JPH0538298A - Detection of aminoglycoside antibiotic - Google Patents

Abstract

PURPOSE:To highly sensitively detect the subject antibiotic by growing a microorganism sensitive to an aminoglycoside antibiotic in an agar medium containing the antibiotic in a concentration below a minimum growth-inhibiting concentration, bringing the medium into contact with a specimen and judging the extent of the growth inhibition. CONSTITUTION:A method for highly sensitively detecting an aminoglycoside antibiotic, especially spectinomycin, in a specimen comprises growing a microorganism [e.g. Klebsiella.pneumoniae (ATCC 10031)] sensitive to the aminoglycoside antibiotic in an agar medium containing the aminoglycoside antibiotic in a concentration below the minimum growth-inhibiting concentration against the microorganism, forming an agar well in the agar medium with a punch, injecting the specimen into the agar well for the contact of the specimen with the agar medium, culturing the microorganism, and subsequently measuring the diameter of the formed inhibition circular zone for the judgment of the extent of the growth inhibition.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a detection method capable of detecting aminoglycoside antibiotics, particularly spectinomycin with high sensitivity.

[0002]

2. Description of the Related Art As a method for detecting and measuring the concentration of an antibiotic in a sample, a bioassay method using a microorganism sensitive to the antibiotic and an ultraviolet absorption spectrum using high performance liquid chromatography (HPLC) Have been developed. These detection / quantification methods are widely used as a study of the pharmacokinetics when the antibiotic is administered to a living body, and a method of detecting the antibiotic remaining in the meat of livestock to which the antibiotic has been administered.

However, no microorganisms having high sensitivity to aminoglycoside antibiotics, particularly spectinomycin widely used as a veterinary drug have been found, and detection by a bioassay has been difficult. Further, since aminoglycoside antibiotics do not exhibit strong spectroscopic absorption, it has been difficult to develop a quantitative method with high detection sensitivity using an ultraviolet absorption spectrum or the like.

[0004]

Accordingly, the present invention provides a detection method capable of detecting aminoglycoside antibiotics, particularly spectinomycin, with high sensitivity.

[0005]

Means for Solving the Problems As a result of diligent efforts to solve the above problems, the present inventor used microorganisms sensitive to aminoglycoside antibiotics and added aminoglycoside antibiotics to a medium in advance at a constant concentration. It was found that aminoglycoside antibiotics can be detected with high sensitivity by performing a bioassay using the above method, and the present invention has been completed.

That is, the present invention is a method for detecting an aminoglycoside antibiotic in a sample, comprising: (a) a microorganism sensitive to an aminoglycoside antibiotic, wherein the aminoglycoside antibiotic is contained at a concentration lower than the minimum inhibitory concentration for the microorganism. Growing on an agar medium containing antibiotics, and (b)
The present invention provides a detection method including a step of contacting a sample with the agar medium and then culturing the sample to determine the presence or absence of growth inhibition of microorganisms.

The aminoglycoside antibiotic which can be detected by the method of the present invention is a basic antibiotic having a glycoside of an amino sugar and a sugar or a pseudo sugar of cyclitol or aminocyclitol sugar in the structure. Yes, for example, neomycin, paromomycin, ribidomycin, ribostamycin, xylostacin, butyrosine, kanamycin,
Gentamicin, sisomicin, netilmicin, streptomycin, dihydrostreptomycin, spectinomycin, dihydrospectinomycin, hotimicin,
Examples thereof include sporalysin, istamycin, dactimicin, kasugamycin, viomycin and the like. Of these, spectinomycin, kasugamycin, viomycin, streptomycin, kanamycin, neomycin, and gentamicin are preferable, and spectinomycin is particularly preferable. These preferred aminoglycoside antibiotics act at the initiation stage of protein synthesis on the ribosome to inhibit protein synthesis. The method of the present invention includes urine of humans and animals administered with these aminoglycoside antibiotics, liquid samples such as serum, lymph fluid, bone marrow fluid, and biological samples such as solid samples such as tissue pieces and meat pieces. It can be used for the purpose of detecting or quantifying aminoglycoside antibiotics. These samples may be directly used in the method of the present invention, but preferably, these biological samples are extracted with water, buffer solution, organic solvent or the like, and sample solution or sample suspension is prepared by deproteinization. May be adjusted and then assayed by the method of the present invention.

