JPWO2018181997A1 - Method for detecting carbapenemase producing bacteria - Google Patents

Abstract

Step 1: a step of adding an antimicrobial agent to the sample and culturing it. Step 2: About the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, A step of measuring the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength, step 3: applying the absorbance (B-Abs) obtained in step 2 to a calibration curve, and applying a blank absorbance (C-Abs). ) And step 4: if the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is less than a preset threshold, the sample may be carbapenemase. A method for detecting a carbapenemase-producing bacterium, the method comprising: According to the detection method of the present invention, a carbapenemase-producing bacterium can be easily and quickly detected with high sensitivity and specificity, which is useful for clinical and nosocomial infection control.

Description

  The present invention relates to a method for detecting a carbapenemase-producing bacterium and an apparatus used for the method, a method for determining the hydrolysis performance of a carbapenemase-producing bacterium, and a method for screening for an inhibitor of a carbapenemase-producing bacterium.

  The worldwide spread of carbapenemase-producing Enterobacteriaceae (CPE) bacteria is a major problem, as they show resistance by degrading carbapenem antibiotics, a trump card for the treatment of severe infections. Therefore, establishing a CPE detection system has great clinical significance, and some reports have been made so far.

  For example, Patent Document 1 relates to a disk diffusion method, and specifically, a disk test containing at least three kinds of antibacterial agents consisting of a carbapenem antibacterial agent, a fluoroquinolone antibacterial agent and an aminoglycoside antibacterial agent. Simple and accurate multidrug resistance in a short time by simply observing the presence or absence of colonies in the drug diffusion area on the medium using a piece and a solid medium that can grow Pseudomonas aeruginosa, Acinetobacter, etc. It is disclosed that multidrug-resistant bacteria such as Pseudomonas aeruginosa and multidrug-resistant Acinetobacter can be detected.

  Patent Document 2 discloses that, by using a composition in which faropenem and cloxacillin are combined in a microfluidic dilution method, only carbapenemase-producing resistant bacteria selectively grow, and carbapenemase-producing resistant bacteria can be easily and accurately detected. ing.

  There is also a method of detecting the presence or absence of resistant bacteria by performing PCR using primers specific to the carbapenemase gene (for example, see Patent Document 3). A method in which a probe capable of being selectively cleaved is brought into contact with a carbapenemase to detect a label bound to the probe (for example, see Patent Document 4), and a pH of a reaction mixture generated by reacting with a substrate having specificity. There is also a method of evaluating the change with an indicator (for example, see Patent Document 5).

JP-A-2006-168058 JP-A-2006-010399 JP-A-2006-146818 JP-T-2017-505612 JP 2014-533951 A

  However, the prior art requires a complicated operation and time for preparation of a measurement sample, or requires the evaluator's skill for determination, and can be easily and quickly performed with high sensitivity and specificity. The establishment of a new system was required.

  An object of the present invention is to provide a method for detecting a carbapenemase-producing bacterium and a device for detecting a carbapenemase-producing bacterium that can be easily and quickly performed with high sensitivity and specificity, a method for determining the hydrolysis performance of a carbapenemase-producing bacterium, and a carbapenemase-producing bacterium To provide a method of screening for an inhibitor of the compound.

The present invention relates to the following [1] to [9].
[1] A method for detecting carbapenemase-producing bacteria, comprising the following steps.
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating step 4: If the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold, the sample contains carbapenemase-producing bacteria. [2] The detection method according to [1], wherein the wavelength (Bnm) is 45 to 400 nm higher than the wavelength (Anm).
[3] The detection method according to [1] or [2], wherein imipenem, panipenem, meropenem, biapenem, doripenem, tebipenem, ertapenem, or faropenem is used as the antibacterial agent.
[4] The detection method according to any one of [1] to [3] above, wherein the sample is a specimen derived from a living body, a specimen derived from an environment, or a specimen derived from food of a human or another animal.
[5] The detection method according to any one of [1] to [4], wherein the culture time in step 1 is at least 15 minutes.
[6] The detection method according to any one of [1] to [5] above, wherein the carbapenemase-producing bacterium is a gram-negative bacterium that produces carbapenemase.
[7] A spectrophotometer, including a computer having a processor and a memory under the control of the processor, wherein the memory includes the following steps:
For the culture solution in which the sample and the antibacterial agent were added and cultured, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent by a spectrophotometer, and a wavelength at least 40 nm higher than the wavelength. Measuring the absorbance (B-Abs) at (B nm),
Step of calculating the blank absorbance (C-Abs) by applying the absorbance (B-Abs) obtained above to a calibration curve, and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) obtained above is calculated. An apparatus for detecting carbapenemase-producing bacteria, wherein a computer program for causing the computer to execute a step of determining that a sample contains carbapenemase-producing bacteria when the value is lower than a preset threshold value is recorded.
[8] A method for determining the hydrolysis performance of carbapenemase-producing bacteria, comprising the following steps.
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating Step 4: When the difference between the absorbance (A-Abs) obtained in Step 2 and the absorbance (C-Abs) obtained in Step 3 is lower than a preset threshold, the carbapenemase-producing bacteria contained in the sample (9) the absorbance (A-Abs) at the wavelength (Anm) of the sample obtained by allowing the test substance to act on the carbapenemase-producing bacterium, and the wavelength (Bnm) at least 40 nm higher than the wavelength (Bnm). )), Absorbance (B-Abs) is measured, and the absorbance (B-Abs) is The absorbance (C-Abs) is calculated and the test substance is selected as the substance that suppresses the activity of the carbapenemase-producing bacteria when the difference between the absorbance (A-Abs) and the absorbance (C-Abs) exceeds a preset threshold. Screening method for a substance that suppresses the activity of a carbapenemase-producing bacterium.

