JP2010213598A - Method for examining efficacy of antimicrobial agent to microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply examining efficacy of antimicrobial agent to microorganisms, which has high reliability and quickly obtains results since microorganisms are directly measured. <P>SOLUTION: A microorganism-containing liquid is prepared. A region of an object (a) to be examined for a control, which is composed of the microorganism-containing liquid or a sample obtained by diluting the liquid, and a region of an object (b) to be examined, which is composed of the microorganism-containing liquid or a sample obtained by diluting the liquid and a sample containing an antimicrobial agent of examination object, are formed on a carrier. The microorganisms in the regions are cultured in the vicinity of the optimum temperature of the microorganisms under the same conditions for 2.5-8.0 hours. After the culture, the microorganisms are dyed. The dyed images of the microorganisms in the region of the object (a) and the dyed images of the microorganisms in the region of the object (b) are compared and observed. The amount of the microorganisms in the dyed images of the region of the object (a) is used as a standard. The efficacy of the antimicrobial agent is judged valid when the amount of the microorganisms in the dyed image of the object (b) is ≤10%, intermediate when the amount is >10% and <50% and invalid when the amount is ≥50%. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for determining the effectiveness of an antibacterial agent against microorganisms, a kit used for carrying out the method, and a method for producing a carrier used for carrying out the method. According to the present invention, the effectiveness of antibacterial drugs against microorganisms can be determined quickly and easily using an inexpensive kit. Here, “determining the effectiveness of an antibacterial agent against microorganisms” means determining whether or not the test microorganism is sensitive to a specific antibacterial agent, and testing microorganisms against a specific antibacterial agent Determination of whether or not is resistant, determining the minimum inhibitory concentration (MIC) of a specific antibacterial agent against the test microorganism, and the like.

  Patients whose conditions have suddenly changed due to infections need to be treated early. However, the test for identifying the causative microorganism of the infectious disease and examining the susceptibility to the antibacterial drug takes at least 1.5 days, and in some cases, about 3 days, until the result is obtained. In the clinical setting, it is too late to wait for the results of such a test. Therefore, the current situation is that doctors estimate the causative bacteria of infectious diseases, select and administer antibacterial drugs effective for the bacteria.

  However, highly specialized knowledge is required for accurate estimation of the causative bacteria of infectious diseases. For this reason, a drug having a broad antibacterial spectrum is usually selected as the first-line drug. That is, specific antibacterial drugs that are not necessarily appropriate are frequently used.

  Inappropriate use of antibacterial drugs not only causes a rise in medical costs, but also MRSA (methicillin-resistant Staphylococcus aureus), PRSP (penicillin-resistant pneumococci), VRE (vancomycin-resistant enterococci), ESBL (substrate expansion) Type β-lactamase-producing bacteria) and various resistant bacteria represented by MDRP (multidrug-resistant Pseudomonas aeruginosa) were caused to appear and increase. Therefore, in recent years, there is a strong demand for proper use of antibacterial drugs.

  By the way, at present, drug susceptibility testing of microorganisms is carried out using a test kit based on the measurement principle such as a disk diffusion method or a micro liquid dilution method. The disk diffusion test is a test method of CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI) standard, which is the US clinical laboratory standardization committee. In this method, a suspension of a test microorganism isolate, which is a McFarland no. Inoculate a suspension of 0.5 turbidity on a gel plate, place a disk impregnated with a test antibacterial agent at a predetermined concentration on it, and incubate the suspension. The radius (if present) is measured to determine the sensitivity of the test microorganism inoculated on the gel plate to the test antimicrobial drug impregnated in the disk. The micro liquid dilution method is also a CLSI standard test method. In this method, a fixed amount of a test microorganism that has been separately cultured is inoculated into a culture solution in which a test antibacterial agent is diluted in stages, and after culturing, the turbidity, cloudiness, and / or precipitates of the medium are quantitatively determined. The degree of growth of the test microorganism is measured, and the minimum concentration (MIC) of the test antibacterial agent that inhibits the growth of the test microorganism is determined. In these drug susceptibility tests, the presence or absence of growth of a test microorganism is determined with the naked eye or measured with an automatic analyzer, but it takes about 18 to 48 hours from the start of the test to the determination.

  If the results of tests to identify the causative microorganisms of infectious diseases and susceptibility to antibacterial drugs are obtained in a short time, the inappropriate use of the above inappropriate antibacterial drugs can be prevented and the appearance of various resistant bacteria can also be suppressed. it can. Therefore, a drug sensitivity test method has been proposed that can obtain results in a shorter time than the conventional standard method. Such a method is disclosed in Patent Documents 1 to 9. Patent Document 10 discloses a sensitivity test plate in which an antibacterial drug, a color reagent and a stabilizer are solid-phased on a microplate, and an example of a drug sensitivity test using the plate. Yes. Furthermore, in Non-Patent Documents 1 and 2, it is possible to perform a drug susceptibility test in a minimum of 3 hours by the rapid method by using a fully automatic rapid identification sensitivity measurement device “Lythus”, and a culture time in accordance with the CLSI method. It is described that a susceptibility test can be performed (usually 18 hours), and bacteria can be identified. Furthermore, Non-Patent Document 3 describes that by using the “Lisas”, many bacterial species can be identified within 5 hours, and the sensitivity test can be performed within 4 to 7 hours.

  Here, the two standard test methods of CLSI described above basically determine the growth of microorganisms, that is, whether or not the microorganisms grow, whereas the methods described in Patent Documents 1 to 10 Then, measure the metabolic activity, that is, whether the activity of an enzyme that is a component of a microorganism individual is high or low, or measure the amount of a specific metabolite, metabolic degradation product, nutrient source, etc. ing. That is, rather than confirming the growth of the microorganism itself, the presence or absence of the growth of the microorganism is indirectly determined using the activity of the enzyme as an index. However, there is no strict correlation between these indicators and the growth of microorganisms, and therefore pseudo-resistance may occur, for example, in drug sensitivity tests. Thus, in terms of reliability of results, it can be said that the methods described in Patent Documents 1 to 10 are inferior to the standard test method of CLSI.

Non-Patent Document 2 has the following description as a measurement principle of a rapid method performed using “Lisa”:
(In the case of identification of bacterial species) The bacterial substance is released by decomposing the fluorescent substrate formed by binding the fluorescent substance to the substrate. Therefore, the change in fluorescence intensity due to the release of the fluorescent substance is measured.
(In the case of a drug susceptibility test) During the growth of bacteria, intracellular dehydrogenase oxidizes organic acids. At this time, the redox coloring reagent is reduced through a coenzyme such as NADH to form a water-soluble formazone, which develops a yellow color. Therefore, this color development is measured by absorbance.

  In other words, the measurement principle of the rapid method performed using “Lisa” is to determine the presence or absence of microbial growth indirectly using the enzyme activity or the like as an index, as in the methods described in Patent Documents 1 to 10. It is.

  Furthermore, the methods described in Patent Documents 1 to 9 and the method using “Lythus” described in Non-Patent Documents 1 to 3 require expensive reagents and devices.

  As described above, it is necessary to quickly select an appropriate antibacterial agent for the treatment of infectious diseases, but the current standard method, a drug sensitivity test that directly examines the growth and growth of microorganisms, gives results There is a problem that the time until is too long, can not cope with urgent use. On the other hand, the rapid methods proposed so far do not directly look at the growth and growth of microorganisms and require a measuring device with an expensive reagent.

U.S. Pat. No. 4,311,794 US Pat. No. 4,622,297 US Pat. No. 5,164,301 U.S. Pat.No. 4,014,745 JP 7-313193 A Special table 2000-500340 JP-A-10-210998 Japanese Patent No. 3636849 JP2007-222182A JP 2001-321156 A

http: // www. nissui-pharm. co. jp / diagnostics / raiss. html (December 8, 2008), Nissui Pharmaceutical Rice "Nissui" Hirofumi Sagara, Ryuichi Endo, Masatoshi Sanna, “Fully Automatic Rapid Identification Sensitivity Measuring Device RAISUS“ Lisas ”System”, Clinical and Microbiology, Vol. 33, no. 1, 027-031, 2006 Isao Nakasone, “Operation of the fully automated rapid identification and sensitivity measurement device RAISUS“ Lisas ””, Clinical and Microbiology, Vol. 33, no. 1, 033-037, 2006

  An object of the present invention is to enable appropriate antibacterial drugs to be administered from the beginning everywhere, especially for patients with infectious diseases that require urgency. More specifically, although it is a simple method, the microorganism is directly measured as in the CLSI standard test method, so the result is highly reliable and the result can be obtained quickly. It is to provide a method for examining sex, a test kit that can be used for carrying out the method, and a method for producing a carrier that can be used for carrying out the method.

  The present inventors determine whether or not a test microorganism is sensitive to various antibacterial drugs and / or determine whether or not a test microorganism is resistant, or against a test microorganism. We have intensively studied to develop a simple and rapid test method that makes it possible to determine the minimum inhibitory concentration (MIC) of a specific antibacterial drug. As a result, the present invention has been made.

That is, the present invention relates to a method for examining the effectiveness of an antibacterial agent against microorganisms, which includes the following steps (1) to (6):
(1) preparing a microorganism-containing liquid;
(2) A test sample (a) region for control consisting of a microorganism-containing liquid or a sample obtained by diluting the microorganism-containing liquid on a carrier; a sample obtained by diluting the microorganism-containing liquid or the same; A test sample (b) region composed of a sample containing a drug is formed, where it can be ensured that the test sample (a) region and the test sample (b) region are not mixed with each other. They may be formed on the same carrier or different carriers, and the concentration and / or number of microorganisms in the test sample (a) region and the test sample (b) region are substantially the same;
(3) On the carrier, the microorganisms in the test sample (a) region and the test sample (b) region are cultured for 2.5 to 8.0 hours under the same conditions near the optimum temperature of the microorganisms;
(4) staining microorganisms in the test sample (a) region and the test sample (b) region after culture;
(5) The microorganism-stained image in the test sample (a) region is compared with the microorganism-stained image in the test sample (b) region; and (6) As a result of the comparative observation, the test sample (a) Based on the amount of microorganisms in the stained image of the region, it is effective if the amount of microorganisms in the stained image of the sample (b) region is 10% or less, intermediate if 50% or more and less than 50%, 50% If it is above, it is determined to be invalid.

  In the method for examining the effectiveness of the antibacterial agent of the present invention against microorganisms, “the concentration of microorganisms is substantially the same” means that the sample present in the test sample (a) region and the test sample (b) region, For example, it means that some value used as an index indicating the concentration of microorganisms, such as turbidity of McFarland, is the same. Therefore, even when the microorganism-containing sample showing a certain turbidity is the origin and the dilution ratio of the sample is the same, the “microorganism concentration is substantially the same”.

  “The number of microorganisms is substantially the same” means that for a sample existing in the test sample (a) region and the test sample (b) region, for example, a microorganism-containing sample showing a certain McFarland turbidity is When the number of microorganisms in the (a) region and the (b) region is almost the same as in the case of adding to the region (B) and adding the double amount to the region (B). Point to.

