Method for detecting drug resistance of ceftazidime avibactam paper method grey area

Abstract

The invention discloses a method for detecting the drug resistance of a ceftazidime avibactam paper-based grey area, which comprises the following steps: preparing ceftazidime avibactam drug sensitive solutions with different concentrations by adopting a bouillon paper-based elution method: finally respectively obtaining a 6 / 4 mu g / ml concentration group, a 8 / 4 mu g / ml concentration group, a 12 / 4 mu g / ml concentration group, a 16 / 4 mu g / ml concentration group and a 18 / 4 mu g / ml concentration group, and taking CAMHB as a growth control group; and then preparing an inoculum, incubating, and observing the result to judge whether the drug resistance exists. According to the invention, two different drug sensitive paper sheets are combined and mixed according to a certain proportion at high and low concentrations to achieve the concentration required by the MIC method break point of a ceftazidime avibactam drug sensitive test, and the method is a supplementary verification method for an inhibition zone diameter gray region (20-22mm) of a CZA paper sheet method.

Description

Technical Field

[0001] The invention belongs to the technical field of medical inspection and detection, and in particular relates to a method for detecting drug resistance in the gray zone of a ceftazidime avibactam paper disc method. Background Art

[0002] Carbapenem-resistant Enterobacteriaceae (CRE) are widely prevalent in clinic, and antibiotics for the treatment of CRE are limited. Ceftazidime avibactam (CZA) was approved for use by the US Food and Drug Administration in 2015. It is a new enzyme inhibitor combination preparation that combines ceftazidime (CAZ) and a new β-lactamase inhibitor avibactam (AVI), which has activity against multiple β-lactamases produced by multidrug-resistant bacteria. Ceftazidime avibactam can more widely and effectively fight infections caused by multidrug-resistant bacteria, providing a safe treatment option for clinicians. In order to prevent the long-term overuse of antibiotics, it is necessary to conduct drug resistance testing.

[0003] To date, the ceftazidime avibactam automated susceptibility system has not been widely used in China, and the reference broth microdilution method (BMD) is difficult to perform in most routine laboratories. Although the Etest method for ceftazidime avibactam has been shown to be useful for ceftazidime avibactam susceptibility testing of Enterobacter isolates, it is very expensive. Currently, the disc diffusion test (KB method) is the most widely used susceptibility method for ceftazidime avibactam because it is convenient, economical, and practical. However, the disc diffusion test also has some disadvantages. According to the M100 Antimicrobial Susceptibility Test Implementation Standard 2024 (abbreviated as CLSI M100 2024), the KB method for ceftazidime avibactam for Enterobacteriaceae strains with a diameter of 20-22 mm needs to be confirmed by the minimum inhibitory concentration (MIC) test to avoid reporting false sensitive or false resistant results. It is expensive to purchase commercial ceftazidime avibactam micro broth susceptibility panels or e-test strips, and the preparation of micro broth in the laboratory is cumbersome and expensive, which is not easy to carry out in clinical laboratories, especially in grassroots hospitals. Therefore, there is an urgent need for a low-cost, local, and convenient method for clinical laboratories to detect the resistance of Enterobacteriaceae to ceftazidime avibactam in the 20-22mm gray range of the KB method. Summary of the invention

[0004] The purpose of the present invention is to provide a simple, operable and low-cost method for testing the resistance of ceftazidime avibactam in the gray zone of the disc method. The broth disc elution method (BDE) is an alternative method recommended by the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for the detection of colistin MICs in Enterobacteriales and Pseudomonas aeruginosa. At present, the broth disc elution method is used for single drug superposition to prepare drug concentration for drug sensitivity testing, or different discs are superimposed for combined drug sensitivity testing. Ceftazidime avibactam is a compound preparation. Its drug sensitivity test breakpoint is MIC≤8 / 4μg / ml (ceftazidime drug concentration is 8μg / mL, avibactam drug concentration is 4μg / mL) for sensitive, and MIC≥16 / 4μg / ml (ceftazidime drug concentration is 16μg / mL, avibactam drug concentration is 4μg / mL) for resistance. Its drug sensitivity test must ensure that the concentration of avibactam remains fixed. By changing the concentration of ceftazidime, it is impossible to adjust the breakpoint required for drug sensitivity judgment using only one drug sensitivity disc.

