JP2014200200A - β-LACTAMASE DETECTION METHOD - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of detailed classification of enzymes in a short period of time based on the substrate specificity of β-lactamase, as well as reagents therefor.SOLUTION: A method for detecting and determining microbial β-lactamase comprises the steps of: (a) bringing each of the following six classes of β-lactam in contact with a microorganism to be tested to determine the presence or absence of decomposition of respective drugs by acidmetry, where the six classes of β-lactam are: 1. penicillin, 2. the first generation cephem, 3. the third generation cephem and/or the fourth generation cephem, 4. monobactam, 5. cephamycin and/or oxacephem, and 6. carbapenem; (b) determining the enzyme classification of the target microorganism to be tested on the basis of the results of decomposition obtained in the step (a).

Description

  The present invention relates to a method for detecting β-lactamase. More specifically, the present invention relates to a method for determining a detailed enzyme classification based on the substrate specificity of β-lactamase by an acidometry method and a reagent used therefor. In the present specification, the scientific name and gene name of a bacterium are described in an ordinary typeface, not a commonly used italic type.

  β-lactam antibacterial drugs (hereinafter referred to as β-lactam drugs) are generic names of antibacterial drugs having a β-lactam ring in the molecular structure of the nucleus, and since the discovery of penicillin, many β-lactam drugs have been developed. ing. With the development of this β-lactam drug, resistant bacteria that produce an enzyme (β-lactamase) that hydrolyzes the β-lactam ring in its molecular structure and invalidates its antibacterial action (β-lactamase producing bacteria) Has emerged and has become an important issue for measures against nosocomial infections.

  β-lactamases have been classified into classes A to D by Ambler et al. based on the homology of the primary amino acid sequence of the enzyme protein. Β-lactamase belonging to class A is a serine peptidase having a serine residue at the active center. The original enzyme is penicillinase that hydrolyzes penicillin antibacterials, but its substrate specificity expands to hydrolyze not only penicillin antibacterials but also cephalosporin and monobactam antibacterials. An extended-spectrum β-lactamase (hereinafter referred to as ESBL), which has also been shown, is also included in the class A β-lactamase. In addition, the production of KPC (Klebsiella pneumoniae carbapenemase) type carbapenemase belonging to class A has become a problem overseas. Β-lactamase belonging to class B is called metallo-β-lactamase (hereinafter referred to as MBL) because it has a zinc ion at the active center. Many β-lactamases belonging to this class hydrolyze a wide range of penicillin antibacterials, cephem antibacterials and carbapenem antibacterials. Β-lactamase belonging to class C is a cephalosporinase having a serine residue at the active center and hydrolyzing mainly cephalosporin antibacterial agents. AmpC, which is a gene encoding β-lactamase belonging to class C, is present in the chromosomes of bacterial species belonging to non-fermentative bacteria from the Enterobacteriaceae except for some bacterial species, and such β- Strains that produce lactamase are called chromosomal AmpC producers and are sensitive to cephamycin antibiotics. On the other hand, a plasmid-like AmpC in which ampC is encoded on a plasmid (for example, overproduced AmpC) is resistant to cephamycin antibacterials and oxacephem antibacterials because AmpC is efficiently produced. Β-lactamase belonging to class D has a serine residue at the active center like enzymes belonging to class A, and the prototype enzyme mainly hydrolyzes penicillin antibacterial drugs. The difference from class A β-lactamases is that the efficiency of degrading oxacillin is higher than the efficiency of penicillin degradation, and β-lactamases belonging to class D are called oxacillinases. OXA (oxacillinase) type carbapenemase is known as a carbapenemase belonging to class D (Non-patent Document 1).

  As described above, since β-lactamase hydrolyzes and inactivates β-lactam drug as its substrate, β-lactam drug as its substrate is administered to patients infected with β-lactamase producing bacteria. Often makes treatment difficult. On the other hand, administration of an antibacterial agent having a wider antibacterial spectrum than necessary also causes new resistant bacteria to appear. Therefore, there is a need for a rapid test method that can determine whether or not an infected bacterium produces β-lactamase and, if so, which anti-bacterial agent the β-lactamase inactivates before administering the antibacterial agent. It is rare.

  Currently, chromogenic methods, UV methods, iodometry methods, acidometry methods, and the like are known as methods capable of rapid detection of β-lactamase (Patent Documents 1, 2, and 3). The chromogenic method uses a substrate that changes color when the β-lactam ring is cleaved by hydrolysis with β-lactamase. This method is highly sensitive because the substrate itself changes color, but the substrate specificity cannot be determined because the β-lactamase class is classified by combining a single substrate and multiple β-lactamase inhibitors. . The UV method is generally a method performed by purifying an enzyme from bacterial cells, and therefore cannot be used for a simple test method.

  The iodometry method is based on the fact that an acid generated by hydrolysis of a substrate by β-lactamase reduces iodine, and detects a change in color due to an iodo starch reaction as an index. On the other hand, the acidometry method is a method for detecting a pH change derived from an acid generated by hydrolysis of a substrate by β-lactamase as a color change of a pH indicator. However, a method that can determine the detailed enzyme classification of β-lactamase using these detection principles has not been known so far.

JP 2004-166694 A International Publication No. 2002/024707 International Publication No. 2006/043558

Yoshikazu Ishii, Clinical Examination, 54 (5), 473-480, 2010

  The present invention has been made in view of the above-described present situation, and an object thereof is to provide a method capable of determining a detailed enzyme classification based on the substrate specificity of β-lactamase in a short time and a reagent used therefor. .

  In order to solve the above problems, the present inventors first examined a method for discriminating the substrate specificity of many types of β-lactamases. Although it is possible to use an iodometry method for detection, there is a concern that in bacteria that show a positive result in the oxidase test, the oxidation-reduction reaction affects the coloration and cannot be accurately determined. As a result of various investigations, by selecting the acidometry method as the detection principle, the influence on the determination can be reduced even in bacteria that are positive in the oxidase test, and the first drug: penicillin, second First generation cephem, third drug; third generation cephem and / or fourth generation cephem, fourth drug; monobactam, fifth drug; cephamycin and / or oxacephem, And a sixth drug; each of at least six β-lactam drugs comprising a carbapenem series is brought into contact with the detection target bacteria, and the presence or absence of decomposition of each drug is determined by an acidometry method. Presence / absence of production, penicillinase, cephalosporinase, ESBL, overproduced AmpC, MBL, KPC carbapenemase or OXA (oxacillinase) carbape Found that can determine whether a Maze. Furthermore, the present inventors also have a reaction in which β-lactamase, which is slow in reaction and takes a long time to detect, is selected from polymyxin B (PL-B), colistin (CL), and n-octyl-β-D-glucoside. It has been found that detection can be performed in a short time by adding an accelerator, and the present invention has been completed.