  According to the present invention, a carbapenemase-producing bacterium can be easily and quickly detected with high sensitivity and specificity.

1 shows the results of measuring the absorbance spectrum of each antibacterial drug, FIG. 1a shows the results of measurement at a wavelength of 220 to 400 nm, and FIG. 1b shows the correlation between the concentration of imipenem and the absorbance at 297 nm. FIG. 2 shows the results of measuring the spectrum of each antibacterial agent in the presence of bacteria, and FIG. 2 a shows the results obtained when a carbapenemase producing bacterium (CPE strain) or a carbapenemase non-producing bacterium (non-CPE strain) was present. FIG. 2B shows the results of the measurement at 400 nm. FIG. 2B is a diagram comparing the spectrum in the case where the antimicrobial agent and the bacterium coexist with the spectrum in the case where the bacterium exists in the PBS for each antimicrobial agent. FIG. 3 is a diagram showing the absolute absorbance of imipenem in the bacterial mixture, and FIG. 3a is a diagram schematically showing the absolute absorbance from the absorbance spectra of the bacterial mixture containing imipenem and the bacterial mixture not containing imipenem. FIG. 3b shows the absolute absorbance spectrum obtained by subtracting the absorbance of the bacterial mixture containing no imipenem. FIG. 4 is a diagram schematically showing a solution prepared for measurement of absorbance. FIG. 4a shows a case where a solution containing bacteria and imipenem (experimental solution) and a solution containing only bacteria at the same concentration (background) are prepared. FIG. 4b shows a case where the preparation of the background solution is omitted. FIG. 5 is a diagram showing the correlation between the background absorbance at 297 nm (C-Abs) and the absorbance at a wavelength different from 297 nm (B-Abs), and FIG. 5a shows the absorbance at a wavelength of 350 nm (B-Abs). ), FIG. 5B is a diagram showing the correlation when the absorbance at a wavelength of 450 nm (B-Abs) is used, and FIG. 5C is a diagram showing the correlation when the absorbance at a wavelength of 600 nm (B-Abs) is used. FIG. 6 is a diagram showing the hydrolysis rate of imipenem, FIG. 6a shows the result of comparing the hydrolysis rate between the CPE strain and the non-CPE strain (55 strains), and FIG. 6b shows the hydrolysis rate in the CPE strain. It is a figure which shows the level of performance. FIG. 7 is a diagram showing the results of comparing the hydrolysis rates between CPE strains and non-CPE strains using each antibacterial agent. FIG. 8 is a diagram showing the hydrolysis rate of imipenem, FIG. 8a shows the result of comparing the hydrolysis rate between the CPE strain and the non-CPE strain (149 strains), and FIG. 8b shows the hydrolysis rate in the CPE strain. It is a figure which shows the level of performance. FIG. 9 is a diagram showing the results of comparing the hydrolysis rate between the CPE strain and the non-CPE strain at each incubation time.

  The present invention utilizes a hydrolysis reaction of an antibacterial drug by a carbapenemase-producing bacterium, and is based on the fact that the specific ultraviolet region absorption of the antibacterial drug changes by hydrolysis.

  As a conventional method utilizing a hydrolysis reaction by a carbapenemase-producing bacterium, a method using an enzyme activity of carbapenemase as an index can be exemplified. In this case, if the enzyme activity is low, it becomes difficult to detect the enzyme, and there is a problem in detection sensitivity such as false negative. On the other hand, the present invention has been found for an antimicrobial agent hydrolyzed by carbapenemase, focusing on the fact that the ultraviolet absorption spectrum of the antimicrobial agent changes with the hydrolysis. That is, when measuring the absorbance of the antibacterial agent, a wavelength at which the absorbance fluctuates due to hydrolysis, and a specific wavelength different from the above-described wavelength are set as the background, and the absorbance at these wavelengths is measured from the same sample. Thus, a carbapenemase-producing bacterium can be easily and quickly detected with high sensitivity and specificity. In this specification, detecting a carbapenemase-producing bacterium means detecting whether or not a sample contains a carbapenemase-producing bacterium, and it can also be said that detecting the presence or absence of a carbapenemase-producing bacterium.

1. Method for detecting carbapenemase-producing bacteria The detection method of the present invention includes the following steps.
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating step 4: If the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold, the sample contains carbapenemase-producing bacteria. Step to determine

  In step 1, a sample for measuring absorbance is prepared. Specifically, it can be prepared by adding an antibacterial agent to a sample and culturing the sample. Here, as a sample that can be used, since the presence or absence of a carbapenemase-producing bacterium is detected, a sample that may contain the bacterium can be used. Specifically, a specimen derived from a living body, a specimen derived from an environment, and a specimen derived from food of humans and other animals are exemplified. Examples of the biological specimen include clinical materials such as urine, blood, sputum, and stool. Examples of environmentally-derived samples include water and soil in water and sewage, hospital equipment and medical equipment surfaces (eg, bedside fences, floors, sinks, artificial respirators, etc.). The concentration of the carbapenemase-producing bacterium in the sample is not particularly limited.

