JP7166772B2 - Method for detecting class A carbapenemase-producing bacteria and multi-well plate for detection - Google Patents

Description

The present invention relates to a method for detecting a carbapenem-degrading enzyme (hereinafter referred to as carbapenemase) that hydrolyzes carbapenem antibacterial drugs, and particularly to a method for detecting carbapenemase belonging to class A (hereinafter referred to as class A carbapenemase)-producing bacteria. More specifically, the present invention relates to a method for detecting class A carbapenemase-producing bacteria by the broth microdilution method and a multi-well plate used therefor.

β-Lactam antibacterial agents (hereinafter referred to as β-lactam drugs) is a general term for antibacterial agents that have a β-lactam ring in the core of their molecular structure, and many β-lactam drugs have been developed since the discovery of penicillin. ing. Along with the development of this β-lactam drug, resistant bacteria have emerged that produce an enzyme (hereinafter referred to as β-lactamase) that hydrolyzes the β-lactam ring in its molecular structure and nullifies its antibacterial action. , making infections difficult to treat. Among them, carbapenemase-producing bacteria are sometimes resistant to multiple strains of antibacterial agents including carbapenems, and the increase in carbapenemase-producing bacteria has become a global problem in recent years.

β-lactamases were classified into classes A to D by Ambler et al. based on the homology of the primary amino acid sequences of the enzyme proteins. β-lactamases belonging to class A are serine peptidases having a serine residue at the active center. The prototypic enzyme is penicillinase, which hydrolyzes penicillin antibiotics, but by expanding the substrate specificity, it is possible to hydrolyze not only penicillin antibiotics but also cephalosporins and monobactam antibiotics. Extended-spectrum β-lactamase (hereinafter referred to as ESBL) is also included in class A β-lactamases. β-lactamases belonging to class B are called metallo-β-lactamases (hereinafter referred to as MBL) because they have a zinc ion in the active center. Many β-lactamases belonging to this class hydrolyze a wide range of penicillin antibiotics, cephalosporin antibiotics and carbapenem antibiotics. β-Lactamase belonging to class C is a cephalosporinase that has a serine residue in the active center and mainly hydrolyzes cephalosporin antibiotics. ampC , a gene encoding a β-lactamase belonging to class C, is present on the chromosomes of bacteria belonging to the family Enterobacteriaceae to non-glucose-fermenting bacteria, with the exception of some strains. - Lactamase-producing strains are called chromosomal AmpC-producing strains and are sensitive to cephamycins. On the other hand, plasmidic AmpC in which ampC is encoded on a plasmid (for example, overproduced AmpC) exhibits resistance to cephamycin and oxacephem antibiotics due to efficient production of AmpC. β-lactamases belonging to class D have a serine residue in the active center like enzymes belonging to class A, and the prototype enzyme mainly hydrolyzes penicillin antibiotics. The difference from class A β-lactamase is that the efficiency of degrading oxacillin is higher than that of penicillin, and β-lactamase belonging to class D is called oxacillinase (Non-Patent Document 1).

Carbapenemases have been found in classes A, B, and D, but in the United States, KPC ( Klebsiella pneumoniae carbapenemase)-type carbapenemase-producing bacteria, which belong to class A, have become a problem. Bacteria that produce OXA (oxacillinase)-48 and its mutant enzymes have become a problem. On the other hand, IMP (imipenemase) type, which is commonly detected in Japan, NDM (New Delhi metallo-β-lactamase) type, which is prevalent in Southeast Asian countries, and VIM (Verona imipenemase) type, which is commonly detected in Europe and the United States, are carbapenemase-producing bacteria. Both belong to class B (Non-Patent Document 2).

Carbapenemase-producing Enterobacteriaceae bacteria are called Carbapenemase-producing Enterobacteriaceae (hereafter referred to as CPE). Among them, there is a so-called "stealth type CPE" that shows sensitivity to carbapenem antibiotics. It is known. On the other hand, CPE shows resistance to multiple strains of antibiotics, including carbapenems, and the carbapenemase gene is transmitted to other Enterobacteriaceae bacteria via plasmids, etc., so not only hospital infections but also The detection and control of CPE is also important in terms of measures against community-acquired infections, and it is necessary to test for CPE, including KPC, OXA, and NDM, even in patients with no overseas travel history. (Non-Patent Document 2).

For CPE, as the first step, a drug susceptibility test is performed by the microdilution method or the disk method, and if carbapenemase production is suspected, the second step is the Carba NP Test based on the principle of the acidometry method (Patent Document 1). , It is detected by a general CPE test such as the mCIM method (modified Carbapenem Inactivation Method) using a disc (Non-Patent Document 3), and a differential test combining a carbapenem antibacterial drug and a carbapenemase inhibitor. However, stealthy CPE may be overlooked because it is sensitive to carbapenem antibiotics in routine first-tier drug susceptibility testing.

Studies using carbapenemase inhibitors include discrimination of MBL using carbapenem antibiotics and sodium mercaptoacetate (SMA) as an inhibitor (Patent Document 2), Cica combining a chromogenic cephalosporin and a specific inhibitor Detection of MBL by β-Test, carbapenemase differential disk using a combination of faropenem and EGTA, faropenem and cloxacillin (Patent Document 3), and the like are known. However, although these methods can make a qualitative determination, they cannot determine a quantitative Minimum Inhibitory Concentration (hereinafter referred to as MIC).

In addition to the above methods, it is also possible to detect the genotype of carbapenemase by genetic testing using the PCR method. However, since this method requires expensive equipment and a high degree of expertise, a simpler method is desired.

Japanese translation of PCT publication No. 2014-519833 JP-A-2000-224998 WO2017/098206

Clinical Laboratory, 54(5), 473-480, 2010 Journal of Japanese Society of Chemotherapy, 66(1), 119-128, 2017 Journal of Clinical Microbiology, 55(8), 2321-2333, 2017

The present invention has been made in view of the above-mentioned current situation, and aims to provide a method capable of detecting class A carbapenemase-producing bacteria simply, accurately and quantitatively, and a multi-well plate used therefor.

As a result of intensive studies to solve the above problems, the present inventors used meropenem (MEPM) as a substrate in combination with clavulanic acid (CVA) as an inhibitor, and particularly meropenem containing 0.015 to 256 μg / mL and a liquid medium containing 0.015 to 256 μg/mL of meropenem and 1 to 64 μg/mL of clavulanic acid to perform a liquid microdilution method to detect class A carbapenemase-producing bacteria with high sensitivity and specificity. We found that it can be detected effectively. Furthermore, the inventors used meropenem as substrate in combination with tazobactam (TAZ) as inhibitor, in particular liquid medium containing 0.015-256 μg/mL meropenem and 0.015-256 μg/mL meropenem and The inventors have found that class A carbapenemase-producing bacteria can be detected with high sensitivity and specificity by performing a broth microdilution method using a liquid medium containing 1 to 64 µg/mL of tazobactam, and have completed the present invention.