In step (a) of the method of the present invention, a microorganism sensitive to an aminoglycoside antibiotic is grown on an agar medium containing the aminoglycoside antibiotic at a concentration below the minimum inhibitory concentration for the microorganism. A microorganism sensitive to an aminoglycoside antibiotic is a microorganism having a minimum inhibitory concentration (MIC) of 10 μg / ml or less when the above aminoglycoside antibiotic is acted on. For example, when spectinomycin is detected by the method of the present invention, microorganisms that are highly sensitive to spectinomycin, such as Escherichia col.
i) It is preferable to use B2029 or Escherichia coli B2030. The minimum inhibitory concentrations of these microorganisms when using Mueller Hinton Agar are shown in Table 1 below.

[0009]

[Table 1] Strain pH 7.0 8.0 8.5 9.0 E. coli B 2029 12.5 1.56 1.56 0.39 E. coli B 2030 12.5 3.13 1.56 0.39 Escherichia coli B2029 and Escherichia
Kori B2030 was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as of October 31, 1988, and Microtechnical Laboratory No. 10376 (FERM P-10376) and 10 respectively.
Deposited as 378 (FERM P-10378). However, the method of the present invention is not limited to these microorganisms, and it is easily understood by those skilled in the art that any microorganism susceptible to aminoglycoside antibiotics can be used in the method of the present invention.

The agar medium used in the method of the present invention is a medium containing the above aminoglycoside antibiotic at a concentration lower than the minimum inhibitory concentration against the above microorganisms. The minimum inhibitory concentration of the aminoglycoside antibiotic against the microorganism used can be easily determined by a method well known to those skilled in the art, for example, the standard method of the Japanese Society of Chemotherapy. The same aminoglycoside antibiotic as the aminoglycoside antibiotic to be detected by the method of the present invention is preferably added to the medium, but an aminoglycoside antibiotic of a different type from the aminoglycoside antibiotic to be detected is added to the medium. May be added to. In this case, the two kinds of aminoglycoside antibiotics are preferably selected from the group consisting of spectinomycin, kasugamycin, viomycin, streptomycin, kanamycin, neomycin, and gentamicin, which have similar action mechanisms to each other. Furthermore, it is particularly preferable that both the antibiotic to be detected and the antibiotic contained in the medium are spectinomycin.

Examples of the agar medium used in the method of the present invention include ordinary agar medium, Mueller Hinton agar medium,
Heart infusion agar medium or the like can be used. Additives such as Ca ²⁺ and Mg ²⁺ may be added to the agar medium as long as they do not inhibit the growth of the microorganism used. The pH of the medium can take various values depending on the microorganism used,
For example, when Escherichia coli B2029 or Escherichia coli iB2030 is grown in a Mueller Hinton medium, the pH of the medium is preferably about 9. In this case, the minimum inhibitory concentration of spectinomycin was 0.39 μg / m 2 for the above microorganisms.
l, 0.39 μg / ml, so 0.2 μg / m
Spectinomycin at a concentration of 1 to 0.3 μg / ml, preferably 0.3 μg / ml may be added to the medium. Generally 50 to 7 based on the minimum inhibitory concentration of the microorganism used
An aminoglycoside antibiotic at a concentration of 0% may be added to the medium. If the concentration of aminoglycoside antibiotics in the medium is below this range, the detection sensitivity will be inadequate, and if the concentration of aminoglycoside antibiotics exceeds the above range, it will be difficult for microorganisms to grow on the medium. Occurs.

