Microorganism broad-spectrum dynamic phenotype drug sensitivity rapid detection method and device

Abstract

The invention relates to the technical field of microbial drug resistance detection, in particular to a microbial broad-spectrum dynamic phenotype drug sensitivity rapid detection method and device. According to the method provided by the invention, the drug resistance of the microorganisms is detected based on the application of the measurement time and the application threshold value, a long culture period of culturing to macroscopic turbidity or a stable end point of a fluorescence signal in a traditional method is abandoned, and rapid judgment is realized by capturing early metabolism or growth response difference of bacteria under drug pressure. The method for detecting the drug resistance of the microorganisms has high accuracy, the detection time is greatly shortened compared with that of a traditional method, meanwhile, the obvious cost advantage is achieved, and the method has good application prospects in microorganism drug resistance detection and equipment development.

Description

Technical Field

[0001] This invention relates to the field of microbial drug resistance detection technology, and in particular to a method and apparatus for rapid detection of broad-spectrum dynamic phenotypic drug susceptibility in microorganisms. Background Technology

[0002] Currently, methods for detecting bacterial resistance mainly include traditional classical methods, molecular biology techniques, and high-throughput sequencing technologies. Traditional classical methods encompass disk diffusion, agar dilution, broth dilution, and concentration gradient methods, with dilution being widely considered the "gold standard" for bacterial resistance detection. Molecular biology techniques, utilizing flow cytometry, mass spectrometry, Raman spectroscopy, immunohybridization, nucleic acid hybridization, and amplification, achieve detection at the physical, chemical, and molecular levels. However, most of these techniques are still in the research exploration stage, with limited practical applications, and are typically only applicable to specific bacterial species or classes of antibiotics, lacking universality. High-throughput sequencing technologies, such as whole-genome sequencing (WGS), metagenomic sequencing (mNGS), and microbial single-cell transcriptome sequencing (MSC RNA-seq), have been gradually applied to the field of resistance detection. However, their application presupposes that the strain carries known resistance sites, relying on the correlation between genotype and phenotype. In practical applications, the effectiveness of molecular biology techniques and high-throughput sequencing technologies highly depends on known and stable resistance sites. The absence, addition, or mutation of resistance sites can lead to missed detections or false positives, limiting the versatility and reliability of these methods. A comparison of bacterial resistance detection methods is shown in Table 1.

[0003] Table 1. Comparison of methods for detecting bacterial resistance In the detection of fungal resistance, traditional phenotypic detection methods (such as disk diffusion and dilution methods) are dominant because genotype and phenotype are difficult to accurately correspond. Molecular biology techniques have not yet developed into mature and effective detection methods.

[0004] The core bottleneck in current bacterial and fungal resistance testing lies in the excessively long testing time. Common methods and commercial equipment (such as bioMérieux VITEK2 in France) typically require 8-24 hours, resulting in high costs. Therefore, there is an urgent need to develop resistance testing methods that combine timeliness and universality. Summary of the Invention

[0005] This invention provides a method and apparatus for detecting microbial drug resistance.