That is, the present invention consists of the following configurations.
(1) A method for detecting a carbapenemase-producing bacterium by a liquid microdilution method using a liquid medium, wherein the liquid medium is the following (a) and (b), and the carbapenemase-producing bacterium is class A A method for detecting a carbapenemase-producing bacterium, which is a type carbapenemase-producing bacterium.
(a) a liquid medium containing 0.015-256 μg/mL of meropenem;
(b) Liquid medium containing 0.015-256 μg/mL meropenem and 1-64 μg/mL clavulanic acid or 1-64 μg/mL tazobactam as inhibitors.
(2) In addition to the liquid medium, the following (c) is used, and the carbapenemase-producing bacterium is a class A carbapenemase-producing bacterium and a carbapenemase belonging to class B (hereinafter referred to as class B carbapenemase)-producing. The method for detecting a carbapenemase-producing bacterium according to (1), which is a bacterium.
(c) Liquid medium containing 0.015-256 μg/mL meropenem and 100-1600 μg/mL dipicolinic acid (2,6-pyridinedicarboxylic acid) (DPA) as inhibitor.
(3) In addition to the liquid medium, the following (d) and (e) are used, and the carbapenemase-producing bacterium is a class A carbapenemase-producing bacterium and a class B carbapenemase-producing bacterium. The method for detecting carbapenemase-producing bacteria according to (2).
(d) a liquid medium containing imipenem (IPM) 0.015-256 μg/mL;
(e) Liquid medium containing imipenem 0.015-256 μg/mL and dipicolinic acid 100-1600 μg/mL as an inhibitor.
(4) In addition to the liquid medium, the following (f) is used, and the carbapenemase-producing bacterium is a class A carbapenemase-producing bacterium and a carbapenemase belonging to class D (hereinafter referred to as class D carbapenemase)-producing. The method for detecting a carbapenemase-producing bacterium according to (1), which is a bacterium.
(f) Liquid medium containing 0.015-256 μg/mL meropenem and 1-64 μg/mL avibactam (AVI) as an inhibitor.
(5) In addition to the liquid medium, the following (f) is used, and the carbapenemase-producing bacteria are class A carbapenemase-producing bacteria, class B carbapenemase-producing bacteria and class D carbapenemase-producing bacteria. A method for detecting a carbapenemase-producing bacterium according to (2) or (3).
(f) Liquid medium containing 0.015-256 μg/mL meropenem and 1-64 μg/mL avibactam as inhibitor.
(6) The method for detecting a carbapenemase-producing bacterium according to any one of (1) to (5), wherein the following (g) is used in addition to the liquid medium.
(g) Liquid medium containing 0.06-128 μg/mL latamoxef (LMOX).
(7) The liquid medium is selected from Mueller Hinton broth medium, cation-adjusted Mueller Hinton broth medium, hemolyzed Mueller Hinton broth medium, heart infusion broth medium, normal broth medium and tryptic soy broth medium. The method for detecting a carbapenemase-producing bacterium according to any one of (1) to (6).
(8) A multi-well plate comprising a plurality of holding portions consisting of a plurality of wells for holding a liquid medium, and used in the method for detecting carbapenemase-producing bacteria according to any one of (1) to (7). The multi-well plate, wherein the plurality of housing portions includes a first housing portion containing the following (a) in each well and a second housing portion containing the following (b) in each well: .
(a) a liquid medium containing 0.015-256 μg/mL of meropenem;
(b) Liquid medium containing 0.015-256 μg/mL meropenem and 1-64 μg/mL clavulanic acid or 1-64 μg/mL tazobactam as inhibitors.
(9) The multi-well plate according to (8), wherein the liquid medium contained in each well is frozen.
(10) The multi-well plate according to (8), wherein the liquid medium contained in each well is dried and immobilized.
(11) A multi-well plate for use in the method according to any one of (1) to (7), comprising a plurality of holding portions each composed of a plurality of wells for holding a liquid medium, wherein the plurality of contains the following (a) in each well, a first container in which the drug in each well is dry-immobilized, and each well includes the following (b), and the drug in each well is dry-immobilized said multi-well plate, comprising a second reservoir that is fused.
(a) a reagent composition prepared to give 0.015 to 256 μg/mL meropenem at the time of use;
(b) A reagent composition prepared to provide 0.015-256 μg/mL meropenem and 1-64 μg/mL clavulanic acid or 1-64 μg/mL tazobactam as inhibitors at the time of use.

According to the present invention, a liquid medium containing 0.015 to 256 μg/mL of meropenem and a liquid medium containing 1 to 64 μg/mL of clavulanic acid or 1 to 64 μg/mL of tazobactam as an inhibitor in the broth microdilution method. By using them in combination, the first step required in the conventional method (as the first step, a drug susceptibility test by the liquid microdilution method or disk method is performed, and the presence or absence of bacteria suspected of producing carbapenemase is examined) ) becomes unnecessary, MIC can be obtained, and class A carbapenemase-producing bacteria can be detected easily, accurately and quantitatively one day earlier than the conventional method.

In addition to a combination of liquid medium containing 0.015-256 μg/mL of meropenem and liquid medium containing 1-64 μg/mL of clavulanic acid or 1-64 μg/mL of tazobactam as inhibitors, 0.015-256 μg/mL of meropenem was added to the liquid medium. 015-256 μg/mL and a liquid medium containing 100-1600 μg/mL of dipicolinic acid as an inhibitor; In addition to class A carbapenemases, class B carbapenemases and class D carbapenemases can be inhibited by using a combination of medium, liquid medium containing 0.015-256 μg/mL meropenem and 1-64 μg/mL avibactam as an inhibitor. A type carbapenemase can also be detected at the same time. On the other hand, by adding a liquid medium containing 0.06 to 128 μg/mL latamoxef, it is possible to simultaneously screen for carbapenemase-producing bacteria and detect class A, B, and D-type carbapenemases, making it more reliable. It is possible to detect carbapenemase in

FIG. 1-1 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in the method for detecting carbapenemase-producing bacteria in one embodiment of the present invention. FIG. 1-2 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in a method for detecting carbapenemase-producing bacteria according to another embodiment of the present invention. FIG. 2-1 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in a method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 2-2 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in a method for detecting carbapenemase-producing bacteria according to still another embodiment of the present invention. FIG. 3-1 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in a method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 3-2 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in the method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 4-1 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in the method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 4-2 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in the method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 5-1 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in a method for detecting carbapenemase-producing bacteria according to still another embodiment of the present invention. FIG. 5-2 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in the method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 6-1 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in a method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 6-2 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in the method for detecting carbapenemase-producing bacteria according to still another embodiment of the present invention. FIG. 7-1 is a diagram schematically showing an example of a drug arrangement of a multi-well plate used in a method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 7-2 is a diagram schematically showing an example of a drug arrangement in a multi-well plate used in the method for detecting carbapenemase-producing bacteria in still another embodiment of the present invention. FIG. 8 is a diagram showing the drug arrangement in the microplate prepared in Example 1(1). FIG. 9 is a diagram showing drug arrays in the microplate prepared in Example 2(1). FIG. 10 is a diagram showing drug arrays in the microplate prepared in Example 3(1). FIG. 11 is a diagram showing drug arrays in the microplate prepared in Example 4(1). FIG. 12 is a diagram showing drug arrays in the microplate prepared in Example 5(1).

In the present invention, class A carbapenemase-producing bacteria are detected by performing a drug susceptibility test by the broth microdilution method using meropenem, which is a carbapenem antibacterial drug, as a substrate and clavulanic acid or tazobactam as an inhibitor. Specifically, as exemplified in FIGS. 1-1 and 1-2, a liquid medium containing meropenem in a 2-fold dilution series is dispensed into each well of the first container of the multi-well plate, and the second Prepare a multi-well plate in which a liquid medium containing a 2-fold dilution series of meropenem and a fixed amount of inhibitor (clavulanic acid or tazobactam) is dispensed into each well of the storage part, and the liquid medium in each well inoculate with the test bacteria and culture. After culturing, the MIC is measured, and if the MIC in the liquid medium containing meropenem and the inhibitor is reduced compared to the MIC in the liquid medium containing meropenem without the inhibitor, the class A carbapenemase-producing strain Determine that there is.

The concentration range of meropenem contained in the liquid medium of each well of the multi-well plate can include 0.001-256 μg/mL, but may include 0.015-256 μg/mL. For example, when Enterobacteriaceae bacteria are to be detected, the concentration range of meropenem can be 0.015 to 64 μg/mL, and may include 0.015 to 32 μg/mL. In particular, by conducting the test at a concentration of 1 μg / mL or less, which is lower than the concentration of meropenem tested in a normal drug susceptibility test, it is judged as sensitive (S) in the normal drug susceptibility test, and the carbapenemase cannot be detected. Producers such as stealth CPE can also be detected. Furthermore, in order to detect stealth CPE with high sensitivity, tests should be conducted at a meropenem concentration of 1 µg/mL or less, and a standard should be set such that, for example, meropenem MICs greater than 0.125 µg/mL are carbapenemase-producing bacteria. is more preferable.

