JP3466735B2 - A rapid screening method for clinical bacterial drug sensitivity - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention aims to rapidly select a drug effective for clinical bacteria and to contribute to early treatment of disease treatment. It has a very important medical significance. The invention of the present application is mainly for a patient's tongue, gastric juice, urine,
In selecting a drug effective against pathogenic bacteria isolated from others, adenosine triphosphate (hereinafter abbreviated as ATP) derived from clinical bacteria and a solution reagent containing luciferin and luciferase (hereinafter referred to as luciferin / luciferase luminescence reagent) are reacted. A method for rapidly and highly sensitively examining the drug sensitivity of clinical bacteria by light-enhancing the weak light produced by an image analyzer, for example, RMDS (trade name, manufactured by Nippon Millipore Co., Ltd.), and capturing live bacteria as bright spots. It is something to offer. 2. Description of the Related Art As a method for determining the drug sensitivity of clinical bacteria, bacteria collected from a specimen are cultured in a nutrient medium containing various drugs, and the presence or absence of proliferation of the bacteria is observed. It is a general method to determine the sensitivity based on the presence or absence of a growth inhibition circle generated when the inserted disk is cultured on a nutrient agar medium. However, these methods have the drawback that a long period of culture is required before their efficacy is determined, and are not satisfactory methods for realizing early treatment. SUMMARY OF THE INVENTION The present invention is intended to reduce the time required for judging drug sensitivity of clinical bacteria, to select an effective drug more quickly than before, and to contribute to early treatment of disease. Utilizing a combination of a high-sensitivity rapid viable cell analyzer (trade name: RMDS: manufactured by Nippon Millipore Co., Ltd.) and an incubator suitable for examination of clinical bacteria proposed in Japanese Patent Application Laid-Open No. 4-505080 and Japanese Patent Application Laid-Open No. 5-328959. That was the task. [0004] As a result of various studies based on the above-mentioned problems, in a method for selecting a drug effective for a clinical bacterium, a filtration membrane is placed on an incubator comprising a medium containing the drug of interest. Is placed on top of it, and clinical bacteria are dropped thereon to proliferate according to a conventional method.
The luciferase luminescence reagent is supplied by spraying or other methods to react and emit light, and the presence or absence of light emission is measured by an image analyzer to determine the effectiveness of the drug from the absence or substantial absence of light emission. The present inventors have found a method for selecting a drug for determining the effectiveness of a drug by determining that the drug has no effect based on the observation of luminescence, and have completed the present invention. [0005] In particular, when there are many types of samples to be tested, it is desired to make many judgments at once, and for juxtaposed comparison, seeding, cultivation, extraction, reaction, and luminescence must be performed adjacent to each other. It is desirable to be able to do it. In such a case, it is desirable to use an aggregate of culture media provided with compartments so that the media components do not mix. When a large number of compartments are provided in a certain area, the environment where the bacteria on the filtration membrane are placed on it tends to dry out and hinders accurate determination. Properly supplying water is an indispensable operation. Further, it is preferable that a portion of the filter membrane covering the upper portion of the culture medium that is in contact with the culture medium is hydrophilic and the medium components are smoothly provided on the filtration membrane. On the other hand, the upper part of the filtration membrane is equipped with a grid that acts as a hydrophobic partition so that it does not mix during processing operations such as extraction and emission of adjacent samples on the membrane to determine the number of samples collected. Is also preferred. The present invention will be described in further detail.
2, nutrient agar containing various chemicals at various concentrations was poured into an incubator 1 composed of a plastic or glass column or plate 3 having a large number of small holes 2, mixed and solidified. After the clinical bacterium collected from the specimen is dropped or adhered to a portion on the opposite side of the filtration membrane that comes into contact with the agar, that is, on the filtration membrane, the incubator 1 is placed in a petri dish and cultured. After a predetermined time, the filtration membrane 4 is removed, air-dried, and A
To extract TP, an extraction reagent (RMD reagent: manufactured by Nippon Millipore Co., Ltd.) and a luciferin / luciferase luminescence reagent (RMD reagent: manufactured by Nippon Millipore Co., Ltd.) were used with an ultrasonic inhaler EW622 (Matsushita Electric Works). Spray to emit light. Immediately, the bright spot is observed using an image analyzer, for example, RMDS, and the presence or absence of bacterial growth is determined, thereby making it possible to determine the drug effect. Repeatedly, according to this method, when the reagent is ineffective, clinical bacteria grow normally, so RMDS
Bioluminescence based on ATP derived from live bacteria is detected as a bright spot by the method, and when the reagent is effective, clinical bacteria do not grow normally.
Bioluminescence based on P cannot be detected, and the effectiveness of the drug on the collected bacteria can be determined. A specific example of the incubator used in the present invention is as follows. A glass or plastic cylinder having a diameter of 50 to 110 mm and a height of 10 to 30 mm has a diameter of 3 to 10 mm.
Holes that do not necessarily have to have a circular cross section, which may be called 6 mm depressions, are provided at selected intervals, and a cylinder having such holes is used in a petri dish or used with a lid attached. It may be used as an incubator. Further, as shown in FIGS. 3 and 4, the filtration membrane 4 used is, for example, 3 to 6 mm in accordance with the diameter of the hole provided in the column of the incubator.