[0006] The purpose of this invention is to address the key problems of long detection cycles and insufficient timeliness in current microbial drug resistance detection technologies and equipment. The drug resistance detection method provided by this invention is a rapid drug resistance detection technology system based on Application Measurement Time (AMT) and Application Threshold (AT). It overcomes the limitations of traditional drug susceptibility testing modes that rely on minimum inhibitory concentration (MIC) endpoint interpretation and growth curve analysis. This significantly improves detection efficiency, increasing the speed of drug resistance detection by 2-5 times, and has broad adaptability to various bacterial species, applicable to all types of bacteria and fungi. It fundamentally overcomes the shortcomings of existing methods in terms of detection timeliness and universality, providing a technical path for developing a new generation of efficient and rapid drug resistance detection and analysis equipment.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a method for detecting microbial drug resistance, the method comprising: inoculating a bacterial suspension of the microorganism to be tested into culture media containing the drug to be tested and those without the drug to be tested, culturing them until the application measurement time, determining the total number of microorganisms in the culture system, and calculating the percentage of drug-consuming bacteria; The proportion of drug-consuming bacteria is compared with the application threshold, and the drug resistance of the test microorganism is determined based on the comparison results. The application measurement time and the application threshold are determined based on the known drug-resistant strains and known drug-sensitive strains of the microorganism to be tested. The application threshold is a percentage value of (Smin + Rmax) / 2, where Smin and Rmax are measured at the same culture time point and satisfy Smin - Rmax > 10%~15%; Rmax is the maximum percentage of the known drug-resistant strain that consumes the drug at a certain culture time point, and Smin is the minimum percentage of the known susceptible strain that consumes the drug at the same time point as Rmax. The formula for calculating the percentage of the drug consumed is: Percentage of the drug consumed = (Total number of microorganisms in the culture system excluding the test drug - Total number of microorganisms in the culture system containing the test drug) / Total number of microorganisms in the culture system excluding the test drug × 100%. In the formula for calculating the percentage of the drug consumed, the total number of microorganisms in the culture system excluding the test drug and the total number of microorganisms in the culture system containing the test drug both originate from the same culture time point. The application measurement time is the cultivation time corresponding to the application threshold; The application thresholds include the drug resistance breakpoint application threshold (ATR) with the drug resistance breakpoint concentration as the working concentration and / or the drug sensitivity breakpoint application threshold (ATS) with the drug sensitivity breakpoint concentration as the working concentration; wherein, the drug resistance breakpoint application threshold is obtained by using the drug resistance breakpoint concentration as the working concentration of the culture system containing the drug to be tested; and the drug sensitivity breakpoint application threshold is obtained by using the drug sensitivity breakpoint concentration as the working concentration of the culture system containing the drug to be tested.

[0009] In the above method, the resistance breakpoint application threshold is obtained by setting the concentration of the test drug in the culture system containing the test drug to the resistance breakpoint concentration (R-BP); the sensitivity breakpoint application threshold is obtained by setting the concentration of the test drug in the culture system containing the test drug to the sensitivity breakpoint concentration (S-BP).

[0010] Depending on the needs and purposes of determining drug resistance, one can choose either the Application Threshold for Resistance (ATR) or the Application Threshold for Sensitivity (ATS) as a single threshold, or both the Application Threshold for Resistance (ATR) and the Application Threshold for Sensitivity (ATS) can be selected simultaneously for drug resistance determination.

[0011] In this invention, the method for determining the drug resistance of the test microorganism is as follows: If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consumed by the drug is less than the drug resistance breakpoint application threshold when the time for application measurement is reached; or, in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consumed by the drug is less than the sensitivity breakpoint application threshold when the time for application measurement is reached, then the culture is determined to be drug resistant. If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consumed by the drug is greater than the drug resistance breakpoint application threshold when the time for application measurement is reached; or, in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consumed by the drug is greater than the sensitivity breakpoint application threshold when the time for application measurement is reached, then the system is judged to be sensitive. If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consumed by the drug is greater than the drug resistance breakpoint application threshold when the time for application measurement is reached, and in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consumed by the drug is less than the sensitivity breakpoint application threshold when the time for application measurement is reached, then the system is judged to be intermediate.

[0012] This invention, through the analysis of over 11,000 test results involving four pathogens (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa), 15 antibiotics, determined a drug resistance detection method based on the resistance breakpoint concentration (R-BP) and sensitivity breakpoint concentration (S-BP) as the working drug concentration, and on application measurement time and application threshold. The method was validated through extensive testing with Acinetobacter baumannii and Staphylococcus aureus, demonstrating high accuracy and good universality. The detection method and drug resistance judgment rules of this invention are based on the results of numerous research and development experiments, and all specific parameters involved are within experimentally verified effective ranges.