Clavulanic acid coexisting with meropenem in the liquid medium of each well of the second container of the multi-well plate illustrated in FIG. Class A carbapenemase-producing bacteria can be detected at a constant concentration. A constant concentration is more preferable, a constant concentration within a concentration range of 2 to 32 μg/mL is more preferable, and a constant concentration within a concentration range of 2 to 16 μg/mL is even more preferable. .

On the other hand, tazobactam coexisting with meropenem in the liquid medium of each well of the second container of the multi-well plate illustrated in FIG. Class A carbapenemase-producing bacteria can be detected at a constant concentration. A constant concentration is more preferable, a constant concentration within a concentration range of 2 to 32 μg/mL is more preferable, and a constant concentration within a concentration range of 2 to 16 μg/mL is even more preferable. .

When tested, MIC in liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) compared to MIC in liquid medium containing meropenem without inhibitor, as shown later in the Examples. When the MIC has decreased by 2 tubes (4 times) or more, more preferably when it has decreased by 3 tubes (8 times) or more, it can be determined to be a class A carbapenemase-producing bacterium. Here, "tube" means ^a multiplier of the concentration ratio of the concentration dilution series tube. ^2x , concentration ratio of ³ tubes means 23x.

In one embodiment of the present invention, in addition to the combination of liquid medium containing meropenem and liquid medium containing clavulanic acid or tazobactam as inhibitor, a liquid medium containing meropenem and dipicolinic acid as inhibitor The combination is used to detect class A carbapenemase producers and class B carbapenemase producers. That is, as illustrated in FIGS. 2-1 and 2-2, using a multi-well plate containing a liquid medium containing meropenem and dipicolinic acid as an inhibitor in each well of the third container of the multi-well plate Class A carbapenemase-producing bacteria and class B carbapenemase-producing bacteria can be detected.

Dipicolinic acid coexisting with meropenem in the liquid medium of each well of the multi-well plate has a constant concentration within the concentration range of 100 to 1600 μg/mL, making it possible to detect class B carbapenemase-producing bacteria. During the test, the MIC value in liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) was reduced by more than 2 tubes compared to the MIC value in liquid medium containing meropenem without inhibitor. In this case, it can be determined that the strain is a class A carbapenemase-producing bacterium, more preferably when it is reduced by 3 or more tubes. In addition, when the MIC value in the liquid medium containing meropenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing the inhibitor. In some cases, it can be determined to be a class B type carbapenemase-producing bacterium.

In addition, in addition to the combination of meropenem and a liquid medium containing dipicolinic acid as an inhibitor, imipenem, which is a carbapenem antibiotic, and a liquid medium containing dipicolinic acid as an inhibitor, meropenem It is possible to comprehensively detect class B carbapenemase-producing bacteria, including class B carbapenemase-producing bacteria that cannot be detected with the combination of dipicolinic acid and dipicolinic acid. That is, each well of the fourth container of the multi-well plate contains a liquid medium containing imipenem, and each well of the fifth container contains a liquid medium containing imipenem and dipicolinic acid as an inhibitor. -1 and 3-2, class A carbapenemase-producing bacteria and class B carbapenemase-producing bacteria can be detected with high accuracy.

The concentration range of imipenem contained in the liquid medium of each well of the multi-well plate can include 0.001-256 μg/mL, but can also include 0.015-256 μg/mL, and can include 0.015 μg/mL. ~32 μg/mL may be included. On the other hand, the concentration of dipicolinic acid coexisting with imipenem in the liquid medium of each well of the multi-well plate should be within the concentration range of 100-1600 μg/mL, making it possible to detect class B carbapenemase-producing bacteria. be. During the test, the MIC value in liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) was reduced by more than 2 tubes compared to the MIC value in liquid medium containing meropenem without inhibitor. In this case, it can be determined that the strain is a class A carbapenemase-producing bacterium, more preferably when it is reduced by 3 or more tubes. In addition, when the MIC value in the liquid medium containing meropenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing the inhibitor. and/or when the MIC value in the liquid medium containing imipenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes, compared to the MIC value in the liquid medium containing imipenem without the inhibitor If the level is decreased as much as above, the bacterium can be determined to be a class B type carbapenemase-producing bacterium.

In one embodiment of the present invention, in addition to the combination of liquid medium containing meropenem and liquid medium containing clavulanic acid or tazobactam as inhibitor, a combination of liquid medium containing meropenem and avibactam as inhibitor to detect class A carbapenemase-producing bacteria and class D carbapenemase-producing bacteria. That is, as illustrated in FIGS. 4-1 and 4-2, by using a multi-well plate containing a liquid medium containing meropenem and avibactam as an inhibitor in each well of the third container of the multi-well plate , class A carbapenemase-producing bacteria and class D carbapenemase-producing bacteria can be detected.

Avibactam, which coexists with meropenem in the liquid medium of each well of the multi-well plate, has a constant concentration within the concentration range of 1 to 64 μg / mL, so that class A carbapenemase-producing bacteria and class D carbapenemase-producing bacteria Detection may be at a constant concentration within the concentration range of 2-16 μg/mL, or at a constant concentration within the concentration range of 2-8 μg/mL. When the MIC value in the liquid medium containing meropenem and avibactam is reduced by 2 tubes or more, more preferably 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing the inhibitor during the test If it decreases, it is determined to be a class A-type or D-type carbapenemase-producing bacterium. Therefore, if the MIC value in liquid medium containing meropenem and an inhibitor (clavulanic acid or tazobactam) is reduced by more than 2 tubes compared to the MIC value in liquid medium containing meropenem but without inhibitor, It can be determined to be a class A carbapenemase-producing bacterium, preferably when it is reduced by 3 or more tubes. In addition, the MIC value in a liquid medium containing meropenem and an inhibitor (clavulanic acid or tazobactam) does not decrease, compared with the MIC value in a liquid medium containing meropenem but not containing an inhibitor, and the inhibitor Class D when the MIC value in the liquid medium containing meropenem and avibactam is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing It can be determined to be a type carbapenemase-producing bacterium.

In one embodiment of the present invention, in addition to the combination of a liquid medium containing meropenem and a liquid medium containing clavulanic acid or tazobactam as an inhibitor, a liquid medium containing meropenem and dipicolinic acid as an inhibitor, In addition, class A carbapenemase-producing bacteria, class B carbapenemase-producing bacteria and class D carbapenemase-producing bacteria are detected by using a combination of a liquid medium containing meropenem and avibactam as an inhibitor. That is, as illustrated in FIGS. 5-1 and 5-2, each well of the third container of the multi-well plate contains meropenem and a liquid medium containing dipicolinic acid as an inhibitor; Class A carbapenemase-producing bacteria, class B carbapenemase-producing bacteria and class D carbapenemase-producing bacteria can be detected by using a multi-well plate containing a liquid medium containing meropenem and avibactam as an inhibitor in each well. be. During the test, the MIC value in liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) was reduced by more than 2 tubes compared to the MIC value in liquid medium containing meropenem without inhibitor. In this case, it can be determined that the strain is a class A carbapenemase-producing bacterium, more preferably when it is reduced by 3 or more tubes. In addition, class A carbapenemase-producing bacteria do not contain inhibitors, and compared to the MIC value in the liquid medium containing meropenem, the MIC value in the liquid medium containing meropenem and avibactam is reduced by 2 tubes or more. Preferably, it can also be detected by dropping 3 tubes or more. In addition, when the MIC value in the liquid medium containing meropenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing the inhibitor. In some cases, it can be determined to be a class B type carbapenemase-producing bacterium. Furthermore, the MIC value in a liquid medium containing meropenem and an inhibitor (clavulanic acid or tazobactam) does not decrease as compared to the MIC value in a liquid medium containing meropenem without an inhibitor, and the inhibitor Class D when the MIC value in the liquid medium containing meropenem and avibactam is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing meropenem but not containing It can be determined to be a type carbapenemase-producing bacterium.

