Preparation method and application of flat plate for detecting minimum inhibitory concentration of fosfomycin by simple agar dilution method

Abstract

The invention discloses a preparation method and application of a flat plate for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method. The method comprises the following steps: dissolving fosfomycin and glucose-6-phosphoric acid dry powder in a small amount of sterile water in advance according to a target concentration, quantitatively coating the surface of solidified agar with the dissolved fosfomycin and glucose-6-phosphoric acid dry powder, and uniformly infiltrating the fosfomycin into the agar after drying so as to form a flat plate with a specific concentration gradient. The minimum inhibitory concentration of fosfomycin is detected through the flat plate provided by the invention, and compared with a standard agar dilution method, the basic consistency of the method reaches 100%, the classification consistency reaches 99%, and medicine distribution is uniform through verification of 100 strains of clinical urinary tract infection Escherichia coli. The method abandons tedious steps of preparing agar from zero in a traditional method, is simple and convenient to operate, is accurate and reliable, solves the technical problems that a traditional agar dilution method is tedious in operation and short in plate storage life, and provides an efficient and standardized fosfomycin drug sensitivity detection scheme for a clinical microbiological laboratory.

Description

Technical Field

[0001] This invention belongs to the field of clinical microbiology testing technology, specifically relating to a simple agar dilution method for preparing plates for detecting the minimum inhibitory concentration of fosfomycin and its application. Background Technology

[0002] Fosfomycin, an ancient antibacterial drug, has regained its important clinical status in recent years due to the serious situation of multidrug-resistant Gram-negative bacterial infections, especially in the treatment of urinary tract infections. Its rational use depends on accurate detection of the minimum inhibitory concentration (MIC).

[0003] Currently, the agar dilution (AD) method recommended by the Clinical and Laboratory Standards Institute (CLSI) is the reference method for fosfomycin MIC detection. However, the traditional agar dilution method has the following technical limitations: 1. Cumbersome operation: It requires preparing agar from scratch, dissolving drugs, and pouring plates, involving a large number of pipetting and dilution steps, which is time-consuming and labor-intensive; 2. Short shelf life of agar plates: Pre-prepared drug-containing agar plates typically have a shelf life of only about one week, requiring immediate preparation and resulting in reagent waste; 3. Prone to errors: Multi-step operation increases the risk of contamination and human error, and it is difficult to guarantee reproducibility between different batches; 4. Difficult to routinely implement: The above-mentioned defects have led to clinical laboratories rarely using the agar dilution method in their daily work, and instead using the paper disc diffusion method or gradient diffusion method, etc., but there are reports of unstable results when these methods are used to detect fosfomycin.

[0004] Therefore, developing a simple, accurate, reliable, and standardized method for detecting fosfomycin susceptibility is of significant clinical value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the existing agar dilution method, such as cumbersome operation, complicated plate preparation and short shelf life, and to provide a simple agar dilution method for detecting the minimum inhibitory concentration of fosfomycin and its application. The method is based on commercial MH agar plates.

[0006] This application provides a method for preparing plates for determining the minimum inhibitory concentration of fosfomycin using a simplified agar dilution method, which includes the following steps: (1) Prepare fosfomycin stock solution; (2) Prepare glucose-6-phosphate stock solution; (3) Mix fosfomycin stock solution with glucose-6-phosphate stock solution to obtain working solution; (4) Pour the working solution directly onto the surface of a commercial MH agar plate; (5) Spread the working solution evenly on the surface of the agar; (6) Drying treatment to allow the liquid to be completely absorbed, to obtain fosfomycin susceptibility plates.

[0007] Furthermore, in step (1), the ratio N of the concentration of fosfomycin stock solution to the final concentration of fosfomycin in the fosfomycin susceptibility plate in step (6) is calculated based on the volume V1 of the fosfomycin stock solution in the working solution and the agar volume V2 of the commercial MH agar plate in step (4). The value of N is... .

[0008] Furthermore, in step (6), the final concentration of glucose-6-phosphate in the fosfomycin susceptibility plate is 25 mg / L, and the final concentration of fosfomycin is one or more of 0.03125-2048 mg / L.

