A method for rapidly detecting the synergistic effect of amikacin combined with ceftazidime-avibactam

Abstract

The application discloses a combined drug sensitivity test method for rapidly detecting the synergistic effect of amikacin and ceftazidime-avibactam, and the method comprises the following steps: uniformly coating a to-be-detected bacterial suspension on an MH agar plate, firstly pasting two ATM paper sheets and two CZA paper sheets respectively, and ensuring that the four paper sheets are uniformly distributed on the plate; secondly, pasting a to-be-detected ATM drug sensitivity paper sheet on a to-be-detected CZA paper sheet, and indicating the CZA ATM ; thirdly, pasting a to-be-detected CZA drug sensitivity paper sheet on a to-be-detected ATM paper sheet, and indicating the ATM CZA ; fourthly, respectively dropping 20 μL of sterile normal saline on the CZA ATM and ATM CZA ; and finally, placing the plate in a 35℃ incubator for 8 hours, measuring the diameters of the bacteriostatic rings of ATM, CZA, ATM CZA and CZA ATM , and determining the synergistic effect according to the diameters of the bacteriostatic rings and the drug sensitivity breakpoint standard. The application has the advantages of simple operation, rapidness, accuracy, low cost and the like.

Description

Technical Field

[0001] This invention relates to the field of combined drug susceptibility testing technology, and is a rapid method for combined drug susceptibility testing to detect the synergistic effect of aztreonam and ceftazidime-avibactam. Background Technology

[0002] Carbapenem antibiotics, belonging to the β-lactam class, are important antibacterial drugs for treating Enterobacterial infections and are widely used in clinical practice. However, with the frequent use of these antibiotics, the isolation rate of carbapenem-resistant Enterobacterales (CRE) is also increasing year by year, posing a significant challenge to clinical anti-infective therapy.

[0003] β-lactamases can be classified into four classes (A, B, C, and D) based on their amino acid sequences. Classes A, C, and D rely on the serine residue at their active site for catalytic activity and are called serine-β-lactamases (SBLs). Class B β-lactamases require the participation of zinc ions at their active site to produce catalytic activity and are therefore called metallo-β-lactamases (MBLs). Major metallo-β-lactamases include New Delhi metallo-β-lactamase, imipenem hydrolase, and verona integrin, which encode metallo-β-lactamases. These enzymes can hydrolyze all β-lactam antibiotics except aztreonam (ATM) and are an important molecular mechanism mediating multidrug resistance phenotypes in Enterobacteriaceae.

[0004] Ceftazidime-avibactam (CZA) is a novel combination therapy of β-lactamase inhibitors. Avibactam effectively inhibits sclerosing basal blue blood cells (SBLs) but is ineffective against medullary blue blood cells (MBLs). While acitretin (ATM) can resist MBL hydrolysis, the vast majority of MBLs-positive cerebral recurrent dermal infections (CREs) also produce SBLs, which effectively hydrolyze ATM. Therefore, the efficacy of using ATM or CZA alone to treat MBLs-positive CREs is very limited.

[0005] Recent clinical studies have shown that the combination of ATM and CZA has a good therapeutic effect on MBLs-positive CRE. However, there is currently a lack of a rapid and convenient in vitro validation method to evaluate this synergistic effect and provide clinicians with faster and more accurate medication guidance, thereby reducing the formation of drug-resistant bacteria due to clinicians' experience-based medication.

[0006] Currently available combined antimicrobial susceptibility testing methods, including the ordinary disk diffusion method, the E-test multidrug combination method, and the checkerboard dilution method, cannot meet clinical needs. The ordinary disk diffusion method combined with antimicrobial susceptibility testing is one of the simplest and most commonly used methods, judging the interaction between drugs based on the intersection morphology of the inhibition zones of two antimicrobial disks. However, the interpretation of results largely depends on the subjective judgment of the test personnel, lacking strict judgment criteria and resulting in significant errors. While the E-test multidrug combination method is simple to operate, it is expensive and inconvenient for routine laboratory testing. Although the checkerboard microdilution method combined with antimicrobial susceptibility testing offers high accuracy, it is technically difficult to operate and requires expensive materials, making it unsuitable as a routine procedure in clinical microbiology laboratories.

[0007] The invention patent application CN114990189A published by the State Intellectual Property Office on September 2, 2022 discloses a method for multi-drug combined drug sensitivity testing. Although the method is simple, economical and has high accuracy, it requires incubation for 18 to 24 hours before the results can be read, which still cannot meet the clinical demand for rapid and accurate combined drug sensitivity testing results. Summary of the Invention

[0008] To address the shortcomings of existing combined drug susceptibility testing methods, this invention proposes a simple, easy-to-interpret, and inexpensive combined drug susceptibility testing method for rapid detection of whether there is a synergistic effect between aztreonam (ATM) and ceftazidime-avibactam (CZA), providing a basis for precise clinical anti-infective treatment.

