JPH09117299A - Bacterial antibiotic susceptibility testing method - Google Patents

Abstract

(57) Abstract: [PROBLEMS] To provide a test method for bacteria susceptibility to antibiotics, which has a high test accuracy, is simple, and can be processed in a short time. SOLUTION: Escherichia coli is collected from a bacterial solution containing E. coli by centrifugation, an aqueous solution of antibiotics is added to the obtained bacterial precipitate, mixed well, and then left at room temperature for a reaction time of 1 hour. The treatment liquid is dropped on a cover glass, air-dried, and then observed with an atomic force microscope. Escherichia coli not reacted with antibiotics is also observed with an atomic force microscope to compare the morphology of both. Those whose morphology is changed by the reaction with antibiotics are judged to have antibiotic sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for rapidly and simply testing the susceptibility of bacteria isolated from various clinical test materials such as urine, blood, sputum, cerebrospinal fluid, feces and pus to antibiotics. . This test method is useful in the medical field, especially in the diagnosis and treatment of bacterial infections, and is used to provide clinicians with the test results required for optimal antibiotics in order to carry out appropriate treatment of bacterial infections. can do.

[0002]

2. Description of the Related Art Testing of bacteria for susceptibility to antibiotics is generally carried out by a disc test method. In the disc inspection method, a filter paper disc (diameter 5 to 10 mm) which has absorbed a certain amount of antibiotics is placed on an agar medium having a diameter of about 90 mm coated with a test bacterium and cultured. If the test bacterium is resistant to the antibiotic, the test bacterium grows on the entire surface of the agar medium. On the other hand, when the test bacterium is sensitive to the antibiotic, a concentric area with the filter paper disc, called a “blocking circle”, is formed in which the test bacterium has not grown. The diameter of this inhibition circle is the minimum inhibitory concentration (MIC) of the test organism.
Therefore, the diameter of the inhibition circle is measured, and whether the test bacterium is a susceptible bacterium or a resistant bacterium is determined by measuring the diameter of the inhibition circle.

[0003]

The disk diffusion method has the following problems. (1) The antibiotic contained in the filter paper disc exudes into the agar medium, but the exudation amount is affected by the water content in the medium and the medium temperature (or humidity), so the conditions cannot be clearly defined. Therefore, even when using the same test strain,
There is a high possibility that the size of the stop circle will be different. As a result, the inspection accuracy must be low. (2) Since the formation of the inhibition circle is affected by many factors such as the components of the medium used, the amount of inoculated (coated) bacteria, the thickness of the medium, the culture time, and the CO ₂ concentration in the atmosphere, it is highly accurate and effective. In order to obtain accurate inspection results, detailed condition setting is necessary, and the convenience is poor. (3) Since it is difficult to make individual inspection conditions constant, it is impossible to compare each data. (4) Since culture is performed for inspection, the inspection requires a long time. Therefore, an object of the present invention is to solve such problems of the disk diffusion method, and to provide an inspection method which has a high inspection accuracy, is simple, and can be processed in a short time.

[0004]

Means for Solving the Problems A predetermined amount of antibiotics is added to a liquid containing a predetermined amount of test bacteria and reacted for a predetermined time. If the test bacterium is sensitive to the antibiotic, the morphology of the test bacterium undergoes changes such as swelling and elongation due to the action of the antibiotic. However, this morphological change occurs when the reaction time with antibiotics is short, such as about 1 hour,
It is difficult to find with an optical microscope. Therefore, in the present invention, a scanning probe microscope observable in the atmosphere such as an atomic force microscope is used to observe the bacterial solution treated with the antibiotic and the untreated bacterial solution, and the morphology is compared to determine whether the antibiotic is not treated. A test bacterium in which a morphological change is recognized as compared with the treated test strain is determined as a susceptible bacterium.