[0009] Furthermore, in step (3), the mixing ratio of fosfomycin storage solution to glucose-6-phosphate storage solution is 5:2.

[0010] Furthermore, in step (6), there are 17 fosfomycin susceptibility plates, and the final concentration of fosfomycin in the 17 fosfomycin susceptibility plates is distributed in 17 multiples of 0.03125-2048 mg / L.

[0011] Furthermore, the drying process described in step (6) involves placing the coated plate in a 37°C incubator and letting it stand for 2 hours.

[0012] The application of fosfomycin susceptibility plates prepared using the plate preparation method described above in detecting the minimum inhibitory concentration of fosfomycin against Escherichia coli.

[0013] An application of a kit for detecting the minimum inhibitory concentration of fosfomycin based on a simplified agar dilution method, prepared using the plate preparation method described above, comprising fosfomycin lyophilized powder, G-6-P, multiple commercially available MHA plates for preparing multiple fosfomycin susceptibility plates to a series of final concentrations, and instructions.

[0014] Furthermore, the fosfomycin lyophilized powder is packaged according to a gradient concentration, so that the fosfomycin lyophilized powder is mixed with a specific volume of dispersing solvent to form a fosfomycin solution with a final concentration 60 times greater than the target concentration. The G-6-P is packaged in individual packages corresponding to the quantity of fosfomycin lyophilized powder.

[0015] The application of the kit described above in detecting the minimum inhibitory concentration of fosfomycin against Escherichia coli.

[0016] The beneficial effects of this application are: Compared with the prior art, the present invention has the following significant advantages: 1. Simple operation: It eliminates the tedious steps of preparing agar from scratch in the traditional method. It directly uses commercial plates as carriers and only requires coating the surface with drug solution, which greatly simplifies the operation process and enables the preparation and use of drug sensitivity plates on the spot. 2. High accuracy: Validated on 100 strains of clinical Escherichia coli causing urinary tract infections, this method showed 100% basic agreement (EA) and 99% classification agreement (CA) compared to the standard agar dilution method. All error rates met the CLSI method validation criteria. 3. Uniform drug distribution: The MIC consistency test of the quality control strain on the surface of the plate and the bottom of the plate confirmed that the drug achieved rapid and uniform penetration and distribution in the agar, ensuring the reliability of the test results; 4. Wide detection range: The method covers an ultra-long MIC detection range of 17 fold concentration gradients from 0.03125 to 2048 mg / L, covering the detection needs of common clinical sensitive and drug-resistant strains; 5. Good stability: By utilizing the high stability of fosfomycin raw material dry powder, the shortcomings of short shelf life of pre-prepared drug-containing plates are overcome. The storage of experimental consumables has changed from the original drug sensitivity plates with a shelf life of 1 week to fosfomycin raw material with much higher stability. 6. High practicality: It provides a highly efficient and standardized fosfomycin susceptibility testing solution for clinical microbiology laboratories, especially suitable for laboratories with small testing volumes or limited technical resources, and is expected to promote the precise clinical application of fosfomycin. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0018] Figure 1 This is a simplified preparation process diagram of the fosfomycin susceptibility plate of the present invention; Figure 2 This is a schematic diagram illustrating the steps of testing drug uniformity using fosfomycin plates prepared by the simplified method of this invention. Figure 3This is a schematic diagram of the drug uniformity test results of the present invention. The number in the upper left corner of each plate represents the concentration of fosfomycin in that plate (unit: mg / L). The plate is divided into upper and lower halves. The lower half of the agar is flipped so that its bottom surface (B) faces upward; the upper half is left unchanged with its surface (S) facing upward. Four standard bacteria (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212) are sequentially spotted on the surface of the agar in four regions, namely S1 (upper surface region 1), S2 (upper surface region 2), B1 (lower bottom region 1), and B2 (lower bottom region 2). Each region has Escherichia coli ATCC 25922 in the upper left, Staphylococcus aureus ATCC 29213 in the upper right, Pseudomonas aeruginosa ATCC 27853 in the lower left, and Enterococcus faecalis ATCC 29212 in the lower right, as indicated by the growth control (GC) plate labeling in the figure. The remaining 11 drug-containing plates are arranged identically. The black arrows pointing right, down, up, and left indicate the minimum inhibitory sites (MICs) of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212 on each plate, respectively. The results show that the MICs of the quality control strains are completely consistent across different regions of the plate (surfaces S1 and S2 and the flipped bottom surfaces B1 and B2), indicating that the fosfomycin agar plates prepared in this invention have good drug uniformity.