  The sample is added to a medium and cultured under conditions suitable for culturing carbapenemase-producing bacteria, for example, at pH 6.0 to 8.0 and 10 to 42 ° C. The culturing time may be 15 minutes, and from the viewpoint of improving accuracy, the lower limit is exemplified by 30 minutes, 1 hour, 1 hour 30 minutes, and 2 hours. The upper limit is, for example, 6 hours, 4 hours, or 3 hours from the viewpoint of speed. A known medium can be appropriately selected and used.

  The antibacterial agent to be added is not particularly limited as long as it can be hydrolyzed by carbapenemase-producing bacteria. For example, carbapenem antibacterial agents, penem antibacterial agents include, for example, imipenem (IPM), panipenem (PAPM), meropenem (MEPM), biapenem (BIPM), doripenem (DRPM), tebipenem (TBPM), Ertapenem (ETPM), faropenem (FRPM) and the like are exemplified. The addition concentration is a concentration at which hydrolysis can be detected by the method described below when the microorganism is brought into contact with a carbapenemase-producing bacterium. Specifically, 0.1-100 μg / mL is exemplified in the culture solution.

  In step 2, the absorption in the ultraviolet region of the culture solution thus obtained, that is, the sample for measuring the absorbance is measured.

  The wavelength to be measured is a wavelength (Anm) that is the maximum wavelength of the maximum absorption of the antibacterial agent used in Step 1, and at least 40 nm, preferably 42 nm or more, more preferably 45 nm or more, and still more preferably 50 nm or more from the wavelength. Although the upper limit is not particularly set, from the viewpoint of specificity, preferably 400 nm or less, more preferably 300 nm or less, more preferably 200 nm or less, further preferably 150 nm or less, more preferably 100 nm or less wavelength (Bnm ). Table 1 shows an example of combinations of the wavelength (Anm) and the wavelength (Bnm) that can be set in the present invention for the antibacterial agent. For example, when imipenem is used as a carbapenem antibacterial, the wavelength (Anm) can be set to 297 nm and the wavelength (Bnm) can be set to 350 nm, but is not limited thereto. The wavelength (Anm) may be set as a maximum wavelength by measuring a solution obtained by dissolving only an antibacterial agent in a culture solution in advance, or may be set from known technical information. Further, the wavelength (Anm) may be set to a value slightly deviated from the maximum wavelength, for example, the maximum wavelength ± 10 nm, preferably the maximum wavelength ± 5 nm, and the wavelength (Anm) and the wavelength (Bnm) are as described above. It suffices if the wavelengths are separated.

  The measurement of the absorbance is not particularly limited as long as the absorbance at the above-described wavelength can be measured. For example, the measurement can be performed using a known spectrophotometer. The absorbance (A-Abs) at the wavelength (Anm) and the absorbance (B-Abs) at the wavelength (Bnm) may be measured separately or simultaneously, as long as they are measured for the same sample. In the present invention, since a control solution containing only bacteria as a background is not separately prepared, there is a possibility that the bacteria may grow and the absorbance may fluctuate with time, so that there is no time difference when measuring separately. Are preferably measured at the same time. Specifically, for example, it is preferably within 5 minutes, more preferably within 1 minute. Here, simultaneous measurement includes not only a mode in which two wavelengths are measured at the same time without a time difference, but also a mode in which measurements are continuously performed if the time is such that bacteria do not grow.

  In the present invention, the sample for measuring the absorbance may be appropriately concentrated or diluted according to the absorbance at the wavelength (Anm). The method of concentration and the method of dilution are not particularly limited, and examples thereof include a method of concentration under reduced pressure at a temperature of room temperature or lower, and a method of adding and diluting a culture solution.

  In step 3, the absorbance (B-Abs) obtained in step 2 is applied to a calibration curve to calculate a blank absorbance (C-Abs).

  The blank absorbance (C-Abs) is characterized in that it is calculated as a blank absorbance at the above-mentioned wavelength (Anm) using a separately prepared calibration curve. That is, in the present invention, since the type and amount of the bacterium, the composition of the culture solution, and the like differ depending on the sample for measuring the absorbance, the blank value varies depending on the sample and cannot be set unconditionally, and the absorbance (A-Abs) is measured. One characteristic is that the blank absorbance (C-Abs) can be set without being affected by the blank value by measuring the absorbance (B-Abs) in the same sample as above and applying it to the calibration curve.

  The calibration curve is created as follows. First, the amount of antimicrobial agent added, the type and amount of bacteria, the type and amount of culture solution, etc. were prepared in advance, and samples were prepared in advance, and various culture solutions were cultured by changing the culture conditions, etc., at the wavelength (Bnm). Measure the absorbance. Then, except that the antimicrobial agent is not added, a sample can be prepared under the same conditions as described above, and the culture solution cultured under the same conditions can be prepared by measuring the absorbance at a wavelength (Anm) and plotting the correlation. . The calibration curve shows a favorable linear relationship (approximate expression) when, for example, the X-axis is prepared as the absorbance at the wavelength (Bnm) and the Y-axis is the blank absorbance at the wavelength (Anm). It should be noted that the calibration curve is such that the approximate expression can be recalculated as needed after the data is additionally updated.

  The determination in step 4 is performed using the blank absorbance (C-Abs) thus obtained.