A hole having a size of about 3 to 6 mm and a grid 6 provided with a filtration portion 5 having an interval equal to the interval between the holes is 0.1 to 0.1 mm.
The grid 6 can be formed by impregnating a hydrophilic filter membrane with a hydrophobic resin by printing or the like, for example. If clinical bacteria need to be cultured for a long time, agar will dry out due to evaporation of water during the culture and adversely affect the growth of bacteria.Therefore, it is necessary to add water to the petri dish to keep it dry to prevent drying is there. By adopting such a method, a large amount of samples can be treated with various kinds of drugs extremely quickly as compared with the conventional method, and the drug sensitivity of clinical bacteria can be quickly determined. [0009] A clinical bacterium is dropped on a porous membrane placed in contact with an incubator having at least one agar medium containing the drug of interest, and cultured at an appropriate temperature for an appropriate time. ATP is extracted with an extractant, a luminescent reagent containing luciferin and luciferase is applied by spraying or other means, and luminescence is detected by an image analyzer, so that the effectiveness of the drug against the collected bacteria can be determined. The present invention will be described below with reference to examples. Example 1 Escherichia col (Escherichia col)
i ATCC 2592), Staphylococcus aureus
ATCC 25923), Salmonella enteritidis.
116-54), Pseudomonas aeruginosa (P
pseudomonas aeruginosa ATC
C27853), Klebsiella pneumonia (Kl
ebsiella pneumonia ATCC 8
329), Shigella frezeneri (Shigella f.
lexeneri 1b) SCD medium (Nippon Pharmaceutical)
The overnight culture was diluted with physiological saline to about 10 ⁵ cells / ml to obtain test bacteria. Aminobenzylpenicillin (A
BPC), piperacillin (PIPC), (cefazolin (CEZ), cefmetazole (CMZ), gentamicin (GM), erythromycin (EM)
0 μg / ml of the solution and 1.0% SCD agar were placed in a 5 mm diameter, 10 mm deep hole of the incubator at 0.1 ml and 0.1%, respectively.
Add 1 ml, mix and solidify. On top of that, a hydrophilic portion of a filtration membrane with a special lattice (diameter 47 mmφ, pore diameter 0.45 μm, manufactured by Nippon Millipore) is placed so as to be in contact with agar. After dropping 10 μl of each test bacterial solution onto the contact area, 37
Incubate at 4 ° C for 4-5 hours. After the culture, the filter membrane is removed and air-dried, and the ATP extraction reagent is spray-supplied onto the filter membrane at a flow rate of 5 μl / sec for 10 to 20 seconds, and the luminescent reagent is supplied at a flow rate of 10 μl / sec for 5 to 10 seconds to emit light. The luminescence due to the reaction is immediately increased by RMDS, photon counting is performed for 30 to 120 seconds, and the light is captured as a bright spot. As a comparison, Table 1 shows the results obtained by culturing the same strain for 48 hours using the disk containing the same concentration of the same drug by the Kirby Bowell method. [Table 1] A: Conventional method by growth of bacteria-: does not grow, susceptible; +: growth, resistance B: Invention method by luminance-: no luminescence, susceptible; +: luminescence, resistant Example 2 Miko Bacterium tuberculosis (Micobac)
terium tubeclosis Aoyama strain)
% 2 weeks culture in Ogawa medium saline about 10 ⁵ /
This was diluted to make the test bacteria. Rifampicin (RFP), isonicotinic hydrazide (INH), ethanebutanol (EB), penicillin (PC) 30μ
Bacterial solution added to Ogawa medium (manufactured by Far East Pharmaceutical Co.)
(V / v) and cultured at 37 ° C. for 6 days. 1 of culture solution
0 μl is dropped on the filtration membrane as in Example 1. After air drying, A
The TP extraction reagent and the luminescent reagent are sprayed to emit light. Immediately, the presence or absence of light emission is measured by RMDS. As a comparison, Table 2 shows the results obtained by culturing the Ogawa medium containing the above drug and bacterial solution for 2 weeks and examining the presence or absence of growth. [Table 2] A: Conventional method by growth of fungus-: No growth, susceptible; +: Growth, resistance B: Invention method by luminance-: No luminescence, susceptible; +: Luminescent, resistant According to the present invention, it is possible to detect a drug effective for clinical bacteria by using a highly sensitive image analyzer to react and emit the extracted ATP even if the culturing time is short. Since it is possible to make a determination, it is possible to realize early treatment, which is significant.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view showing an example of a porous culture vessel that can be used in the present invention. FIG. 2 is a sectional view taken along line AA of FIG. FIG. 3 is a plan view showing an example of a filtration membrane that can be used in the present invention. FIG. 4 is a sectional view taken along line BB of FIG. 2; [Description of Signs] 1: Porous incubator 2: Hole 3: Plastic or glass cylinder 4: Filtration membrane 5: Filtration unit 6: Grid

Continuation of front page (56) References JP-A-4-370100 (JP, A) JP-A-5-328995 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1 / 18 C12M 1/18 C12M 1/34 C12Q 1/66 BIOSIS / CA / MEDLINE / W PIDS (STN)

Claims (1)

(57) [Claim 1] One or more clinical bacteria against a plurality of drugs
Methods for simultaneously detecting different drug sensitivities
Medium consisting of a plate or disk with multiple holes
Prepare a nutrient agar containing nutrient agar containing several different drugs
Pour the ground into each of the holes and allow it to solidify,
The nutrient agar medium solidified on the disc is mixed with the
The number of hydrophilic filtration sections and the surrounding hydrophobic compartments
A single filtration membrane so that the filtration section contacts the medium.
On the filtration unit in contact with the nutrient agar medium.
Clinical bacteria are dropped or adhered, and in the incubator on the filtration section
After culturing clinical bacteria for a certain period, the filtration membrane is removed from the incubator.
, And air-dry the filter membrane.
After extracting ATP with an ATP extractant, the filtration membrane
Spray luciferin luciferase on the
Use an image analyzer to detect the presence or absence and
Multiple drugs of one or more clinical bacteria characterized by examining
A method for detecting drug sensitivity to an agent .