[0019] Figure 4 The scatter plot of fosfomycin MIC values ​​determined by the method of this invention and the standard agar dilution method for 100 clinical Escherichia coli strains shows a high degree of consistency between the two methods. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application. The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0022] This application provides a simplified agar dilution method for preparing plates for detecting the minimum inhibitory concentration of fosfomycin, comprising the following steps: (1) Prepare fosfomycin stock solution; (2) Prepare glucose-6-phosphate stock solution; (3) Mix fosfomycin stock solution with glucose-6-phosphate stock solution to obtain working solution; (4) Pour the working solution directly onto the surface of a commercial MH agar plate; (5) Spread the working solution evenly on the surface of the agar; (6) Drying treatment to allow the liquid to be completely absorbed, to obtain fosfomycin susceptibility plates.

[0023] Compared with the traditional standard agar dilution method, this method avoids preparing agar plates from scratch and instead uses commercially available MH agar plates as a carrier, significantly simplifying the operation process. At the same time, it takes advantage of the high stability of fosfomycin dry powder to overcome the short shelf life of freshly prepared plates.

[0024] The plates prepared using the above method achieve rapid and uniform penetration and distribution of drugs in agar, with effects comparable to those prepared by traditional methods from scratch. However, it eliminates the cumbersome steps of traditional methods for preparing agar from scratch, greatly simplifying the operation process and enabling faster preparation and use of drug sensitivity plates.

[0025] Specifically, in step (1), the ratio N of the concentration of fosfomycin stock solution to the final concentration of fosfomycin in the fosfomycin susceptibility plate in step (6) is calculated based on the volume V1 of the fosfomycin stock solution in the working solution and the agar volume V2 of the commercial MH agar plate in step (4). The value of N is... That is, the initial concentration is calculated based on the ideal state of being completely diluted into commercial MH agar plates, and then gradually diluted to obtain the target final concentration.

[0026] The fosfomycin susceptibility testing plates prepared based on the embodiments of this application demonstrate performance comparable to plates prepared from scratch using traditional methods. Therefore, in detecting the minimum inhibitory concentration (MIC) of fosfomycin against Escherichia coli, this method can directly replace the traditional method of preparing fosfomycin susceptibility testing plates from scratch, significantly simplifying the operational process. Extensive data validation shows that this method retains the detection accuracy of the agar dilution method, with classification consistency and basic consistency reaching 99% and 100%, respectively, both exceeding the acceptable standard of 90%.

[0027] The above findings are not readily apparent. The agar dilution method, as the gold standard for bacterial fosfomycin MIC determination, has long been considered an insurmountable technical barrier due to its operational complexity. This invention, however, breaks with conventional thinking by coupling "finished plates" with "stable dry powder drugs," simplifying the operational process without sacrificing accuracy. This result directly overturns the inherent paradigm of "must be prepared and used immediately" in drug susceptibility testing. Furthermore, to address the issue of unclear drug penetration within solidified agar, potentially leading to uneven drug diffusion and inaccurate concentrations (surface concentration greater than bottom concentration), thus affecting the accuracy of MIC determination for the tested bacteria, this application provides example data to assess the reliability of the method.

[0028] The method described in this application has been tested on MHA flat panels from multiple different manufacturers. There are some deviations, but these deviations are within an acceptable range.

[0029] In addition, embodiments of this application also provide a kit for preparing plates for detecting the minimum inhibitory concentration of fosfomycin based on the simplified agar dilution method as described above. The kit includes fosfomycin lyophilized powder, G-6-P, multiple commercially available finished MHA plates for preparing multiple fosfomycin susceptibility plates of a series of final concentrations, and instructions, the instructions of which describe the above method.

