JPS61249400A - Determination of beta-lactamase and material used therefor - Google Patents

Abstract

PURPOSE:A solid culture medium for gram-negative bacteria to which a specific quantitative indicator is combined with a specimen, subjected to cultivation to determine beta-lactamase whereby the resistance of a gram-negative bacterium of beta-lactamse is quantitatively measured. CONSTITUTION:A solid culture medium for gram-negative bacteria which contains 3-(2-azo-p-dimethylanilino-1-pyridiummethyl)-(6R, 7R)-(2-thienylacet amide)-3-cephem-4-carboxylate which changes its color from violet to yellow by hydrolysis with beta-lactamase is inoculated with a specimen and cultured. When the specimen produces beta-lactamase, a ring of color-changing zone is formed around the colony and the size of the color-changing zone is logarithmi cally proportional to the production of beta-lactamase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for quantitatively measuring β-lactamase produced by Gram-negative bacteria and a material used for the measurement.

[Prior art]

Among antibiotics, penicillins and cephalosporins are β-lactam antibiotics that are widely used clinically. Among the mechanisms by which bacteria exhibit resistance to these β-lactam antibiotics, β-lactamase production accounts for a significant proportion.

β-lactamase is an enzyme that specifically hydrolyzes the β-lactam ring of β-lactam antibiotics and deactivates the β-lactam antibiotics.

If the causative bacteria isolated from a patient with an infectious disease produces β-lactamase, it often shows resistance to β-lactam antibiotics. - Lactam antibiotics or antibiotics other than β-lactams must be selected. To this end, it is important to know, prior to treatment, whether the causative bacterium exhibits resistance to β-lactam antibiotics, that is, whether it produces β-lactamase, and in particular, the degree of resistance. Qualitative and quantitative measurement of β-lactamase provides important guidelines for selecting the type and amount of antibiotic to be administered during treatment.

Conventional simple β-lactamase measurement methods basically belong to the iodometric method, the acid metric method, or the chromogenic cephalosporin method, but all of these methods require a reagent solution or a test strip containing the reagent. This is a method of visually examining color changes such as, etc., and is not quantitatively sufficient. In the chromogenic cephalosporin method, conventionally, a bacterial colony is rubbed on a piece of paper impregnated with a chromogenic cephalosporin, and the presence or absence of β-lactamase production is determined by the change in color of the paper piece. However, since such a method can only perform qualitative detection, it has hardly been put into practical use in hospital laboratories or the like.

In addition, in the case of Gram-negative bacteria, the β-lactamase produced is located in the space between the outer and inner membranes of the bacteria (periplasm).
Measuring the amount of β-lactamase produced requires destruction of the outer membrane, such as crushing the bacterial cells, and the β-lactamase measurement operation is complicated, labor-intensive, and time-consuming.

〔the purpose〕

An object of the present invention is to provide a method for simply and quantitatively measuring the degree of resistance of Gram-negative bacteria to β-lactam antibiotics, and a material for the measurement.

〔composition〕

According to the present invention, as a first invention, 3-(2-azoP-dimethylanilino-1-pyridiniummethyl)-(
6R,7R)-7-(2-thienylacetamide)-3
- Adding the specimen to a solid medium for Gram-negative bacteria containing cefem-4-carboxylate as a quantitative indicator,
Provided is a method for culturing bacteria in a sample and quantitatively measuring β-lactamase produced by Gram-negative bacteria in the sample based on the size of a discolored zone formed by the reaction of the quantitative indicator around the developed colonies. As invention 2, 3-(
A solid for Gram-negative bacteria containing 2-azo-p-dimethylanilino-1-pyridinium methyl)-(6R,7R)-7-(2-thienylacetamide)-3-cephem-4-carboxylate as a quantitative indicator. A measurement material for quantifying β-lactamase produced by Gram-negative bacteria is provided, which comprises a culture medium.

In the present invention, 3-(2-azoP-dimethylanilino-1-pyridiniummethyl)-(6R,7R)-7-(2-thienylacetamido)-3-cepheme-4-carboxylate used as a β-lactamase quantitative indicator PADAC (hereinafter abbreviated as PADAC) is a chromogenic cephalosporin that has a structure represented by the following formula and is known to change color from purple to yellow when subjected to a hydrolysis reaction by β-lactamase.

In the present invention, a sample is added to a solid medium for Gram-negative bacteria containing PADAC, and the bacteria in the sample are cultured. In this case, when the sample produces β-lactamase, the production According to the research conducted by the present inventors, the discolored band formed around the colony is due to the combination of β-lactamase and PADAC produced by Gram-negative bacteria contained in the specimen. It is formed as a result of hydrolysis of the lactam ring of PADAC, and the size of the discolored zone (large area) and the amount of β-lactamase produced are almost logarithmically proportional. was discovered.