That is, the present invention has the following configuration.
(1) A method for detecting and determining a β-lactamase of a microorganism, which comprises the following steps.
(A) first drug; penicillin series,
Second drug; first generation cephem system,
A third agent; a third generation cephem system and / or a fourth generation cephem system;
Fourth drug: monobactam,
A fifth drug; cephamycins and / or oxacephems, and
Sixth drug; carbapenem series,
A step of contacting each of the six β-lactam drugs comprising a microorganism to be detected and determining the presence or absence of decomposition of each drug by an acidometry method,
(B) A step of determining the enzyme classification of the detection target microorganism based on the determination result of the presence or absence of decomposition of each drug in the step (a).
(2) In step (a),
Penicillinase producing bacteria are determined that the first drug is decomposed, the second drug is determined to be decomposed or not decomposed, and the third drug, the fourth drug, the fifth drug, and the sixth drug are It is determined that there is no disassembly,
For cephalosporinase-producing bacteria (chromosomal), the first drug and the second drug are determined to be decomposed, and the third drug, the fourth drug, the fifth drug, and the sixth drug are not decomposed. Is determined,
ESBL-producing bacteria are determined that the first drug and the second drug are decomposed, the fifth drug and the sixth drug are determined not to be decomposed, and the third drug and the fourth drug are decomposed or It is determined that there is no disassembly,
Overproduction AmpC producing bacteria, the first drug, the second drug, the third drug, the fourth drug and the fifth drug are determined to be decomposed, the sixth drug is determined to be no decomposition,
MBL-producing bacteria are determined that the first drug, the second drug, the third drug, the fifth drug and the sixth drug are decomposed, the fourth drug is determined not to be decomposed,
The method according to (1), wherein the KPC-type carbapenemase-producing bacterium and the OXA-type carbapenemase-producing bacterium are discriminated that all the six drugs are decomposed.
(3) The method according to (1) or (2), wherein in step (a), the β-lactam drug is contacted with the microorganism to be detected in a medium containing a pH indicator.
(4) The method according to (3), wherein the medium brought into contact with the detection target microorganism in step (a) further contains a reaction accelerator selected from polymyxin B, colistin, and n-octyl-β-D-glucoside.
(5) The method according to (3) or (4), wherein the pH indicator is selected from bromocresol purple, bromothymol blue, nitrazine yellow, and phenol red.
(6) The first drug is penicillin G or piperacillin, the second drug is cefazolin, cephalotin, cefapirin, cephalexin or cefloxazine, and the third drug is cefotaxime, ceftriaxone, ceftizoxime, cefthrosin, cefteram or cefmenoxime The method according to any one of (1) to (5), wherein the fourth drug is aztreonam, the fifth drug is flomoxef or cefmetazole, and the sixth drug is imipenem or panipenem.
(7) The method according to any one of (3) to (6), wherein the medium brought into contact with the detection target microorganism in step (a) is a liquid, a semi-fluid substance, a disk, or a filter paper.
(8) The medium to be contacted with the detection target microorganism in the step (a) is a liquid, and the β-lactam drug and the detection target microorganism are contacted at 20 to 40 ° C. in the well of the multi-well plate. The method according to any one of 7).
(9) A multi-well plate comprising a β-lactamase detection formulation containing one type of β-lactam drug and a pH indicator in each well in a liquid, dry or frozen state, wherein the β-lactam drug is
First drug; penicillin system,
Second drug; first generation cephem system,
A third agent; a third generation cephem system and / or a fourth generation cephem system;
Fourth drug: monobactam,
A fifth drug; cephamycins and / or oxacephems, and
Sixth drug; carbapenem series,
A multi-well plate consisting of 6 lines.
(10) The multi-well plate according to (9), wherein the β-lactamase detection formulation further contains a reaction accelerator selected from polymyxin B, colistin, and n-octyl-β-D-glucoside.
(11) The multiwell plate according to (9) or (10), wherein the pH indicator is selected from bromocresol purple, bromothymol blue, nitrazine yellow, and phenol red.
(12) The first drug is penicillin G or piperacillin, the second drug is cefazolin, cephalotin, cefapirin, cephalexin or cefloxazine, and the third drug is cefotaxime, ceftriaxone, ceftizoxime, cefthrosin, cefteram or cefmenoxime The fourth drug is aztreonam, the fifth drug is flomoxef or cefmetazole, and the sixth drug is imipenem or panipenem (9) to (11) plate.

  The β-lactamase detection method, determination method and reagent used therefor according to the present invention are simple methods in which each of the six β-lactam drugs is brought into contact with a detection target bacterium and the presence or absence of decomposition of each drug is determined by an acidometry method. It has an effect not found in the conventional method that it is possible to determine a detailed enzyme classification based on the substrate specificity of β-lactamase in a short time with a simple operation. In addition, it is possible to make a quicker determination by using a reaction accelerator, including β-lactamases such as Staphylococcus sp. Shortening the judgment time avoids the negative coloration of positive coloration due to the change in pH in the reaction system by prolonging the contact time, and the problem of positive coloration of negative bacteria, resulting in more accurate results. This also brings about an effect that the determination is possible.

FIG. 1 is a diagram showing the results of screening studies for reaction accelerators. (Example 2)

  In the present invention, first drug; penicillin system, second drug; first generation cephem system, third drug; third generation cephem system and / or fourth generation cephem system, fourth drug; monobactam system, A fifth drug; cephamycin and / or oxacephem, and sixth drug; a β-lactam drug selected from carbapenems is contacted with the microorganism to be detected, and the presence or absence of decomposition of each drug is determined by acidometry Discriminate and determine enzyme classification based on substrate specificity. In order to make a more detailed determination of the enzyme classification, the drug to be selected consists of the first drug, the second drug, the third drug, the fourth drug, the fifth drug, and the sixth drug. Six lines of β-lactam drugs are preferred.