[0030] Furthermore, the fosfomycin lyophilized powder is dispensed according to a gradient concentration, so that after mixing the fosfomycin lyophilized powder with a specific volume of dispersing solvent, it becomes a fosfomycin solution with a final concentration 60 times higher than the target concentration; the G-6-P dispensing is done in individual packages corresponding to the number of fosfomycin lyophilized powders dispensed. This pre-dispensing method directly reduces the operation time in clinical microbiology laboratories.

[0031] The method described in this application was successful only in the fosfomycin test. This result may be due to the following reasons: 1. Fosfomycin, a small molecule (molecular weight 138.1) organic acid, has extremely high solubility in water in its sodium salt form and does not contain strong hydrophobic groups, which is a prerequisite for its uniform diffusion in agar; 2. MHA plates usually contain about 98-99% water. Although they are in a solidified state, their microstructure is a water-rich three-dimensional network gel. This network structure contains a large number of micropores with sizes much larger than water molecules and fosfomycin molecules, providing channels for the free diffusion of small molecules; 3. The uniform spreading of the fosfomycin solution on the agar surface causes the slow evaporation of water to continuously generate a slight concentration difference and osmotic pressure at the liquid film-agar interface, realizing the migration from high concentration areas to low concentration areas until a dynamic equilibrium is reached.

[0032] It is unclear whether the method described in this application can be used to obtain accurate results for the determination of MIC of other antibacterial drugs, and systematic methodological validation experiments and professional evaluation are required.

[0033] Example 1: This invention provides a simple method for preparing fosfomycin susceptibility testing plates, such as... Figure 1 As shown, the process includes the following steps: (1) Prepare 60-fold fosfomycin stock solution: Accurately weigh fosfomycin disodium salt (Shenyang No.1 Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group), dissolve it in sterile water, and prepare a series of stock solutions with a concentration range of 1.875 to 122880 mg / L (corresponding to a final concentration of 0.03125-2048 mg / L, a total of 17 multiple concentration gradients of 60 times). (2) Prepare a 3750 mg / L G-6-P stock solution: accurately weigh glucose-6-phosphate (BIOISCO Biotechnology Co., Ltd.) and dissolve it in sterile water; (3) For each target concentration, mix 500 μL of fosfomycin stock solution with 200 μL of G-6-P stock solution to obtain 700 μL of working solution; (4) Pour the working solution directly onto the surface of a commercial MH agar plate (Zhengzhou Antu Bioengineering Co., Ltd., agar volume of about 30 mL / plate), and immediately use a disposable sterile inoculation loop to evenly spread the working solution on the entire agar surface to ensure uniform liquid coverage. Place the plate in a 37℃ incubator and let it stand for 2 hours to allow the liquid to be completely absorbed and dried, to obtain a fosfomycin susceptibility plate with a final concentration range of 0.03125-2048 mg / L and containing 25 mg / L G-6-P. Take another commercial MH agar plate without the drug as a growth control. (5) Prepare the test bacteria into an inoculum according to the standard, and inoculate it on the surface of fosfomycin susceptibility plates with different final concentrations. After inoculation, culture it according to the standard.

[0034] In steps (1), (2), and (3) of this embodiment, the mixture of 500 μL of fosfomycin solution (60 times the target final concentration) and 200 μL of G-6-P solution (3750 mg / L) is obtained by combining the commercially available MH agar plates (approximately 30 mL / plate) used to ensure that after the drug solution is absorbed into the commercially available MH agar plates, the resulting fosfomycin susceptibility plate has the target final concentration and the final G-6-P concentration of each plate is 25 mg / L.

[0035] In step (4) of this embodiment, a disposable sterile inoculation loop is immediately used to evenly spread the agar to the entire surface of the agar. Then the plate is placed in a 37°C incubator and left to stand until the liquid is completely absorbed and dried. In order to ensure that the liquid penetrates evenly and without local enrichment, and to avoid distortion of the concentration gradient due to uneven spreading or uncontrolled drying speed.