In general, β-lactamase produced by Gram-negative bacteria is not secreted outside the bacterial body, unlike Gram-positive bacteria. The method for measuring the amount of β-lactamase produced in the body is an amazing method that has not been thought of in the past.

That is, the advantageous and excellent point of the present invention is that by directly adding a specimen collected from a patient with an infectious disease to the medium according to the present invention and culturing it for a certain period of time, the causative bacteria can be separated from the specimen, and at the same time, β-lactamase can be isolated from the specimen. It is possible to know whether or not there is production. Furthermore, the feature of using PADAC is that β-
The discolored zone produced by the reaction with lactamase is very stable in the solid medium and does not change in size, so accurate measurements can be made even if it takes a long time after culture. The point is that it is possible. PADAC
With other chromogenic cephalosporins, the stability of the discolored band formed is poor; therefore, it has been confirmed that PADAC has a great advantage from a practical point of view compared to other chromogenic cephalosporins. It was done.

To carry out the method of the present invention, a solid medium containing PADAC (usually purple in color) is prepared, a specimen collected from a patient, such as blood or urine, is added to this solid medium, and the mixture is usually heated at 37°C. Culture for a certain period of time (usually 18 to 24 hours). As a result of this culture, if the bacteria isolated from the specimen are β-lactamase producing bacteria, along with the development of colonies,
The color of the solid medium surrounding the colony changes from purple to yellow. Production of β-lactamase was qualitatively confirmed by the formation of a yellow annular discolored zone around the colony, and the diameter or area of the yellow discolored zone was measured.

The amount of β-lactamase produced can be determined quantitatively by comparing this measured value with a calibration curve separately prepared from a strain with a known amount of β-lactamase produced.

In the method of the present invention, if the diameter of the discolored zone formed around the colony is small and it is difficult to measure the amount of β-lactamase produced with high accuracy, it is desirable to pre-culture the specimen before applying the method.

The operation in that case is as follows.

Place the specimen containing the bacteria to be tested in liquid culture overnight.
Culture at 7°C, and use the stock solution or diluted solution of the bacteria at 5°C.
It was added dropwise to a solid medium containing PADAC using a μQ-10μa micropipette and kept at 37℃ for a certain period of time (usually 18~18℃).
24 hours). According to this method, a discolored zone of approximately 8 mm to 40 mm is formed around one developed colony in proportion to the amount of β-lactamase produced, allowing for highly accurate measurements.

In the present invention, a method for adding a specimen to a solid medium is to take a predetermined amount of the specimen, for example, 5 μQ to 10 μQ, with a pipette or the like and drop it onto the surface of the solid medium. In this case, the number of bacteria in the sample dropped onto the solid medium is adjusted to a predetermined number of bacteria, usually 10' to 10 degrees per 1 mA. When the number of bacteria contained in the specimen is small and it is difficult to perform highly accurate measurement, it is preferable to pre-culture in a liquid medium and use the resulting bacterial solution or diluted solution as the specimen, as described above.

It is understood from the results shown in the drawings that the diameter of the discolored zone is proportional to the amount of β-lactamase produced within the bacterial cells.

The solid medium used in the present invention is for general Gram-negative bacteria, and for visibility, it is sufficient that the medium itself is close to transparent or close to white.

Specific examples of media include ``Heart Infusion Agar Medium'' (manufactured by Eiken Chemical Co., Ltd. and Nissui Pharmaceutical Co., Ltd., hereinafter referred to as Eiken, Japan), and ``Trypto Soy Agar Medium'' (manufactured by Eiken Chemical Co., Ltd. and Nissui Pharmaceutical Co., Ltd., respectively). ), “Ordinary agar medium” (Eiken 1
Nissui, manufactured by Kyokuto Pharmaceutical Industry Co., Ltd. (hereinafter referred to as Kyokuto)
, "Agar medium for sensitivity measurement" (Japan), "Standard agar medium" (Eiken, Japan, Kyokuto), etc. are commercially available in powder form.
You can use these things.

The measurement material used in the present invention is composed of the above-mentioned general solid medium for Gram-negative bacteria mixed with PADAC. In this case, the amount of PADAC mixed is as follows:
The medium may normally contain a predetermined amount selected from the range of 25 to 100 μN, for example, 50 μN.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example A: Preparation of solid medium for measurement (1) Heart infusion agar medium (Eiken) was adjusted to twice the specified concentration (80 g/Q) and sterilized by heat.