  “The concentration and number of microorganisms are substantially the same” is used as an index indicating the concentration of microorganisms, for example, turbidity of McFarland, for samples present in the test sample (a) region and the test sample (b) region. This is a case in which some values are the same and the total volume of the sample in the test sample (a) region and the test sample (b) region is almost the same. It is preferable that “the concentration and number of microorganisms are substantially the same”.

  The following methods (A) and (B) are also included in the method for examining the effectiveness of the antimicrobial agent of the present invention against microorganisms.

(A) A method for examining the susceptibility of microorganisms to antibacterial agents, comprising the following steps (A1) to (A6):
(A1) A microbial suspension having a microbial concentration of no. Prepare to be 50 to 1 dilution of 0.5 turbidity;
(A2) On the carrier, a test sample (a ′) region for control consisting of the microorganism suspension or a sample obtained by diluting the microorganism suspension, and a sample and inspection obtained by diluting the microorganism suspension or the same. A sample containing an antibacterial agent of interest, wherein a test sample (b ′) region containing an antibacterial agent of a publicly defined breakpoint concentration is formed, wherein the test sample (a ′) region And the test sample (b ′) region may be formed on the same carrier or different carriers as long as it can be ensured that they do not mix with each other, and the test sample (a ′) The concentration and / or number of microorganisms in the region and the sample (b ′) region are substantially the same;
(A3) On the carrier, the microorganisms in the test sample (a ′) region and the test sample (b ′) region are cultured for about 2.5 to 8.0 hours under the same conditions near the optimum temperature of the microorganism. ;
(A4) staining microorganisms in the test sample (a ′) region and the test sample (b ′) region after culture;
(A5) Comparative observation of the stained image of the microorganism in the region of the test sample (a ′) and the stained image of the microorganism in the region of the test sample (b ′); and (A6) As a result of the comparative observation, the test sample The amount of microorganisms in the stained image of the test sample (b ′) region is 10% or less, preferably 5% or less, more preferably 3% or less, based on the amount of microorganisms in the stained image of the region (a ′). If there is, it is determined that the test microorganism is sensitive to the test antibacterial drug.

(B) A method for testing the resistance of microorganisms to antibacterial agents, the method comprising the following steps (B1) to (B6):
(B1) A microbial suspension having a microbial concentration of no. Prepare to be 50 to 1 dilution of 0.5 turbidity;
(B2) On the carrier, the control sample (a ′) region for control comprising the microorganism suspension or a sample obtained by diluting the microorganism suspension, and the sample and test obtained by diluting the microorganism suspension or the same. A sample containing an antibacterial agent of interest, wherein a test sample (b ′) region containing an antibacterial agent of a publicly defined breakpoint concentration is formed, wherein the test sample (a ′) region And the test sample (b ′) region may be formed on the same carrier or different carriers as long as it can be ensured that they do not mix with each other, and the test sample (a ′) The concentration and / or number of microorganisms in the region and the sample (b ′) region are substantially the same;
(B3) On the carrier, the microorganisms in the test sample (a ′) region and the test sample (b ′) region are cultured for about 2.5 to 8.0 hours under the same conditions near the optimum temperature of the microorganism. ;
(B4) staining microorganisms in the test sample (a ′) region and the test sample (b ′) region after culture;
(B5) The microorganism-stained image in the test sample (a ′) region and the microorganism-stained image in the test sample (b ′) region are compared and observed; and (B6) As a result of the comparative observation, the test sample Based on the amount of microorganisms in the stained image of the region (a ′), the amount of microorganisms in the stained image of the region (b ′) of the test sample is 50% or more, preferably 60% or more, more preferably 70% or more. If there is, it is determined that the test microorganism is resistant to the test antibacterial drug.

  In the above methods (A) and (B), there are a plurality of test sample (b ′) regions, which are test sample regions containing antibacterial drugs, and these test sample (b′-1) regions to test samples The sample (b′-n) region (where n is an integer indicating the number of test sample (b ′) regions containing the antibacterial agent), each differently defined as an antibacterial agent. It may be contained at a breakpoint concentration. In this case, the test sample (b′-1) region to the test sample (b′-n) region are formed on the carrier so as not to mix with each other.

  The following method (C) is also included in the method for examining the effectiveness of the antimicrobial agent of the present invention against microorganisms.

(C) A method for examining the minimum inhibitory concentration (MIC) of an antibacterial agent against microorganisms, the method comprising the following steps (C1) to (C6):
(C1) The microbial suspension had a microbial concentration of no. Prepare to be 50 to 1 dilution of 0.5 turbidity;
(C2) a test sample (a ″) region for control consisting of the microorganism suspension or a sample obtained by diluting the microorganism suspension on a carrier; a sample obtained by diluting the microorganism suspension or the same; A plurality of test sample (b ″ -1) region to test sample (b ″ -n) region (where n is a test sample containing an antibacterial agent (b) ″), Which is an integer indicating the number of the test sample (a ″) region and the test sample (b ″ -1) region to the test sample (b ″ -n) region, As long as it can be ensured that they do not mix with each other, they may be formed on the same carrier or on different carriers, and the test sample (a ″) region and the test sample (b ″ -1) The concentration and / or number of microorganisms in each of the region to the test sample (b ″ -n) region is substantially the same;
(C3) On the carrier, microorganisms in the test sample (a ″) region and the test sample (b ″ -1) region to the test sample (b ″ -n) region have the same conditions in the vicinity of the optimum temperature of the microorganism. Incubate for about 2.5 to 8.0 hours below;
(C4) staining the microorganisms in the test sample (a ″) region and the test sample (b ″ -1) region to the test sample (b ″ -n) region after culturing;
(C5) Comparative observation of the microorganism-stained image in the test sample (a ″) region and the microorganism-stained image in each of the test sample (b ″ -1) to the test sample (b ″ -n) regions And (C6) As a result of the comparative observation, with reference to the amount of microorganisms in the stained image of the test sample (a ″) region, the test sample (b ″ -1) region to the test sample (b ″ -n ) In the test sample (b ″) containing the antibacterial agent in which the amount of microorganisms in each stained image of the region is 10% or less, preferably 5% or less, more preferably 3% or less, the antibacterial agent concentration is The antimicrobial concentration of the thinnest sample is determined as the minimum inhibitory concentration (MIC).

  The present invention also includes at least (I) an antibacterial agent and (II) a microorganism-containing liquid or a test sample (a) which is a diluted sample of the microorganism, and the microorganism-containing liquid or the diluted sample. And the test sample (b) containing the antibacterial drug to be tested, each containing one or more carriers that can be held so as not to mix with each other and can be used for culturing microorganisms, Providing a test kit for the effectiveness of antibacterial agents against microorganisms, which may hold both test sample (a) and test sample (b) or only one of them To do.

  The test kit of the present invention is further selected from the group consisting of (III) microorganism staining reagent, (IV) test method instructions, (V) a rack for holding the carrier, and (VI) a container for holding the carrier. At least one kind may be included.

  Furthermore, the present invention relates to a carrier selected from the group consisting of glass slides, glass slides for microarrays, multi-well plates and multiple modules having two or more areas for use in testing, and at least one control. Adding a predetermined amount of an antibacterial agent to each of the other regions excluding the region by adding a predetermined amount of a test sample when using the carrier with a solution of the antibacterial agent, and removing the solvent; A method for producing a carrier for testing the effectiveness of antibacterial agents against microorganisms is provided.

  According to the present invention, a method for determining whether or not a specific antibacterial drug is effective against a test microorganism, that is, a microorganism contained in a sample collected from a patient or the like, a kit for carrying out the method, And a method for producing a carrier for use in carrying out the method.

  In addition, according to the present invention, the drug sensitivity and drug resistance of a test microorganism can be detected simply, inexpensively and rapidly with high accuracy. Furthermore, the present invention makes it possible to know the minimum inhibitory concentration of a specific antibacterial agent against a test microorganism simply, inexpensively and quickly with high accuracy.

  Since the method of the present invention can be carried out simply, inexpensively and quickly, and the results are highly accurate, appropriate antibacterial drugs should be administered to patients with infectious diseases from the beginning based on the test results of the method of the present invention. Is possible. As a result, useless medication is reduced, and medical costs are suppressed and resistant bacteria are suppressed.

FIG. 1 shows an overview of the method of the present invention carried out on a glass slide. FIG. 2 shows MPIPC (4 μg / mL) against ATCC 29213 strain known to be sensitive to oxacillin (MPIPC) and ATCC 43300 strain known to be resistant to MPIP in S. aureus. ) Shows the results of testing the effectiveness of the method of the present invention. FIG. 3 shows CFX (4 μg / mL) against ATCC 29213 strain known to be sensitive to cefoxitin (CFX) and ATCC 43300 strain known to be resistant to CFX in S. aureus. ) Shows the results of testing the effectiveness of the method of the present invention. FIG. 4 shows VCM (4 μg / mL) against ATCC 29212 strain known to be sensitive to vancomycin (VCM) and ATCC 51299 strain known to be resistant to VCM in E. faecalis. The result of having tested the effectiveness of this by the method of this invention is shown. FIG. 5 shows ATCC 25922 strain known to be sensitive to ceftazidime (CAZ) in E. coli and K. cerevisiae. The result of having tested the effectiveness of CAZ (8 microgram / mL) with respect to ATCC700603 strain known to be resistant to CAZ in pneumoniae by the method of the present invention is shown. FIG. 6 shows each of these three agents against the ATCC 27853 strain of P. aeruginosa known to be sensitive to imipenem (IPM), amikacin (AMK), and ciprofloxacin (CPFX). The results of testing the effectiveness of the present invention by the method of the present invention are shown (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). FIG. 7 shows imipenem (IPM), amikacin (AMK), clinical isolate (No. 433) of P. aeruginosa known to be resistant only to imipenem (IPM), The results of testing the efficacy of ciprofloxacin (CPFX) with the method of the present invention are shown (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). FIG. 8 shows imipenem (IPM) versus clinical isolate (No. 431) of P. aeruginosa known to be resistant to imipenem (IPM) and ciprofloxacin (CPFX). ), The results of testing the efficacy of amikacin (AMK) and ciprofloxacin (CPFX) by the method of the present invention (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). FIG. 9 shows a clinical isolate (No. 204) of P. aeruginosa known to be resistant to imipenem (IPM), amikacin (AMK), ciprofloxacin (CPFX). The results of testing the effectiveness of each of these three agents by the method of the present invention are shown (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). FIG. 10 shows the results of testing the efficacy of levofloxacin (LVFX) 2 μg / mL against clinical isolates of E. coli with the method of the present invention. FIG. 11 shows the effectiveness of MPIPC 4 μg / mL against oxacillin (MPIPC) sensitive S. aureus clinical isolates and MPIPC resistant S. aureus clinical isolates according to the method of the present invention. The test results are shown. FIG. 12 tested the efficacy of VCM 2 μg / mL against vancomycin (VCM) sensitive E. faecium clinical isolates and VCM resistant E. faecium clinical isolates with the method of the present invention. Results are shown. FIG. 13 shows CFX (4 μg / mL) against ATCC 29213 strain known to be sensitive to cefoxitin (CFX) and ATCC 43300 strain known to be resistant to CFX in S. aureus. ) Shows the results of testing the effectiveness of the method of the present invention. In this experiment, five types of dyes were used. FIG. 14 shows each of these three agents against the ATCC 27853 strain of P. aeruginosa known to be sensitive to imipenem (IPM), amikacin (AMK), and ciprofloxacin (CPFX). The results of testing the effectiveness of the present invention by the method of the present invention are shown (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). In this experiment, five types of dyes were used. FIG. 15 shows imipenem (IPM), amikacin (AMK), ciprofloxacin (P. aeruginosa) clinical isolates known to be resistant only to imipenem (IPM). The results of testing the effectiveness of CPFX by the method of the present invention are shown (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). In this experiment, five types of dyes were used. FIG. 16 shows imipenem (IPM), amikacin (AMK) against clinical isolates of P. aeruginosa known to be resistant to imipenem (IPM) and ciprofloxacin (CPFX). ) Shows the results of testing the efficacy of ciprofloxacin (CPFX) by the method of the present invention (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). In this experiment, five types of dyes were used. FIG. 17 shows each of these three agents against the ATCC 27853 strain of P. aeruginosa known to be resistant to imipenem (IPM), amikacin (AMK), and ciprofloxacin (CPFX). 2 shows the results of testing the effectiveness of the method of the present invention (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL). In this experiment, five types of dyes were used. FIG. 18 shows each of these three agents against the ATCC 27853 strain of P. aeruginosa known to be sensitive to imipenem (IPM), amikacin (AMK), ciprofloxacin (CPFX). 3 shows the results of tests conducted by changing the microbial concentration of the microbial suspension (IPM: 16 μg / mL, AMK: 32 μg / mL, CPFX: 4 μg / mL).