The β-lactam drug used in the present invention is a first drug; penicillin system, second drug; first generation cephem system, third drug; third generation cephem system and / or fourth generation cephem system, There is no particular limitation as long as the antibacterial agent is selected from the following four agents: monobactams, fifth agents; cephamycins and / or oxacephems, and sixth agents; carbapenems. Penicillin antibiotics include, for example, penicillin G (PCG), oxacillin (MPIPC), methicillin (DMPPC), flucloxacillin (MFIPC), nafcillin (NFPC), pheneticillin (PEPC), cloxacillin (MCIPC), dicloxacillin (MDIPC) ), Ampicillin (ABPC), cyclacillin (ACPC), amoxicillin (AMPC), lenampicillin (LAPC), aspoxillin (ASPC), piperacillin (PIPC), sulbenicillin (SBPC), sultamicillin (SBTPC), tarampicillin (TAPC), bacampicillin (BAC) ), Pibmesilinum (PMPC), calindacillin (CIPC), calfecillin (CFPC), and hetacillin (IPABPC).
First-generation cephem antibiotics include, for example, cephalotin (CET), cephaloridine (CER), cefapirin (CEPR), cefazolin (CEZ), ceftezol (CTZ), cefacetril (CEC), cephalexin (CEX), cefradine (CED) ), Cefatrizine (CFT), cefloxazine (CXD), cefadroxyl (CDX), cefazedone, cefurodaxin.
Third-generation cephem antibiotics include, for example, cefteram (CFTM), cefmenoxime (CMX), ceftizoxime (CZX), cefoperazone (CPZ), cefotaxime (CTX), cefpyramid (CPM), ceftazidime (CAZ), ceftriaxone ( CTRX), cefodizime (CDZM), cefsulodin (CFS), cefixime (CFIX), ceftibbutene (CETB), cefpodoxime proxetyl (CPDX-PR), cefcapene pivoxil (CFPN-PI), ceftizoxime alla Pivoxil (CZX-AP).
The fourth generation cephem antibacterial agents are, for example, cefepime (CFPM) and cefpirom (CPR).
Monobactam antibacterial agents are, for example, aztreonam (AZT) and carmonam (CRMN).
Cefamycin antibacterial agents are, for example, cefoxitin (CFX), cefmetazole (CMZ), cefotetan (CTT), cefbuperazone (CBPZ), and cefminox (CMNX).
Oxacephem antibacterial agents are, for example, latamoxef (LMOX) and flomoxef (FMOX).
The carbapenem antibacterial agents are, for example, imipenem (IPM), panipenem (PAPM), meropenem (MEPM), biapenem (BIPM), doripenem (DRPM), tevipenem (TBPM), and ertapenem.
In the examples shown later, abbreviations were used. In order to make a quicker and more accurate determination, a drug with strong reaction intensity and stable results, i.e., penicillin G or piperacillin as the first drug, cefazolin, cephalotin, cefapirin, cephalexin or as the second drug Cefloxazine, cefotaxime, ceftriaxone, ceftizoxime, ceftrosin, cefteram or cefmenoxime as the third drug, aztreonam as the fourth drug, fromoxef or cefmetazole as the fifth drug, imipenem or panipenem as the sixth drug Particularly preferred. The present invention is a method using a β-lactam drug selected from at least a first drug, a second drug, a third drug, a fourth drug, a fifth drug and a sixth drug, It is also possible to use other drugs in addition to the β-lactam drugs. For example, a second-generation cephem antibacterial drug can be used as the seventh drug. Specific examples of second-generation cephem antibacterial agents include cefuroxime (CXM), cefamandole (CMD), cefoniside, cefotiam (CTM), loracarbeve (LCBF), cefprozil (CFPZ), and cefolanide.

  The concentration of each β-lactam drug used in the present invention is a concentration at which hydrolysis of the β-lactam drug can be detected by an acidometry method when contacted with a bacterium that produces β-lactamase that degrades the drug. Such a concentration is determined depending on the kind of medium in which the β-lactam drug is brought into contact with the microorganism to be detected, that is, the hydrolysis reaction of the β-lactam drug is performed in a solution or a semi-fluid substance, or a disk or a test piece is used. It is set to an appropriate amount in consideration of whether or not a suitable carrier is used and further considering conditions such as contact time. For example, when reacting in solution, the final concentration of β-lactamase detection reaction solution is 0.1-100 mg / mL penicillin antibacterial agent, 0.1-100 mg / mL first-generation cephem antibacterial, third-generation cephem 0.1-100 mg / mL for antibacterial agents, 0.1-100 mg / mL for fourth-generation cephem antibiotics, 0.1-100 mg / mL for monobactam antibiotics, 0.1-100 mg / mL for cephamycin antibiotics, oxacephem antibiotics The drug is preferably 0.1-100 mg / mL, the carbapenem antibiotic is preferably 0.1-100 mg / mL, the penicillin antibiotic is 0.5-50 mg / mL penicillin G or 0.5-50 mg / mL piperacillin, the first generation cephem antibiotic Cefazolin 0.5-50 mg / mL, cephalotin 0.5-50 mg / mL, cefapirin 0.5-50 mg / mL, cephalexin 0.5-50 mg / mL or cefloxazine 0.5-50 mg / mL, third-generation cephem antibiotics cefotaxime 0.5-50 mg / mL mL, ceftriaxone 0.5-50mg / m L, ceftizoxime 0.5-50 mg / mL, cefsulodin 0.5-50 mg / mL, cefteram 0.5-50 mg / mL or cefmenoxime 0.5-50 mg / mL, monobactam antibacterial agent aztreonam 0.5-50 mg / mL, oxacephem antibacterial agent flomoxef 0.5 More preferably, the cefamycin antibacterial agent is ˜50 mg / mL, the cefamycin antibacterial agent is cefmetazole 0.5 to 50 mg / mL, and the carbapenem antibacterial agent is imipenem 0.5 to 25 mg / mL or panipenem 0.5 to 25 mg / mL.

  The microorganism to be detected of the present invention is a bacterium that may produce β-lactamase. Specific examples include Escherichia coli (hereinafter referred to as E. coli), Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Proteus mirabilis (Proteus mirabilis) , Proteus vulgaris, Morganella morganii, Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Enterobacter cloacae (Enterobacter aerogenes), Pseudomonas aeruginosa (hereinafter referred to as Pseudomonas aeruginosa), Pseudomonas putida, Stenotrophomonas mutofilia (Stenotrophomonas mu Staphylococcus gram-negative and gram-positive bacteria such as ltophilia, Acinetobacter baumannii, Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis (Staphylococcus aureus, hereinafter referred to as Staphylococcus aureus), Enterococcus faecalis, Bacillus cereus, and the like, but are not limited thereto.

Although the microorganisms to be detected are not particularly limited, they may be contained in specimens derived from living organisms, environments, and foods of humans and other animals. Therefore, in the present invention, samples derived from these specimens are used. be able to. A sample solution that can be prepared by dissolving a specimen in a solvent such as physiological saline, phosphate buffered saline, distilled water, or purified water has a concentration of the microorganism to be detected contained in the liquid of 3.0 × 10 ⁴ to Although it can be used as 3.0 × 10 ¹⁰ CFU / mL, the concentration of the microorganism to be detected in the solvent for dissolving the specimen and the sample liquid is not limited to the above description.

