JP2001299381A - Bacterial identification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for capable of discriminating bacteria quickly. SOLUTION: This method for discriminating the bacteria by their biochemical properties is provided by culturing a specimen to be tested, making a colony growing on a medium in contact with a substrate substance, holding them under an atmosphere of >=40 deg.C temperature and detecting the signals derived from substances generated from the above substrate substance by the enzymatic activity of the bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for distinguishing bacteria by biochemical properties, and provides a method for rapidly distinguishing bacteria.

[0002]

BACKGROUND ART Differentiation of bacteria in clinical tests is usually performed by
After purely cultivating the isolated colonies on the medium, the biochemical properties of the isolated colonies are checked against the identification table. For example, if a haemophilus-suspecting colony forms on the separation medium, it must be biochemically identified to confirm its identity (eg, Haemophilus influenza or Haemophilus parainfluenza). The determination is generally based on the requirements for factor X (hemin) and factor V (nicotinamide adenine dinucleotide), but the relationship between the addition of both factors to the medium and the growth of the bacteria is determined. It takes 18 to 24 hours to get results because it is checked by the method.

To solve the above problems, Kirian emphasized that instead of the factor X requirement test, a test for the ability to synthesize porphyrin from 5-aminolevulinic acid should be confirmed [Kilian, M .; (1974). A
cta Path. microbiol. scand.
B82, 835], and this method also requires a culture time of 4 to 20 hours to distinguish Haemophilus.

[0004]

As described above, the conventional techniques for diagnosing infectious diseases are not fast enough and clinicians have to start diagnosis and treatment without waiting for the results. As such, treatment often relies on antibiotics with a broad antimicrobial spectrum. The use of such antibiotics is likely to unnecessarily accelerate the emergence of resistant bacteria. The biggest cause of bacterial testing not helping to diagnose infections and determine treatment strategies is simply the long time it takes to get results. An object of the present invention is to provide means for rapidly performing such a bacterial test, and to solve a fundamental problem of the bacterial test.

[0005]

SUMMARY OF THE INVENTION The above-mentioned problems can be solved at once by the present invention. That is, the present invention, in the identification of bacteria by biochemical properties, contacting the isolated colonies grown on a culture medium by culturing a test sample with a substrate substance,
The present invention relates to a method for identifying a bacterium, which comprises maintaining an atmosphere of 40 ° C. or higher and detecting a signal derived from a substance generated from the substrate substance by an enzyme activity of the bacterium. Hereinafter, the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is based on the determination of isolated bacteria by examining the presence or absence of enzyme activity of bacteria present in a test sample forming an isolated colony on a medium. The activity of the specific enzyme is confirmed using fluorescence or coloring with a coloring reagent. For example, an isolated colony on a medium obtained by separately culturing a test sample is collected and suspended in a solution (a buffer solution or the like) containing a substrate substance suitable for the enzyme activity of the bacterium, A substance produced by the enzymatic activity of the bacteria while being kept in an atmosphere is detected as fluorescence by ultraviolet irradiation or color development by the addition of a coloring solution.

The substrate used in the present invention includes 5-aminolevulinic acid or its hydrochloride. This compound can be identified by the biochemical properties of bacteria, which are deficient in certain bacteria (eg, Haemophilus influenzae) but act on enzymes that many other bacteria have.
A substance (for example, porphyrin) generated by the enzyme activity has a property of emitting fluorescence when irradiated with ultraviolet light. Therefore, a test sample is added to a solution containing this compound in advance on an appropriate plate medium (for example, a chocolate agar medium).
The isolated colonies grown on the medium by culturing at an appropriate temperature (for example, 37 ° C.) are suspended, and the fluorescence, which is a signal derived from the substance contained in the solution and generated by the enzyme activity of the bacteria, is visually observed. Alternatively, observation and detection can be performed using a device.

[0008] The substrate substance is used after being dissolved in purified water, physiological saline, or an appropriate buffer. The buffer that can be used in the present invention is not particularly limited as long as the enzyme activity of bacteria can be expressed. Specifically, for example, phosphate buffer, Tris-HCl buffer, MOPS (MOPS)
PS) and the like. The concentration and pH are not particularly limited. For example 0.01m
Buffers having a concentration of ol / l to 0.5 mol / l and a pH of 5.0 to 10.0 can be mentioned. Preferably, a 0.1 mol / l phosphate buffer pH 6.9 can be used.

A method for preparing a solution in which 5-aminolevulinic acid (hydrochloride) is dissolved may be a known method, and is not particularly limited. Specifically, a buffer solution having a pH of around 7.0 in which 5-aminolevulinic acid is dissolved to a final concentration of 0.001 to 2% is prepared and used. The amount of the liquid at the time of use (when the isolated colony is suspended and reacted) is also not particularly limited, and is 0.01 to
Although it can be widely used up to 10.0 ml, it is preferably 0.05 to 5.0 ml, more preferably 0.1 to 5.0 ml.
1.0 ml.