The culture medium (A) is obtained by incubating at about 50°C.

(2) 50m of PADAC powder to 100μN
PADA was dissolved in M phosphate buffer (pF17.0).
C solution (B) is obtained.

(3) Mix the above medium (A) and PADAC solution CB) at a volume ratio of Nl, 4 mQ for a petri dish with a height of 6 mm x a diameter of 15 mm, and 10 m for a petri dish with a length of 90 mm and a height of 15 mm.
Dispense Q to prepare a purple agar plate.

B: Measurement of β-lactamase Next, an example will be shown in which the amount of β-lactamase produced by Enterobacter cloacae isolated from patient urine was measured.

The test bacterium Enterobacter cloacae isolated from the specimen was cultured overnight at 37°C in heart infusion broth. In addition, in order to prepare a calibration curve, we crushed the bacterial cells and
- 10 standard strains whose lactamase production has been measured in advance were also added to heart infusion broth.
Cultured overnight at 7°C.

Dilute the test bacteria and standard bacterial strain in the same medium at a rate of 10' bacteria per 1 mA, and drop the diluted bacterial solution using a 5 μΩ micropipette onto the agar medium containing the PADAC prepared in Example (A) above. and cultured at 37°C for 24 hours.

The calibration curve shown in the drawing was prepared from the diameter of the annular discolored zone formed around the colony of the standard strain and the amount of β-lactamase produced. On the other hand, the diameter of a ring-shaped yellow discolored zone formed around the colony of the test bacteria cultured at the same time was 30 mm. Based on this value, the amount of β-lactamase produced by the test bacteria was determined using a calibration curve, and was found to be 0.12 units/g total protein.

Furthermore, in order to compare the values obtained with the conventional β-lactamase measurement method, we disrupted the test bacteria, isolated intracellular β-lactamase, and then measured β-lactamase using a spectrophotometric method.
- As a result of measuring the amount of lactamase produced, it was found that the amount of total protein was 0.09 units/mg, which was close to the value read from the calibration curve.

The graph shown in the drawing was created using the 10 standard bacterial strains mentioned above, and the horizontal axis is the diameter of the annular yellow discoloration zone (
X) (ml) is shown, and the vertical axis shows the β-lactamase production amount (Y) (unit/mg total protein). The straight line in the drawing is expressed by the formula Q nY = 0.309X-11,4, and the correlation coefficient r in this case is 0.920, indicating that there is a correlation between X and Y. is verified. Moreover, the black circles indicated by 1 to 10 in the graph indicate the measured values obtained using the standard strains, and the specific names of the strains 1 to 10 are as follows.

1: Escherichia coli w3630/Rms 212
2: Klebsiella pneumoniae GN 693: Escherichia coli v3630/Rms 2134: Escherichia coli W3630/Rte 165: Pseudomonas aeruginosa MI/Rms 1396:
Enterobacter cloacae GN 74717: Cytolobacter freundii GN 73918: Serratia marcescens GN 108579: Pseudomonas aeruginosa GN 1036210: Proteus vulgaris GN 76゜[Effect] According to the present invention, it is difficult to quantify in the past. β-lactamases produced by Gram-negative bacteria can be quantitatively measured relatively easily, and this allows for the accurate selection of the type and amount of antibiotics required for administration prior to treating patients with infectious diseases. be able to.

[Brief explanation of the drawing]

The figure is a graph showing the relationship between the diameter of the yellow discolored zone formed after culturing β-lactamase-producing bacteria on an agar medium containing PADAC for 24 hours and the amount of β-lactamase produced.

Claims (2)

[Claims] (1) Contains 3-(2-azo-p-dimethylanilino-1-pyridiniummethyl)-(6R,7R)-7-(2-thienylacetamido)-3-cephem-4-carboxylate as a quantitative indicator. The sample is added to a solid medium for Gram-negative bacteria, which has been prepared, and the bacteria in the sample are cultured. The production of Gram-negative bacteria in the sample is determined from the size of the discolored zone formed by the reaction of the quantitative indicator around the developed colonies. A method for quantitatively measuring β-lactamase. (2) Contains 3-(2-azo-p-dimethylanilino-1-pyridiniummethyl)-(6R,7R)-7-(2-thienylacetamido)-3-cephem-4-carboxylate as a quantitative indicator A measurement material for quantifying β-lactamase produced by Gram-negative bacteria, comprising a solid medium for Gram-negative bacteria.