  In the present invention, after the β-lactam drug is brought into contact with the detection target microorganism, the presence or absence of decomposition of each drug is determined by an acidometry method. The medium for bringing the β-lactam drug into contact with the detection target microorganism is not particularly limited as long as the β-lactam drug can be decomposed by β-lactamase produced by the detection target microorganism. A medium obtained by impregnating a solid substance such as a semi-fluid substance, a disk, or a filter paper with a β-lactamase detection formulation liquid can be used as a medium, or the medium can be used as a liquid. When the medium is a liquid, the β-lactamase detection formulation liquid itself is used as the medium, or the prepared β-lactamase detection formulation liquid is frozen and stored and then thawed back into a liquid, Alternatively, the β-lactamase detection formulation solution can be used as a medium after being dried by a general method such as natural drying, air drying, heat drying, reduced pressure drying, or freeze drying, and then dissolved by adding a dissolving solution.

  The medium for bringing the β-lactam drug into contact with the microorganism to be detected contains at least a pH indicator. However, a buffer can be further added to the medium for the purpose of stabilizing the optimum pH of the pH indicator. In addition, components that support the growth of microorganisms can be added, but since the acidometry method is used as the detection principle in the present invention, the pH may be increased by factors other than the hydrolysis reaction of β-lactam drugs by β-lactamase. It is not preferable to fluctuate. When a microorganism grows, the pH varies depending on its metabolized components, which hinders accurate discrimination of the hydrolysis reaction according to the present invention by the acidometry method. Therefore, it is preferable that the medium of the present invention does not contain components that support the growth of microorganisms, that is, components that serve as nutrient sources for the growth of microorganisms, such as peptone, extracts, carbohydrates, and minerals.

  The kind and concentration of the pH indicator used in the present invention are not particularly limited. For example, bromocresol purple, bromothymol blue, nitrazine yellow, phenol red, etc. can be used as a pH indicator, but when polymyxin B is used as a reaction accelerator as described later, phenol red having good compatibility with solubility Is preferably used. When the medium is liquid, the concentration of phenol red is preferably 0.01 to 10 ng / mL as the final concentration of the β-lactamase detection reaction solution.

  In the present invention, the β-lactam drug and the microorganism to be detected can be contacted without using a buffer to detect and determine β-lactamase, and for the purpose of stabilizing the optimum pH of the pH indicator. It is also possible to add a buffer to the medium. In the present invention, when a buffering agent is used, the type and concentration of the buffering agent accurately detects a pH change accompanying the hydrolysis of the β-lactam drug by β-lactamase produced by the detection target microorganism as a color change of the pH indicator. It can be. By using the buffer, the optimum pH of the pH indicator can be stabilized, but on the other hand, the pH fluctuation is less likely to occur as the concentration of the buffer increases. Therefore, the type and concentration of the buffering agent are set in consideration of the type of pH indicator, the number of target microorganisms, detection sensitivity, whether or not to contain components that support the growth of microorganisms, and the like. Especially if the pH is kept stable until the hydrolysis of the substrate by β-lactamase starts and the pH can be changed sensitively based on the production of acid accompanying the hydrolysis of the β-lactam drug. Although not limited, for example, when phenol red is used as a pH indicator, a buffering agent that maximizes the buffering ability around pH 6.8 to 8.0 is preferable. As final concentrations of such a buffer and β-lactamase detection reaction solution, for example, 3-morpholinopropanesulfonic acid (hereinafter referred to as MOPS) 0.01 to 100 mM, phosphate buffer 0.01 to 100 mM, Tris-HCl buffer 0.01 ˜100 mM, acetate buffer solution 0.01-100 mM, citrate buffer solution 0.01-100 mM, and the like can be used.

  In the present invention, first drug; penicillin system, second drug; first generation cephem system, third drug; third generation cephem system and / or fourth generation cephem system, fourth drug; monobactam system, A fifth drug; cephamycin and / or oxacephem, and sixth drug; a β-lactam drug selected from carbapenems is contacted with the microorganism to be detected, and the presence or absence of decomposition of each drug is determined by acidometry Discrimination is performed, and detailed enzyme classification is determined based on the obtained substrate specificity data. It is possible to test whether all the drugs are decomposed at once, or to test in multiple times. In the case where the test is divided into a plurality of times, for example, it is possible to first test the three systems of drugs and further test the remaining three systems of drugs as necessary. However, in order to perform detailed enzyme classification in a short time, it is preferable to test 6 drugs in a single test. Table 1 shows the classification and classification table of enzymes. When a typical enzyme determination example is described, penicillinase-producing bacteria are identified as having the first drug decomposed and the second drug having decomposed or not decomposed. It is determined that the third drug, the fourth drug, the fifth drug, and the sixth drug are not decomposed, and the cephalosporinase producing bacterium (chromosomal) is the first drug and the second drug. The drug is determined to be decomposed, the third drug, the fourth drug, the fifth drug, and the sixth drug are determined not to be decomposed, and the ESBL-producing bacteria are decomposed by the first drug and the second drug. It is determined that the fifth and sixth drugs are not decomposed, the third drug and the fourth drug are determined to be decomposed or not decomposed, and the overproducing AmpC producing bacteria are , The second drug, the third drug, the fourth drug, and the fifth drug are determined to be decomposed, It is determined that there is no decomposition, and MBL-producing bacteria are determined that the first drug, the second drug, the third drug, the fifth drug, and the sixth drug are decomposed, and the fourth drug is It is discriminated that there is no degradation, and it is discriminated that all six strains of the KPC-type carbapenemase-producing bacteria and OXA-type carbapenemase-producing bacteria are decomposed. However, the enzyme that can be determined in the present invention is not limited to the above.

  The temperature and time for the decomposition reaction of the β-lactam drug may be set to appropriate conditions according to the type of microorganism to be detected and the medium, but for example, an anaerobic bacterium requires a longer contact time. As the condition setting, the reaction temperature is preferably 20 to 40 ° C, more preferably 30 to 37 ° C. The contact time is preferably 1 second to 48 hours, more preferably 5 minutes to 24 hours.

  The determination of the presence or absence of decomposition of the β-lactam drug of the present invention can be carried out visually or by measuring the presence or absence of a change in the color tone of the medium after contact at an appropriate temperature and time. For example, when bromocresol purple is used as a pH indicator, yellow: decomposed, bluish purple: no degradation, and when bromothymol blue is used as a pH indicator, yellow: decomposed, blue; decomposed In the case of using nitrazine yellow as a pH indicator, yellow: with decomposition, blue: When it is determined that there is no decomposition, and when phenol red is used as a pH indicator, yellow: with decomposition, red; It is determined that there is no disassembly. When an apparatus is used, the presence or absence of decomposition can be determined by measuring the change in absorbance at a specific wavelength before and after the hydrolysis reaction, the change in RGB (Red, Green, Blue), or the change in luminance. The measurement wavelength of absorbance may be set to an appropriate wavelength according to the pH indicator to be used.For example, when phenol red is used as a pH indicator, the absorption maximum wavelength of red is around 500 nm or yellow. The measurement wavelength is preferably around 400 nm which is the maximum wavelength.