In one embodiment of the present invention, liquid media containing meropenem and dipicolinic acid, meropenem and avibactam are added to the combination of liquid media containing meropenem and liquid media containing clavulanic acid or tazobactam as inhibitors. and class A carbapenemase-producing bacteria, class B carbapenemase-producing bacteria and class D carbapenemase-producing bacteria by using a liquid medium containing imipenem and a liquid medium containing dipicolinic acid as an inhibitor. Detect bacteria. That is, as illustrated in FIGS. 6-1 and 6-2, each well of the third container of the multi-well plate contains meropenem and a liquid medium containing dipicolinic acid as an inhibitor; Each well contains a liquid medium containing meropenem and avibactam as an inhibitor, each well of the fifth container contains a liquid medium containing imipenem, each well of the sixth container contains imipenem and an inhibitor class A carbapenemase-producing bacteria, class B carbapenemase-producing bacteria and class D carbapenemase-producing bacteria can be detected by using a multi-well plate containing a liquid medium containing dipicolinic acid. Therefore, by using this combination, it is possible to distinguish whether the carbapenemase-producing strain is of class A, B or D. During the test, the MIC value in liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) was reduced by more than 2 tubes compared to the MIC value in liquid medium containing meropenem without inhibitor. In this case, it can be determined that the strain is a class A carbapenemase-producing bacterium, more preferably when it is reduced by 3 or more tubes. In addition, class A carbapenemase-producing bacteria do not contain inhibitors, and compared to the MIC value in the liquid medium containing meropenem, the MIC value in the liquid medium containing meropenem and avibactam is reduced by 2 tubes or more. Preferably, it can also be detected by dropping 3 tubes or more. In addition, the MIC value in the liquid medium containing meropenem and inhibitor (clavulanic acid or tazobactam) does not decrease, and meropenem and If the MIC value in the avibactam-containing liquid medium is reduced by 2 tubes or more, preferably by 3 tubes or more, the bacterium can be determined to be a class D carbapenemase-producing bacterium. Furthermore, when compared to the MIC value in the liquid medium containing meropenem, the MIC value in the liquid medium containing meropenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes or more, and/or inhibition If the MIC value in the liquid medium containing imipenem and dipicolinic acid is reduced by 2 tubes or more, more preferably by 3 tubes or more, compared to the MIC value in the liquid medium containing imipenem without the agent, class It can be determined to be a type B carbapenemase-producing bacterium.

In one embodiment of the present invention, carbapenemase-producing bacteria are detected (screened) regardless of class classification by combining a liquid medium containing latamoxef, an oxacephem antibiotic, in addition to each drug combination. . That is, by using the multi-well plates exemplified in FIGS. can be detected (screened). By setting the concentration range of 0.06 to 128 μg / mL for the liquid medium of each well of the multi-well plate, carbapenemase-producing bacteria can be detected with high sensitivity regardless of the class classification. A concentration range of ~64 μg/mL may be used. For determination, for example, in the case of Enterobacteriaceae bacteria, carbapenemase-producing bacteria can be detected based on a constant concentration within the concentration range of 8 to 16 μg/mL.

Additionally, other antimicrobial agents may be used in addition to the combination of agents of the present invention. For example, a liquid medium containing the penicillin antibiotics piperacillin (PIPC) and tazobactam can be combined with a liquid medium containing latamoxef without an inhibitor in addition to the combination of agents of the invention. In that case, the MIC value in the liquid medium containing meropenem without inhibitors, the MIC value in the liquid medium containing piperacillin and tazobactam, and the MIC value in the liquid medium containing latamoxef without inhibitors should be established. , CPE can be detected (screened) regardless of class classification by judging that the bacterium is a carbapenemase-producing bacterium if any of the criteria is met. In addition, a combination of a liquid medium containing the cephem antibiotic cefpodoxime (4-128 μg/mL) and a fixed amount of clavulanic acid in the range of 1-64 μg/mL as an inhibitor was added. By detecting ESBL as a bacterium, it becomes possible to easily distinguish between carbapenemase-producing bacteria and ESBL.

Microorganisms to be detected in the present invention are carbapenemase-producing bacteria. For example, class A carbapenemase-producing bacteria, Escherichia coli (hereinafter referred to as Escherichia coli ), Klebsiella pneumoniae , Klebsiella oxytoca , Serratia marcescens , Citrobacter freundii , Enterobacter cloacae , Enterobacter aerogenes , Enterobacter asburiae , Pseudomonas aeruginosa (hereinafter referred to as Pseudomonas aeruginosa) and Salmonella enteritidis, class B carbapenemase-producing bacteria Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Proteus vulgaris , Proteus rettgeri ( Proteus rettgeri ), Providencia stuartii , Morganella morganii , Citrobacter freundii , Citrobacter amalonaticus, Citrobacter youngae , Enterobacter Cloacae, Enterobacter erogenes, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii , Aeromonas junii , Bacillus cereus , Bacteroides fragilis ( Bacteroides fragilis ) and Shigella flexneri , class D type carbapenemers Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Pandoraea pnomenusa , Ralstonia picketti and Shewanella algae , etc., are specific examples. be done.

Microorganisms to be detected in the present invention are particularly preferably Enterobacteriaceae bacteria, for example, Escherichia spp., Klebsiella spp., Enterobacter spp., Serratia spp., Citrobacter spp., Salmonella spp., Proteus spp. Specific examples include fungi, genus Morganella, genus Providencia, genus Shigella, and genus Yersinia.

The test sample used in the present invention is not particularly limited as long as the presence of bacteria is suspected. Specific examples include biological samples such as human and animal urine, blood, sputum, and stool, environment-derived samples, food-derived samples, and the like.

In the present invention, each antibacterial drug or inhibitor (meropenem, imipenem, ratamoxef, clavulanic acid, clavulanic acid, tazobactam, dipicolinic acid, avibactam, etc.) is contained (dissolved) and used as a liquid medium. Specific examples of the liquid basal medium include Mueller Hinton broth medium, cation-adjusted Mueller Hinton broth medium, hemolyzed Mueller Hinton broth medium, heart infusion broth medium, normal broth medium and tryptic soy broth medium. The pH of the liquid medium is not particularly limited as long as it is a pH at which the microorganisms to be detected by the present invention grow.

In the present invention, there is provided a multi-well plate having a plurality of holding portions composed of a plurality of wells for holding a liquid medium, each well containing a liquid medium containing a carbapenem antibacterial drug. A multi-well plate is used that contains a first reservoir and a second reservoir containing liquid medium containing a carbapenem antibacterial agent and an inhibitor in each well. Antibacterial agents and inhibitors contained in each well of the multi-well plate of the present invention are illustrated in FIGS. 1-1 to 7-2. The position of the accommodating portion within the plate is not limited to those illustrated.

INDUSTRIAL APPLICABILITY The multi-well plate of the present invention can be stored frozen until use while containing a liquid medium, and has long-term stability. When stored at -70°C, it does not lose its potency for at least 6 months.

When using a commercially available powdered medium when preparing a liquid medium, dissolve the powder in a predetermined amount of purified water and sterilize with high pressure such as autoclaving. After preparing the necessary components (medium components other than antibacterial agents and inhibitors), dissolve in purified water (or add each component to purified water and dissolve), and perform high-pressure sterilization such as autoclaving. Next, after confirming that the medium has cooled sufficiently, each antibacterial agent and inhibitor (meropenem, imipenem, ratamoxef, clavulanic acid, tazobactam, dipicolinic acid, avibactam, etc.) is added as appropriate and stirred. Subsequently, the medium is dispensed into each well of the multi-well plate housing section. The multi-well plate is then stored at room temperature, refrigerated (eg, 2-10°C), or frozen (eg, -4 to -80°C). In principle, all operations for preparing multi-well plates are performed under a sterile environment.