[0010] The substrate solution thus prepared can be tested by directly suspending the test bacterium. The substrate solution is impregnated with a water-absorbing carrier (for example, a cotton swab or filter paper) and dried.
In use, the test can be carried out by bringing the test bacteria into contact with the portion where the substrate is retained, or redissolving the substrate with purified water or a buffer, and then suspending the test bacteria.

An important requirement of the present invention is that the reaction for confirming the enzymatic activity of bacteria using the substrate is carried out in an atmosphere at 40 ° C. or higher. With the exception of specifically searching for heat-resistant enzymes such as those used in PCR, it is common knowledge that enzyme reactions generally performed in the field of biochemistry are measured at 37 ° C. Because, in many cases, the enzyme is inactivated by heating, and the measurement of the enzyme activity itself,
Alternatively, there is no idea to raise the temperature more than necessary in the analysis utilizing the enzyme activity. Conversely, it is common practice to control the temperature strictly because it is likely to cause disadvantages. In such a technical background, performing the enzymatic reaction under an atmosphere of 40 ° C. or more of the present invention
It cannot be easily conceived and has a special effect. In other words, the general procedure for testing pathogenic bacteria in clinical material is to isolate the bacteria from the test sample by culturing, and then determine the results after culturing in the biochemical property test medium for 1 to 7 days. It takes a long process of checking the bacterial species by comparing it with the identification table, and even if the Kirian method that improves this point does not provide sufficient bacterial mass, it takes 20 or more hours for the final enzyme reaction alone. In contrast, the present invention provides that a test sample can be applied to a suitable plate medium (eg, chocolate agar medium), cultured under normal conditions (eg, 37 ° C., 16 hours), and the bacteria can be isolated. The isolated colonies on the culture medium are suspended in the substrate solution prepared as described above, and the reaction of the bacteria with the enzyme at this time is carried out at a temperature of 40 ° C. or more, specifically, a temperature higher than 42 ° C., more specifically, By performing at 47 to 72 ° C, a sufficient amount of bacteria can be obtained. Kutomo compared to conventional means (37 ° C.),
The time required to obtain a discrimination result can be greatly reduced. For example, when the reaction is performed at 62 ° C., a clear discrimination result can be obtained within 30 minutes. Specifically, the isolated colony is brought into contact with 5-aminolevulinic acid under the above-described conditions, and the presence or absence of a signal derived from the generated substance (for example, when detecting porphyrin, the presence or absence of fluorescent light under ultraviolet irradiation) is determined in a very short time. It is possible to make a determination. This signal is visual observation,
Alternatively, fluorescence or coloring may be detected mechanically.

For example, when discriminating whether the isolated bacterium is Haemophilus influenza or Haemophilus parainfluenza, the colonies that have grown on the medium are, for example, 0.03%.
0.1 mo with 35% 5-aminolevulinic acid hydrochloride
1 / l of a phosphate buffer (pH 6.9) and reacted at, for example, 62 ° C. for 30 minutes.
60 nm), and the presence or absence of red fluorescence emitted from the porphyrin contained in the buffer is visually determined. As a result, when bacteria that cannot produce porphyrin (for example, Haemophilus influenza) are suspended, red fluorescence is not emitted,
When a porphyrin-producing bacterium (eg, Haemophilus parainfluenza) is suspended, red fluorescence is emitted, so that the two can be quickly distinguished.

The equipment used for adjusting the reaction temperature in the present invention is not particularly limited, and may be, for example, a water bath or an air bath.
An incubator, a heating block and the like can be mentioned, but preferably a water bath can be used.
Further, it is not necessary to perform an operation for gradually increasing the temperature until the target temperature is reached, and the temperature of the reaction solution may be quickly raised using a device such as a water bath that has reached the target temperature.

The test sample in the present invention includes human,
Bacteria isolated from all clinical materials such as blood, urine, feces, cerebrospinal fluid, pharyngeal fluid, sputum, bronchial secretions, nasopharyngeal fluid, and eye fluid of animals such as domestic animals can be used.

According to the present invention, Haemophilus influenza (Haemophilus influenza)
e), Haemophilus egypius
rus aegyptius, Haemophilus haemolytici
cus) can be rapidly differentiated from other hemophilus.

With such a configuration, for example, as shown in the following embodiments, the time until the determination can be greatly reduced. This is probably because increasing the reaction temperature increased the amount of porphyrin produced and increased the fluorescence intensity. Enzyme reaction is also a kind of catalytic reaction, and not only does the reaction speed increase with increasing temperature, but also the rapid change in fluorescence intensity at an appropriate temperature (for example, around 52 ° C) It is possible that the change is caused in the cell membrane and that the permeation amount of 5-aminolevulinic acid is increased. In addition, since an increase in temperature causes denaturation of the protein at the same time, when a certain temperature (for example, 77 ° C.) is reached, the enzyme activity decreases, and the temperature falls from 47 ° C. to 72 ° C.
It seems that the optimum temperature up to has been established.