  The medium for contacting the β-lactam drug and the microorganism to be detected of the present invention can contain at least one reaction accelerator selected from polymyxin B, colistin, and n-octyl-β-D-glucoside. By using these reaction accelerators, the membrane permeability of the microorganism to be detected is increased, and the time required for the hydrolysis reaction of the β-lactam drug by β-lactamase can be shortened. As a result, it is possible to detect β-lactamase such as Staphylococcus genus that reacts slowly in a short time, and the problem that positive coloration becomes negative due to change in pH of the medium due to prolonged contact time, It is also possible to avoid the problem of positive coloration of negative bacteria. In the present invention, the reaction accelerator is used to increase the permeability of the cell membrane and / or cell wall of the microorganism to be detected. For example, when the medium is a liquid, it is preferable that polymyxin B be used alone at 1 to 40,000 U / mL as the final concentration of the β-lactamase detection reaction solution. In the present invention, however, polymyxin B, colistin, and n-octyl-β-D-glucoside are selected so long as the presence or absence of degradation of six β-lactam drugs can be determined in a short time by the acidometry method. In addition to the types of reaction accelerators, it is also possible to add further reaction accelerators.

  When the medium for contacting the β-lactam drug of the present invention and the microorganism to be detected is a liquid or semi-fluid substance, a medium containing one type of β-lactam drug, buffer, pH indicator, etc. in each well It can be set as the multiwell plate provided with. When the medium in which the microorganism to be detected is brought into contact in a multi-well plate is a liquid, it can be stored in a dry fixed state or a frozen state until use, and returned to the liquid as needed during use. Furthermore, if necessary, a multi-well plate having a control well containing a buffer and a pH indicator without a β-lactam drug, or a negative control solution containing no microorganism, β -It is also possible to set a positive control solution containing lactamase, a sample dilution solution, a pretreatment solution and the like. On the other hand, when the medium for contacting the β-lactam drug and the microorganism to be detected is a carrier such as a disk or filter paper, similarly, a negative control solution, a positive control solution, a sample diluent, a pretreatment solution, etc. should be used as a set. Can do.

Examples of the present invention are shown below, but the present invention is not limited to these examples.
The list of test strains used in each example is shown in Table 2 below.

(Determination of enzyme classification of β-lactamase)
Escherichia coli (β-lactamase non-producing strain and ESBL producing strain), Staphylococcus aureus (penicillinase producing strain), Pseudomonas aeruginosa (cephalosporinase producing strain), Serratia marcescens (MBL producing strain), Klebsiella pneumoniae (carbapenemase producing) The enzyme classification was determined.

1. Preparation of β-lactamase detection formulation solution and plate preparation When 100 μL of liquid was added and dissolved, the concentration of β-lactam drug and buffer solution in the basic reagent composition shown below were (1) 4 mg / mL PCG , 0.5 mM phosphate buffer, (2) 4 mg / mL CEZ, 0.5 mM phosphate buffer, (3) 4 mg / mL CFTM, 0.5 mM phosphate buffer, (4) 4 mg / mL CMX, 0.5 mM phosphate Buffer, (5) 4 mg / mL AZT, 0.25 mM phosphate buffer, (6) 4 mg / mL FMOX, 1 mM phosphate buffer, (7) 2 mg / mL IPM, 1.25 mM phosphate buffer, (8) Prepare β-lactamase detection formulation solutions (1) to (8) so that they are 0.25 mM phosphate buffer (negative control) without β-lactam, and dispense appropriate amounts into a 96-well microplate. A plate was prepared by drying at 60 ° C. for 60 minutes and immobilizing and drying the β-lactamase detection formulation solution.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15, 60 and 180 minutes, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 4 to 6 show the results of determination of the presence or absence of decomposition after 15, 60, and 180 minutes. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  The gray shaded column in the above table indicates the expected β-lactam drug degradability of the β-lactamase that was previously determined to be produced by the tested strain to be detected (determination of enzyme classification in Table 1). This is a case where the result of this example is different from that predicted from the table. From the results of Tables 4 to 6, it is possible to correctly determine the presence or absence of degradation of CEZ, CFTM, CMX, and IPM after 15 minutes of incubation, and the degradation of all drugs other than PCG after 60 minutes of incubation. It was shown that the presence or absence can be correctly determined, and that the presence or absence of degradation of PCG, CEZ, CMX, and IPM can be correctly determined after 180 minutes of incubation.

(Screening of reaction accelerators)
About the substance which may accelerate | stimulate the hydrolysis reaction of (beta) -lactam drugs, such as an antibacterial agent and surfactant, E. coli (ESBL production strain) was made into the object, and the influence on a hydrolysis reaction was investigated.

1. Preparation of β-lactamase detection formulation and preparation of plate When 100 μL of liquid was added and dissolved, the reaction promoting screening substance in the basic reagent composition shown below was (1) 0.125 million to 80,000 U / mL polymyxin B, (2) 0.325 million to 240,000 U / mL colistin, (3) 0.625 to 40 mg / mL n-octyl-β-D-glucoside, (4) 0.625 to 40 mg / mL CHAPS, (5) 0.625 to 40 mg / mL MYS -45 (manufactured by Nikko Chemicals Co., Ltd.), (6) 0.625-40 mg / mL Adekatol N-1000 (manufactured by ADEKA), (7) 0.625-40 mg / mL Adekatol N-1100 (manufactured by ADEKA) (8) 0.625-40 mg / mL Nonion NS-270 (manufactured by NOF Corporation), (9) 0.625-40 mg / mL Pluronic F-68 (Sigma), (10) 0.625-40 mg / mL Pluronic F -127 (manufactured by Sigma), (11) 0.625-40 mg / mL Formulation solutions (1)-(11) for detection of β-lactamase were prepared so as to be sodium cholate, and there was no reaction accelerator as a control Β-lactamase test A dispensing formulation solution (12) was prepared, dispensed in an appropriate amount to a 96-well microplate, and dried at 45 ° C. for 60 minutes to prepare a plate on which the β-lactamase detection formulation solution was immobilized and dried.

2. Bacterial inoculation and incubation The pre-cultured strain was suspended in sterile physiological saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Then, it incubated at 35 degreeC for 24 hours, and measured with the brightness | luminance measuring apparatus at intervals of 15 minutes from the time of a contact start to 24 hours later. Since this measured value decreases as the phenol red changes from red to yellow, the measured value 200 was cut off, and the influence on the hydrolysis reaction was evaluated according to the time until the measured value reached 200.

3. Results FIG. 1 shows a graph summarizing the “time until the measured value reaches 200” at the minimum concentration at which each substance exhibited a high reaction promoting effect. Substances other than sodium cholate showed some reaction promoting action, but polymyxin B, colistin, and n-octyl-β-D-glucoside showed remarkable reaction promoting action. In the absence of the reaction accelerator, the time to reach 200 was 6.75 hours, whereas with polymyxin B, colistin, and n-octyl-β-D-glucoside, the time was shortened to 0.5 hours.