Furthermore, the multi-well plate of the present invention may contain a reagent composition in each well and may be supplied by drying and fixing each reagent composition in each well. The reagent composition includes a reagent composition (a) prepared so that meropenem will be 0.015 to 256 μg / mL at the time of use (when performing a drug susceptibility test for detecting carbapenemase of the present invention), and meropenem at the time of use It may contain a reagent composition (b) prepared to 0.015-256 μg/mL and 4 μg/mL clavulanic acid as an inhibitor. The reagent composition may consist solely of the drug (antibacterial drug and/or inhibitor) or may include the drug and basal medium components.
In the multi-well plate of the present invention, for example, a reagent composition prepared so that meropenem at the time of use is 0.015 to 256 μg/mL is dried and immobilized in each well of the first container of the multi-well plate, A multi-well plate in which a reagent composition prepared to give 0.015 to 256 μg/mL of meropenem and 4 μg/mL of clavulanic acid as an inhibitor at the time of use is dry-immobilized in each well of the second container. and so on. When the reagent composition contains basal medium components, purified water or the like is added to each well of the multi-well plate to dissolve the reagent composition into a drug-containing liquid medium, which is used in the carbapenemase detection method of the present invention. . On the other hand, when the reagent composition does not contain basal medium components, a liquid basal medium is added to each well of the multi-well plate to dissolve the reagent composition into a drug-containing liquid medium, and the carbapenemase detection method of the present invention is used. used for The method for drying and immobilization is not particularly limited as long as the drug and medium components are not degraded, and general drying methods such as air drying, air drying, freeze drying, and vacuum drying can be used. As a general rule, all operations for preparation of dry-immobilized multi-well plates are also performed in a sterile environment.

The multi-well plate of the present invention may be provided as a carbapenemase detection plate, but may also be provided as a carbapenemase determination plate, a carbapenemase discrimination plate, or a drug susceptibility test plate.

The multi-well plate of the present invention includes, for example, a plastic plate having a plurality of wells as a container, and specific examples include microplates such as 24-well plates, 96-well plates, and 192-well plates. The number is not limited.

In the method for detecting a carbapenemase-producing bacterium of the present invention, for example, after preculturing a test sample, a colony of the strain is caught and suspended in sterilized physiological saline to prepare a suspension of the test bacterium. A liquid medium contained in each well of the multi-well plate of the invention is inoculated with a test bacterium. Next, the medium inoculated with the test bacterium is cultured for a predetermined time (16 to 24 hours) under temperature conditions (35±2° C.) suitable for the growth of the test bacterium. Next, observe the medium after culture, observe the presence or absence of growth of the test bacteria, determine the MIC from the growth status of the test bacteria and the concentration of the carbapenem antibacterial drug, and If the MIC in the liquid medium containing the carbapenem antibacterial drug and inhibitor is lower than the MIC in the liquid medium containing, it is determined to be a carbapenemase-producing bacterium. As described above, the detection method of the present invention enables detection and discrimination of various carbapenemase-producing bacteria including class A in a single drug susceptibility test (liquid microdilution method). In addition, simpler and quicker detection is possible by performing the determination using an automatic analyzer.

Examples of the present invention are shown below, but the present invention is not limited to these examples.

(Confirmation of class A carbapenemase-producing and non-producing bacteria by the microdilution method 1)
3 strains of Klebsiella pneumoniae, 1 strain of Escherichia coli, and 3 strains of Klebsiella pneumoniae, which have been confirmed to be class A carbapenemase-producing bacteria in a liquid microdilution method using meropenem (MEPM) as a substrate and clavulanic acid (CVA) as an inhibitor. 2 strains of Escherichia coli and 1 strain of Pseudomonas aeruginosa, which have been confirmed as non-class A carbapenemase-producing bacteria, were used as test bacteria to examine the detection performance of class A carbapenemase-producing bacteria. Although positive controls are not described below, cation-adjusted Muller-Hinton bouillon medium containing no antibacterial drugs or inhibitors was used as a positive control, and it was confirmed that the test bacteria grew without problems.

(1) Preparation of microplate
Two-fold dilution series of cation-adjusted Mueller-Hinton broth medium containing 0.015 to 32 μg/mL of meropenem according to the standard microdilution method set by the Clinical and Laboratory Standards Institute (hereinafter referred to as CLSI) , and dispensed 100 μL each into a 96-well microplate. Similarly, 100 μL each of a 2-fold dilution series of cation-adjusted Muller-Hinton bouillon medium supplemented with 1 to 64 μg/mL of clavulanic acid was dispensed into a 96-well microplate. The drug arrangement of the prepared microplate is as shown in FIG. 8 (drug arrangement 1). The prepared microplate was stored at −70° C. and thawed before use.

(2) Bacteria inoculation and culture A colony of each pre-cultured strain was picked and suspended in sterile physiological saline to prepare a McFarland No. 1 bacterial solution, which was then diluted 10-fold with sterile physiological saline. It was used as an inoculum solution. After that, 2.5 μL of the inoculum solution was inoculated into each well of the microplate prepared in (1) (therefore, the final inoculum amount per 100 μL of medium in each well was about 5×10 ⁴ CFU ( colony forming unit)/well) and aerobically cultured at 35±2° C. for 18 hours.

(3) MIC measurement and determination result After culturing, each MIC was measured and determined. The MIC value results are shown in Table 1 below.

From the results in Table 1, in the tested meropenem concentration range of 0.015 to 32 μg / mL, the threshold for the difference between the MIC value of meropenem/clavulanic acid and the MIC value of meropenem alone should be 2 tubes (4 times) or more. showed that class A carbapenemase-producing bacteria, typified by KPC type, can be detected in the concentration range of 1 to 64 μg/mL of clavulanic acid added.

(Confirmation of class A carbapenemase-producing and non-producing bacteria by microdilution method 2)
Three strains of Klebsiella pneumoniae, one strain of E. coli, and one strain of Klebsiella pneumoniae, one strain of E. coli, and three strains of Klebsiella pneumoniae, one strain of E. coli, and three strains of Klebsiella pneumoniae, one strain of E. coli, and three strains of Klebsiella pneumoniae that have been confirmed to be class A carbapenemase-producing bacteria in a broth microdilution method using meropenem (MEPM) as a substrate and tazobactam (TAZ) as an inhibitor in combination with tazobactam (TAZ) as an inhibitor Two strains of Escherichia coli and one strain of Pseudomonas aeruginosa, which have been confirmed as class A carbapenemase non-producing bacteria, were used as test bacteria, and the detection performance of class A carbapenemase producing bacteria was investigated. Although positive controls are not described below, cation-adjusted Muller-Hinton bouillon medium containing no antibacterial drugs or inhibitors was used as a positive control, and it was confirmed that the test bacteria grew without problems.

(1) Preparation of microplate
A 2-fold dilution series of cation-adjusted Muller Hinton bouillon medium containing 0.015-32 μg/mL of meropenem was prepared according to the standard microdilution method established by CLSI, and 100 μL of each was added to a 96-well microplate. Dispensed. Similarly, 100 μL each of a 2-fold dilution series of cation-adjusted Muller-Hinton bouillon medium supplemented with 1 to 64 μg/mL of tazobactam was dispensed into a 96-well microplate. The drug arrangement of the prepared microplate is as shown in FIG. 9 (drug arrangement 2). The prepared microplate was stored at −70° C. and thawed before use.

(2) Bacteria inoculation and culture A colony of each pre-cultured strain was picked and suspended in sterile physiological saline to prepare a McFarland No. 1 bacterial solution, which was then diluted 10-fold with sterile physiological saline. It was used as an inoculum solution. After that, 2.5 μL of the inoculum solution was inoculated into each well of the microplate prepared in (1) (therefore, the final inoculum amount per 100 μL of medium in each well was about 5×10 ⁴ CFU/ wells) and aerobically cultured at 35±2° C. for 18 hours.

(3) MIC measurement and determination result After culturing, each MIC was measured and determined. The MIC value results are shown in Table 2 below.

From the results in Table 2, in the tested meropenem concentration range of 0.015 to 32 μg / mL, by setting the threshold of the difference between the MIC value of meropenem / tazobactam and the MIC value of meropenem alone to 1 tube (double) or more, It was shown that class A carbapenemase-producing bacteria, typified by KPC type, can be detected at concentrations of tazobactam added in the range of 1 to 64 μg/mL.