  In step 4, depending on whether or not the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a predetermined threshold value, if the difference is lower than step 1, If the sample used in step 1 contains a carbapenemase-producing bacterium, and is equal to or more than that, it is determined that the sample used in step 1 does not contain carbapenemase-producing bacterium.

  The difference between the absorbance (A-Abs) and the absorbance (C-Abs) is compared with a preset threshold. The threshold is an appropriate cut-off value for identifying whether or not hydrolysis has occurred, and by comparing the difference between the threshold and the absorbance obtained above, the bacteria contained in the sample are carbapenemase-producing bacteria. Can be determined.

  The threshold can be set as follows. For a plurality of known carbapenemase-producing bacteria and carbapenemase non-producing bacteria, the difference between the absorbance (A-Abs) and the absorbance (C-Abs) is determined as described above, and the difference can be set by grasping the tendency from the values. it can. For example, the difference in absorbance can be set to 0.2, preferably 0.15, more preferably 0.1. Therefore, when the difference between the absorbance (A-Abs) and the absorbance (C-Abs) is lower than the threshold value of 0.2, preferably 0.15, more preferably 0.1, it is determined that the sample contains carbapenemase-producing bacteria. If they are equal to or greater than each other, it can be determined that they are not included.

  Further, in the present invention, the presence or absence of bacteria can be confirmed from a comparison between a value obtained by directly calculating the difference between the absorbance (A-Abs) and the absorbance (C-Abs) and a threshold, but from the viewpoint of ease of determination. Alternatively, a numerical value calculated by applying to the relational expression may be used for the determination.

For example, the determination can be made by calculating the hydrolysis rate of the antibacterial agent from the absorbance (A-Abs) and the absorbance (C-Abs) using the following formula.
Hydrolysis rate (%) = 100 − [(A-Abs) − (C-Abs)] / [(D-Abs) − (E-Abs)] × 100
A-Abs: Absorbance at the wavelength (Anm) of the culture solution measured in step 2
C-Abs: Blank absorbance of the culture solution obtained in step 3
D-Abs: Absorbance at the wavelength (Anm) of antimicrobial drug-containing PBS at the same concentration as in step 1.
E-Abs: Absorbance at the wavelength of PBS (Anm) The absorbance (D-Abs) and the absorbance (E-Abs) may be measured each time the absorbance (A-Abs) is measured. The measured data may be used. In addition, although PBS (phosphate buffer) is used here, if the sample for measuring the absorbance (D-Abs) and the absorbance (E-Abs) is prepared using the same solution, a known solution is used. A buffer can be used without particular limitation.

  The obtained hydrolysis rate is compared with a preset threshold value. The threshold used here can be set by calculating the hydrolysis rate for a plurality of known carbapenemase-producing bacteria and carbapenemase-non-producing bacteria using the above-described method, and grasping the tendency from the values. For example, a hydrolysis rate of 10%, preferably 15%, more preferably 20% can be set as the threshold. Therefore, when determining the hydrolysis rate from the difference between the absorbance (A-Abs) and the absorbance (C-Abs), when the hydrolysis rate is equal to or greater than the threshold value of 10%, preferably 15%, more preferably 20% Can be determined to contain a carbapenemase-producing bacterium in the sample, and if less, it can be determined that it is not contained.

  Note that the threshold value may be acquired separately and simultaneously when measuring the absorbance of the sample, or may be acquired in advance. Further, when comparing with the hydrolysis rate of the sample, the analysis result obtained up to that time may be additionally updated as needed and acquired.

  By comparing the preset threshold value with the absorbance or the hydrolysis rate calculated from the absorbance in this manner, it can be determined whether or not the sample contains carbapenemase-producing bacteria.

  The bacteria thus detected are not particularly limited as long as they are carbapenemase-producing bacteria, and examples include NDM-type carbapenemase-producing bacteria, KPC-type carbapenemase-producing bacteria, OXA-type carbapenemase-producing bacteria, and IMP-type carbapenemase-producing bacteria. Specific examples include gram-negative bacteria that produce carbapenemase, and include Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Pseudomonas aeruginosa, Vibrio cholera, Acinetobacter, Aeromonas, and the like. .

2. Method for Determining Hydrolysis Performance of Carbapenemase-Producing Bacteria In addition, the level of hydrolysis performance of carbapenemase-producing bacteria can be evaluated in comparison with the threshold value in step 4 in the detection method. Specifically, the smaller the difference in the calculated absorbance or the higher the hydrolysis rate, the higher the hydrolysis performance of the carbapenemase-producing bacteria contained in the sample can be determined to be higher. It can be evaluated that the lower the absorbance at the wavelength (Anm) of the culture broth, the higher the hydrolysis activity of the carbapenemase-producing bacteria is. Therefore, the present invention also provides a method for determining the hydrolysis performance of a carbapenemase-producing bacterium, comprising the following steps, since the degree of hydrolysis of a carbapenemase-producing bacterium can be determined from the absorbance at a wavelength serving as an index.
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating Step 4: When the difference between the absorbance (A-Abs) obtained in Step 2 and the absorbance (C-Abs) obtained in Step 3 is lower than a preset threshold, the carbapenemase-producing bacteria contained in the sample For judging that the hydrolysis performance of is high

  For specific operations and specifications in Steps 1 to 4 in the determination method, the section in the detection method of the present invention can be referred to. In addition, the bacteria whose hydrolysis performance is determined include bacteria detected by the detection method of the present invention.