[0036] Step (5) in this embodiment involves inoculating and culturing the bacterial suspension according to the standard operating procedure of the traditional agar dilution method (Clinical and Laboratory Standards Institute (CLSI) M100 guideline). This application has not made any modifications to this procedure to ensure comparability of results and consistency with clinical interpretation. Specifically, the agar dilution method requires an inoculation volume of 1×10⁻⁶. 4 CFU / spotting, standard procedure is as follows: pick 3-5 fresh single colonies with consistent morphology (incubate for 16-24 h), inoculate into MH broth / 0.85% sterile physiological saline, correct the turbidity to 0.5 McFarland turbidity standard tube, dilute 1:10, and then spot inoculate. After spotting, incubate at 35±2 ℃ for 16-20 h.

[0037] Results interpretation also follows CLSI standards: 1. First, observe the growth control: Obvious colony growth is required; otherwise, the experiment is invalid. 2. MIC interpretation: The MIC is the lowest drug concentration that completely inhibits colony growth (allowing only a single colony / very thin film covering ≤10% of the inoculum area). 3. Tailing treatment: For only 1–2 points of trace growth, the concentration of the first point with no growth is taken; multi-well tailing requires re-testing.

[0038] Example 2: This example assesses the uniformity of fosfomycin distribution in agar and the quality control of standard strains, such as... Figure 2 As shown: (1) Take fosfomycin plates of different concentrations prepared according to the method of Example 1, and cut the agar in the middle of the plate with a sterile knife; (2) Use sterile tweezers to pick up one half of the agar and turn it over so that the bottom side is facing up; (3) Using a calibrated inoculator, apply approximately 2 μL of the adjusted inoculation solution (0.5 standard McFarland turbidity, further diluted 1:10 with physiological saline) to the surface of each agar plate. Inoculate the four quality control strains into the four regions of the plate: surface region 1 (S1), surface region 2 (S2), inverted bottom region 1 (B1), and inverted bottom region 2 (B2). Inoculate each region according to a fixed layout: Escherichia coli ATCC 25922 in the upper left position, Staphylococcus aureus ATCC29213 in the upper right position, Pseudomonas aeruginosa ATCC 27853 in the lower left position, and Enterococcus faecalis ATCC 29212 in the lower right position; (4) Incubate the inoculated plates at 35℃±2℃ for 16-20 hours and observe the results; (5) The results showed that the MIC values ​​of each control strain were completely consistent in the four different spot areas: Escherichia coli ATCC 25922 was 1 mg / L, Staphylococcus aureus ATCC 29213 was 2 mg / L, Pseudomonas aeruginosa ATCC 27853 was 4 mg / L, and Enterococcus faecalis ATCC 29212 was 64 mg / L, which confirmed that the drug achieved uniform penetration and distribution in the agar. (6) All results were within the quality control allowable range specified in the CLSI M100 document, preliminarily confirming that the SAD method is comparable to the reference method in terms of detection accuracy: the MIC for Escherichia coli ATCC 25922 was 1 mg / L (allowable range: 0.5-2 mg / L), the MIC for Staphylococcus aureus ATCC 29213 was 1 mg / L (allowable range: 0.5-4 mg / L), the MIC for Pseudomonas aeruginosa ATCC 27853 was 4 mg / L (allowable range: 2-8 mg / L), and the MIC for Enterococcus faecalis ATCC 29212 was 64 mg / L (allowable range: 32-128 mg / L). All results are shown in Table 1 below. Figure 3 As shown.