(Detection of carbapenemase-producing bacteria by microdilution method 1)
Microdilution using meropenem (MEPM) as substrate in combination with clavulanic acid (CVA) as inhibitor, dipicolinic acid (DPA) as inhibitor, and avibactam (AVI) as inhibitor 3 strains of Klebsiella pneumoniae and 1 strain of E. coli confirmed to be class A carbapenemase-producing bacteria, 1 strain of Klebsiella pneumoniae confirmed to be class B carbapenemase-producing bacteria, and class D One strain of Escherichia coli and one strain of Klebsiella pneumoniae, which have been confirmed to be type carbapenemase-producing bacteria, were used as test bacteria to examine the detection performance of carbapenemase-producing bacteria.

(1) Preparation of microplate
According to the standard microdilution method established by CLSI, a 2-fold dilution series of cation-adjusted Muller-Hinton broth medium containing 0.001-256 μg/mL of meropenem was prepared, and 100 μL each was placed in a 96-well microplate. Dispensed. Similarly, 100 μL each of a 2-fold dilution series of cation-adjusted Muller-Hinton bouillon medium supplemented with 4 μg/mL of clavulanic acid, 175 μg/mL of dipicolinic acid, or 4 μg/mL of avibactam is dispensed into a 96-well microplate. did. The drug arrangement of the prepared microplate is as shown in FIG. 10 (drug arrangement 3). The prepared microplate was stored at −70° C. and thawed before use.

(2) Bacteria inoculation and culture A colony of each pre-cultured strain was picked and suspended in sterile physiological saline to prepare a McFarland No. 1 bacterial solution, which was then diluted 10-fold with sterile physiological saline. It was used as an inoculum solution. After that, 2.5 μL of the inoculum solution was inoculated into each well of the microplate prepared in (1) (therefore, the final inoculum amount per 100 μL of medium in each well was about 5×10 ⁴ CFU/ wells) and aerobically cultured at 35±2° C. for 18 hours.

(3) MIC measurement and judgment results After culturing, each MIC was measured, and the threshold for the difference between the MIC value of meropenem/clavulanic acid and the MIC value of meropenem alone was set to 2 tubes or more, and the MIC value of meropenem/dipicolinic acid and the The threshold for the difference in MIC value for meropenem alone was set at 2 tubes or more, and the threshold for the difference between the MIC value for meropenem/avibactam and the MIC value for meropenem alone was set at 2 tubes or more for determination.

The MIC value results are shown in Table 3 below. All of the class A carbapenemase-producing bacteria tested showed that the MIC value of meropenem/clavulanic acid was lower than that of meropenem alone by more than 2 tubes, and the combination of meropenem and clavulanic acid reduced the MIC value of class A carbapenemase-producing bacteria. was shown to be detectable. The class A carbapenemase-producing bacteria tested showed that the MIC value of meropenem/avibactam was lower than the MIC value of meropenem alone by two or more tubes, indicating that the combination of meropenem and avibactam can also be detected. rice field. That is, the MIC value of meropenem/clavulanic acid is lower than the MIC value of meropenem alone by 2 tubes or more, and the MIC value of meropenem/avibactam is lower than the MIC value of meropenem alone by 2 tubes or more. It can be identified as a class A carbapenemase-producing bacterium. In addition, the class B carbapenemase-producing bacteria tested showed that the MIC value of meropenem/dipicolinic acid was lower than the MIC value of meropenem alone by two tubes or more, and the combination of meropenem and dipicolinic acid reduced the class B carbapenemase-producing bacteria. shown to be detectable. Furthermore, in the two class D-type carbapenemase-producing strains tested, the MIC value of meropenem/clavulanic acid did not decrease by more than 2 tubes compared to the MIC value of meropenem alone, and compared to the MIC value of meropenem alone, The MIC value of meropenem/avibactam was reduced by two tubes or more, indicating that class D carbapenemase-producing bacteria can be detected by using the combination of meropenem and clavulanic acid and the combination of meropenem and avibactam.

(Detection of carbapenemase-producing bacteria by microdilution method 2)
Microdilution using meropenem (MEPM) as substrate in combination with clavulanic acid (CVA) as inhibitor, dipicolinic acid (DPA) as inhibitor, and avibactam (AVI) as inhibitor 1 strain of Escherichia coli that has been confirmed to be a class A carbapenemase-producing bacterium, 1 strain of Klebsiella pneumoniae that has been confirmed to be a class B carbapenemase-producing bacterium, and a class D-type carbapenemase-producing bacterium. Using 1 strain of E. coli and 1 strain of Klebsiella pneumoniae, for which it has been confirmed, as test bacteria, the detection performance of carbapenemase-producing bacteria was examined.

(1) Preparation of microplate
A 2-fold dilution series of cation-adjusted Muller Hinton bouillon medium containing 0.015-32 μg/mL of meropenem was prepared according to the standard microdilution method established by CLSI, and 100 μL of each was added to a 96-well microplate. Dispensed. Similarly, 100 μL each of a 2-fold dilution series of cation-adjusted Muller-Hinton bouillon medium supplemented with 4 μg/mL of clavulanic acid, 175 μg/mL of dipicolinic acid, or 4 μg/mL of avibactam is dispensed into a 96-well microplate. did. The drug arrangement of the prepared microplate is as shown in FIG. 11 (drug arrangement 4). The prepared microplate was stored at −70° C. and thawed before use.

(2) Bacteria inoculation and culture A colony of each pre-cultured strain was picked and suspended in sterile physiological saline to prepare a McFarland No. 1 bacterial solution, which was then diluted 10-fold with sterile physiological saline. It was used as an inoculum solution. After that, 2.5 μL of the inoculum solution was inoculated into each well of the microplate prepared in (1) (therefore, the final inoculum amount per 100 μL of medium in each well was about 5×10 ⁴ CFU/ wells) and aerobically cultured at 35±2° C. for 18 hours.

(3) MIC measurement and judgment results After culturing, each MIC was measured, and the threshold for the difference between the MIC value of meropenem/clavulanic acid and the MIC value of meropenem alone was set to 2 tubes or more, and the MIC value of meropenem/dipicolinic acid and the The threshold for the difference in MIC value for meropenem alone was set at 2 tubes or more, and the threshold for the difference between the MIC value for meropenem/avibactam and the MIC value for meropenem alone was set at 2 tubes or more for determination.

The MIC value results are shown in Table 4 below. In the class A carbapenemase-producing bacteria tested, the MIC value of meropenem/clavulanic acid was lower than the MIC value of meropenem alone by more than 2 tubes. shown to be detectable. The class A carbapenemase-producing bacteria tested showed that the MIC value of meropenem/avibactam was lower than the MIC value of meropenem alone by two or more tubes, indicating that the combination of meropenem and avibactam can also be detected. rice field. That is, the MIC value of meropenem/clavulanic acid is lower than the MIC value of meropenem alone by 2 tubes or more, and the MIC value of meropenem/avibactam is lower than the MIC value of meropenem alone by 2 tubes or more. It can be identified as a class A carbapenemase-producing bacterium. In addition, the class B carbapenemase-producing bacteria tested showed that the MIC value of meropenem/dipicolinic acid was lower than the MIC value of meropenem alone by two tubes or more, and the combination of meropenem and dipicolinic acid reduced the class B carbapenemase-producing bacteria. shown to be detectable. Furthermore, in the two class D-type carbapenemase-producing strains tested, the MIC value of meropenem/clavulanic acid did not decrease by more than 2 tubes compared to the MIC value of meropenem alone, and compared to the MIC value of meropenem alone, The MIC value of meropenem/avibactam was reduced by two tubes or more, indicating that class D carbapenemase-producing bacteria can be detected by using the combination of meropenem and clavulanic acid and the combination of meropenem and avibactam.

(Detection of carbapenemase-producing bacteria by microdilution method 3)
In a broth microdilution method using meropenem (MEPM) as substrate in combination with tazobactam (TAZ) as inhibitor, dipicolinic acid (DPA) as inhibitor, and avibactam (AVI) as inhibitor , 1 strain of Escherichia coli confirmed to be a class A carbapenemase-producing bacterium, 1 strain of Klebsiella pneumoniae confirmed to be a class B carbapenemase-producing bacterium, and a class D carbapenemase-producing bacterium. One strain of Escherichia coli and one strain of Klebsiella pneumoniae that have been confirmed were used as test bacteria, and the detection performance of carbapenemase-producing bacteria was examined.