  Hereinafter, based on the form for inventing, this invention is demonstrated in detail.

1: Method for examining effectiveness of antibacterial agent against microorganisms A method for examining the effectiveness of an antibacterial agent against microorganisms of the present invention includes the following steps (1) to (6).
(1) preparing a microorganism-containing liquid;
(2) A test sample (a) region for control consisting of a microorganism-containing liquid or a sample obtained by diluting the microorganism-containing liquid on a carrier; a sample obtained by diluting the microorganism-containing liquid or the same; A test sample (b) region composed of a sample containing a drug is formed, where it can be ensured that the test sample (a) region and the test sample (b) region are not mixed with each other. They may be formed on the same carrier or different carriers, and the concentration and / or number of microorganisms in the test sample (a) region and the test sample (b) region are substantially the same;
(3) On the carrier, the microorganisms in the test sample (a) region and the test sample (b) region are cultured for 2.5 to 8.0 hours under the same conditions near the optimum temperature of the microorganisms;
(4) staining microorganisms in the test sample (a) region and the test sample (b) region after culture;
(5) The microorganism-stained image in the test sample (a) region is compared with the microorganism-stained image in the test sample (b) region; and (6) As a result of the comparative observation, the test sample (a) Based on the amount of microorganisms in the stained image of the region, it is effective if the amount of microorganisms in the stained image of the sample (b) region is 10% or less, intermediate if 50% or more and less than 50%, 50% If it is above, it is determined to be invalid.

1-1: Microorganism The test microorganism to be examined by the method of the present invention is not particularly limited. For example, various microorganisms classified as “general bacteria” in clinical examinations, specifically, yellow grapes S. aureus, E. faecalis, E. coli, P. aeruginosa, K. pneumoniae, and drug-resistant bacteria thereof.

1-2: Collection of test microorganisms and preparation of microorganism-containing liquids Test microorganisms are collected from biological samples such as sputum, blood, urine, pus, puncture fluid, stool, etc. collected from patients, and environments such as hospitals. It originates from microorganisms present in the wiped sample. Among the above biological samples, sputum, blood, urine, pus, puncture solution, etc. may be prepared as it is or pre-treated by a known method and dispersed in a liquid to prepare a microorganism-containing liquid. The microorganism-containing liquid may be prepared by diluting with a special medium. When a solid sample such as stool is used, it is preferable to prepare a microbial suspension by suspending the sample in an appropriate culture medium for microbial culture, and use it as a microorganism-containing liquid.

In general, in order to avoid contamination with resident bacteria and to test only pathogenic bacteria, a biological sample is cultured in a solid or liquid medium such as an agar medium by a conventional method to prepare a microbial suspension. This is used as a microorganism-containing liquid. When using a solid medium, for example, a microorganism suspension is prepared as follows. First, an agar medium containing a biological sample is prepared, and the biological sample is applied to the agar medium and cultured. Among the colonies formed on this agar medium, fish are killed from the colonies of pathogenic bacteria. The cultivated bacteria are applied to the surface of another agar medium and cultured for enrichment. The bacteria are picked from the agar medium after enrichment and added to a suitable medium to prepare a microorganism suspension. At this time, the turbidity of the microbial suspension was determined by Macfarland No. The turbidity is preferably adjusted to 0.5, and the concentration of microorganisms in the test sample (a) region and the test sample (b) region in step (2) of the method of the present invention is Land No. The concentration of 0.5 turbidity is preferably 50 to 1 times diluted, more preferably 50 to 2 times diluted, and more preferably 30 to 3 times diluted. More preferably, the concentration is preferably 20 to 5 times diluted. In other words, when starting the culture of microorganisms in the method of the present invention, the concentration of microorganisms is preferably about 2 × 10 ⁶ cfu / mL to 1 × 10 ⁸ cfu / mL, preferably 2 × 10 ⁶ cfu / mL. More preferably, it is about 5 to 10 × ⁷ cfu / mL, preferably about 3.3 × 10 ⁶ cfu / mL to about 3.3 × 10 ⁷ cfu / mL, and preferably about 5 × 10 ⁶ cfu / mL. More preferably, it is about 2 × 10 ⁷ cfu / ml.

  In carrying out the method of the present invention, if the microbial concentration is too high, a pseudo-resistance result may be obtained. On the other hand, if the microbial concentration is too low, it may be difficult to determine in a short period of culture. is there.

  When using a liquid medium, for example, a microorganism suspension is prepared as follows. First, a biological sample such as blood is diluted with an appropriate medium. The resulting microbial suspension was obtained from McFarland No. When the turbidity is smaller than 0.5 turbidity, the bacterium is enriched by culturing. A turbidity suspension of 0.5 turbidity is obtained. On the other hand, the diluted solution of the prepared biological sample and the microbial suspension after the enrichment are No. If turbidity greater than 0.5 turbidity is indicated, dilution is performed and McFarland no. Adjust to 0.5 turbidity. Then, in the same manner as when a microorganism suspension is prepared by fishing from an agar medium and added to an appropriate medium, the test sample (a) region or test sample (b) in step (2) in the method of the present invention. ) The concentration of microorganisms in the region is determined by McFarland No. The concentration of 0.5 turbidity is preferably 50 to 1 times diluted, more preferably 50 to 2 times diluted, and more preferably 30 to 3 times diluted. More preferably, the concentration is preferably 20 to 5 times diluted.

  The medium used for preparing the microbial suspension is not particularly limited as long as the target microorganism can grow. For example, in the case of aerobic bacteria such as staphylococci, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter, Salmonella, etc., Cation-adjusted Mueller-Hinton broth (CAMHB; cation-adjusted Mueller Hinton used in the CLSI standard micro-liquid dilution method) Bromo) is preferably used, and Haemophilus Test Medium (HTM) is preferably used in the case of Haemophilus spp., Which is highly auxotrophic, and in the case of Streptococcus spp. Hinton broth is preferably used, equine hemolyzed Brucella broth is preferably used for anaerobic bacteria, Middlebrook 7H9 broth is preferably used for Mycobacterium tuberculosis and non-tuberculous mycobacteria, yeast, For fungi and fungi, RPMI 1640 Body medium is preferably used. However, it is not limited to these.

1-3: Antibacterial drug In the present invention, any antibacterial drug may be used as long as it has antibacterial activity. Examples of antibacterial drugs include macrolides, new quinolones, tetracyclines, lincomycins, penicillins, cephem, monobactams, carbapenems, fosfomycins, aminoglycosides, glycopeptides System drugs and β-lactamase inhibitors. Among them, levofloxacin (LVFX), oxacillin (MPIPC), vancomycin (VCM), imipenem (IPM), amikacin (AMK), ciprofloxacin (CPFX), cefoxitin (CFX), ceftazidime (CAZ), etc. are preferably used. Can do. Many of these antibacterial drugs are commercially available and can be easily obtained. In the present invention, new antibacterial drugs not yet marketed or developed at the present stage can also be used in the future.

1-4: Formation of test sample (a) region and test sample (b) region The microorganism-containing liquid is obtained by the method described in the column “1-2: Collection of test microorganism and preparation of microorganism-containing liquid”. Prepare. In addition, “the test sample (a) region and the test sample (b) region should not be mixed with each other” will be described in the section “1-5: Carrier”.

  The test sample (a) region can be formed by dropping (i) a microorganism-containing liquid, such as a microorganism suspension, at an appropriate location on the carrier, or (ii) the microorganism-containing liquid and the same. It can be formed by dropping a medium, a culture solution, a buffer solution, or the like for diluting.

An example of a method for forming the test sample (b) region is as follows.
(I) A certain amount of a microorganism-containing liquid (this may be a diluted one, hereinafter the same), for example, a microorganism suspension (provided that the microorganism concentration is the same as the microorganism concentration at the start of culture) and an antibacterial agent A test sample (b) prepared by mixing is collected on a carrier.
(II) A certain amount of microorganism-containing liquid (however, the microorganism concentration is about twice the microorganism concentration at the start of culture) and the antibacterial agent containing the antibacterial agent at the concentration at the start of culture, for example, about twice The drug solution is taken in one of the areas to be used in the inspection on the carrier in approximately the same amount.
(III) In the case where the carrier has “an area for use in an inspection in which an antibacterial agent is fixed”, a certain amount of microorganism-containing liquid (provided that the microorganism concentration is the same as the microorganism concentration at the start of culture) It is only necessary to add the same).
(IV) When the carrier has “an area for use in an inspection in which an antibacterial agent is fixed”, a certain amount of a microorganism-containing liquid and a certain amount of a diluent are respectively added to the area. The microbial concentration at the start of culture. In this case, it is preferable to first add a diluent to dissolve the fixed antibacterial agent.

  In the case of the above (III), for example, a certain concentration of microbial suspension is dropped (preferably, the same volume is dropped) at the location to be the test sample (a) region and the test sample (b) region. Just do it. When a certain concentration of microbial suspension is dropped in the same volume, the liquid present in the test sample (a) region and the liquid present in the test sample (b) region are the same except for the presence or absence of an antibacterial agent. It becomes. In the cases (I), (II) and (IV), the liquid present in the test sample (a) region and the liquid present in the test sample (b) region are the same except for the presence or absence of the antibacterial drug. Needless to say, this condition can be used.