3. Apparatus for detecting carbapenemase-producing bacteria The detection apparatus of the present invention includes a spectrophotometer and a computer having a processor and a memory under the control of the processor, wherein the memory includes the following steps:
For the culture solution in which the sample and the antibacterial agent were added and cultured, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent by a spectrophotometer, and a wavelength at least 40 nm higher than the wavelength. Measuring the absorbance (B-Abs) at (B nm),
Step of calculating the blank absorbance (C-Abs) by applying the absorbance (B-Abs) obtained above to a calibration curve, and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) obtained above is calculated. A computer program for causing the computer to execute a step of determining that the sample contains a carbapenemase-producing bacterium when the value is below a preset threshold value is recorded.

  Hereinafter, an example of an apparatus suitable for performing the method of the present invention will be described. However, the present embodiment is not limited to only this example.

  The present embodiment includes a spectrophotometer and a computer system connected to the spectrophotometer.

  The measuring device is, for example, a UV measuring device that measures the absorbance of a culture solution obtained by adding a sample and an antibacterial agent, based on two wavelengths that have been set. The measurements at the two wavelengths may be performed simultaneously or separately. The interval in the case of separate is not particularly limited, but it is preferable to perform the interval sequentially. The wavelength is automatically changed to the maximum absorption maximum wavelength (Anm) and the wavelength (Bnm) away from the wavelength by a certain wavelength to the higher wavelength side by inputting information on the antibacterial agent used in the computer system described later. Can also be set. Further, in the measurement device, the mixed solution containing the sample and the antibacterial agent was subjected to a pH of 6.0 to 8.0 and a temperature of 10 to 42 ° C. for about 15 minutes (the lower limit was 30 minutes, 1 hour, 1 hour 30 hours). Minutes and 2 hours are exemplified, and as an upper limit, 6 hours, 4 hours and 3 hours are exemplified), and then the absorbance can be automatically measured as it is. The detection method of the present invention can be referred to for the type and amount of the antibacterial agent, the method of setting the wavelength, and the like. The container for storing the sample may be a container capable of UV measurement, and may be used, for example, with reference to the section of the detection kit of the present invention. The obtained absorbance is transmitted to a computer system.

  The computer system executes a program for determining the presence or absence of carbapenemase-producing bacteria in the sample based on the obtained absorbance.

  The program for determining the presence or absence of carbapenemase-producing bacteria calculates a blank absorbance from the obtained absorbance based on an approximate expression stored in a hard disk or the like, and then calculates a hydrolysis rate.

  Thereafter, the presence or absence of carbapenemase-producing bacteria in the sample is determined using a preset threshold value described on a hard disk or the like. The determination result can be separately printed by a printer or displayed on a display unit (display).

  In the detection device of the present invention, the spectrophotometer and the computer system may be integrated or may be separately configured as connectable. In addition, an apparatus from the absorbance measurement to the determination may be automated, for example, an apparatus in which the determination result is displayed on an accompanying display by adding a sample.

  The size of the detection device of the present invention is not particularly limited, and, for example, a spectrophotometer and a computer system are integrated, and preferably, a small-sized automatic measurement and simultaneous display of two-wavelength absorbance, and the display of the result are automated. It may be a device or a device that can be attached to an existing spectrophotometer.

Further, in the present invention, as a program recorded in the computer,
The following steps:
For the culture solution in which the sample and the antibacterial agent were added and cultured, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent by a spectrophotometer, and a wavelength at least 40 nm higher than the wavelength. Measuring the absorbance (B-Abs) at (B nm),
Step of calculating the blank absorbance (C-Abs) by applying the absorbance (B-Abs) obtained above to a calibration curve, and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) obtained above is calculated. A program for causing a computer to execute a step of determining that a carbapenemase-producing bacterium is contained in a sample if the value is less than a preset threshold, or
The following steps:
Separately measured by a spectrophotometer, the absorbance (A-Abs) at the maximum wavelength of the maximum absorption of the antibacterial agent (Anm), and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher wavelength side from the wavelength. Using, to calculate the blank absorbance (C-Abs) by applying the absorbance (B-Abs) to the calibration curve, and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) obtained above in advance Since the program for causing a computer to execute the step of determining that the sample contains carbapenemase-producing bacteria when the value is below the set threshold value can also be mounted on a computer known as determination software, the present invention can be used. Included as one embodiment.

  As described above, the present invention is based on the principle that bacteria contained in a sample are determined based on the change in ultraviolet absorption of an antibacterial agent to be used. For example, those skilled in the art can fully understand that the present invention is also applicable to detection of β-lactamase-producing bacteria.

4. Screening method for inhibitors of carbapenemase-producing bacteria The present invention determines the presence or absence of bacteria based on the change in ultraviolet absorption of the added antimicrobial agent, so if known carbapenemase-producing bacteria are present, they were added. It can be determined whether or not the compound is hydrolyzed, and thus, it can be determined whether or not the added compound can suppress the activity of the carbapenemase-producing bacterium. That is, the present invention also provides a method for screening a compound that inhibits a carbapenemase-producing bacterium.

  The screening method of the present invention uses a change in absorbance at a specific wavelength as an index and a substance that suppresses the activity of a carbapenemase-producing bacterium as a candidate compound. By comparing the absorbance with the absorbance of the sample that does not act, if the absorbance of the sample that has acted with the test substance is higher than the absorbance of the sample that does not act, the test substance may be selected as a substance that suppresses the activity of carbapenemase-producing bacteria. it can.