(Effect of reaction accelerator 1)
E. coli (ESBL production strain), Staphylococcus aureus (penicillinase production strain), Pseudomonas aeruginosa (cephalosporinase production strain), Serratia marcescens (MBL production strain), Klebsiella pneumoniae (carbapenemase production strain) The effect of polymyxin B as a promoter was investigated.

1. Preparation of β-lactamase detection formulation solution and plate preparation When 100 μL of liquid was added and dissolved, the concentration of β-lactam drug and buffer solution in the basic reagent composition shown below were (1) 4 mg / mL PCG , 0.5 mM phosphate buffer, (2) 4 mg / mL CEZ, 0.5 mM phosphate buffer, (3) 4 mg / mL CFTM, 0.5 mM phosphate buffer, (4) 4 mg / mL CMX, 0.5 mM phosphate Buffer, (5) 4 mg / mL AZT, 0.25 mM phosphate buffer, (6) 4 mg / mL FMOX, 1 mM phosphate buffer, (7) 2 mg / mL IPM, 0.25 mM phosphate buffer, (8) Preparation solutions (1) to (8) for detection of β-lactamase were prepared so that a 0.25 mM phosphate buffer solution (negative control) was obtained without β-lactam, and the same as in Example 1 for comparison. Prepare 8 types of β-lactamase detection formulation solution that does not contain polymyxin B, dispense appropriate amount into a 96-well microplate, and dry at 45 ° C for 60 minutes to immobilize and dry the β-lactamase detection formulation solution The plates were made.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15, 30, 60, and 180 minutes, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation. Sterile physiological saline was used as a negative control.

3. result
Tables 9 to 12 show the results of determination of the presence or absence of decomposition after 15, 30, 60, and 180 minutes. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  The gray shaded column in the above table indicates the expected β-lactam drug degradability of the β-lactamase that was previously determined to be produced by the tested strain to be detected (determination of enzyme classification in Table 1). This is a case where the result of this example is different from that predicted from the table. As shown in the results of Tables 9 to 12, by adding polymyxin B to the β-lactamase detection formulation solution, the hydrolysis reaction of the β-lactam drug was promoted, and after 30 minutes of incubation, all the substances except PCG The positive coloration was 2+ or more, and the incubation time required for the determination was shortened, and the ease of determination was improved. After 60 minutes of incubation, it is possible to correctly determine whether or not all drugs have been degraded, and the reaction is particularly slow, and the time required for degradation of PCG by Staphylococcus aureus, which took 180 minutes without polymyxin B, is reduced to 60 minutes. It was done. In addition, after 180 minutes, those that should be negative, including the negative control, showed positive coloration in several drugs, while coloration of AZT degradation reaction by E. coli (ESBL producing strain) The coloration of the degradation reaction of FMOX by Serratia marcescens (MBL producing strain) turned orange after 180 minutes compared to the yellow color after 60 minutes, and the positive color tended to weaken. When using the McFarland No. 1 bacterial solution as a sample like this time, it is considered that the possibility of false positive and false negative coloration will increase after 180 minutes. It was suggested that the time can be shortened, and erroneous determination of β-lactamase based on false positive coloration and false negative coloration can be reduced by setting the determination time short (in the case of the present example, setting to 60 minutes).

(Effect 2 of reaction accelerator)
For E. coli (ESBL producing strain), Pseudomonas aeruginosa (cephalosporinase producing strain), Serratia marcescens (MBL producing strain), the reaction promoting action of polymyxin B was examined when the amount of bacteria in the sample was small.

1. Preparation of β-lactamase detection formulation solution and preparation of plate The same procedure as in Example 3 was performed.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile physiological saline, prepared to McFarland No. 1, and then diluted 10-fold with sterile physiological saline. 100 μL each was dispensed to the plate prepared in (1). Then, it incubated at 35 degreeC for 15 and 30 minutes, the change from red of red of phenol red to yellow was discriminate | determined visually, and the presence or absence of (beta) -lactam drug degradation was determined. Sterile physiological saline was used as a negative control.

3. result
Tables 13 and 14 show the results of determination of the presence or absence of decomposition after 15 and 30 minutes. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  The gray shaded column in the above table indicates the expected β-lactam drug degradability of the β-lactamase that was previously determined to be produced by the tested strain to be detected (determination of enzyme classification in Table 1). This is a case where the result of this example is different from that predicted from the table. As shown in the results of Tables 13 and 14, even when the amount of bacteria in the sample is small, the degradation reaction of the β-lactam drug by polymyxin B is observed, and the degradation of PCG that cannot be determined even after 30 minutes without polymyxin B, It was shown that it can be correctly discriminated after 15 minutes of incubation.

(Examination of penicillin antibacterials as enzyme substrates)
β-lactam drugs PCG, MPIPC, ABPC, AMPC, ASPC, PIPC were transformed into E. coli (β-lactamase non-producing strain, penicillinase producing strain and ESBL producing strain), Staphylococcus aureus (β-lactamase non-producing strain), Pseudomonas aeruginosa ( A β-lactamase non-producing strain and a cephalosporinase producing strain) and Serratia marcescens (MBL producing strain) were contacted to examine the hydrolysis reaction.

1. Preparation of β-lactamase detection formulation solution and plate preparation When 100 μL of liquid was added and dissolved, β-lactam drug was added to (1) PCG, (2) MPIPC, (3) ABPC in the basic reagent composition shown below. , (4) AMPC, (5) ASPC, (6) Prepare β-lactamase prescription liquids (1) to (6) to be PIPC, dispense appropriate amount into 96-well microplate, 45 ° C Was dried for 60 minutes to prepare a plate on which β-lactamase detection formulation solution was immobilized and dried.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15 minutes, 60 minutes, 180 minutes, and 24 hours, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 16 to 19 show the results of determination of the presence or absence of degradation after 15 minutes, 60 minutes, 180 minutes, and 24 hours. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  E. coli (EKN4496), Staphylococcus aureus (EKN2088), and Pseudomonas aeruginosa (EKN3089), which are known to be non-producing strains of β-lactamase, can be identified as having no degradation, and other strains can be identified as having degradation The time to test was the shortest for PCG and the shortest for PIPC. This suggests that PCG and PIPC are suitable for rapid detection.

(Investigation of first-generation cephem antibacterials as enzyme substrates)
β-lactam drugs CEZ, CET, CEPR, CEX, CXD are brought into contact with E. coli (β-lactamase non-producing strain, penicillinase producing strain and ESBL producing strain), Pseudomonas aeruginosa (β-lactamase non-producing strain), and hydrolysis reaction I investigated.