  Since the test sample (a) region is for taking control data, the concentration of microorganisms in each of the test sample (a) region and the test sample (b) region at the start of the culture. And / or the numbers are substantially the same. The identity of the microorganism concentration is ensured by, for example, matching the turbidity of McFarland, and the number of microorganisms is obtained by matching the product of the total volume of the sample and the microorganism concentration in each region. It can be substantially the same. The total volume of the sample in one test sample (a) region and the total volume of the sample in one test sample (b) region are preferably substantially the same. If the concentration of microorganisms is approximately the same and the total volume of the sample is approximately the same, the conditions other than the presence or absence of the antibacterial drug are approximately the same in the test sample (a) region and the test sample (b) region. Similarly, if the number of microorganisms is approximately the same and the total volume of the sample is approximately the same, the conditions other than the presence or absence of the antibacterial drug are approximately the same in the test sample (a) region and the test sample (b) region. It becomes.

  The concentration of the antibacterial drug in the region of the test sample (b) at the start of culture varies depending on the purpose of the test. For example, when testing the sensitivity or resistance of a test microorganism to a test antibacterial drug, it is preferable to adopt a publicly defined determination breakpoint concentration. Here, examples of laws and methods for determining the determination breakpoint concentration include the “Infectious Diseases Law” in Japan, the regulations of the Japanese Society of Chemotherapy, and the CLSI method, which is an international standard method for drug susceptibility testing. . Further, when obtaining the minimum inhibitory concentration (MIC), a plurality of test sample (b-1) to test sample (bn) regions (where n is a sample) containing antibacterial drugs at various concentrations. , A test sample (b) containing an antibacterial agent is an integer indicating the number of regions).

When forming a plurality of test sample (b-1) regions to test sample (bn) regions containing antibacterial agents at various concentrations, test samples (b) containing antibacterial agents at individual concentrations -1) to (bn) may be prepared, or a test sample (b ₀ ) having the highest antibacterial drug concentration may be prepared first and serially diluted. In addition, when the carrier has a plurality of “areas for use in inspection where antibacterial drugs are fixed” and different amounts of antibacterial drugs are fixed to individual areas, It is only necessary to add a certain amount of microorganism-containing liquid or a dilution thereof (where the microorganism concentration is the same as the microorganism concentration at the start of culture).

  The total volume of the sample in one “region for use in examination” on the carrier is not particularly limited, but is, for example, 20 to 250 μL, and may be 30 to 100 μL. If a means for preventing the evaporation of moisture from the medium during culture is employed, the total volume of the sample may be smaller.

  As described above, the same antibacterial drug is used for each “region for use in the inspection”, but different weights may be fixed, or different types of antibacterial drugs may be fixed.

  The method for fixing the antibacterial agent in the “region for use in the inspection” is described in the description of the method for producing the carrier.

1-5: Carrier The carrier used in the present invention is not particularly limited as long as it is capable of culturing and staining microorganisms thereon, and thereafter, for example, observing a stained image under a microscope. Absent. Examples include slide glass (bacteria test slide glass, hole slide glass, sediment plate, etc.), microarray slide glass, multi-hole plate, multiple module, and the like. The material is preferably glass or plastic.

  When a slide glass is used as a carrier, it is not limited to a standard size (dimension: approximately 76 × 26 mm; 3 ″ × 1 ″), but is used for a large slide glass (dimension: approximately 76 × 52 mm) or geology (dimension: (About 46 × 27 mm) can also be used. Of course, the slide glass may have a size other than the existing size.

  The material of the carrier is generally transparent glass or plastic, but may be colored and translucent. Moreover, when dyeing | staining with a fluorescent dye, the thing made from the glass by which background fluorescence was suppressed, and the colored plastic of white and black may be sufficient.

  The carrier has a partitioned area that is used for inspection. The number of areas is usually two or more. In the test method of the present invention, the system (test sample (a) region) containing only the test sample that does not contain the antibacterial agent as the control and the test sample (b) region containing the antibacterial drug are the same. This is because processing is performed under conditions. Therefore, if there are three or more compartments, a test using two or more types of antibacterial agents and a test using two or more types of antibacterial agents can be performed on one carrier. In the method of the present invention, a carrier having only one sectioned area used for inspection can be used. In such a case, two or more carriers are used, and at least one of them is used for the system (a) containing only the test sample.

  The “compartmented area used for inspection” in the carrier will be described. When the carrier is a glass slide, the “partitioned area used for inspection” may be specified in advance or may not be specified. In the simplest example, a test sample (a) region and a test sample (b) region can be formed by dripping a microorganism-containing liquid or the like in regions separated from each other on the slide glass, and the microbes in both regions are mixed. If the cells can be cultured so that they do not match, the portions where the microorganism-containing liquid or the like is dropped become the “divided areas used for the inspection”. In this case, the “partitioned area used for inspection” is not specified in advance.

  An example in the case where the “partitioned area used for inspection” is specified in advance is as follows. Form a ridge or bank-like enclosure of a predetermined height on the surface of the slide glass, and the inside of the crease or enclosure as a "partitioned area used for inspection", on the surface of the slide glass, Printed with a highly water-repellent material, leaving a "divided area to be used for inspection", and a shallow depression (hole) is formed on the surface of the slide glass. "A partitioned area to be used". In addition, the slide glass provided with the "partition area | region used for a test | inspection" by printing a highly water-repellent material on the surface is marketed.

  When a multi-hole plate or a multi-module is used as a carrier, the “compartmental area used for inspection” is a part of the hole.

  The “compartmented area used for inspection” should be transparent or slightly transparent in color even though it is slightly colored. In the region, a colored thin grid line may be further provided. However, for example, when a stained image is taken with a CCD (Charge Coupled Device) camera, the “division area used for the inspection” is a condition that the color density is known (the stained microorganism can be identified). If it satisfies, it does not have to be transparent.

  The volume and size of the “compartmented area used for examination” are not particularly limited as long as microorganisms can be cultured. For example, the volume and size can hold about 20 to 250 μL of a sample to be cultured. Further, when the planar shape of the “partitioned region used for inspection” is a circle, the size thereof is, for example, about 4 to 10 mm in diameter, but may be smaller. Of course, the planar shape is not limited to a circle, and may be square, rectangular, or other shapes.

1-6: Cultivation Cultivation is carried out by subjecting the test sample (a) region and the test sample (b) region to the same conditions except for the total volume of the sample and the presence or absence of the antibacterial agent in both regions. Under the same conditions except for the presence or absence, it is performed for 2.5 to 8.0 hours near the temperature suitable for the growth of microorganisms (optimum temperature). The optimum temperature varies depending on the type of microorganism, and for some microorganisms, it is lower or higher than usual, but for many microorganisms, it is around 35 ° C. The environment is usually preferably aerobic or microaerobic, but if it is an anaerobic bacterium, the culture is performed in an anaerobic environment.

  The time required for the culture is about 2.5 to 8 hours. In about 2 hours, correct determination may not be possible. On the other hand, even if culturing is performed for more than 8 hours, there is almost no change in the results. Usually 3 to 5 hours of culture is sufficient, but in the case of some slow-growing species or combinations of specific microorganisms and specific antibacterial agents, culture should be performed for about 6 to 8 hours. Is preferred.

  In order to prevent the water in the medium from evaporating during culture, place the carrier in a storage case with a lid, or cover the `` partitioned area to be used for inspection '' and put it in the incubator. It is preferable to culture by putting. Moreover, when using a some support | carrier, it is good to use the rack holding a support | carrier.

  At the start of culture, the concentration or number of microorganisms in each of the test sample (a) region and the test sample (b) region is substantially the same, and the total volume of the samples contained in each region is also substantially the same. It is preferable that

1-7: Staining In order to determine the effectiveness of antibacterial agents by comparing the growth of microorganisms in the presence and absence (control) of antibacterial agents, specifically the difference in number, Perform staining. “Dyeing” refers to staining microorganisms by performing an action including all or part of normal operations in dyeing methods such as dyeing-mordanting-washing-decoloring after culturing.

  Before the staining, the water in the medium may be evaporated, or the microorganism may be fixed with alcohol or formalin without evaporating the water. Examples of methods for evaporating moisture include standing, air drying (including heating and air drying using a dryer), heat drying (for example, drying at about 40 ° C. on a hot plate), and the like. The moisture evaporation process is usually performed indoors, in a safety cabinet, in a dryer, or in a dehumidifier.

  Any staining method can be used as long as it is a method usually used for staining microorganisms, but simpler single staining is preferred. Staining reagents are commercially available and can be easily obtained. Microbial staining methods are known in the art (for example, the color edition “All about staining”, Monthly MEDICAL TECHNOLOGY separate volume, Ishiyaku Shuppan Publishing Co., Ltd. (See Examination and Technology 2001, Vol. 29, No. 7, “Guideline for Daily Staining for Pathological Tissue and Cytology”, Medical Shoin, published on June 15, 2001, etc.) It can implement suitably.

  In addition, a staining reagent used for staining microorganisms is not particularly limited, but examples include methylene blue, malachite green, crystal violet, basic fuchsin, acridine orange, 4 ', 6-diamino-2-phenylindole, and the like. Examples of the staining reagent include those that stain only the surface of microorganisms, those that enter the cell membrane and stain the cell membrane, and those that stain the nucleus.

  When a fluorescent reagent is used as the staining reagent, for example, using a fluorescence microscope, the fluorescent substance is excited by applying light of a specific wavelength extracted by a filter, and fluorescence of a specific wavelength separated by an absorption filter is measured.

1-8: Comparative Observation of Microbe Stained Image In the present invention, it is the “region where microorganisms are cultured and stained” on the carrier, and is the test sample (b) region (hereinafter referred to as “antibacterial agent region”) The stained image of the microorganism in the sample (a) is compared and observed with the stained image of the microorganism in the region (a) of the test sample (hereinafter sometimes referred to as “control region”).

  The stained image is observed with the naked eye, for example. In particular, in the case of a drug resistance test, since the antibacterial drug region is stained as strongly as the target region if it is resistant, it is often possible to make a determination with the naked eye without observing with a microscope. In the case of a sensitivity test, the stained image is usually observed visually under a microscope. Specifically, it is microscopically examined using an oil immersion lens at a magnification of about 1000 times, which is the magnification employed in Gram staining. In addition, stained images and fluorescence (when a fluorescent dye is used) in the antibacterial region and the control region may be mechanically read and compared. Alternatively, the antibacterial drug region and the control region may be photographed with a CCD (Charge Coupled Device Image Sensor) camera or the like, and the enlarged images thereof may be compared and observed. In addition, imaging with a microscope or a CCD camera or the like may be performed for a representative one field of view after observing a stained image in a plurality of fields for each region. Preferably, photographing is performed for three or more fields of view. Thereby, the reliability of determination increases. Further, a combination of the optical system magnifying lens group and a personal computer may be used to project the magnified image on the screen of the personal computer and observe the stained image.