[0013] In this invention, the known drug-resistant and known susceptible strains of the test microorganism can be determined based on the known drug resistance profiles of the test microorganism in the prior art, or by using existing methods to determine the drug resistance profiles of the strains. This invention does not impose any special limitations on known drug-resistant and known susceptible strains; however, to ensure the effectiveness of the application measurement time and application threshold, the number of known drug-resistant and known susceptible strains can be appropriately increased.

[0014] In the above-mentioned drug resistance detection method, the method for determining the application measurement time and the application threshold based on the known drug-resistant strains and known susceptible strains of the test microorganism includes: inoculating bacterial suspensions of known drug-resistant strains and known susceptible strains into culture media containing the test drug and those without the test drug, respectively, and culturing them; measuring the total number of microorganisms in each culture system at different time points; calculating the proportion of drug-consuming bacteria for each known drug-resistant strain and known susceptible strain; determining the maximum proportion of drug-consuming bacteria for all known drug-resistant strains detected at the same culture time point; and determining the minimum proportion of drug-consuming bacteria for all known susceptible strains detected at the culture time point.

[0015] In the above-mentioned drug resistance detection method, the total number of microorganisms is characterized by absorbance (i.e., OD value).

[0016] The wavelength corresponding to the absorbance used to detect the total number of microorganisms is a commonly used measurement wavelength in this field, such as 600 nm.

[0017] In the above-mentioned method for detecting drug resistance, the concentration of the bacterial suspension is 0.5 × 10⁻⁶. 8 ~ 5.0×10 8 CFU / mL; preferably 1.0 × 10⁻⁶ 8 ~ 2.0×10 8 CFU / mL.

[0018] In the above-mentioned methods for detecting drug resistance, the microorganisms include bacteria or fungi. Specifically, the bacteria include, but are not limited to, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0019] In this invention, the resistance breakpoint concentration (R-BP) and the susceptibility breakpoint concentration (S-BP) are preferably determined with reference to the antimicrobial susceptibility testing breakpoint standards specified in the Clinical and Laboratory Standards Institute (CLSI) M100 document (32nd edition).

[0020] In this invention, the drug is preferably an antibiotic.

[0021] Preferably, the above-mentioned drug resistance detection method is for non-disease diagnosis and treatment purposes. Non-disease diagnosis and treatment purposes include, but are not limited to, monitoring the prevalence of drug-resistant strains, monitoring environmental drug resistance, and monitoring the drug resistance of strains contained in food or agricultural products.

[0022] Secondly, the present invention provides a microbial drug resistance detection device, the device comprising a retrieval module, a detection module, an analysis module, and an output module; The retrieval module is used to retrieve the corresponding application measurement time and application threshold based on the microorganism to be tested and the drug to be tested; wherein the application measurement time and the application threshold are determined based on the known drug-resistant strains and known sensitive strains of the microorganism to be tested and the drug to be tested. The application threshold is a percentage value of (Smin + Rmax) / 2, where Smin and Rmax are measured at the same culture time point and satisfy Smin - Rmax > 10%~15%; Rmax is the maximum percentage of drug-consuming bacteria of the known drug-resistant strain at a certain culture time point, and Smin is the minimum percentage of drug-consuming bacteria of the known sensitive strain at the same time point as Rmax; the formula for calculating the percentage of drug-consuming bacteria is: percentage of drug-consuming bacteria = (total number of microorganisms in the culture system excluding the test drug - total number of microorganisms in the culture system containing the test drug) / total number of microorganisms in the culture system excluding the test drug × 100%; in the formula for calculating the percentage of drug-consuming bacteria, the total number of microorganisms in the culture system excluding the test drug and the total number of microorganisms in the culture system containing the test drug come from the same culture time point; The application measurement time is the cultivation time corresponding to the application threshold; The application thresholds include the drug resistance breakpoint application threshold (ATR) with the drug resistance breakpoint concentration as the working concentration and / or the drug sensitivity breakpoint application threshold (ATS) with the drug sensitivity breakpoint concentration as the working concentration; wherein, the drug resistance breakpoint application threshold is obtained by using the drug resistance breakpoint concentration as the working concentration in the culture system containing the drug to be tested; and the drug sensitivity breakpoint application threshold is obtained by using the drug sensitivity breakpoint concentration as the working concentration in the culture system containing the drug to be tested. The detection module is used to detect the growth of the microorganisms to be tested based on the application measurement time corresponding to the microorganisms to be tested and the drug to be tested retrieved by the retrieval module; including: inoculating the bacterial suspension of the microorganisms to be tested into culture media containing the drug to be tested and those without the drug to be tested, culturing them until the application measurement time, determining the total number of microorganisms in the culture system, and calculating the proportion of bacterial count consumed by the drug; The analysis module is used to determine the drug resistance of the test microorganism to the test drug based on the application thresholds corresponding to the test microorganism and the test drug retrieved by the retrieval module; including: comparing the proportion of drug consumption bacteria determined by the detection module with the application threshold, and determining the drug resistance of the test microorganism to the test drug based on the comparison result; The output module is used to output the results of the drug resistance of the tested microorganism to the tested drug.