1. Preparation of β-lactamase detection formulation solution and plate preparation When 100 μL of liquid was added and dissolved, β-lactam drug was added to the following basic reagent composition: (1) CEZ, (2) CET, (3) CEPR , (4) CEX, (5) Prepare β-lactamase detection formulation solutions (1) to (5) so as to be CXD, dispense an appropriate amount into a 96-well microplate, and dry at 45 ° C for 60 minutes. A plate was prepared by immobilizing and drying the formulation solution for detecting β-lactamase.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15 minutes, 60 minutes, 180 minutes, and 24 hours, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 21 to 24 show the results of determination of the presence or absence of decomposition after 15 minutes, 60 minutes, 180 minutes, and 24 hours. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  Escherichia coli (EKN4496), which is known to be a non-producing strain of β-lactamase, Pseudomonas aeruginosa (EKN3089) and E. coli (EKN3737), which is known to be a penicillinase producing strain, are not degraded, In E. coli (EKN7133), which is known to be present, CEZ was the shortest time before it could be determined that there was degradation, but for any β-lactam drug tested, β -It was shown that the presence or absence of lactam drug degradation can be determined. However, although data are not shown, CET and CEPR may be false positives even in a negative control (sterile saline), and stable results could not be obtained.

(Examination of third-generation cephem antibiotics as enzyme substrates)
β-lactam drugs CTX, CTRX, CAZ, CZX, CMX, CDZM, CPZ, CFIX, CETB, CFS, CFTM were transformed into Escherichia coli (β-lactamase non-producing strain, penicillinase producing strain and ESBL producing strain), Pseudomonas aeruginosa (β- It was contacted with non-lactamase producing strain), and the hydrolysis reaction was examined.

1. Preparation of β-lactamase detection formulation and preparation of plate When 100 μL of liquid was added and dissolved, β-lactam drugs in the basic reagent composition shown below were (1) CTX, (2) CTRX, (3) CAZ , (4) CZX, (5) CMX, (6) CDZM, (7) CPZ, (8) CFIX, (9) CETB, (10) CFS, (11) For β-lactamase detection Formulation solutions (1) to (11) were prepared, dispensed in appropriate amounts to a 96-well microplate, and dried at 45 ° C. for 60 minutes to prepare a plate on which the β-lactamase detection formulation solution was immobilized and dried.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15 minutes, 60 minutes, 180 minutes, and 24 hours, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 26 to 29 show the results of determination of the presence or absence of decomposition after 15 minutes, 60 minutes, 180 minutes, and 24 hours. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  Due to the known nature of β-lactamase production, the time until only E. coli ESBL-producing strain (EKN7133) is degraded and other strains can be identified as not degraded is CTX, CTRX, CZX, CMX, Within 15 minutes for CFS and CFTM. Among them, CZX, CMX, CFS and CFTM showed a strong positive of 3+ after 15 minutes, and showed excellent results in the rapidity of the reaction. On the other hand, CPZ was determined to be degraded after 60 minutes in the E. coli penicillinase producing strain (EKN6677) that should be identified as having no degradation, resulting in inferior stability compared to other drugs.

(Investigation of cefamycin and oxacephem antibacterials as enzyme substrates)
Cefamycin antibiotics CFX, CMZ, CMNX, Oxacephem antibiotics LMOX, FMOX are E. coli (ESBL production strain), Klebsiella pneumoniae (carbapenemase production strain), Serratia marcescens (MBL production strain), green It was contacted with Pseudomonas aeruginosa (cephalosporinase and MBL producing strain) and Staphylococcus aureus (penicillinase producing strain), and the hydrolysis reaction was examined.

1. Preparation of β-lactamase detection formulation and plate preparation When 100 μL of liquid was added and dissolved, β-lactam drug was added to (1) CFX, (2) CMZ, (3) CMNX in the basic reagent composition shown below. , (4) LMOX, (5) Prepare β-lactamase prescription liquids (1) to (5) so as to be FMOX, dispense an appropriate amount into a 96-well microplate, and dry at 45 ° C for 60 minutes. A plate was prepared by immobilizing and drying the formulation solution for detecting β-lactamase.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15 minutes, 60 minutes, 180 minutes, and 24 hours, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 31 to 34 show the results of determination of the presence or absence of decomposition after 15 minutes, 60 minutes, 180 minutes, and 24 hours. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  Escherichia coli (EKN7133), which is known to be an ESBL-producing strain, and S. aureus (EKN3088), which is known to be a penicillinase-producing strain, are not degraded and Klebsiella is known to be a carbapenemase-producing strain. Pneumonie (EKN9491), Serratia marcescens (EKN4636), which is known to be an MBL-producing strain, Pseudomonas aeruginosa (EKN7170), which is known to be a cephalosporinase and MBL-producing strain The time until discernment became possible was the shortest FMOX among the tests, followed by CMZ. This suggested that FMOX and CMZ are suitable for rapid detection.

(Study of carbapenem antibacterials as enzyme substrates 1)
β-lactam drugs IPM, MEPM, BIPM, and TBPM were brought into contact with Serratia marcescens (MBL producing strain) to examine the hydrolysis reaction.

1. Preparation of formulation liquid for β-lactamase detection When 100 μL of liquid was added and dissolved, β-lactam drug in the basic reagent composition shown below was (1) IPM, (2) MEPM, (3) BIPM, (4) Prepare β-lactamase prescription solution (1) to (4) so that it becomes TBPM, dispense an appropriate amount into a 96-well microplate, dry at 45 ° C. for 60 minutes, and prepare β-lactamase detection prescription solution. Immobilized and dried plates were prepared.

2. Bacterial inoculation and incubation The pre-cultured strain was suspended in sterile physiological saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Then, it incubated at 35 degreeC for 1 hour, 3 hours, and 20 hours, The change from violet to yellow of bromocresol purple was discriminate | determined visually, and the presence or absence of (beta) -lactam drug decomposition | disassembly was investigated.

3. result
IPM exhibited a positive color (yellow) after 1 hour and 3 hours of incubation, and a weak positive color after 20 hours. Both MEPM and BIPM showed a negative color (blue-purple) after 1 hour incubation, and a positive color (yellow) after 3 hours and 20 hours. Further, TBPM exhibited a negative color (blue purple) after 1 hour and 3 hours of incubation, and a weakly positive color after 20 hours. This result suggests that the IPM with the fastest hydrolysis reaction is suitable for use in the present invention.

(Study of carbapenem antibacterials as enzyme substrates 2)
β-lactam drugs IPM, PAPM, MEPM, BIPM, DRPM, TBPM are transformed into E. coli (ESBL producing strain), Klebsiella pneumoniae (carbapenemase producing strain), Serratia marcescens (MBL producing strain), Pseudomonas aeruginosa (cephalosporinase and MBL producing strain) and Staphylococcus aureus (penicillinase producing strain) were contacted to examine the hydrolysis reaction.