In order to grow a microorganism on an agar medium containing an aminoglycoside antibiotic, an aminoglycoside antibiotic is added to a predetermined medium composition at a predetermined concentration and an agar medium serving as a base layer is prepared by a conventional method. Examples of the method include a method in which a liquid medium containing the microorganisms produced and applied to the surface of the agar is applied and then cultured. As a more preferable method, a layered medium containing an aminoglycoside antibiotic is prepared in the same manner as the base layer medium, a culture solution of a microorganism or a dilution thereof is added before the medium is solidified, and the layer is solidified by overlaying on the base layer medium. A method of producing and culturing a multi-layered agar medium by carrying out the above can be mentioned.

In order to bring the above sample into contact with the agar medium on which the microorganisms obtained as described above are grown, a paper disk impregnated with a fixed amount of the above liquid sample, a cylinder filled with the sample, etc. A method of contacting the agar surface on which the microorganism has grown, or a method of directly applying a liquid sample to the agar surface, dripping, spraying or the like can be mentioned, but preferably, after forming an agar well in the above-mentioned layered agar medium, A method of injecting a liquid sample into the well may be used. The agar well having a diameter of, for example, about 6 to 8 mm, preferably about 8 mm, and a depth of about 2.5 to 3 mm, preferably about 3 mm may be formed on an agar medium by a conventional method. A sample of, for example, about 80 to 120 microliters, preferably about 100 microliters, can be injected into the agar well with a micropipette. Spectinomycin in the sample diffuses into the medium due to the concentration gradient with the medium. At the same time, the test bacterium grows independently of the diffusion of the drug, and under these conditions, a zone of inhibition is formed at the boundary around the MIC value of the drug in the medium against the test bacterium.

After the sample is brought into contact with the above-mentioned agar medium on which the microorganisms have been grown as described above, for example, at about 35 ° C., about 20 to
It may be cultured for 24 hours to determine the degree of growth of microorganisms. When the sample to be assayed contains aminoglycoside antibiotics, the growth inhibition zone such as an inhibition zone is observed on the agar due to the death or growth inhibition of the microorganisms in contact with the sample, This case may be determined as positive. When the method of the present invention is used for the purpose of quantifying aminoglycoside antibiotics, the above assay is carried out under the same conditions for samples containing known different concentrations of aminoglycoside antibiotics, and the inhibition circle for each sample is determined. The concentration of the aminoglycoside antibiotic in the sample can be easily determined by preparing the calibration curve (for example, antibiotic concentration-diameter of inhibition circle) by determining the diameter of the.

[0015]

INDUSTRIAL APPLICABILITY According to the method of the present invention, aminoglycoside antibiotics, particularly spectinomycin, which cannot be easily detected by conventional methods, can be easily detected.
For example, K. pneumoiae A, a standard strain commonly used for titration of spectinomycin.
When TCC10031 was used to detect spectinomycin in Mueller Hinton agar without the addition of aminoglycoside antibiotics, the detection limit was 25 μg / ml, whereas in the method of the present invention, Escherichia coli B2029 was used to add spectinomycin ( Mueller Hinton Agar (pH: 0.3 μg / ml)
The detection limit when spectinomycin is detected in 9) is
It is 0.5 μg / ml, and a detection sensitivity increase of about 50 times can be obtained. Therefore, a trace amount of aminoglycoside antibiotics in a biological sample can be accurately detected. Furthermore, the method of the present invention is useful because it can be used as a method for quantifying aminoglycoside antibiotics.