  More specifically, instead of separately preparing the sample as the absorbance of the sample on which the test substance does not act, in the present invention, when measuring the absorbance of the sample on which the test substance has acted, the absorbance at a wavelength set separately is used. It is characterized by measuring. Specifically, the absorbance (A-Abs) at the wavelength (Anm) of the sample in which the test substance was allowed to act on the carbapenemase-producing bacterium, and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength (Bnm) Is measured, the absorbance (B-Abs) is applied to a calibration curve to calculate a blank absorbance (C-Abs), and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) is a preset threshold, for example, , 0.1, preferably 0.15, more preferably 0.2, the test substance is selected as a substance that suppresses the activity of carbapenemase-producing bacteria. Incidentally, even if the maximum wavelength (Anm) is not measured in advance, after estimating from the structure based on the common general knowledge of those skilled in the art and setting it, the wavelength (Bnm) may be set by referring to the section of the detection method of the present invention. Good. After measuring the absorbance (A-Abs) at the wavelength (Anm) and the absorbance (B-Abs) at the wavelength (Bnm), refer to the section of the detection method of the present invention, and calculate the blank absorbance (C-Abs). be able to.

  The carbapenemase-producing bacterium that can be used in the screening method of the present invention is not particularly limited as long as it can be detected by the detection method of the present invention. Culture conditions and the like can be appropriately set based on the bacteria used.

  The absorbance can be compared by calculating the hydrolysis rate by applying the equation to the hydrolysis rate used in the detection method of the present invention. In this case, when the hydrolysis rate is 20% or less, preferably 15% or less, and more preferably 10% or less, the test substance can be selected as a substance that suppresses the activity of the carbapenemase-producing bacterium. The test substance exceeding% cannot be selected as a substance that suppresses the activity of the carbapenemase-producing bacterium.

  Thus, compounds that inhibit carbapenemase-producing bacteria can be screened.

5. Kit for detecting carbapenemase-producing bacteria The present invention determines the presence or absence of bacteria based on the change in ultraviolet absorption of the added antimicrobial agent, so that the determination can be made by adding the antimicrobial agent It can also be said. Therefore, the present invention also provides a kit for detecting a carbapenemase-producing bacterium, which comprises an antibacterial agent.

  The kit of the present invention is preferably a kit which can be provided for absorbance measurement by adding a sample to a container, and specifically, for example, a container (plate type, card type, test tube type, etc.) A substrate to which an antibacterial agent serving as a substrate is added in advance may be used. Further, the container may be provided with a well, and the antibacterial agent may be dried and fixed on the side surface and / or the bottom surface of the container or each well. In this case, a solvent (for example, PBS) that dissolves the antibacterial agent may be included as a component of the kit. Such a kit includes, for example, containers for positive control, negative control, and sample preparation, and these containers are desirably disposable in consideration of safety because they come into direct contact with bacteria. Further, it is preferable that the present invention can be applied to the detection device of the present invention.

  As the antibacterial agent, the antibacterial agent added in the detection method of the present invention can be used. If the concentration of the antibacterial agent in the culture solution at the time of measuring the ultraviolet absorption is, for example, 0.1 to 100 μg / mL, the concentration of the antibacterial agent in the kit is not particularly limited and can be set as appropriate.

  Further, in addition to the antibacterial agent, a material used for preparing a sample for measuring absorbance can be included. Specifically, a known medium such as a culture medium can be applied as long as the bacterium to be measured can be cultured.

  Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are merely examples of the present invention, and the present invention is not limited thereto. In addition, imipenem used Wako Pure Chemical Industries, Ltd .; Meropenem used Tokyo Chemical Industry Co., Ltd .; doripenem, biapenem, ertapenem, faropenem, and PBS (phosphate buffer) purchased from Sigma Aldrich.

Test example 1
In a UV-permeable 96-well plate (Zell-kontakt, Germany), each antibacterial agent was prepared in a PBS solution so as to have various concentrations, and a corona plate reader SH-9000 (HITACHI, Japan) was used at room temperature (25 ° C.). Below, analysis was performed at a wavelength of 220 nm to 400 nm (FIG. 1a).

  Each antimicrobial solution showed a characteristic absorbance between 250 nm and 350 nm, and showed a peak absorbance around 297 nm. No other peak was identified when the spectrum was analyzed up to 1000 nm. For example, for the imipenem solution, the absorbance at 297 nm (A-Abs) showed a linear relationship with the concentration in the solution (FIG. 1b), and the amount of imipenem in the solution could be estimated from the absorbance.

Test example 2
The spectrum of imipenem in the bacterial mixture was measured to determine whether differences in hydrolytic activity could be found between carbapenemase-producing bacteria (CPE strains) and carbapenemase-non-producing bacteria (non-CPE strains) . Specifically, using a CPE strain (Klebsiella pneumoniae, ATCC BAA-2470 strain) and a non-CPE strain (Klebsiella pneumoniae, ATCC 4352 strain), a mixture of imipenem and CPE strains (CPE), imipenem and non-CPE strains (Non-CPE) and bacterium-free imipenem dissolved in PBS (PBS) were measured for the spectrum at 220 nm to 400 nm in the same manner as in Test Example 1. In addition, for each antibacterial agent, a mixture of the antibacterial agent and the CPE strain and a suspension of the CPE strain in PBS were prepared, and the spectra at 200 nm to 1000 nm were also measured.