(1) Preparation of microplate
A 2-fold dilution series of cation-adjusted Muller Hinton bouillon medium containing 0.015-32 μg/mL of meropenem was prepared according to the standard microdilution method established by CLSI, and 100 μL of each was added to a 96-well microplate. Dispensed. Similarly, 100 μL of 2-fold serial dilutions of cation-adjusted Mueller-Hinton broth medium supplemented with 4 μg/mL of tazobactam, 175 μg/mL of dipicolinic acid, or 4 μg/mL of avibactam were dispensed into 96-well microplates. The drug arrangement of the prepared microplate is as shown in FIG. 12 (drug arrangement 5). The prepared microplate was stored at −70° C. and thawed before use.

(2) Bacteria inoculation and culture A colony of each pre-cultured strain was picked and suspended in sterile physiological saline to prepare a McFarland No. 1 bacterial solution, which was then diluted 10-fold with sterile physiological saline. It was used as an inoculum solution. After that, 2.5 μL of the inoculum solution was inoculated into each well of the microplate prepared in (1) (therefore, the final inoculum amount per 100 μL of medium in each well was about 5×10 ⁴ CFU/ wells) and aerobically cultured at 35±2° C. for 18 hours.

(3) MIC measurement and judgment results After culturing, each MIC was measured. The threshold for the difference in the MIC values of meropenem/avibactam and the MIC value for meropenem alone was set at 2 tubes or more.

The MIC value results are shown in Table 5 below. In the class A carbapenemase-producing bacteria tested, the MIC value of meropenem/tazobactam is lower than the MIC value of meropenem alone by two tubes or more, and the combination of meropenem and tazobactam can detect class A carbapenemase-producing bacteria. was shown. The class A carbapenemase-producing bacteria tested showed that the MIC value of meropenem/avibactam was lower than the MIC value of meropenem alone by two or more tubes, indicating that the combination of meropenem and avibactam can also be detected. rice field. That is, the MIC value of meropenem/clavulanic acid is lower than the MIC value of meropenem alone by 2 tubes or more, and the MIC value of meropenem/avibactam is lower than the MIC value of meropenem alone by 2 tubes or more. It can be identified as a class A carbapenemase-producing bacterium. In addition, the class B carbapenemase-producing bacteria tested showed that the MIC value of meropenem/dipicolinic acid was lower than the MIC value of meropenem alone by two tubes or more, and the combination of meropenem and dipicolinic acid reduced the class B carbapenemase-producing bacteria. shown to be detectable. In addition, the MIC value of meropenem/tazobactam did not decrease by more than 2 tubes compared to the MIC value of meropenem alone, and the MIC value of meropenem/tazobactam compared to the MIC value of meropenem alone did not decrease in any of the two tested class D-type carbapenemase-producing strains. The MIC value of avibactam was reduced by two tubes or more, indicating that class D carbapenemase-producing bacteria can be detected by using a combination of meropenem and tazobactam and a combination of meropenem and avibactam.

The method for detecting class A carbapenemase-producing bacteria of the present invention and the multi-well plate used therefor comprise a liquid medium containing 0.015 to 256 μg/mL of meropenem and 1 to 64 μg/mL of clavulanic acid as an inhibitor in a liquid microdilution method. mL or a liquid medium containing 1 to 64 μg/mL of tazobactam can be used in combination to eliminate the first step required in the conventional method (drug susceptibility test by the broth microdilution method or disk method as the first step). and checking for the presence of bacteria suspected of producing carbapenemase) is unnecessary, and class A carbapenemase-producing bacteria represented by KPC type can be detected easily, accurately and quantitatively one day earlier than the conventional method. It becomes possible. Furthermore, in addition to class A carbapenemases, the combination of meropenem and dipicolinic acid, the combination of imipenem and dipicolinic acid, the combination of meropenem and avibactam, and the oxacephem antibiotic latamoxef are used in liquid media containing Since it is possible to specifically detect type B carbapenemase and class D carbapenemase at the same time, it is useful in a wide range of fields such as clinical practice and epidemiological research.

Claims (11)