[0016]

The present invention will be described in more detail with reference to the following examples, but the method of the present invention is not limited to these examples. Example Each of the microorganisms was inoculated into a Mueller Hinton medium (manufactured by Difco, pH unadjusted) from the preserved cells, and statically cultured at 35 ° C. for 18 to 20 hours. Mueller Hinton agar medium (pH 8.9-9.0) containing 1.5% agar (manufactured by Wako Pure Chemical Industries) as a base layer medium
: Adjusted with 1N NaOH) and add spectinomycin so that the final concentration will be the specified concentration after sterilization.
The solution was poured into a 4th-version-sized test plate (made by Iwashiya, microbial culture test box) and solidified. Then, as a layered medium, Mueller Hinton agar medium (pH 1.0) containing 1.0% agar (manufactured by Wako Pure Chemical Industries, Ltd.)
8.9-9.0: adjusted with 1N NaOH) and sterilized after 48.
While maintaining the temperature at 5 ° C, spectinomycin was added to the above-mentioned substratum medium so as to have the same concentration, and further, the above culture solution was diluted 50 times with physiological saline to inoculate it to 1%, Layered on the above-mentioned base layer medium. After the agar solidified, an agar well with a diameter of 8 mm was prepared using a perforator (manufactured by System Science) or a cork borer, and 100 microliters of a sample containing spectinomycin was injected. The assay plate was incubated at 35 ° C. for 20-24 hours, and the diameter of the formed inhibition circle was determined. The results are shown in Table 2 below.

[0017]

[Table 2] Concentration of spectinomycin added to spectino assay plate No mycin (μg / ml) 0.08 0.15 0.3 0.6 1.2 E. coli NIHJ 20.6 mm--22.0 25.2 25 16.0--17.6 21.1 12.5 11.8--13.4 16.2 6.25 ---9.6 12.7 3.13-----No growth 1.56-----0.78----- Standard curve correlation coefficient 1.000--0.999 0.998 E. coli B2029 25 24.8 26.2 27.8 38.8 12.5 20.1 21.6 23.4 35.0 6.25 16.0 17.6 19.7 30.8 3.13 12.0 13.8 15.5 27.7 No growth 1.56 trace 9.6 11.6 23.0 0.78 0 0 0 18.3 Standard curve correlation coefficient 0.999 0.999 1.000 0.998 E. coli B2030 25 24.4 25.7 27.0 36.6 12.5 19.7 21.3 23.0 31.8 6.25 15.2 16.8 18.9 28.4 3.13 11.4 12.8 14.6 23.4 No growth 1.56 0 0 10.6 18.8 0.78 0 0 0 14.2 Correlation coefficient of calibration curve 0.999 1.000 1.000 0.999 In Fig. 1, Mueller Hinton supplemented with 0.3 μg / ml spectinomycin. The results of the above experiments carried out in culture medium, and using the Klebsiella pneumoniae ATCC 10031 as a control, Shows the results of similar experiments in Mueller Hinton agar medium Chinomaishin no additives. Figure 1 and Table 2
From the results shown in, it is possible to significantly improve the detection sensitivity by using the medium containing spectinomycin, and the phase relationship of the calibration curve when the assay was performed using the medium containing spectinomycin. It is clear that the numbers are very good.

[Brief description of drawings]

FIG. 1 shows the results of carrying out the method of the present invention in Mueller Hinton medium supplemented with 0.3 μg / ml spectinomycin.
FIG. 3 is a diagram showing the results of a similar experiment performed using Klebsiella pneumoniae ATCC 10031 as a target on a Mueller Hinton agar medium without spectinomycin.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Rokuro Okamoto 2-18 Hananoki, Fujisawa City, Kanagawa Prefecture

Claims (4)

[Claims] 1. A method for detecting an aminoglycoside antibiotic in a sample, comprising: (a) containing a microorganism sensitive to an aminoglycoside antibiotic at a concentration less than a minimum inhibitory concentration for the microorganism. A detection method comprising a step of growing on an agar medium, and (b) a step of contacting the sample with the agar medium and then culturing the sample to determine the presence or absence of growth inhibition of a microorganism. 2. The method according to claim 1, wherein the aminoglycoside antibiotic in the agar medium and the aminoglycoside antibiotic in the sample are the same antibiotic. 3. The method according to claim 2, wherein the aminoglycoside antibiotic is spectinomycin. 4. The method according to claim 1, wherein the sample is contacted with an agar medium by the Agarwell method.