  As shown in FIG. 2a, it was difficult to compare the peak absorbance at 297 nm between CPE and non-CPE solutions to show different baseline absorbance for different types of bacteria. From FIG. 2b, on the longer wavelength side than around 350 nm, any antibacterial agent was obtained by mixing the antibacterial agent and the CPE strain (antibacterial drug + Bac) and suspending the CPE strain in PBS (PBS + Bac) shows that the spectra are almost the same.

Test example 3
Since it was thought that the background changed due to different types of bacteria, the "absolute absorbance of imipenem" in the bacterial mixture was examined. As shown in FIG. 3a, for example, at a wavelength of 297 nm, the absolute absorbance of imipenem is obtained by subtracting the absorbance (point C) of the bacterial mixture containing no imipenem from the absorbance of the bacterial mixture containing imipenem (point A). Therefore, the absolute absorbance of the sample of Test Example 2 was calculated as described above.

  From FIG. 3b, the hydrolysis of imipenem was clearly shown as the disappearance of the characteristic spectrum of imipenem in the solution containing CPE, and it was found that the presence or absence of CPE can be detected by the difference in absorbance. On the other hand, non-CPE solutions, for example, had a relatively stable peak absorbance at 297 nm, similar to bacterial-free imipenem solutions.

Test example 4
In the method of Test Example 3, it was necessary to prepare two types of solutions for measuring the absorbance. Specifically, a solution containing bacteria and imipenem (experimental solution) and a solution containing only bacteria at the same concentration (background) were required (FIG. 4a). However, considering the application to the detection system, it was ideal to skip the step of preparing the background solution to save time and simplify the system. Also, the degree of bacterial growth in the two solutions was different, suggesting that the background solution could no longer be used as "background". Therefore, as shown in FIG. 4b, a method of estimating the background absorbance using the experimental solution was studied.

In the measurement of imipenem, the absorbance at 297 nm (A-Abs) is used as an index, so the focus is on the relationship between the background absorbance at 297 nm (C-Abs) and the absorbance at 350 nm (B-Abs). (Relationship between points B and C in FIG. 3a). Using the strains shown in Table 2, the absorbance of these two types was measured and plotted. As a result, it was found that R ² = 0.9987 and a good linear relationship was exhibited (FIG. 5a). The absorbance at 350 nm (B-Abs) was not affected by the absorbance of imipenem, suggesting that it can be used in place of the imipenem-free bacterial mixture. The same applies to the case where the background absorbance (C-Abs) is calculated from the absorbance at 450 nm (B-Abs) and the case where the background absorbance (C-Abs) is calculated from the absorbance at 600 nm (B-Abs). 5b and 5c show the results of the studies described above, suggesting that both can be used in place of the imipenem-free bacterial mixture. The carbapenemase type shown in Table 2 indicates NDM: New Delhi Metallo-b-lactamase, KPC: Klebsiella pneumonia Carbapenemase, OXA: Oxacillinase, IMP: Imipenemase.

Test example 5
Based on Test Example 4, the hydrolysis activity of 55 known strains shown in Table 3 was measured. The control strain used was purchased from ATCC, and the other was a human clinical isolate from the laboratory.

Specifically, the bacteria were cultured overnight on an agar plate, and the colonies were added to 100 μL of an imipenem solution (25 μg / mL) so that the turbidity was equivalent to that of the 1.0 McFarland standard. After incubation, spectrophotometric measurements were taken. From the obtained absorbance, the hydrolysis rate was calculated using the following equation.
Hydrolysis rate (%) = 100 − [(A-Abs) − (C-Abs)] / [(D-Abs) − (E-Abs)] × 100
A-Abs: absorbance at 297nm
C-Abs: blank absorbance calculated from the relational expression (350 nm) obtained in Test Example 4
D-Abs: Absorbance of antimicrobial-containing PBS at wavelength (297nm)
E-Abs: Absorbance at the wavelength of PBS (297nm)

  FIG. 6a shows that the hydrolysis rates are clearly separated between CPE and non-CPE strains. In addition, although the MIC value of imipenem is shown using a color scale, some CPEs have a low MIC value, but the difference in carbapenemase activity from non-CPE strains by using the detection method of the present invention. Is clear. In addition, it has been confirmed that the OXA type, which is known to have a weak hydrolysis effect in a short time, shows relatively lower activity than other CPE strains, and the detection method of the present invention is applicable to such detection. It was found to be excellent (FIG. 6b).

Test example 6
The carbapenemase producing bacteria (CPE strain: Klebsiella pneumoniae, ATCC BAA-2470 strain) and the carbapenemase non-producing bacteria (non-CPE strain: Klebsiella pneumoniae, ATCC 4352 strain) used in Test Example 5 were tested using each antibacterial agent. The hydrolysis rate was calculated in the same manner as described above. In this test, the approximate formula for determining the blank absorbance was that of imipenem prepared in Test Example 4, and the wavelength (Anm) was set to 297 nm and the wavelength (Bnm) was set to 350 nm for all antibacterial agents for convenience.