[0005] The present invention is achieved through the following technical solution: a method for testing drug resistance in the gray zone of the ceftazidime avibactam paper disc method, the steps of which are as follows: Step 1: Prepare ceftazidime avibactam susceptibility solutions with concentrations of 6 / 4μg / ml, 12 / 4μg / ml, and 18 / 4μg / ml by broth disc elution method: Step 2, using the broth paper elution method to prepare ceftazidime avibactam drug sensitivity solutions with concentrations of 8 / 4μg / ml and 16 / 4μg / ml: finally obtaining 6 / 4μg / ml concentration group, 8 / 4μg / ml concentration group, 12 / 4μg / ml concentration group, 16 / 4μg / ml concentration group, and 18 / 4μg / ml concentration group; using CAMHB as the growth control group; Step 3, inoculum preparation: adjust the fresh bacteria to be tested to the standard inoculum, add the standard inoculum to the growth control group, 6 / 4μg / ml concentration group, 8 / 4μg / ml concentration group, 12 / 4μg / ml concentration group, 16 / 4μg / ml concentration group, and 18 / 4μg / ml test tubes, so that the final inoculum concentration is about 7.5*10^5 CFU / ml; use an inoculation loop to take the original inoculum and inoculate it on a blood plate for purity test; Step 4. Incubation: Cover the test tube tightly and vortex at low speed to mix each inoculated test tube. Gently loosen the lid before incubation and place in an air environment at 33-35℃ for 16-20h; Step 5. Observe the results: Check the purity test plate to confirm that the inoculum is a pure culture; test the growth control group, there must be obvious turbidity to confirm the test is valid; read the lowest concentration that inhibits the growth of the test bacteria as MIC; the 8 / 4μg / ml concentration group and the 16 / 4μg / ml concentration group both do not grow and are judged as sensitive, MIC<8 / 4μg / ml; the 8 / 4μg / ml concentration group grows and the 16 / 4μg / ml concentration group does not grow, MIC=8μg / ml, and is judged as sensitive; the 8 / 4μg / ml and 16 / 4μg / ml concentration groups both grow, MIC≥16μg / ml, and are judged as resistant.

[0006] Further preferably, step one specifically comprises: Place a piece of ceftazidime avibactam drug sensitivity paper (30 / 20 μg) into 5 ml of cationic adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam drug sensitivity solution with a concentration of 6 / 4 μg / ml; Place one ceftazidime avibactam susceptibility paper disc and one ceftazidime susceptibility paper disc into 5 ml of cation adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam susceptibility solution with a concentration of 12 / 4 μg / ml; Place one ceftazidime avibactam susceptibility paper disc and two ceftazidime susceptibility paper discs into 5 ml of cation adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam susceptibility solution with a concentration of 18 / 4 μg / ml; Vortex the test tube with the added drug sensitivity paper to ensure that the ceftazidime, avibactam and ceftazidime in the paper are eluted at room temperature for at least 30 minutes.

[0007] Further preferably, in the step 2, 2 ml of a 6 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 1 ml of a 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution are mixed to prepare a 8 / 4 μg / ml ceftazidime avibactam drug sensitivity solution; 1 ml of a 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 2 ml of a 18 / 4 μg / ml ceftazidime avibactam drug sensitivity solution are prepared to prepare a 16 / 4 μg / ml ceftazidime avibactam drug sensitivity solution.

[0008] Further preferably, in the incubation process of step 4: tightly cover the test tube lid and vortex at a low speed to mix each inoculated test tube, gently loosen the lid before incubation, and culture in an air environment at 33-35°C for 16-20h.

[0009] More preferably, Escherichia coli ATCC25922 is used as the quality control strain.

[0010] The present invention is improved on the basis of the broth disc elution method of colistin, and the concentration required for the breakpoint of the MIC method of the ceftazidime avibactam susceptibility test is achieved by combining two different drug sensitivity discs (using ceftazidime avibactam and ceftazidime avibactam to prepare drug sensitivity concentrations) and mixing high and low concentrations in a certain proportion, so as to serve as a supplementary verification method for the gray area (20-22 mm) of the inhibition zone diameter of the CZA disc method. DETAILED DESCRIPTION