1-9: Determination In the present invention, the growth of microorganisms in the presence of antibacterial drugs (test sample (b) region) is referred to as the growth of microorganisms in the absence of antibacterial drugs (test sample (a) region) (control). In comparison, the effectiveness of the test antibacterial agent against the test microorganism is determined. Specifically, if the amount of microorganisms present in the antibacterial region is 10% or less with respect to the amount of microorganisms present in the control region, the antibacterial agent is effective, and if the amount exceeds 10% and less than 50%, intermediate, 50% If this is the case, antibacterial drugs are ineffective.

  Here, the “microbe amount” is not limited to the number of microorganisms actually counted. For example, it may be the fluorescence intensity within one visual field of the stained image or the ratio of the area of the stained portion in the entire area of one visual field. Alternatively, the number of microorganisms can be counted using a personal computer in which program software capable of calculating the number of microorganisms in one field of view is incorporated.

1-9-1: Evaluation of Drug Sensitivity The method of the present invention was applied to McFarland No. 1 as the microbial concentration at the start of culture. Using a microorganism-containing liquid adjusted to a microorganism concentration corresponding to a 50-fold to 1-fold dilution of a 0.5-turbidity microorganism suspension, the test sample (b ') region was officially defined by the CLSI method, etc. When the test was carried out under the condition that the sample containing the antibacterial agent at the breakpoint concentration was contained, the amount of microorganisms in the stained image in the antibacterial region was 10% based on the amount of microorganisms in the stained image in the control region. Hereinafter, if it is preferably 5% or less, more preferably 3% or less, it can be determined that the test microorganism is sensitive to the test antibacterial drug. The microbial concentration of the microbial suspension in the target region and the antibacterial region at the start of the culture was determined by McFarland No. Those corresponding to 50 times to 1 times dilution of 0.5 turbidity are preferred, those corresponding to 50 times to 2 times dilution are more preferred, those corresponding to 30 times to 3 times dilution are still more preferred, and 20 times Those corresponding to a 5-fold dilution are particularly preferred.

1-9-2: Evaluation of drug resistance According to the method of the present invention, McFarland No. 1 was used as the microbial concentration at the start of culture. Using a microorganism-containing liquid adjusted to a microorganism concentration corresponding to a 50-fold to 1-fold dilution of a 0.5-turbidity microorganism suspension, the test sample (b ') region was officially defined by the CLSI method, etc. When the test was conducted under the condition that a sample containing an antibacterial agent at a breakpoint concentration was contained, the amount of microorganisms in the stained image in the antibacterial region was 50% based on the amount of microorganisms in the stained image in the control region. As described above, preferably 60% or more, more preferably 70% or more, it is determined that the test microorganism is resistant to the test antibacterial drug or that the test antibacterial drug is ineffective against the test microorganism. it can. The microbial concentration of the microbial suspension in the target region and the antibacterial region at the start of the culture was determined by McFarland No. Those corresponding to 50 times to 1 times dilution of 0.5 turbidity are preferred, those corresponding to 50 times to 2 times dilution are more preferred, those corresponding to 30 times to 3 times dilution are still more preferred, and 20 times Those corresponding to a 5-fold dilution are particularly preferred.

  Non-limiting examples of drug resistant bacteria include methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), multidrug resistant Pseudomonas aeruginosa (MDRP) and the like. For example, if it is determined that oxacillin (MPIPC) or cefoxitin (CFX) is ineffective for a test microorganism in an experiment using a specimen in which the presence of staphylococci can be estimated in Gram staining findings, the microorganism is MRSA It is suggested that there is a high possibility of Similarly, in an experiment using a specimen in which the presence of enterococci can be estimated in Gram staining findings, if it is determined that vancomycin (VCM) is ineffective against the test microorganism, the microorganism has a high possibility of VRE. Is suggested. In addition, Pseudomonas aeruginosa (clinical isolate) in which all of imipenem (IPM), amikacin (AMK), and ciprofloxacin (CPFX) were determined to be ineffective is suggested to have a high possibility of MDRP. .

1-9-3: Determination of Minimum Inhibitory Concentration (MIC) The method of the present invention was used as the microbial concentration at the start of culturing according to MacFarland No. 1-9. A plurality of test samples (b ″ -1) each containing an antibacterial agent at a different concentration using a microorganism-containing liquid adjusted to a microorganism concentration corresponding to a 50 to 1 dilution of a 0.5 turbidity microorganism suspension When a test is performed by forming a region to a test sample (b ″ -n) region (where n is an integer indicating the number of test samples (b ″) containing an antibacterial drug), The minimum inhibitory concentration (MIC) of the test antibacterial agent against the test microorganism can be examined, that is, the amount of microorganism in the stained image of the antibacterial region is 10 based on the amount of microorganism in the stained image of the control region. %, Preferably 5% or less, more preferably 3% or less, and the antibacterial drug concentration in the test sample (b ″ -x) region containing the sample with the lowest antibacterial drug concentration is Determined as minimum inhibitory concentration (MIC). The microbial concentration of the microbial suspension in the target region and the antibacterial region at the start of the culture was determined by McFarland No. Those corresponding to 50 times to 1 times dilution of 0.5 turbidity are preferred, those corresponding to 50 times to 2 times dilution are more preferred, those corresponding to 30 times to 3 times dilution are still more preferred, and 20 times Those corresponding to a 5-fold dilution are particularly preferred.

2: Test kit for testing effectiveness of antibacterial agent against microorganisms The test kit of the present invention includes at least the following (I) and (II):
(I) antibacterial drugs;
(II) A test sample (a) which is a microorganism-containing liquid or a sample obtained by diluting it, and a test sample containing the microorganism-containing liquid or a sample obtained by diluting it and an antibacterial agent to be examined ( One or more carriers that can be held so as not to mix with each other and can be used for culturing microorganisms.

Moreover, the test kit of the present invention may include at least one selected from the group consisting of the following (III) to (VI) in addition to the above (I) and (II). Good:
(III) Reagent for microbial staining;
(IV) Instructions for inspection methods;
(V) a rack for holding the carrier; and (VI) a container for holding the carrier.

2-1: Antibacterial Agent The antibacterial agent used in the test kit of the present invention is as described in the description of the above-mentioned “Method for Inspecting Effectiveness of Antibacterial Agent against Microorganism”. The antibacterial agent may be present alone as a component of the kit, or may be adhered to the “compartmental area used for testing” of the carrier. Moreover, the kind of antibacterial agent may be only 1 type, or 2 or more types may be sufficient as it.

2-2: Carrier The carrier used in the test kit of the present invention is as described in the description of the above-mentioned “Method for testing the effectiveness of antibacterial drugs against microorganisms”. The number of carriers contained in the test kit of the present invention may be only one, or two or more. When only one “area for use in inspection” can be formed on the carrier, the number of carriers is two or more. When two or more “regions for use in inspection” can be formed on the carrier, the number of carriers is one or more.

  The structure in the case where the antibacterial agent is fixed to the carrier will be described in the description of “Method for producing test carrier for effectiveness of antibacterial agent against microorganisms”.

2-3: Reagent for microbial staining Among the reagents for microbial staining used in the test kit of the present invention, the dye was described in the explanation of the above-mentioned "Method for examining the effectiveness of antibacterial agents against microorganisms". It is as follows.

  The concept of the dyeing reagent includes, in addition to a dyeing liquid containing a dye, various liquids used for dyeing such as a fixing liquid, a mordant, a washing liquid, and a decoloring liquid used in the dyeing operation.

2-4: Instructions The kit of the present invention contains instructions for carrying out a method for examining the effectiveness of antibacterial drugs against microorganisms using the above-mentioned antibacterial drugs, carriers, and reagents for staining microorganisms. The instructions describe the procedure for carrying out the method for examining the effectiveness of the antimicrobial agent of the present invention against microorganisms.

  If an antibacterial drug is attached to the area to be used for inspection on the carrier, the name of the antibacterial drug, the amount of adhesion (or the antibacterial drug concentration when a specified amount of sample is added), and the antibacterial drug on the carrier The arrangement of and the arrangement of the fixing amount may also be described in the manual.

2-5: Rack for holding a carrier A rack for holding a carrier is, for example, when two or more carriers are cultured under the same conditions when a carrier that can form only one "area for use in inspection" is used. Used to do. Of course, a rack can be used even when only one carrier is used.

  The rack may be any rack that can hold or place the carrier horizontally. Specific examples include metal pipes and plastic shelves (those that allow multiple slide glasses to be stacked vertically with a space between each other (each placed on a shelf)). There is a support body that can sandwich a portion (for example, both side portions).

2-6: Container in which carrier is put In carrying out the method of the present invention, if the water in the medium evaporates more than necessary during cultivation, appropriate cultivation may not be performed. Therefore, it is preferable to use a sealed container in which a carrier such as a slide glass is placed. Such a container is not particularly limited as long as it can contain a carrier, has a sealing property, can be used in an incubator, and does not adversely affect the culture of microorganisms. In addition, about airtightness, it does not necessarily need to be perfect and it should just be able to suppress evaporation of a water | moisture content. An example of such a container is a plastic square container.

  When loading the carrier into such a container, the carrier may be loaded or placed directly in the container, or a plurality of carriers may be loaded into the rack and the entire rack placed in the container. Also good.

  Moreover, the container in which the carrier is placed includes a container in which a partition is formed so that a plurality of carriers can be charged.

2-6: Others It is desirable that the kit of the present invention further includes a microorganism culture medium for diluting or suspending the biological sample or the environmental wipe sample. The medium is not particularly limited as long as it is suitable for culturing microorganisms, and may be liquid or powder. It may also include materials for use in wiping the environment, such as a swab.

  The kit of the present invention may further include a cover glass. The cover glass serves as a lid and can suppress evaporation of moisture in the medium during culture.

  Moreover, it is good also as a kit which does not bundle the reagent for dyeing | staining. In this case, the staining reagent may be sold separately, or a staining reagent widely used in a pathology laboratory may be used.

3: Method for producing test carrier for testing effectiveness of antibacterial agent against microorganisms The method for producing a test carrier for testing the effectiveness of antibacterial agent against microorganisms has two or more regions for use in testing, In a carrier selected from the group consisting of a slide glass, a slide glass for microarray, a multi-hole plate, and a multiple module, a predetermined amount of test is performed in each of the other regions except at least one region for control. It includes a step of applying an antibacterial agent in an amount to a predetermined concentration by adding a sample with a solution of the antibacterial agent and removing the solvent.

  In the case where the carrier is a slide glass or a slide glass for microarray, the “region for use in inspection” is defined, for example, by forming a ridge or bank-like enclosure of a predetermined height on the surface. , The area inside the cage or enclosure. Alternatively, a portion of the carrier formed by printing a highly water-repellent material while leaving a part on the surface of the slide glass is a “region for use in inspection”. Further, in the simplest example, if an antibacterial drug is fixed at one place on the slide glass, the fixed part, the test sample (b) region around it, and the test sample (b) region The region of the sample (a) to be separated is the “region for use in inspection”.