A method for detecting a carbapenemase-producing bacterium by a liquid microdilution method using a liquid medium, wherein the liquid medium is the following (a), (b) and (f), and the carbapenemase-producing bacterium belongs to the class It is a type A carbapenemase-producing bacterium and a class D carbapenemase-producing bacterium, and includes the step of conducting a drug susceptibility test in all liquid media containing meropenem concentrations of 0.03 μg / mL or more and 16 μg / mL or less. A method for detecting carbapenemase-producing bacteria.
(a) A liquid medium containing serial dilutions of meropenem, wherein the concentration of meropenem is 0.03 μg/mL or more and 16 μg/mL or less within the range of 0.015 μg/mL or more and 256 μg/mL or less a liquid medium that necessarily contains a range of dilution series concentrations and optionally contains a range of dilution series concentrations below 0.03 μg/mL and above 16 μg/mL ;
(b) In each liquid medium containing meropenem at a dilution series concentration, a constant concentration of clavulanic acid within the range of 1 µg/mL or more and 32 µg/mL or less or a range of 2 µg/mL or more and 16 µg/mL or less as an inhibitor A liquid medium containing a constant concentration of tazobactam in
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL a liquid medium optionally containing serial dilution concentrations ranging from less than and greater than 16 μg/mL ;
(f) A liquid medium containing avibactam at a constant concentration within the range of 2 μg/mL or more and 8 μg/mL or less as an inhibitor in each liquid medium containing serial concentrations of meropenem ,
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL A liquid medium optionally containing serial dilution concentrations ranging below and above 16 μg/mL . A method for detecting a carbapenemase-producing bacterium by a liquid microdilution method using a liquid medium, wherein the liquid medium is the following (a), (b) and (f), and the carbapenemase-producing bacterium belongs to the class A carbapenemase characterized by comprising a step of conducting a drug susceptibility test in all liquid media containing a type A carbapenemase-producing bacterium and a class D carbapenemase-producing bacterium and a meropenem concentration of 0.015 µg/mL or more and 32 µg/mL or less. A method for detecting producing bacteria.
(a) A liquid medium containing serial dilutions of meropenem, wherein the concentration of meropenem is 0.015 μg/mL or more and 32 μg/mL or less within the range of 0.015 μg/mL or more and 256 μg/mL or less a liquid medium that necessarily contains a range of dilution series concentrations and optionally contains a range of dilution series concentrations greater than 32 μg/mL;
(b) In each liquid medium containing meropenem at a dilution series concentration, a constant concentration of clavulanic acid within the range of 1 μg / mL or more and 32 μg / mL or less or a constant concentration of 2 μg / mL or more and 16 μg / mL or less as an inhibitor A liquid medium containing a constant concentration of tazobactam,
Concentration of meropenem must include dilution serial concentrations over the entire range of 0.015 μg/mL or more and 32 μg/mL or less in the range of 0.015 μg/mL or more and 256 μg/mL or less, and exceeds 32 μg/mL a liquid medium optionally containing a range of dilution series concentrations;
(f) A liquid medium containing avibactam at a constant concentration within the range of 2 μg/mL or more and 8 μg/mL or less as an inhibitor in each liquid medium containing serial concentrations of meropenem,
Concentration of meropenem must include dilution serial concentrations over the entire range of 0.015 μg/mL or more and 32 μg/mL or less in the range of 0.015 μg/mL or more and 256 μg/mL or less, and exceeds 32 μg/mL A liquid medium that optionally contains a range of dilution series concentrations. In addition to the liquid medium, the following (c) is used, and the carbapenemase-producing bacterium is a class A carbapenemase-producing bacterium, a class B-type carbapenemase-producing bacterium, and a class D-type carbapenemase-producing bacterium. The method for detecting a carbapenemase-producing bacterium according to claim 1.
(c) a liquid medium containing dipicolinic acid at a constant concentration within the range of 100 μg/mL or more and 1600 μg/mL or less as an inhibitor in each liquid medium containing meropenem at a dilution series concentration ,
The concentration of meropenem must be in the range of 0.015 μg/mL or more and 256 μg/mL or less, and must include a serial dilution series concentration within the range of 0.03 μg/mL or more and 16 μg/mL or less, and 0.03 μg/mL A liquid medium optionally containing serial dilution concentrations ranging below and above 16 μg/mL . In addition to the liquid medium, the following (d) and (e) are used, and the carbapenemase-producing bacterium is a class A carbapenemase-producing bacterium, a class B carbapenemase-producing bacterium and a class D carbapenemase-producing bacterium. The method for detecting a carbapenemase-producing bacterium according to claim 3 .
(d) A liquid medium containing imipenem at a dilution series concentration, wherein the concentration of imipenem is in the range of 0.015 μg/mL or more and 256 μg/mL or less, and in the range of 0.03 μg/mL or more and 16 μg/mL or less and optionally containing serial dilutions in the range of less than 0.03 μg/mL and greater than 16 μg/mL ;
(e) A liquid medium containing dipicolinic acid at a constant concentration within the range of 100 μg/mL or more and 1600 μg/mL or less as an inhibitor in each liquid medium containing imipenem at a dilution series concentration ,
Concentration of imipenem must be in the range of 0.015 μg/mL or more and 256 μg/mL or less, and must include a serial dilution series concentration within the range of 0.03 μg/mL or more and 16 μg/mL or less, and 0.03 μg/mL A liquid medium optionally containing serial dilution concentrations ranging below and above 16 μg/mL . 5. The method for detecting a carbapenemase-producing bacterium according to claim 1 , 3 or 4 , wherein the following (g) is used in addition to the liquid medium.
(g) a liquid medium containing latamoxef at serially diluted concentrations within the range of 0.06 μg/mL or more and 128 μg/mL or less; 2. The liquid medium is selected from Mueller-Hinton broth, cation-adjusted Mueller-Hinton broth, hemolyzed Mueller-Hinton broth, heart infusion broth, normal broth and trypto-soy broth. , the method for detecting a carbapenemase-producing bacterium according to any one of 3 to 5 . A multi-well plate comprising a plurality of storage units consisting of a plurality of wells for storing a liquid medium, and used in the method for detecting carbapenemase-producing bacteria according to any one of claims 1 and 3 to 6 , The plurality of storage units include a first storage unit including the following (a) in each well, a second storage unit including the following (b) in each well, and a third storage unit including the following (f) in each well The multi-well plate, comprising:
(a) A liquid medium containing serial dilutions of meropenem, wherein the concentration of meropenem is 0.03 μg/mL or more and 16 μg/mL or less within the range of 0.015 μg/mL or more and 256 μg/mL or less a liquid medium that necessarily contains a range of dilution series concentrations and optionally contains a range of dilution series concentrations below 0.03 μg/mL and above 16 μg/mL ;
(b) In each liquid medium containing meropenem at a dilution series concentration, a constant concentration of clavulanic acid within the range of 1 µg/mL or more and 32 µg/mL or less or a range of 2 µg/mL or more and 16 µg/mL or less as an inhibitor A liquid medium containing a constant concentration of tazobactam in
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL a liquid medium optionally containing serial dilution concentrations ranging from less than and greater than 16 μg/mL ;
(f) A liquid medium containing avibactam at a constant concentration within the range of 2 μg/mL or more and 8 μg/mL or less as an inhibitor in each liquid medium containing serial concentrations of meropenem ,
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL A liquid medium optionally containing serial dilution concentrations ranging below and above 16 μg/mL . 8. The multi-well plate of claim 7 , wherein said liquid medium contained in each well is frozen. 8. The multi-well plate according to claim 7 , wherein the liquid medium contained in each well is dry-immobilized. A multi-well plate used in the method according to any one of claims 1 and 3 to 6, comprising a plurality of holding portions each composed of a plurality of wells for holding a liquid medium, wherein the plurality of holding portions contains the following (a') in each well, the first container in which the drug in each well is dry-immobilized, each well contains the following (b'), and the drug in each well is dry-immobilized The multi-well plate is characterized by comprising a second container and a third container in which each well contains the following (f') and a drug in each well is dried and immobilized.
(a′) A liquid medium containing meropenem at dilution serial concentrations when reagent composition 1 is dissolved by adding a liquid when used for performing a drug susceptibility test by a microdilution method for detecting carbapenemase-producing bacteria wherein the concentration of meropenem must include serial dilutions over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and said reagent composition 1 , prepared to be a liquid medium optionally containing serial dilution concentrations ranging from less than 0.03 μg/mL and greater than 16 μg/mL ;
(b') A liquid medium containing meropenem at dilution serial concentrations when reagent composition 2 is dissolved by adding a liquid when used for drug susceptibility testing by the microdilution method for detecting carbapenemase-producing bacteria Each is a liquid medium containing clavulanic acid at a constant concentration within the range of 1 µg/mL or more and 32 µg/mL or less or tazobactam at a constant concentration within the range of 2 µg/mL or more and 16 µg/mL or less as an inhibitor. hand,
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL Reagent composition 2 , prepared to be a liquid medium optionally comprising serial dilution concentrations ranging from less than and greater than 16 μg/mL ;
(f') A liquid medium containing meropenem at dilution series concentrations when reagent composition 3 is dissolved by adding a liquid when used for drug susceptibility testing by the microdilution method for detecting carbapenemase-producing bacteria A liquid medium containing avibactam at a constant concentration within the range of 2 μg/mL or more and 8 μg/mL or less as an inhibitor,
The concentration of meropenem must include the dilution series concentration over the entire range of 0.03 μg/mL to 16 μg/mL within the range of 0.015 μg/mL to 256 μg/mL, and 0.03 μg/mL Reagent composition 3 prepared to be a liquid medium optionally containing serial dilution concentrations ranging from less than and greater than 16 μg/mL . 3. A multi-well plate for use in the method of claim 2, comprising a plurality of receptacles consisting of a plurality of wells for containing a liquid medium, wherein the plurality of receptacles includes (a '), a first containing portion in which the drug in each well is dry-immobilized, a second containing portion in each well containing the following (b'), in which the drug in each well is dry-immobilized, and each The above multi-well plate characterized by containing the following (f') in the wells and containing a third container in which a drug in each well is dried and immobilized.
(a′) A liquid medium containing meropenem at dilution serial concentrations when reagent composition 1 is dissolved by adding a liquid when used for performing a drug susceptibility test by a microdilution method for detecting carbapenemase-producing bacteria wherein the concentration of meropenem is in the range of 0.015 μg/mL or more and 256 μg/mL or less, necessarily including the dilution series concentration over the entire range of 0.015 μg/mL or more and 32 μg/mL or less, and 32 μg The reagent composition 1 prepared so as to be a liquid medium optionally containing a dilution series concentration in a range exceeding /mL,
(b') A liquid medium containing meropenem at dilution serial concentrations when reagent composition 2 is dissolved by adding a liquid when used for drug susceptibility testing by the microdilution method for detecting carbapenemase-producing bacteria A liquid medium containing clavulanic acid at a constant concentration in the range of 1 μg/mL or more and 32 μg/mL or less or tazobactam at a constant concentration in the range of 2 μg/mL or more and 16 μg/mL or less as an inhibitor,
Concentration of meropenem must include dilution serial concentrations over the entire range of 0.015 μg/mL or more and 32 μg/mL or less in the range of 0.015 μg/mL or more and 256 μg/mL or less, and exceeds 32 μg/mL reagent composition 2, prepared to be a liquid medium optionally comprising a range of dilution series concentrations;
(f') A liquid medium containing meropenem at dilution series concentrations when reagent composition 3 is dissolved by adding a liquid when used for drug susceptibility testing by the microdilution method for detecting carbapenemase-producing bacteria A liquid medium containing avibactam at a constant concentration within the range of 2 μg/mL or more and 8 μg/mL or less as an inhibitor,
Concentration of meropenem must include dilution serial concentrations over the entire range of 0.015 μg/mL or more and 32 μg/mL or less in the range of 0.015 μg/mL or more and 256 μg/mL or less, and exceeds 32 μg/mL A reagent composition 3 prepared to result in a liquid medium optionally containing a range of dilution series concentrations.

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