[0011] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The reagents, kits and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0012] The content of ceftazidime avibactam drug sensitivity disc (CZA) is 30 / 20μg, and the content of ceftazidime avibactam drug sensitivity disc (CAZ) is 30μg. According to the 2024 edition of CLSI M100, the KB method of ceftazidime avibactam for Enterobacteriaceae has an inhibition zone diameter of ≥21mm for sensitive and ≤20mm for resistant, but strains with an inhibition zone diameter of 20-22mm require MIC test confirmation to avoid reporting false positives or false negatives. The MIC breakpoint judgment standard for ceftazidime avibactam in Enterobacteriaceae is MIC≤8 / 4μg / ml for sensitive, and MIC≥16 / 4μg / ml for resistant. Therefore, this embodiment provides a method for checking drug resistance in the gray zone of the ceftazidime avibactam disc method, and the steps are as follows: Step 1: Prepare ceftazidime avibactam susceptibility solutions with concentrations of 6 / 4μg / ml, 12 / 4μg / ml, and 18 / 4μg / ml by broth disc elution method: Place a piece of ceftazidime avibactam drug sensitivity paper (30 / 20 μg) into 5 ml of cationic adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam drug sensitivity solution with a concentration of 6 / 4 μg / ml; Place one ceftazidime avibactam susceptibility paper disc and one ceftazidime susceptibility paper disc into 5 ml of cation adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam susceptibility solution with a concentration of 12 / 4 μg / ml; Place one ceftazidime avibactam susceptibility paper disc and two ceftazidime susceptibility paper discs into 5 ml of cation adjusted broth (CAMHB) for elution to prepare a ceftazidime avibactam susceptibility solution with a concentration of 18 / 4 μg / ml; Vortex the test tube with the added drug sensitivity paper to ensure that the ceftazidime avibactam and ceftazidime in the paper are eluted at room temperature for at least 30 minutes; Step 2: Prepare ceftazidime avibactam susceptibility solutions with concentrations of 8 / 4 μg / ml and 16 / 4 μg / ml by broth disc elution method: 2 ml of 6 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 1 ml of 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution were mixed to prepare 8 / 4 μg / ml ceftazidime avibactam drug sensitivity solution; 1 ml of 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 2 ml of 18 / 4 μg / ml ceftazidime avibactam drug sensitivity solution were mixed to prepare 16 / 4 μg / ml ceftazidime avibactam drug sensitivity solution; finally, 3 ml of 6 / 4 μg / ml concentration group, 8 / 4 μg / ml concentration group, 12 / 4 μg / ml concentration group, 16 / 4 μg / ml concentration group and 18 / 4 μg / ml concentration group were obtained respectively; 3 ml of CAMHB was used as the growth control group; Step 3, inoculum preparation: adjust the standard inoculum of 0.5 McFarland turbidity of the fresh bacteria to be tested, add 15 μl of the standard inoculum to the growth control group, 6 / 4 μg / ml concentration group, 8 / 4 μg / ml concentration group, 12 / 4 μg / ml concentration group, 16 / 4 μg / ml concentration group, and 18 / 4 μg / ml test tubes, so that the final inoculum concentration is about 7.5*10^5 CFU / ml; use an inoculation loop to take the original inoculum and inoculate it on a blood plate for purity test; Step 4. Incubation: Cover the test tube tightly and vortex at low speed to mix each inoculated test tube. Gently loosen the lid before incubation and place in an air environment at 33-35℃ for 16-20h; Step 5. Observation of results: Check the purity test plate to confirm that the inoculum is a pure culture; detect the growth control group, there must be obvious turbidity to confirm that the test is valid; read the lowest concentration that inhibits the growth of the test bacteria as MIC; the 8 / 4μg / ml concentration group and the 16 / 4μg / ml concentration group both do not grow and are judged to be sensitive, MIC<8 / 4μg / ml; the 8 / 4μg / ml concentration group grows and the 16 / 4μg / ml concentration group does not grow, MIC=8μg / ml, and is judged to be sensitive; both the 8 / 4μg / ml and 16 / 4μg / ml concentration groups grow, MIC≥16μg / ml, then it is judged to be resistant. To ensure the accuracy of the results, Escherichia coli ATCC25922 is used as the quality control strain.

[0013] The broth disc elution method (CMD) was evaluated using the broth microdilution method (BMD) of ceftazidime avibactam for each strain as the reference method. Category agreement (CA) indicates the consistency of the results of the broth disc elution method as sensitive and resistant compared with the reference method broth dilution method. Very major error (VME) means that the broth disc elution method is sensitive, while the broth dilution method is resistant, that is, false sensitivity. Major error (ME) means that the broth disc elution method is resistant, while the broth dilution method is sensitive, that is, false resistance. According to the Clinical and Laboratory Standards Institute (2015) Validation of Commercial Microbial Identification and Antimicrobial Susceptibility Testing Systems M52 (CLSI M52), CA ≥ 90%, the incidence of VME is less than 1.5%, and the incidence of ME is less than 3% are acceptable. Compared with the results of BMD, the broth disc elution method of the present invention showed that two of the 87 tested bacteria had extremely serious errors. The reference method detected them as resistant, while the results of the present invention method showed them as sensitive. Overall, the CA of the present invention method was 97.70% (85 / 87), the VME was 2.30% (2 / 87), and the ME was 0%.