  In the case where the carrier is a slide glass with a hole formed on the surface, a slide glass for microarray, a multi-hole plate or a multiple module, the hole portion is an “area for use in inspection”.

  In the test carrier according to the present invention, at least one of the plurality of “regions for use in the test” is used as a control, and therefore, the antibacterial drug is not fixed thereto. Antibacterial drugs are adhered to “areas for use in examination” other than the control. The antibacterial drug is adhered to the whole or a part of each “area for use in the examination”.

  In order to fix the antibacterial agent to the carrier, a solution of the antibacterial agent, for example, an aqueous solution, is dropped on the “region for use in the inspection” of the carrier, and is allowed to stand at room temperature or air dry, heat dry, forced dehumidification dry The solvent is evaporated and the antibacterial agent is fixed on the carrier as a solid content by a method generally known in the art such as drying in a desiccator, drying under reduced pressure, or freeze drying.

  At this time, the amount of the antibacterial agent fixed to each region is an amount that becomes a predetermined concentration by adding a predetermined amount of the test sample when the carrier is used. For example, when 50 μL of a 10% solution of an antibacterial drug is dropped, 5 μg of the antibacterial drug is fixed to the “area for use in the inspection”. If the volume of the bacterial suspension dripped into this region at the time of inspection is 100 μL, the antibacterial agent concentration of the prepared test sample (b) is 5 w / v%.

  When the antibacterial drug lacks stability, for example, a carbapenem compound, it is preferable to use a stabilizer together with the antibacterial drug. An example of a method for stabilizing a carbapenem compound is as follows.

  The carbapenem antibacterial agent is dissolved in a 2-hydroxy-3-morpholinopropane sulfonic acid (MOPSO) buffer (preferably 30 to 120 mM) as a stabilizer, and the resulting solution is dropped onto the carrier. Then, the solvent is removed by drying, and the antibacterial agent and the stabilizer are fixed on the carrier. In addition, refer to Unexamined-Japanese-Patent No. 2005-341827 for the fixing method to the support | carrier when using a stabilizer together with an antibacterial agent, the example of an antibacterial agent with a preferable use of a stabilizer, and the example of a stabilizer.

  As described above, when there are multiple "areas for use in testing" other than the control, different antibacterial drugs may be adhered to these areas, or one type of antibacterial drug may be attached to the areas. The amount may be fixed (ie, so that the concentration in the culture medium is different), or different antibacterial agents may be applied in two or more amounts for each (ie, test sample (b- 1) The region and the test sample (bn) region may be fixed (so that the concentration of the antibacterial drug is different).

  In the test carrier of the present invention, at least one control region exists. Depending on the application, there may be more than one control region. For example, in the case of a multi-hole plate that can perform a series of tests for each row, there is a control region, one for each row.

Hereinafter, the present invention will be specifically described with reference to examples.
[Example 1] Examination using known standard strains The bacterial species and strains shown in Table 1 were used to test whether they were effective at a predetermined concentration against antibacterial drugs. Table 1 also shows the antibacterial agent and culture medium used in the test, that is, the concentration of the antibacterial agent in the test sample (b) region.

(1) Strain For each bacterial species, an international standard strain of ATCC (American Type Culture Collection) was used. P.P. For drug resistant strains of aeruginosa, clinical isolates were used because there is currently no designation of an international standard strain.

(2) Drug sensitivity The drug sensitivity of the used strain is shown in the known judgment result column of Table 1. Specifically, the determination breakpoint concentration (μg / mL) of the MIC criterion described in the CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI) method, which is an international standard method for drug susceptibility testing currently performed in daily tests ( In the column of antibacterial drug concentration, it indicates whether it is drug sensitive or drug resistant. P.P. For aeruginosa, the lower limit concentration determined to be drug resistance specified in “Necessary laboratory findings for notification” in the notification of doctors based on the Infectious Disease Law is shown.

(3) Inspection method A test was performed according to the method shown in FIG. Specifically, it is as follows. On one surface of the slide glass, water-repellent printing is performed, and four circular holes having a diameter of 10 mm are formed as unprinted areas (manufactured by Matsunami Glass; model number TF0410; 4 holes). These holes are areas for use in inspection. In one of the holes, an aqueous solution of the antibacterial drug shown in Table 1 was dropped, and air-dried at room temperature to fix the antibacterial drug, thereby forming an antibacterial drug region. The dropping amount of the aqueous solution of the antibacterial agent was such that the concentration of the antibacterial agent would be the value shown in Table 1 when 50 μL of the microorganism suspension was dropped in a region for use in the test in the subsequent step. One of the other holes is not dripped with an aqueous solution of antibacterial and is used as a control area for the control test. The other two holes were not used.

In each of the antibacterial region and the control region, suspensions of the bacterial species and strains shown in Table 1, which are McFarland No. 0.5 turbidity those of the ^{(1 × 10 8 cfu / mL} ) to which was diluted 10-fold ^{(1 × 10 7 cfu / mL} ), 50μL (5 × 10 5 cfu) was added. The medium used was cation-adjusted Muller Hinton broth.
After adding the suspension of bacterial species and strains shown in Table 1, the slide glass is placed in a sealed container with a lid, and the container is placed in an incubator at 35 ° C. for 1 hour, 2 hours, 4 hours or Cultured for 8 hours.

  The slide glass was taken out from the incubator and naturally dried at 23 ± 3 ° C. for about 30 minutes to fix the microorganisms on the slide glass, and then stained with methylene blue. Staining was performed as follows.

(Preparation of staining solution)
1.5 g of methylene blue was dissolved in 30 mL of 95% ethanol. After the methylene blue was dissolved, the whole amount was mixed with 100 mL of 0.01% aqueous potassium hydroxide solution.

(Dyeing method)
The staining solution was placed on the entire slide glass and left for about 1 minute. No mordant was used. Thereafter, the staining solution was discarded, and the slide glass was washed with water. The slide glass was dried in a safety cabinet for about 30 minutes. Decolorization was not performed.

  The stained image of the microorganism was observed under a microscope at a magnification of 1,000 times with oil immersion. Observation was performed for multiple fields of view. One or more representative fields were selected and microscopic images were taken. The microscope used was a Leica inspection / research microscope DMLB.

(4) Results (4-1) S.I. Efficacy of Oxacillin against Aureus S. aureus is known to be sensitive to this antibacterial agent using oxacillin (MPIPC), a penicillin antibacterial agent. S. aureus ATCC 29213 strain known to be resistant. FIG. 2 shows the results of culturing using the Aureus ATCC 43300 strain.

  As is apparent from FIG. 2, in the ATCC 29213 strain, the number of bacteria in the antibacterial region (described as “drug-containing” in the drawing; the same applies hereinafter) in the control region (in the drawing, “control” after 2 hours of culturing). It was about 10% of the number of bacteria of the same; And after 4 hours or 8 hours of culture, the number of bacteria in the antibacterial drug region was 5% or less of the number of bacteria in the control region. Therefore, it can be seen that MPIPC is effective.

  On the other hand, in the ATCC 43300 strain, the number of bacteria in the antibacterial region was less than 50% of the number in the control region after 2 hours of culture, but the number of bacteria in the antibacterial region after 4 hours or 8 hours of culture. Exceeded 50% of the number of bacteria in the control area (specifically, about 100% after 4 hours and about 80% after 8 hours). Therefore, it can be determined that MPIPC is invalid.

  For example, if MPIPC is determined to be ineffective by testing the effectiveness of MPIPC in this manner against clinically isolated microorganisms, the bacterial species is likely to be methicillin-resistant Staphylococcus aureus (MRSA) Is suggested.

(4-2) S.M. Efficacy of cefoxitin against Aureus FIG. 3 shows the results when cefoxitin (CFX) was used at the concentrations shown in Table 1 instead of oxacillin (MPIPC).

  As is clear from FIG. 3, in the case of the ATCC 29213 strain and the ATCC 43300 strain, there was no significant difference in the number of bacteria in the antibacterial region and the control region after 2 hours of culture. After 4 hours or 8 hours of culturing, the number of bacteria in the ATCC 29213 strain was 10% or less, more specifically 5% or less of the number of bacteria in the control region. Therefore, it can be seen that CFX is effective. On the other hand, in the ATCC 43300 strain, the number of bacteria in the antibacterial region after 4 hours or 8 hours of culture was about 100% of the number of bacteria in the control region. Therefore, it can be determined that CFX is invalid.

  For example, testing the effectiveness of CFX in this way against clinically isolated microorganisms and determining that CFX is ineffective at a concentration of 4 μg / mL suggests that the strain is likely to be MRSA. .

(4-3) E.E. Efficacy of vancomycin (VCM) against faecalis Using vancomycin (VCM), a glycopeptide antibacterial agent, using ATCC 29212 strain known to be sensitive to this antibacterial agent and ATCC 51299 strain known to be resistant FIG. 4 shows the results when the culture was performed.

  As is clear from FIG. 4, in the case of ATCC29212 and ATCC51299, there was no significant difference in the number of bacteria in the antibacterial region and the control region after 2 hours of culture. After 4 hours or 8 hours of culturing, the number of bacteria in the ATCC29212 strain was 10% or less, more specifically 5% or less of the number of bacteria in the control region. Therefore, it can be seen that the VCM is effective. On the other hand, in the ATCC 51299 strain, the number of bacteria in the antibacterial area after 4 hours of culture is about 60% of the number of bacteria in the control area, and after 8 hours, the number of bacteria in the antibacterial area is 100% of the number of bacteria in the control area. That was all. Therefore, it can be determined that the VCM is invalid.

(4-4) E.E. coli and K.K. Efficacy of ceftazidime (CAZ) against Pneumoniae Using ceftazidime (CAZ), a cephalosporin antibacterial agent, E. pneumoniae is known to be sensitive to this antibacterial agent. coli known to be resistant to the ATCC 25922 strain. FIG. 5 shows the results of culturing using pneumoniae ATCC7000060 strain.

  As is clear from FIG. 5, the ATCC 25922 strain had about 30% of the number of bacteria in the antibacterial region after 2 hours of culture, but the number of bacteria in the antibacterial region after 4 hours of culture. Was 10% or less of the number of bacteria in the control region, and after 8 hours of culture, the number of bacteria in the antibacterial region was 5% or less of the number of bacteria in the control region. Therefore, it was determined that CAZ was effective after 4 hours of culture.

  On the other hand, K.K. In pneumoniae ATCC7000060 strain, after 2 hours of culturing, the cells in the antibacterial drug region did not reach lysis, and a greatly elongated filament formation was confirmed. Moreover, the number of bacteria in the antibacterial region was about 60% of the number of bacteria in the control region. After 4 hours and 8 hours of culture, filament formation of the cells was observed, and the number of bacteria in the antibacterial region exceeded 100% of that in the control region. Therefore, it was determined that CAZ was invalid after 2 hours of culture. In addition, when a microbial cell forms a filament, it is known that the bactericidal property of an antibacterial agent is not necessarily excellent.