[0009] A rapid combined drug susceptibility test method for detecting the synergistic effect of aztreonam and ceftazidime-avibactam (hereinafter referred to as the disc stacking microelution method) includes the following steps:

[0010] Step 1: Prepare MH agar plates, 30μg / plate ATM susceptibility testing discs, and 10-4μg / plate CZA susceptibility testing discs;

[0011] Step 2: Prepare a bacterial suspension with a turbidity of 0.5 McFarland using a sterile cotton swab and spread it evenly on an MH agar plate.

[0012] Step 3: On the MH agar plate inoculated with bacterial solution, attach two ATM paper discs and two CZA paper discs respectively, ensuring that the four paper discs are evenly distributed on the MH agar plate.

[0013] Step 4: Take an ATM sensitivity test strip and attach it to one of the CZA test strips placed in Step 3, forming an overlay structure with the CZA test strip at the bottom and the ATM test strip on top, so that the CZA test strip is on top. ATM This means: Take a CZA drug sensitivity test strip and attach it to one of the ATM test strips placed in step 3, forming an overlapping structure with the ATM test strip at the bottom and the CZA test strip at the top, so that the ATM test strip is on top. CZAexpress;

[0014] Step 5, in CZA ATM and ATM CZA Add 20 μL of sterile saline to each of the above.

[0015] Step 6: After incubating in a 35°C incubator for 8 hours, measure ATM, CZA, and ATM separately. CZA CZA ATM The diameter D of the inhibition zone;

[0016] Step 7: Determine the synergistic effect based on the diameter of the inhibition zone and the drug sensitivity breakpoint standard.

[0017] Furthermore, the criteria for determining synergy are as follows:

[0018] ATM CZA <![CDATA[ATM CZA ]]> <![CDATA[CZA ATM ]]> Result determination R(D≤17mm) R (D < 10 mm) S(D≥21mm) S(D≥12mm) Synergistic effect

[0019] Among them, ATM and ATM CZA Sensitivity was determined according to the CLSI rapid drug sensitivity breakpoint criteria, CZA and CZA ATM Sensitivity is determined according to the EUCAST rapid drug sensitivity breakpoint standard, where S indicates sensitivity and R indicates resistance.

[0020] This invention rapidly detects the in vitro efficacy of aztreonam combined with ceftazidime-avibactam using precise inhibition zone diameter and drug sensitivity breakpoint standards. Results are available in just 8 hours, significantly shortening the testing time and providing more timely and accurate medication guidance for clinical use. Verification has shown that its sensitivity and specificity are consistent with the checkerboard microdilution method combined with drug sensitivity testing, while also possessing the advantages of simplicity, speed, and economy, better meeting clinical needs. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the operation steps of the paper stacking micro-washing method disclosed in this invention;

[0022] Figure 2 A schematic diagram of the diameter of the inhibition zone obtained by the paper disc stacking micro-elution method for bacteria ECO1007. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

[0024] Example 1

[0025] This invention discloses a rapid combined drug susceptibility test method for detecting the synergistic effect of aztreonam (ATM) and ceftazidime-avibactam (CZA), such as... Figure 1 As shown, it includes the following steps:

[0026] Step 1: Prepare MH agar plates, 30μg / plate ATM susceptibility testing discs, and 10-4μg / plate CZA susceptibility testing discs;

[0027] Step 2: Prepare a bacterial suspension with a turbidity of 0.5 McFarland using a sterile cotton swab and spread it evenly on an MH agar plate.

[0028] Step 3: On the MH agar plate inoculated with bacterial solution, attach two ATM paper discs and two CZA paper discs respectively, ensuring that the four paper discs are evenly distributed on the MH agar plate.

[0029] Step 4: Take an ATM sensitivity test strip and attach it to one of the CZA test strips placed in Step 3, forming an overlay structure with the CZA test strip at the bottom and the ATM test strip on top, so that the CZA test strip is on top. ATM This means: Take a CZA drug sensitivity test strip and attach it to one of the ATM test strips placed in step 3, forming an overlapping structure with the ATM test strip at the bottom and the CZA test strip at the top, so that the ATM test strip is on top. CZA express;

[0030] Step 5, in CZA ATM and ATM CZA Add 20 μL of sterile saline to each of the above.

