention makes it possible to easily determine bacterial coaggregation reactions by directly measuring skatole in oral samples. In the case of oral bacteria, the presence or absence of a coaggregation reaction of oral bacteria can be confirmed by detecting skatole. Furthermore, by quantifying the amount of skatole in the oral sample, it is possible to grasp the state of coaggregation of oral bacteria.

  Oral health or hygiene, and prevention of oral diseases, by simply measuring saliva and expiration, monitoring oral hygiene and deterioration of the oral environment, further caries, Early detection or prevention of oral diseases such as periodontal disease and bad breath can be achieved.

  Examples of the bacteria according to the present invention include bacteria such as oral bacteria and enteric bacteria. Examples of oral bacteria include Capuocytophaga bacteria, such as C. sputigena, C. ochracea, etc .; Eubacterium bacteria, such as E. alactolyticum; Treponema bacteria, such as T. denticola, etc .; Prevotella bacteria For example, P. intermedia, P. denticola, etc .; Actinomyces group of bacteria, such as A. naeslundii, A. israelli, etc .; Propionibacterium group, such as P. acnes, etc .; Veillonella group of bacteria, such as V. atypica Haemophilus group of bacteria, such as H. parainluenza, etc .; Porphyromonas group of bacteria, such as P. gingivalis, etc .; Fusobacterium group of bacteria, such as F. nucleatum, etc .; Streptococcus group of bacteria, such as S. mutans, Mention may be made of one or more selected from the group consisting of S. sobrinus, S. oralis, S. mitis, S. gordonii, S. sanguis, etc .; Actinobacillus genus bacteria group, such as A. actinomycetemcomitans, etc. it can.

  Furthermore, examples of oral bacteria include one or more selected from the group consisting of Streptococcus mutans, Streptococcus sobrinus, Actinobacillus actinomycetemcomitans, Porphynomonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum. it can.

  The intraoral sample that is a determination target of the present invention may be a sample that can be collected from the oral cavity, and examples thereof include saliva, mouthwash, tongue coating, plaque, exhalation, and mouth breath.

  The detection of skatole according to the present invention is carried out by various analysis using oral samples such as mouthwash liquid washed with 30 to 60 seconds of ion exchange water, distilled water for 30 seconds to 60 seconds, or directly collected saliva. It can be done by the method.

  Although it is preferable to analyze by directly putting the mouthwash liquid or the directly collected liquid into the analytical instrument, the headspace part alone may be analyzed. Moreover, what was liquid-liquid extracted with solvents, such as ether, hexane, and a methylene chloride, can also be analyzed.

  When measuring skatole in exhaled breath, it is possible to directly analyze the exhaled breath collected in an odor bag (Omi Odo Air Service Co., Ltd.) by an analysis method such as gas chromatography-mass spectrometry, AED-mass spectrometry. it can. If the collected sample is concentrated and analyzed according to the analysis method described above, the accuracy can be further improved.

  When measuring skatole in the mouth, in the state that the subject is breathing nasally, it is inserted into the oral cavity into a gas sampling device such as a gas tight syringe or syringe, and the gas in the oral cavity is collected, The collected sample can be directly analyzed by an analysis method such as gas chromatography-mass spectrometry or AED-mass spectrometry. Furthermore, if the collected sample is concentrated and analyzed according to the above-described analysis method, the accuracy can be further improved.

  As an analysis method of skatole, general analysis methods such as thin layer chromatography, gas chromatography, liquid chromatography, infrared spectroscopy, absorbance analysis, and fluorescence analysis can be used. As a method of analyzing with higher accuracy, gas chromatography-mass spectrometry, solid layer adsorption (SPME) -mass spectrometry, AED-mass spectrometry, liquid chromatography-mass spectrometry, infrared spectroscopy-mass spectrometry, A radiation irradiation light emitting method or the like can be used. In addition, as a method for detecting skatol more easily, a method of reacting with skatol to develop a color or a method of using a microbial sensor that specifically absorbs skatole can be used.

  As a method for quantifying skatole according to the present invention, among oral samples such as saliva, mouthwash, tongue coating, plaque, exhalation, mouth breath, etc., preferably 6 to 7 mL of ion-exchanged water and distilled water for 30 seconds. Extract skatole from 1 mL of mouthwash rinsed for -60 seconds or 1 mL of saliva collected directly from subjects using organic solvents such as hexane, methylene chloride, diethyl ether, etc., and analyzed using the various analytical methods described above can do. Particularly preferably, skatole can be extracted from the mouthwash or directly collected sample using hexane and quantified using gas chromatography-mass spectrometry.

  The method for determining the hygiene environment in the oral cavity, periodontal disease and / or caries progress or improvement status according to the present invention can be performed by measuring the amount of skatole in the oral sample. In order to determine the presence or absence of a bacterial coaggregation reaction and the periodontal disease from the measurement result of the amount of skatole, the amount of skatole in the oral sample is compared with that of a healthy person.

  As a determination kit for determining the progress or improvement status of oral hygiene environment, periodontal disease and / or caries according to the present invention, skatole can be detected from saliva, exhaled breath, oral mucosa, etc. Examples thereof include, but are not particularly limited to, for example, those in which a compound that changes color and / or develops color by reacting with skatole is previously supported on a carrier. The carrier is not particularly limited as long as it can support the compound, and examples thereof include paper; a film of nitrocellulose, nylon, PVDF (polyvinylidene difluoride), and the like.