1. Preparation of β-lactamase detection formulation solution and plate preparation When 100 μL of liquid was added and dissolved, β-lactam drug in the basic reagent composition shown below was (1) IPM, (2) PAPM, (3) MEPM , (4) BIPM, (5) DRPM, (6) Prepare β-lactamase prescription liquids (1) to (6) so as to be TBPM, and dispense an appropriate amount into a 96-well microplate at 45 ° C. Was dried for 60 minutes to prepare a plate on which β-lactamase detection formulation solution was immobilized and dried.

2. Bacterial inoculation and incubation Each pre-cultured strain was suspended in sterile saline and prepared to McFarland No. 1. 100 μL each was dispensed to the plate prepared in (1). Thereafter, incubation was performed at 35 ° C. for 15 minutes, 60 minutes, 180 minutes, and 24 hours, and the change of phenol red from red to yellow was visually discriminated to determine the presence or absence of β-lactam drug degradation.

3. result
Tables 37 to 40 show the results of determination of the presence or absence of decomposition after 15 minutes, 60 minutes, 180 minutes, and 24 hours. When it is determined that the β-lactam drug is decomposed, it is displayed stepwise as +, 2+, 3+ depending on the degree, and when it is determined that the β-lactam drug is not decomposed, it is displayed as −.

  Escherichia coli (EKN7133), which is known to be an ESBL-producing strain, and S. aureus (EKN3088), which is known to be a penicillinase-producing strain, are not degraded and Klebsiella is known to be a carbapenemase-producing strain. Pneumonie (EKN9491), Serratia marcescens (EKN4636), which is known to be an MBL-producing strain, Pseudomonas aeruginosa (EKN7170), which is known to be a cephalosporinase and MBL-producing strain The time until it became discriminable was the shortest IPM among the tests, followed by the PAPM. This suggests that IPM and PAPM are suitable for rapid detection.

  The β-lactamase detection method, determination method and reagent used therefor according to the present invention are simple methods in which each of the six β-lactam drugs is brought into contact with the detection target bacterium, and the presence or absence of decomposition of each drug is determined by an acidometry method. In a short time by the operation, detailed enzyme classification based on the substrate specificity of β-lactamase can be determined. It is useful in a wide range of clinical settings, epidemiological studies, and other fields because simple, quick and accurate determination is possible, including the determination of KPC and OXA carbapenemases that cannot be determined by conventional rapid detection methods.

Claims (12)

A method for detecting and determining a β-lactamase of a microorganism, comprising the following steps.
(A) first drug; penicillin series,
Second drug; first generation cephem system,
A third agent; a third generation cephem system and / or a fourth generation cephem system;
Fourth drug: monobactam,
A fifth drug; cephamycins and / or oxacephems, and
Sixth drug; carbapenem series,
A step of contacting each of the six β-lactam drugs comprising a microorganism to be detected and determining the presence or absence of decomposition of each drug by an acidometry method,
(B) A step of determining the enzyme classification of the detection target microorganism based on the determination result of the presence or absence of decomposition of each drug in the step (a). In step (a),
Penicillinase producing bacteria are determined that the first drug is decomposed, the second drug is determined to be decomposed or not decomposed, and the third drug, the fourth drug, the fifth drug, and the sixth drug are It is determined that there is no disassembly,
For cephalosporinase-producing bacteria (chromosomal), the first drug and the second drug are determined to be decomposed, and the third drug, the fourth drug, the fifth drug, and the sixth drug are not decomposed. Is determined,
ESBL-producing bacteria are determined that the first drug and the second drug are decomposed, the fifth drug and the sixth drug are determined not to be decomposed, and the third drug and the fourth drug are decomposed or It is determined that there is no disassembly,
Overproduction AmpC producing bacteria, the first drug, the second drug, the third drug, the fourth drug and the fifth drug are determined to be decomposed, the sixth drug is determined to be no decomposition,
MBL-producing bacteria are determined that the first drug, the second drug, the third drug, the fifth drug and the sixth drug are decomposed, the fourth drug is determined not to be decomposed,
The method according to claim 1, wherein the KPC-type carbapenemase-producing bacterium and the OXA-type carbapenemase-producing bacterium are determined as having decomposed all six strains.   The method according to claim 1 or 2, wherein in step (a), the β-lactam drug is contacted with the microorganism to be detected in a medium containing a pH indicator.   The method according to claim 3, wherein the medium to be contacted with the microorganism to be detected in step (a) further contains a reaction accelerator selected from polymyxin B, colistin, and n-octyl-β-D-glucoside.   The method according to claim 3 or 4, wherein the pH indicator is selected from bromocresol purple, bromothymol blue, nitrazine yellow, and phenol red.   The first drug is penicillin G or piperacillin, the second drug is cefazolin, cephalotin, cefapirin, cephalexin or cefloxazine, the third drug is cefotaxime, ceftriaxone, ceftizoxime, cefthrozine, cefteram or cefmenoxime, The method according to any one of claims 1 to 5, wherein the fourth drug is aztreonam, the fifth drug is flomoxef or cefmetazole, and the sixth drug is imipenem or panipenem.   The method according to any one of claims 3 to 6, wherein the medium to be contacted with the microorganism to be detected in the step (a) is a liquid, a semi-fluid substance, a disk or a filter paper.   The medium to be contacted with the detection target microorganism in the step (a) is a liquid, and the β-lactam drug and the detection target microorganism are contacted at 20 to 40 ° C. in a well of a multi-well plate. 2. The method according to item 1. A multi-well plate having a β-lactamase detection formulation containing one type of β-lactam drug and a pH indicator in each well in a liquid, dry or frozen state, wherein the β-lactam drug is
First drug; penicillin system,
Second drug; first generation cephem system,
A third agent; a third generation cephem system and / or a fourth generation cephem system;
Fourth drug: monobactam,
A fifth drug; cephamycins and / or oxacephems, and
Sixth drug; carbapenem series,
A multi-well plate consisting of 6 lines.   The multi-well plate according to claim 9, wherein the β-lactamase detection formulation further contains a reaction accelerator selected from polymyxin B, colistin, and n-octyl-β-D-glucoside.   The multi-well plate according to claim 9 or 10, wherein the pH indicator is selected from bromocresol purple, bromothymol blue, nitrazine yellow, and phenol red.   The first drug is penicillin G or piperacillin, the second drug is cefazolin, cephalotin, cefapirin, cephalexin or cefloxazine, the third drug is cefotaxime, ceftriaxone, ceftizoxime, cefthrozine, cefteram or cefmenoxime, The multi-well plate according to any one of claims 9 to 11, wherein the fourth drug is aztreonam, the fifth drug is flomoxef or cefmetazole, and the sixth drug is imipenem or panipenem.