[0014] Table 1

[0015] Table 1 shows the results of broth micro MIC values ​​(μg / mL) of ceftazidime avibactam and broth disk elution method MIC (μg / mL) with Enterobacter isolates (n=87); the dotted line indicates the breakpoint of ceftazidime avibactam (CLSI); * indicates that two extremely significant errors occurred in the broth disk dilution method compared with BMD.

[0016] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for testing the drug resistance of ceftazidime avibactam in the gray area of ​​the paper disk method, characterized in that: Here are the steps: Step 1: Prepare ceftazidime avibactam susceptibility solutions with concentrations of 6 / 4μg / ml, 12 / 4μg / ml, and 18 / 4μg / ml by broth disc elution method: Step 2, using the broth paper elution method to prepare ceftazidime avibactam drug sensitivity solutions with concentrations of 8 / 4μg / ml and 16 / 4μg / ml: finally obtaining 6 / 4μg / ml concentration group, 8 / 4μg / ml concentration group, 12 / 4μg / ml concentration group, 16 / 4μg / ml concentration group, and 18 / 4μg / ml concentration group; using CAMHB as the growth control group; Step 3, inoculum preparation: adjust the fresh bacteria to be tested to the standard inoculum, add the standard inoculum to the test tubes of the growth control group, 6 / 4μg / ml concentration group, 8 / 4μg / ml concentration group, 12 / 4μg / ml concentration group, 16 / 4μg / ml concentration group, and 18 / 4μg / ml concentration group, so that the final inoculum concentration is 7.5*10^5 CFU / ml; use an inoculation loop to take the original inoculum and inoculate it on a blood plate for purity test; Step 4. Incubation: Cover the test tube tightly and vortex at low speed to mix each inoculated test tube. Gently loosen the lid before incubation and place in an air environment at 33-35℃ for 16-20h; Step 5. Observe the results: Check the purity test plate to confirm that the inoculum is a pure culture; test the growth control group, there must be obvious turbidity to confirm the test is valid; read the lowest concentration that inhibits the growth of the test bacteria as MIC; the 8 / 4μg / ml concentration group and the 16 / 4μg / ml concentration group both do not grow and are judged as sensitive, MIC<8 / 4μg / ml; the 8 / 4μg / ml concentration group grows and the 16 / 4μg / ml concentration group does not grow, MIC=8μg / ml, and is judged as sensitive; the 8 / 4μg / ml and 16 / 4μg / ml concentration groups both grow, MIC≥16μg / ml, and are judged as resistant.

2. A method for checking drug resistance of ceftazidime avibactam in the gray area of ​​the disk method according to claim 1, characterized in that: Step 1 specifically includes: Place a 30 / 20 μg ceftazidime avibactam drug sensitivity paper disc into 5 ml of cation-adjusted broth for elution to prepare a 6 / 4 μg / ml ceftazidime avibactam drug sensitivity solution; Place one ceftazidime avibactam drug sensitivity paper disc and one ceftazidime avibactam drug sensitivity paper disc into 5 ml of cation-adjusted broth for elution to prepare a ceftazidime avibactam drug sensitivity solution with a concentration of 12 / 4 μg / ml; Place one ceftazidime avibactam drug sensitivity paper disc and two ceftazidime drug sensitivity paper discs into 5 ml of cation-adjusted broth for elution to prepare a ceftazidime avibactam drug sensitivity solution with a concentration of 18 / 4 μg / ml; Vortex the test tube with the added drug sensitivity paper to ensure that the ceftazidime, avibactam and ceftazidime in the paper are eluted at room temperature for at least 30 minutes.

3. A method for checking drug resistance of ceftazidime avibactam in the gray area of ​​the disk method according to claim 1, characterized in that: In the step 2, 2 ml of a 6 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 1 ml of a 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution are mixed to prepare a 8 / 4 μg / ml ceftazidime avibactam drug sensitivity solution; 1 ml of a 12 / 4 μg / ml ceftazidime avibactam drug sensitivity solution and 2 ml of a 18 / 4 μg / ml ceftazidime avibactam drug sensitivity solution are prepared to prepare a 16 / 4 μg / ml ceftazidime avibactam drug sensitivity solution.

4. A method for checking drug resistance of ceftazidime avibactam in the gray zone of the disk method according to claim 1, characterized in that: Incubation process described in step 4: Cover the test tube cap tightly and vortex at low speed to mix each inoculated test tube. Remove the cap before incubation and place in an air environment at 33-35°C for 16-20 hours.

5. A method for checking drug resistance of ceftazidime avibactam in the gray zone of the disk method according to claim 1, characterized in that: Escherichia coli ATCC25922 was used as the quality control strain.

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