Example 1
(Relationship between oral bacterial coaggregates and skatole)
According to the following method, the relationship between oral bacterial coaggregates and skatole was examined.

<Method>
0.1 mL of each bacterial solution of Fn bacteria, Ss bacteria, Pi bacteria, Pg bacteria, and Aa bacteria was mixed with 7 mL of brain heart infusion medium and cultured at 37 ° C. for 20 hours. 0.5 g of sodium chloride was placed in a 10 mL lidded test tube, and 1 mL of the culture solution was added thereto, and then 2 mL of a hexane / ether solution was added and mixed by shaking for 15 seconds. This was separated into an aqueous layer and a solvent layer at 3500 rpm for 10 minutes with a centrifuge, and the solvent layer was separated and subjected to GC-MS analysis.

<Result>
Table 1 shows the analysis results of skatole produced from a mixture of oral bacteria. Compared to a system in which no bacteria are added (control), the production of skatole is also observed when each bacterium is added alone, in particular, Fn and Aa, Fn and Pi, Fn and Ss, Fn It can be confirmed that the mixture of fungus, Aa fungus, Pi fungus, Fn fungus, Aa fungus, Pi fungus, and Ss fungus produced 1.4 to 2 times or more skatole as compared with the case of Fn fungus alone.

On the other hand, according to a conventional method, only Fn bacteria (FIG. 1) or a mixture of Fn bacteria, Aa bacteria, Pi bacteria, and Sa bacteria (FIG. 2) was observed by a bacterial aggregation assay. As shown in FIG. 1, in the case of only Fn bacteria, the solution was transparent or suspended, there was no precipitate, and no bacterial aggregation was observed, but the Fn bacteria and Aa bacteria in FIG. In the case of a mixture of Pi bacteria and Sa bacteria, a precipitate is confirmed in the solution, which clearly means that an agglutination reaction has occurred.
From this, it was found that the coaggregation reaction of oral bacteria such as Fn bacteria, Aa bacteria, Pi bacteria, and Ss bacteria can be confirmed by measuring skatole in the oral sample.

Example 2 (Determination of periodontal disease by skatole measurement)
According to the following method, the relationship between the morbidity of periodontal disease and the amount of skatole was examined.
<Method>
In order to clinically examine the diseased state of periodontal disease in 78 subjects in their 20s to 50s, the Gingival Index (hereinafter referred to as GI) for examining the inflammatory state of the gingiva was measured. The target teeth are the representative 6 teeth shown below in the oral cavity, and the gingival states of 4 teeth (lip (cheek) side, tongue (palate) side, mesial, centrifugal) are 0 (no inflammation), 1 ( Slight changes in gingival color and surface morphology, 2 (bleeding due to moderate gingival surface gloss, redness, edema and swelling compression), 3 (significant redness and swelling, tendency for sudden bleeding and ulceration) Was given a score, and the average of the four sites was defined as the GI of the corresponding tooth.
Further, the amount of skatole was quantified by gas chromatography using as an analytical sample an extract of a mouthwash obtained by washing 6 to 7 mL of ion exchange water for 60 seconds.

<Result>
Table 2 shows the results of the gingival inflammation state and the amount of skatole extracted from the mouthwash. It can be seen that the amount of skatole increases as the gingival inflammation progresses, and the amount of skatole increases rapidly from the point where GI exceeds 1 compared to the system in which no bacteria are added (control).

Example 3 (Relationship between the amount of skatole and the presence of oral disease-related bacteria)
The relationship between the presence of oral disease-related bacteria and the amount of skatole was examined by the following method.

<Method>
Resting saliva was collected from 78 subjects (20's to 50's). The Fn, Pg, and Pi bacteria present in the saliva were qualitatively analyzed using the Nested PCR method. The case where each bacterium was detected was defined as (+), and the case where each bacterium was not detected was defined as (−). Further, the amount of skatole was quantified by gas chromatography using as an analytical sample an extract of a mouthwash obtained by washing 6 to 7 mL of ion exchange water for 60 seconds.

<Result>
Table 3 shows the results of the presence of Fn bacteria and Pg bacteria, and Table 4 the presence of Fn bacteria and Pi bacteria and the amount of skatole. The amount of skatole in the group in which Fn bacteria and Pg bacteria were detected was about 3 times higher, and the same result was obtained in the group in which Fn bacteria and Pi bacteria were detected. This shows that the presence of Fn bacteria and Pg bacteria or Pi bacteria is necessary for skatole production.

It is the figure which showed the state of the solution in case only Fn microbe exists. It is the figure which showed the state of the solution when the mixture of Fn bacteria, Ss bacteria, Pi bacteria, and Aa bacteria exists.

Claims (1)

Extract skatole from the mouthwash or saliva of the subject using an organic solvent and measure the amount of skatole. sobrinus, Actinobacillus actinomycetemcomitans, and more that a combination of the Porufinomonasu gingivalis and Prevotella one selected from the group consisting of intermedia or two or more are compared to those not detected, oral bacteria To determine the presence or absence of plaque deposits or deposits due to coaggregation .

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