(4-5) P.I. Efficacy of imipenem (IPM), amikacin (AMK) and ciprofloxacin (CPFX) against aeruginosa (4-5-1) Sensitivity to three antibacterial drugs: imipenem, amikacin and ciprofloxacin FIG. 6 shows the results of testing the effectiveness of each of these three antibacterial agents against a known Pseudomonas aeruginosa strain (ATCC 27853).

  After 2 hours of culture, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was about 20 to 30% for any antibacterial drug. However, after 4 hours and 8 hours of culturing, the number of bacteria in the antibacterial region was 10% or less, more specifically, 5% or less of the number of bacteria in the control region. Therefore, it can be seen that the three antibacterial agents tested here are all effective.

(4-5-2) A clinical isolate (No. 433) of Pseudomonas aeruginosa that has been determined to be resistant only to IPM among the above three types of antibacterial drugs by the current CLSI method instead of ATCC 27853 strain. FIG. 7 shows the result when using ().

  After 2 hours of culture, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was about 40% for IPM and 20% or less for AMK and CPFX. However, after 4 hours and 8 hours of culturing, the number of bacteria in the antibacterial region was almost equal to or greater than that in the control region for IPM. In addition, for AMK and CPFX, the number of bacteria in the antibacterial region was 10% or less, more specifically 5% or less, after 4 hours and 8 hours of culture. Therefore, even in the method of the present invention, it was determined that AMK and CPFX were effective and IPM was ineffective against this bacterium.

(4-5-3) A clinical isolate of Pseudomonas aeruginosa that has been determined to be resistant to IPM and CPFX among the above three types of antibacterial agents by the current CLSI method instead of ATCC 27853 strain (No. FIG. 8 shows the result when 431) is used.

  After 2 hours of culture, the ratio of the number of bacteria grown in the antibacterial region to the number of cells grown in the control region was about 50% for IPM, about 15 to 20% for AMK, and about 80% for CPFX. . However, after 4 hours and 8 hours of culture, the number of bacteria in the antibacterial region was 70% or more of the number in the control region for IPM and CPFX. For AMK, the number of bacteria in the antibacterial region was 10% or less of the number of bacteria in the control region, more specifically 5% or less. Therefore, it was determined that AMK was effective and IPM and CPFZX were ineffective against this bacterium.

(4-5-4) Instead of ATCC 27853 strain, a clinical isolate (No. 204) of Pseudomonas aeruginosa determined to be resistant to all three types of antibacterial agents by the current CLSI method was used. The case results are shown in FIG.

  Already after 2 hours of culture, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 100% or more when any antibacterial drug was used. Even after 4 hours of culture, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 100% or more when any antibacterial drug was used. After 8 hours of culture, the number of bacteria in the antibacterial area is about 70% of the number of bacteria in the control area for IPM and CPFX, and the number of bacteria in the antibacterial area is 100 times the number of bacteria in the control area for AMK. More than%. Therefore, it was determined that all three antibacterial agents were ineffective against this bacterium.

  From the above results, according to the method of the present invention, it became clear that the effectiveness of the antibacterial agent against the test microorganism can be determined at the same level as the standard method which currently requires 18 hours or more in culture for 4 hours. .

[Example 2] Determination of antibacterial drug efficacy against E. coli clinical isolates Among the two areas for use in the examination of glass carrier similar to Example 1, levofloxacin (LVFX) ) Was fixed in an amount such that the concentration of LVFX was 2 μg / mL when 50 μL of the microorganism suspension was dropped in the subsequent step, thereby producing an antibacterial drug region. On the other side, nothing was fixed and used as a control region.

  In each of the antibacterial and control areas, E. coli. microbial suspension of a clinical isolate of E. coli, McFarland No. 50 μL of 0.5 turbidity diluted 10-fold was added. The medium used was cation-adjusted Muller Hinton broth. E. E. coli clinical isolates can be classified into two: those determined to be sensitive to LVFX by other tests (MIC ≦ 0.25 μg / mL) and those determined to be resistant (MIC> 8 μg / mL). Used types.

  The glass slide was placed in a square jar manufactured by Mitsubishi Gas Chemical Co., Inc., which was placed in an incubator and cultured at 35 ± 1 ° C. for 4 hours.

  The slide glass is taken out and air-dried at 23 ± 3 ° C. for about 30 minutes to fix the test microorganism on the slide glass. Then, in the same manner as described in Example 1, using methylene blue, Staining was performed. The stained image was observed under a microscope at a magnification of 1,000 times with oil immersion, a representative visual field was selected, and a microscopic image was taken. The results are shown in FIG.

  For each of the two types of clinical isolates, the left photograph is a stained image without LVFX (control region), and the right photograph is a stained image with LVFX added (antibacterial agent region). E. The ratio of the number of growing bacteria in the antibacterial drug region to the number of growing bacteria in the control region was 10% or less, more specifically 5% or less, and it was confirmed that LVFX was effective. On the other hand, the lower E.E. In E. coli, the ratio of the number of growth bacteria in the antibacterial drug region to the number of growth bacteria in the control region exceeded 100%, and it was confirmed that LVFX was ineffective against this bacterium.

[Example 3] Detection of methicillin-resistant Staphylococcus aureus (MRSA) and determination of antimicrobial efficacy Instead of fixing LVFX in an amount such that its concentration during culture is 2 μg / mL, MPIPC is The antibacterial region was prepared by adhering in an amount of 4 μg / mL; instead of S. coli clinical isolates. A test for determining the effectiveness of the antibacterial agent against the test microorganism was performed in the same manner as in Example 2, except that a clinical isolate of aureus was used. The results are shown in FIG.

  For each of the two types of clinical isolates, the left photograph is a stained image without MPIPC (control area), and the right photograph is a stained image with MPIPC added (antibacterial drug area). The upper S.M. Aureus had a ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 10% or less, more specifically 5% or less, and it was confirmed that MPIPC was effective. On the other hand, the lower S.M. In aureus, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was over 100%, and it was confirmed that MPIPC was ineffective against this bacterium. That is, the lower clinical isolate was determined to be MRSA.

[Example 4] Determination of effectiveness of antibacterial agents for enterococci and detection of vancomycin-resistant enterococci Instead of fixing LVFX in an amount such that its concentration at the time of culture is 2 μg / mL, VCM has a concentration at the time of culture of An antibacterial region was prepared by adhering in an amount of 2 μg / mL; instead of E. coli clinical isolates. A test to determine the effectiveness of antibacterial agents against bacteria was conducted in the same manner as in Example 2 except that a clinical isolate of faecium was used. The results are shown in FIG.

  For each of the two clinical isolates, the left photograph is a stained image without VCM (control area), and the right photograph is a stained image with VCM added (antibacterial drug area). E. For faecium, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 10% or less, more specifically 5% or less, and it was confirmed that VCM was effective. On the other hand, the lower E.E. In faecium, the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area exceeded 100%, and it was confirmed that MPIPC was ineffective against this bacterium. That is, the lower clinical isolate was determined to be VCM resistant.

[Example 5] Examination of dye (part 1)
In Example 1, S.M. Experiments were performed in a manner similar to the test to determine the efficacy of cefoxitin against Aureus, except that the dye used was crystal violet, malachite green, fuchsin and acridine orange in addition to methylene blue. The culture time was 4 hours.

  The method for preparing the staining solution was as follows. The staining method was the same as that described in Example 1 (methylene blue staining).

(1) Crystal violet staining solution 2 g of crystal violet was dissolved in 20 mL of 95% ethanol. After the crystal violet was dissolved, the whole amount was mixed with 80 mL of distilled water.

(2) Malachite Green Staining Solution 0.08 g of malachite green was dissolved in 1 mL of 95% ethanol. After the malachite green was dissolved, the entire amount was mixed with 99 mL of distilled water.

(3) Fuchsin staining solution First, 1.5 g of basic fuchsin was dissolved in 50 mL of 95% ethanol to prepare a fuchsin stock solution. Separately, 2.5 g of carboxylic acid was dissolved in 50 mL of distilled water to prepare a 5% aqueous carboxylic acid solution. 50 mL of the fuchsin stock solution was mixed with 45 mL of a 5% aqueous solution of carboxylic acid to obtain a fuchsin staining solution.

(4) Acridine orange staining solution 0.01 g of acridine orange was dissolved in 1 mL of distilled water. After the acridine orange was dissolved, the whole amount was mixed with 99 mL of acetate buffer (pH 4.0).

  The stained image was observed under a microscope with oil immersion at a magnification of 1,000 times, a representative visual field was selected, and a microscopic image was taken. The microscope used was a Leica inspection / research microscope DMLB, and an excitation filter (450 to 480 nm) and an absorption filter (515 nm) were used for fluorescence measurement (photographing) in the case of acrylic orange staining. The results are shown in FIG.

  As can be seen from FIG. 13, any of the dyes used here can clearly stain the bacteria, and therefore, no matter which dye is used, the growth in the antibacterial region relative to the number of growth in the control region. It is clear that the proportion of the number of bacteria can be determined.

[Example 6] Examination of dye (part 2)
In Example 1, these against Pseudomonas aeruginosa strains (ATCC 27853) known to be sensitive to three antibacterial agents: imipenem (IPM), amikacin (AMK) and ciprofloxacin (CPFX). The experiment was conducted in the same manner as the test for determining the effectiveness of each of the three antibacterial agents, except that the dye used was crystal violet, malachite green, fuchsin and acridine orange in addition to methylene blue. . The staining method is the same as in Example 5. The culture time was 4 hours. The stained image was observed under a microscope with oil immersion at a magnification of 1,000 times, a representative visual field was selected, and a microscopic image was taken. The results are shown in FIG.

  As can be seen from FIG. 14, any of the dyes used here can clearly stain the bacteria, and therefore, no matter which dye is used, the growth in the antibacterial region relative to the number of growth in the control region. It is clear that the proportion of the number of bacteria can be determined.

[Example 7] Examination of dye (part 3)
In Example 6, instead of the ATCC 27853 strain, a clinical isolate (No. 433) of Pseudomonas aeruginosa determined to be resistant only to IPM among the above three types of antibacterial agents by the current CLSI method. The results when used are shown in FIG.

  As can be seen from FIG. 15, any of the dyes used here can clearly stain bacteria, and therefore, any dye used can be used to develop growth in the antibacterial region relative to the number of growth in the control region. It is clear that the proportion of the number of bacteria can be determined.

[Example 8] Examination of dye (part 4)
In Example 6, Pseudomonas aeruginosa clinical isolate (No. 431) determined to be resistant to IPM and CPFX among the above three antibacterial agents by the current CLSI method instead of ATCC 27853 strain FIG. 16 shows the result obtained when using.

  As can be seen from FIG. 16, any of the dyes used here can clearly stain bacteria, and therefore any dye can be used to develop growth in the antibacterial region relative to the number of growth in the control region. It is clear that the proportion of the number of bacteria can be determined.