[0005]

[Examples] An example of measuring the morphological change of a test bacterium using an atomic force microscope will be described. The atomic force microscope uses a cantilever with a sharp tip on the tip and a size of several hundred μm. The cantilever is brought close to the sample surface, and the atomic force acting on the cantilever is replaced by the cantilever deflection. It is something to detect. The sample is scanned with high precision in the 3D direction by the scanner, the cantilever traces the fine shape of the sample surface, the movement of the cantilever is detected by detecting the reflected light from the back surface of the cantilever, and the surface shape of the sample is measured. It

Escherichia coli H10407
Strain and Staphylococcus aureus FR
Experiments were performed using the I1151-7NG strain. After culturing these strains in a brain heart infusion medium at 37 ° C. overnight, the turbidity (O
D ₆₀₀ ) was measured and the approximate amount of bacteria was quantified. The turbidity of both strains was 3.06 to 3.08 / ml.

1 ml of these bacterial liquids were rotated at 10,000 r
The cells were collected by centrifugation at pm for 5 minutes at room temperature. To the obtained bacterial precipitate, 1 ml of an aqueous antibiotic solution adjusted to the concentration shown in Table 1 below was added, mixed well, and then left at room temperature for a reaction time of 1 hour. Then, 5 μl of the treatment liquid was dropped on a cover glass (15 mm in diameter), air-dried, and then observed with an atomic force microscope (SPM-9500 manufactured by Shimadzu Corporation). The antibiotic 0 μg / ml in the table was used as a control, and 1 ml of distilled water containing no antibiotic was added to the bacterial precipitate.

Table 1 shows the results with E. coli and S. aureus. Both bacteria gave the same results. Three kinds of antibiotics were used: ampicillin, cephalosporin, and tetracycline. + In the table indicates a morphological change,
− Means no morphological change, ± means indeterminate.

[0009]

[Table 1]

Atomic force microscope images of E. coli treated with and without the antibiotic ampicillin are shown in FIG. When ampicillin was not reacted at the left end of the upper row, the Ecoli 2 to 5 arranged from the second row to the lower row of the upper row have concentrations of 3, 10, and 3 shown in Table 1.
This is the case where 0,100 μg / ml of ampicillin was reacted. It can be seen that E. coli is swollen by the action of ampicillin. In addition to the isolated bacterium, the present invention also collects bacteria contained in the sample by centrifuging the sample which is originally sterile such as blood, urine, and cerebrospinal cord by the method of the example. Can be inspected.

[0011]

INDUSTRIAL APPLICABILITY According to the present invention, since an antibiotic is directly reacted with a test bacterium, it is less affected by others, and the effect of the antibiotic on the test bacterium can be directly determined. Can be done easily. Since a scanning probe microscope such as an atomic force microscope is used, the morphological image of bacteria, which is the inspection data, is stored as digital data in a computer and can be used as a database thereafter. In addition, quantitative data such as the average value of the size of bacteria can be obtained from the data, and accuracy management can be facilitated by creating a database of test results. The reaction time required for the inspection may be about 1 hour. This is shorter than the culture time (about one night) for inspection in the conventional disc diffusion method. The simplification of the inspection process, in particular, the fact that the culture is not necessary makes it possible to speed up the inspection.

[Brief description of the drawings]

FIG. 1 is a photograph showing an atomic force microscope image of Escherichia coli,
If the ampicillin is not reacted, the left end of the upper row is the Ecoli located from the second to the bottom of the upper row.
2 to 5 are the cases where ampicillin having a concentration of 3, 10, 30, and 100 μg / ml was reacted.

Claims (1)

[Claims] 1. A step of (A) adding an antibiotic to a liquid containing a test bacterium to react with each other, and (B) a test bacterium reacted with the antibiotic and a non-reacted test bacterium, respectively, to the atmosphere. Observing with a scanning probe microscope observable in
A method of comparing the morphology of both test bacteria, and examining the susceptibility of the test bacteria to the antibiotics depending on the presence or absence of a change in the morphology of the test bacteria due to reaction with the antibiotic.