[0017]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 (Preparation of Substrate-Containing Buffer) The reagent used in the present invention was prepared according to the well-known method "Killian's method". Specifically, 0.097 g of 5-aminolevulinic acid hydrochloride (Wako Pure Chemical Industries, Ltd.) is 1.97 m.
dissolved in 100 ml of a 0.1 mol / l phosphate buffer (pH 6.9) containing g / l magnesium sulfate (0.0
335% solution), and 500 μl thereof was dispensed into a small test tube and used.

(Test Bacteria and Culture of Test Bacteria) As test bacteria used as test samples, 20 clinical isolates identified using ID Test NH-20 Rapid (Nissui Pharmaceutical) were used. The breakdown is Haemophilus influenza (Ha
emophilus influenzae) is 10
Strain, Haemophilus parainfluenza (Haemop)
hilus parainfluenzae). After culturing the 20 test strains on a chocolate agar medium (Nissui Pharmaceutical) at 37 ° C. for 18 hours, isolated colonies (several to several tens) on the medium were prepared as described above. In a test tube into which 500 μl of the aminolevulinic acid hydrochloride-containing buffer was dispensed, MacFarland No. 3 and suspended at 42 ° C, 47 ° C, 52 ° C, 57 ° C,
Water set at 62 ° C, 67 ° C, 72 ° C, 77 ° C
Move the test tube containing the reaction solution into the bath
After reacting for 0 minute, 1 hour and 4 hours, the suspension was returned to room temperature (25 ° C.), and then irradiated with 360 nm ultraviolet light.
The presence or absence of the fluorescent light emitted therefrom was visually determined.

(Conventional Method) As a comparative example, a porphyrin production test was conducted by the conventional method, "Killian's method". The test bacteria and reagents used were the same as those of the present invention, and the operation was the same as that of the present invention except that the operation was carried out at 37 ° C.

Table 1 shows the results of 10 strains of Haemophilus parainfluenza according to temperature and reaction time. The hemophilus influenza was negative for both the present invention and the conventional method (table not shown).
The conventional method (37 ° C., 4 hours) and the method of the present invention (62
(° C., 30 minutes) is shown in Table 2.

[0021]

[Table 1] In case of Haemophilus parainfluenza -Indicates negative, + indicates weak positive, ++ indicates positive, +++ indicates strong positive

[0022]

[Table 2]

From the results, it has been confirmed that by setting the reaction temperature from 47 ° C. to 77 ° C., the time until the judgment result is obtained can be greatly reduced. In addition, the results of discriminating the biochemical properties of the two methods according to the conventional method and the present invention were in agreement.

[0024]

As described above, according to the present invention, in the case of distinguishing a colony grown on a culture medium from clinical materials, the conventional method required 4 to 24 hours to judge the result. According to the present invention, discrimination is possible at least within 30 minutes after the growth of the bacteria is recognized on the medium. In addition, the results were completely consistent with the test results of the conventional Haemophilus differentiation method, and its specificity was confirmed. Thus, the present invention, which is specific and extremely rapid, greatly contributes to the diagnosis and treatment of pneumonia and meningitis, which may be caused, for example, by Haemophilus influenza.

Claims (4)

[Claims] Claims: 1. In distinguishing bacteria by biochemical properties, an isolated colony grown on a medium by culturing a test sample is brought into contact with a substrate substance, kept at an atmosphere of 40 ° C. or more, and the enzyme activity of the bacteria A method for identifying bacteria, comprising detecting a signal derived from a substance generated from the substrate substance by the method. 2. In the step of distinguishing bacteria by biochemical properties, (a) suspending an isolated colony grown on a medium by culturing a test sample in a solution containing 5-aminolevulinic acid; A) a step of maintaining the suspension in an atmosphere of 40 ° C. or higher; (c) a step of producing or generating a substance produced from 5-aminolevulinic acid by the enzymatic activity of the bacterium in the step (b). 2. The method for identifying bacteria according to claim 1, further comprising a step of detecting a color by adding a color liquid. 3. In distinguishing bacteria by biochemical properties, (d) contacting an isolated colony grown on a medium by culturing a test sample with a water-absorbing carrier containing 5-aminolevulinic acid. (E) maintaining the carrier in an atmosphere of 40 ° C. or higher by directly or immersing the carrier in a solution;
(F) detecting the substance produced from 5-aminolevulinic acid by the enzyme activity of the bacterium in the step (e) as fluorescence by ultraviolet irradiation or color development by addition of a color developing solution. The method for identifying bacteria according to claim 1. 4. The method according to claim 1, wherein the bacterium is of the genus Haemophilus.