[0039] Table 1. Minimum inhibitory concentrations (MICs) of fosfomycin against four quality control strains, determined using the standard agar dilution method and the simplified agar dilution method (unit: mg / L). Example 3: This embodiment uses 100 strains of Escherichia coli from clinical urinary tract infections to verify the method of the present invention: (1) Source of strains: 100 non-duplicate clinical isolates of Escherichia coli were obtained from urine samples of outpatients and inpatients of Wenzhou Municipal Hospital of Traditional Chinese Medicine from January 2023 to June 2025. Only one strain was included from each patient. (2) Strain identification: MALDI-TOF MS (Bruker Daltonik GmbH) was used for strain identification; (3) Reference method: The MIC of fosfomycin was determined by the standard agar dilution method according to the CLSI M100 guidelines: Prepare drug-containing plates (containing 25 mg / L G-6-P) with a final concentration of 0.03125-2048 mg / L using the conventional standard method. Inoculate about 2 μL of bacterial suspension (0.5 McFarland turbidity, 1:10 dilution) using a calibrated inoculator and incubate at 35℃±2℃ for 16-20 hours before reading. (4) Method of the present invention: Prepare SAD drug susceptibility plates according to the method of Example 1, and perform parallel detection using the same bacterial suspension, the same inoculator, and the same incubation conditions; (5) Result interpretation: Drug susceptibility classification was determined according to the CLSI M100 S35 breakpoint criteria (sensitive ≤64 mg / L, intermediate 128 mg / L, resistant ≥256 mg / L); (6) Performance evaluation: Using the standard agar dilution method as a reference, calculate basic consistency (EA), classification consistency (CA), minor error (mE), major error (ME), and very major error (VME). According to the CLSI guidelines, the acceptable standards are: CA ≥ 90%, EA ≥ 90%, mE ≤ 10%, ME < 3%, and VME < 3%. (7) Validation results: EA was 100%, CA was 99%, only one small error (1%) occurred, and no major or extremely major errors occurred. All indicators met the CLSI methodology validation criteria. The results are as follows: Figure 4 As shown in Table 2.

[0040] Table 2. Performance evaluation of the SAD method compared with the standard AD method.

[0041] The above embodiments demonstrate that the simplified agar dilution method provided by the present invention is easy to operate, accurate and reliable, and highly consistent with the standard reference method, showing good prospects for clinical application.

[0042] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A simplified agar dilution method for preparing plates for determining the minimum inhibitory concentration of fosfomycin, characterized in that... Includes the following steps: (1) Prepare fosfomycin stock solution; (2) Prepare glucose-6-phosphate stock solution; (3) Mix fosfomycin stock solution with glucose-6-phosphate stock solution to obtain working solution; (4) Pour the working solution directly onto the surface of a commercial MH agar plate; (5) Spread the working solution evenly on the surface of the agar; (6) Drying treatment to allow the liquid to be completely absorbed, to obtain fosfomycin susceptibility plates.

2. The method for preparing a flat plate according to claim 1, characterized in that, In step (1), the ratio N of the concentration of fosfomycin stock solution to the final concentration of fosfomycin in the fosfomycin susceptibility plate in step (6) is calculated based on the volume V1 of the fosfomycin stock solution in the working solution and the agar volume V2 of the commercial MH agar plate in step (4). The value of N is... .

3. The method for preparing a flat plate according to claim 2, characterized in that, In step (6), the final concentration of glucose-6-phosphate in the fosfomycin susceptibility plate is 25 mg / L, and the final concentration of fosfomycin is one or more of 0.03125-2048 mg / L.

4. The method for preparing a flat plate according to claim 3, characterized in that, In step (3), the mixing ratio of fosfomycin storage solution to glucose-6-phosphate storage solution is 5:

2.

5. The method for preparing a flat plate according to claim 3, characterized in that, In step (6), there are 17 fosfomycin susceptibility plates, and the final concentration of fosfomycin in the 17 fosfomycin susceptibility plates is 0.03125-2048 mg / L, which is a total of 17 multiple concentration gradient distributions.

6. The method for preparing a flat plate according to claim 2, characterized in that, The drying process described in step (6) involves placing the coated plate in a 37°C incubator and letting it stand for 2 hours.

7. The application of fosfomycin susceptibility plates prepared by the plate preparation method according to any one of claims 1-6 in the detection of the minimum inhibitory concentration of fosfomycin against Escherichia coli.

8. An application of a reagent kit for detecting the minimum inhibitory concentration of fosfomycin based on a simplified agar dilution method according to the plate preparation method described in any one of claims 1-6, characterized in that, Includes fosfomycin lyophilized powder, G-6-P, multiple commercially available MHA plates for preparing multiple fosfomycin susceptibility testing plates to a series of final concentrations, and instructions.

9. The application according to claim 7, characterized in that, The fosfomycin lyophilized powder is packaged according to a gradient concentration, so that the fosfomycin lyophilized powder is mixed with a specific volume of dispersing solvent to form a fosfomycin solution with a final concentration 60 times greater than the target concentration. The G-6-P is packaged in individual packages corresponding to the quantity of fosfomycin lyophilized powder.

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