  From FIG. 7, it is possible to clearly distinguish between the CPE strain and the non-CPE strain using any of the antibacterial agents. Also, it can be seen that the carbapenemase activity differs depending on the antimicrobial agent even between the same CPE strains. In addition, even if the wavelength (Anm) of each antibacterial drug slightly deviates from its maximum wavelength, the determination can be performed easily and with good accuracy by measuring the absorbance of the wavelength (Anm) and the wavelength (Bnm). Is also suggested.

Test example 7
It was confirmed whether the detection method of the present invention can handle various strains. Specifically, the hydrolysis activity of the strains (149 strains) shown in Table 4 was evaluated in the same manner as in Test Example 4 except that the incubation time was changed to 120 minutes. The strain was purchased from the ATCC or used as a human clinical isolate in the laboratory.

  8a and 8b, it is confirmed that even when the number of strains is increased, the hydrolysis rate is clearly separated between the CPE strain and the non-CPE strain, and the OXA type having weak activity can be sufficiently discriminated. It is clear that. Therefore, it was found that the detection method of the present invention is an excellent detection method applicable to various strains.

Test Example 8
Regarding the detection method of the present invention, the discriminability between the CPE strain and the non-CPE strain at different incubation times (30, 60, 120, and 180 minutes) was examined using the 149 strains used in Test Example 7. Specifically, the hydrolysis activity was evaluated in the same manner as in Test Example 4 except that the incubation time was changed to the above time. In addition, at each incubation time, an ROC curve was created for the hydrolysis rate, and a cutoff value was calculated (Table 5).

  The cut-off value of 9.43 after 30 minutes incubation is 98.25% sensitivity and 100% specificity, indicating that it shows sufficient judgment performance. The incubation time of 60 minutes, 120 minutes, and 180 minutes shows 100% sensitivity and specificity, indicating that the detection method of the present invention can perform determination with good specificity and accuracy (FIG. 9). ).

Test example 9
An antimicrobial agent is added to a colony grown by culturing a biological sample (such as blood) and further culturing. For the obtained culture solution, the absorbance (A-Abs) at the maximum wavelength (Anm) and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength are measured according to the added antimicrobial agent. Then, a blank absorbance (C-Abs) is calculated from the absorbance (B-Abs). Next, it is determined from the difference between the absorbance (A-Abs) and the blank absorbance (C-Abs) whether or not the specimen contains a carbapenemase-producing bacterium.

  According to the detection method of the present invention, a carbapenemase-producing bacterium can be easily and quickly detected with high sensitivity and specificity, which is useful for clinical and nosocomial infection control.

Claims (9)

A method for detecting a carbapenemase-producing bacterium, comprising the following steps:
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating step 4: If the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold, the sample contains carbapenemase-producing bacteria. Step to determine   The detection method according to claim 1, wherein the wavelength (Bnm) is 45 to 400 nm higher than the wavelength (Anm).   The detection method according to claim 1 or 2, wherein imipenem, panipenem, meropenem, biapenem, doripenem, tebipenem, ertapenem, or faropenem is used as the antibacterial agent.   The detection method according to any one of claims 1 to 3, wherein the sample is a biological sample, a human sample, an environmental sample, or a food sample of a human or another animal.   The detection method according to any one of claims 1 to 4, wherein the culture time in step 1 is at least 15 minutes.   The detection method according to any one of claims 1 to 5, wherein the carbapenemase-producing bacterium is a gram-negative bacterium that produces carbapenemase. Including a spectrophotometer and a computer with a processor and a memory under the control of the processor, the memory comprising the following steps:
For the culture solution in which the sample and the antibacterial agent were added and cultured, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent by a spectrophotometer, and a wavelength at least 40 nm higher than the wavelength. Measuring the absorbance (B-Abs) at (B nm),
Step of calculating the blank absorbance (C-Abs) by applying the absorbance (B-Abs) obtained above to a calibration curve, and the difference between the absorbance (A-Abs) and the absorbance (C-Abs) obtained above is calculated. An apparatus for detecting carbapenemase-producing bacteria, wherein a computer program for causing the computer to execute a step of determining that a sample contains carbapenemase-producing bacteria when the value is lower than a preset threshold value is recorded. A method for determining the hydrolysis performance of a carbapenemase-producing bacterium, comprising the following steps.
Step 1: adding an antimicrobial agent to a sample and culturing Step 2: using the culture solution obtained in step 1, the absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antimicrobial agent, and the wavelength Step 3: Measuring Absorbance (B-Abs) at at least 40 nm Higher Wavelength (Bnm) from Step 3: Apply Absorbance (B-Abs) Obtained in Step 2 to a Calibration Curve to Determine Blank Absorbance (C-Abs) Calculating Step 4: When the difference between the absorbance (A-Abs) obtained in Step 2 and the absorbance (C-Abs) obtained in Step 3 is lower than a preset threshold, the carbapenemase-producing bacteria contained in the sample For judging that the hydrolysis performance of is high   The absorbance (A-Abs) at the wavelength (Anm) of the sample in which the test substance was allowed to act on the carbapenemase-producing bacteria, and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength were measured. The absorbance (B-Abs) is applied to a calibration curve to calculate a blank absorbance (C-Abs), and the test substance is determined when the difference between the absorbance (A-Abs) and the absorbance (C-Abs) exceeds a preset threshold. A method for screening a substance that suppresses the activity of a carbapenemase-producing bacterium, which is selected as a substance that suppresses the activity of a carbapenemase-producing bacterium.