[0031] Step 6: After incubating in an incubator at 33-37℃ for 8 hours, measure ATM, CZA, and ATM separately. CZA CZA ATM The diameter D of the inhibition zone;

[0032] Step 7: Determine the synergistic effect based on the diameter of the inhibition zone and the drug sensitivity breakpoint standard. The determination criteria are as follows:

[0033]

[0034]

[0035] Among them, ATM and ATM CZA Sensitivity was determined according to the CLSI rapid drug sensitivity breakpoint criteria, CZA and CZA ATM Sensitivity is determined according to the EUCAST rapid drug sensitivity breakpoint standard, where S indicates sensitivity and R indicates resistance.

[0036] Example 2

[0037] This invention discloses a paper disc stacking microelution method for rapid detection of the synergistic effect of aztreonam and ceftazidime-avibactam, which verifies the effectiveness and accuracy.

[0038] In this embodiment, the paper disc stacking microelution method and the checkerboard microdilution method disclosed in this invention were used to conduct a comparative drug sensitivity test on the combined effects of aztreonam and ceftazidime-avibactam on different Enterobacteriaceae. The comparison results are shown in Tables 1-2 below. Table 1 shows the determination results of the existing checkerboard microdilution method, and Table 2 shows the determination results of the paper disc stacking microelution method disclosed in this invention. In this paper, ECO represents Escherichia coli, ECL represents Enterobacter cloacae, KPN represents Klebsiella pneumoniae, KOX represents Klebsiella acidogenic bacteria, and CFR represents Citrobacter freundii.

[0039] Table 1

[0040]

[0041]

[0042] Table 2

[0043]

[0044] Figure 2 A schematic diagram of the diameter of the inhibition zone obtained by the paper disc stacking micro-elution method for bacteria with the corresponding bacterial number ECO1007.

[0045] Comparing Tables 1 and 2, it can be seen that the disc-stacking microelution method disclosed in this invention differs from the high-precision checkerboard microdilution method only in the determination results for bacteria ECO5061 and ECO8096. The checkerboard microdilution method determined synergy, while the disc-stacking microelution method disclosed in this invention did not. However, if the disc-stacking microelution method determined synergy, the checkerboard microdilution method also determined synergy. Therefore, it can be demonstrated that the disc-stacking microelution method provided by this invention has high accuracy in detecting the synergistic effect of aztreonam and ceftazidime-avibactam, and can obtain results in 8 hours, greatly shortening the test time and providing more timely and accurate medication guidance for clinical use.

[0046] The specific detection steps disclosed in this invention also show that the results can be intuitively determined by the diameter of the inhibition zone. Compared with the ordinary paper diffusion method, this enhances the objectivity of the test, and the steps are simple and the cost is low.

[0047] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

Claims

1. A rapid combined drug susceptibility test method for detecting the synergistic effect of aztreonam and ceftazidime-avibactam, characterized in that, Includes the following steps: Step 1: Prepare MH agar plates, 30μg / plate ATM susceptibility testing discs, and 10-4μg / plate CZA susceptibility testing discs; Step 2: Prepare a bacterial suspension with a turbidity of 0.5 McFarland using a sterile cotton swab and spread it evenly on an MH agar plate. Step 3: On the MH agar plate inoculated with bacterial solution, attach two ATM paper discs and two CZA paper discs respectively, ensuring that the four paper discs are evenly distributed on the MH agar plate. Step 4: Take an ATM sensitivity test strip and stack it on top of one of the CZA test strips placed in Step 3, forming a stacked structure with the CZA test strip at the bottom and the ATM test strip on top, so that the CZA test strip is on top. ATM This means: Take a CZA drug sensitivity test strip and stack it on top of one of the ATM test strips placed in step 3, forming an overlay structure with the ATM test strip at the bottom and the CZA test strip on top, so that the ATM test strip is on top. CZA express; Step 5, in CZA ATM and ATM CZA Add 20 μL of sterile saline to each of the above. Step 6: After incubating in a 35°C incubator for 8 hours, measure ATM, CZA, and ATM separately. CZA CZA ATM The diameter D of the inhibition zone; Step 7: Determine synergistic effects based on the diameter of the inhibition zone and the drug sensitivity breakpoint criteria; the criteria for determining synergistic effects are as follows: Among them, ATM and ATM CZA Sensitivity was determined according to the CLSI rapid drug sensitivity breakpoint criteria, CZA and CZA ATM Sensitivity is determined according to the EUCAST rapid drug sensitivity breakpoint standard, where S indicates sensitivity and R indicates resistance.

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