[Example 9] Examination of dye (part 5)
In Example 6, instead of the ATCC 27853 strain, a clinical isolate (No. 204) of Pseudomonas aeruginosa determined to be resistant to all three types of antibacterial agents by the current CLSI method was used. The results are shown in FIG.

  As can be seen from FIG. 17, any of the dyes used here can clearly stain the bacteria, and therefore, no matter which dye is used, the growth in the antibacterial region relative to the number of growth in the control region. It is clear that the proportion of the number of bacteria can be determined.

[Example 10] Examination of microorganism concentration during culture According to the method described in Example 1, the sensitivity of microorganisms to antibacterial drugs was tested. The microorganism used is a Pseudomonas aeruginosa strain (ATCC 27853) known to be sensitive to three antibacterial agents: imipenem, amikacin and ciprofloxacin, and the antibacterial agents include imipenem (IPM), amikacin (AMK) and Three types of ciprofloxacin (CPFX) were used. Also,
The microbial concentration at the start of the culture is McFarland No. 0.5 turbidity (1 × 10 ⁸ cfu / mL), McFarland No. 0.5 turbidity diluted 2 times (5 × 10 ⁷ cfu / mL), 10 times diluted (1 × 10 ⁷ cfu / mL), 50 times diluted (2 × 10 ⁶ cfu / mL) and 100-fold diluted (1 × 10 ⁶ cfu / mL).

  As a carrier, water-repellent printing is performed on one surface of a slide glass, and 10 circular holes with a diameter of 6 mm are formed as unprinted areas (manufactured by Matsunami Glass; model number TF1006; 10 holes )It was used. The amount of the microorganism suspension used was 30 μL. The culture time was 4 hours. Methylene blue staining was performed in the same manner as in Example 1. The stained image was observed under a microscope at a magnification of 1,000 times, a representative visual field was selected, and a microscopic image was taken. The results are shown in FIG.

  As is clear from FIG. 18, for IPM, the microbial concentration of the microbial suspension at the start of culture was McFarland No. When it was 0.5 turbidity, it resulted in false resistance. The microbial concentration of the microbial suspension at the start of the cultivation is McFarland No. When the turbidity was 2 to 100 times diluted with 0.5 turbidity, it was possible to determine that it was sensitive.

  For AMK, the microbial concentration of the microbial suspension at the start of culture was McFarland No. 0.5 Turbidity and McFarland No. When the turbidity was 0.5 to 50 times diluted, it was possible to determine that it was sensitive. In the 100-fold dilution, the number of bacteria was small, and it was not possible to clearly determine that the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 10% or less.

  For CPFX, the microbial concentration of the microbial suspension at the start of culture was McFarland No. In the case of 0.5 turbidity and two-fold dilutions thereof, the growth number of the control region may be too large, and the ratio of the number of growth bacteria in the antibacterial region to the growth number of the control region is When it was 10% or less, it could not be clearly determined. The microbial concentration of the microbial suspension at the start of the cultivation is McFarland No. When the turbidity was 10 to 50 times diluted, it could be determined that it was sensitive. At 100-fold dilution, the number of bacteria was small, and it was not possible to clearly determine that the ratio of the number of growing bacteria in the antibacterial drug area to the number of growing bacteria in the control area was 10% or less.

  As described above, depending on the combination of the type of microorganism and the type of test antibacterial agent, McFarland No. 1 is generally used as the microorganism concentration of the microorganism suspension at the start of culture. It has been found that a concentration corresponding to a 2 to 50-fold dilution of 0.5 turbidity is preferred.

  INDUSTRIAL APPLICABILITY The present invention is useful for examining the effectiveness of antibacterial agents against microorganisms separated from patient biological samples and the like in hospitals and clinical laboratories.

Claims (24)

A method for examining the effectiveness of antibacterial agents against microorganisms, comprising the following steps (1) to (6):
(1) preparing a microorganism-containing liquid;
(2) A test sample (a) region for control consisting of a microorganism-containing liquid or a sample obtained by diluting the microorganism-containing liquid on a carrier; a sample obtained by diluting the microorganism-containing liquid or the same; A test sample (b) region composed of a sample containing a drug is formed, where it can be ensured that the test sample (a) region and the test sample (b) region are not mixed with each other. They may be formed on the same carrier or different carriers, and the concentration and / or number of microorganisms in the test sample (a) region and the test sample (b) region are substantially the same;
(3) On the carrier, the microorganisms in the test sample (a) region and the test sample (b) region are cultured for 2.5 to 8.0 hours under the same conditions near the optimum temperature of the microorganisms;
(4) staining microorganisms in the test sample (a) region and the test sample (b) region after culture;
(5) The microorganism-stained image in the test sample (a) region is compared with the microorganism-stained image in the test sample (b) region; and (6) As a result of the comparative observation, the test sample (a) Based on the amount of microorganisms in the stained image of the region, it is effective if the amount of microorganisms in the stained image of the sample (b) region is 10% or less, intermediate if 50% or more and less than 50%, 50% If it is above, it is determined to be invalid. The method according to claim 1, wherein in step (2), the test sample (a) region and the test sample (b) region are formed on the same carrier. The carrier to be used has two or more areas for use in the inspection, and an antibacterial drug is adhered to the area excluding at least one of the areas for use in the two or more inspections. A test sample (a) region is formed by taking a microorganism-containing liquid or a sample obtained by diluting it in a region for use in a test in which the drug is not fixed, and the test in which the antibacterial drug is fixed 3. The method according to claim 1 or 2, wherein a microorganism-containing liquid or a sample obtained by diluting the microorganism-containing liquid is taken in an area for use in the step, and an antibacterial drug is dissolved in the sample to form a test sample (b) area. . The method according to claim 1, wherein the comparative observation is performed with the naked eye, under a microscope, or with a magnified image taken. For each of the test sample (a) region and the test sample (b) region, a stained image is comparatively observed for a representative visual field, and the number of stained microorganisms in the visual field or the entire area in the visual field is stained. The method according to claim 4, wherein the determination of the step (6) is performed using the ratio of the area thus obtained as the amount of microorganisms. For each of the test sample (a) region and the test sample (b) region, a magnified image of the entire region is photographed to observe a stained image, and the number of stained microorganisms in the photographed magnified image is calculated. Or the method of Claim 4 which performs determination of a process (6) by making the ratio of the dye | stained area with respect to the whole area in the image | photographed enlarged image into microbe quantity. The microorganism-containing liquid is a sample derived from at least one selected from the group consisting of sputum, blood, urine, pus, puncture liquid, stool, and environmental wipe sample. the method of. A culture suspension of microorganisms obtained by separating and culturing at least one selected from the group consisting of sputum, blood, urine, pus, puncture solution, feces, and environmental wipe samples in a liquid or solid medium. The method of claim 7, wherein Samples present in the test sample (a) region and the test sample (b) region are Nos. Of McFarland. The method according to any one of claims 1 to 8, which has a microorganism concentration adjusted to 50-fold to 1-fold dilution of a 0.5-turbidity culture solution. The sample existing in the test sample (b) region contains an antibacterial agent having a breakpoint concentration publicly defined by the Infectious Disease Method, the Japanese Chemotherapy Society, the CLSI method, etc., and staining the test sample (a) region Based on the amount of microorganisms in the image, if the amount of microorganisms in the stained image of the test sample (b) region is 10% or less, the microorganisms are sensitive to the test antibacterial drug (susceptible), more than 10% and 50% The method according to any one of claims 1 to 8, wherein a microorganism is judged to be resistant to a test antibacterial drug if it is less than%, and is 50% or more. The sample in the test sample (b) region contains an antibacterial agent having a minimum inhibitory concentration (MIC) measured by another method, and the test is performed based on the amount of microorganisms in the stained image of the test sample (a) region. If the amount of microorganisms in the stained image of the sample (b) region is 10% or less, the microorganisms are sensitive to the test antibacterial agent (susceptible), and if it is more than 10% and less than 50%, the intermediate (intermediate), 50 The method according to any one of claims 1 to 8, wherein a microorganism is determined to be resistant to a test antibacterial agent if it is at least%. The test sample (b) region contains antibacterial drugs at a plurality of different concentrations from the test sample (b-1) region to the test sample (bn) region (where n is the test sample ( The method according to claim 1, wherein b) is an integer indicating the number of regions. 13. For an antimicrobial agent, one of a plurality of different concentrations is the minimum inhibitory concentration (MIC) of the antimicrobial agent measured against the microorganism that is predicted to be present in the microorganism-containing liquid, otherwise measured. The method described in 1. At least (I) an antibacterial drug and (II) a test sample (a) which is a microorganism-containing liquid or a sample obtained by diluting it, a sample obtained by diluting the microorganism-containing liquid or a test sample, and a test object The test sample (b) containing an antibacterial drug includes one or more carriers that can be held so as not to mix with each other and can be used for culturing microorganisms, where the carrier is the test sample (a) A test kit for the effectiveness of antibacterial agents against microorganisms, which may hold both the test sample and the test sample (b) or only one of them. The kit according to claim 14, wherein the carrier has two or more regions for use in a test which is a region holding the test sample (a) and / or the test sample (b). The kit according to claim 15, wherein an antibacterial agent is fixed to an area excluding at least one of the areas for use in two or more examinations. The kit according to any one of claims 14 to 16, wherein the carrier is a slide glass, a slide glass for microarray, a multi-hole plate or a multiple module. Antibacterial drugs include macrolides, new quinolones, tetracyclines, lincomycins, penicillins, cephem, monobactams, carbapenems, fosfomycins, aminoglycosides, glycopeptides The kit according to any one of claims 14 to 17, which is at least one selected from the group consisting of a β-lactamase inhibitor. The kit according to claim 18, wherein the antibacterial agent is at least one selected from the group consisting of methicillin, oxacillin, cefoxitin, vancomycin, imipenem, amikacin, and ciprofloxacin. Furthermore, it includes at least one selected from the group consisting of (III) a reagent for microbial staining, (IV) an instruction for an inspection method, (V) a rack for holding a carrier, and (VI) a container for containing the carrier. Item 20. The kit according to any one of Items 14 to 19. The dye in the staining reagent is at least one selected from the group consisting of methylene blue, malachite green, crystal violet, basic fuchsin, acridine orange, and 4 ′, 6-diamino-2-phenylindole. The described kit. Other areas excluding at least one area for control in a carrier selected from the group consisting of glass slides, glass slides for microarrays, multi-well plates and multiple modules, having two or more areas for use in testing The antibacterial agent is effective against microorganisms, including a step of adding an antibacterial agent in a predetermined concentration by adding a predetermined amount of the sample when using the carrier, and removing the solvent. Method for manufacturing sex test carrier. The method for producing a carrier for testing the effectiveness of an antibacterial agent against microorganisms according to claim 22, wherein the antibacterial agent solution also contains a stabilizer for the antibacterial agent. 24. The carrier for testing effectiveness of antibacterial agents against microorganisms according to claim 22 or 23, wherein the step of removing the solvent is a step of drying at room temperature, drying by heating, forced dehumidification (desiccator), drying under reduced pressure or freeze drying. Manufacturing method.