[0023] The method used in the above-mentioned device to determine the drug resistance of the test microorganism is as follows: If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consumed by the drug is less than the drug resistance breakpoint application threshold when the time for application measurement is reached; or, in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consumed by the drug is less than the sensitivity breakpoint application threshold when the time for application measurement is reached, then the culture is determined to be drug resistant. If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consumed by the drug is greater than the drug resistance breakpoint application threshold when the time for application measurement is reached; or, in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consumed by the drug is greater than the sensitivity breakpoint application threshold when the time for application measurement is reached, then the system is judged to be sensitive. If, in a culture system where the drug working concentration is the resistance breakpoint, the percentage of bacteria consuming the drug is greater than the drug resistance breakpoint application threshold when the time for application measurement is reached, and in a culture system where the drug working concentration is the sensitivity breakpoint, the percentage of bacteria consuming the drug is less than the sensitivity application threshold when the time for application measurement is reached, then the system is judged to be intermediate.

[0024] In this invention, a database can be constructed based on the application measurement time and application threshold data determined by the known drug-resistant and known drug-sensitive strains of the test microorganism. The retrieval module retrieves the corresponding application measurement time and application threshold from the database according to the test microorganism and the test drug. As an example, this invention constructs a "bacteria-antibiotic-application threshold" paired database for Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa, which contains application measurement time and application threshold data for different antibiotics for Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0025] Preferably, the method for determining the application measurement time and the application threshold based on the known drug-resistant strains and known susceptible strains of the test microorganisms includes: inoculating bacterial suspensions of known drug-resistant strains and known susceptible strains into culture media containing and without the test drug, respectively; measuring the total number of microorganisms in each culture system at different time points; calculating the proportion of drug-consuming bacteria for each known drug-resistant strain and known susceptible strain; determining the maximum proportion of drug-consuming bacteria for all detected known drug-resistant strains at the same culture time point; and determining the minimum proportion of drug-consuming bacteria for all detected known susceptible strains at the same culture time point.

[0026] Preferably, the total number of microorganisms is characterized by absorbance.

[0027] Preferably, the concentration of the bacterial suspension is 0.5 × 10⁻⁶. 8 ~ 5.0×10 8 CFU / mL; more preferably 1.0 × 10⁻⁶. 8 ~2.0×10 8 CFU / mL.

[0028] Preferably, the microorganisms include bacteria or fungi.

[0029] Thirdly, the present invention provides a computer device, the computer device including a processor and a memory, the memory storing a computer program, and the processor executing the computer program to implement the microbial resistance detection method described in the first aspect above.

[0030] Fourthly, the present invention provides a computer storage medium storing a computer program, which, when executed, implements the microbial resistance detection method described in the first aspect above.

[0031] The beneficial effects of this invention include at least the following: Addressing the current technical bottlenecks of excessively long testing times, low timeliness, and poor universality in microbial drug resistance detection, this invention provides a method for detecting microbial drug resistance. This method detects microbial drug resistance based on application measurement time and application thresholds, representing a novel, broad-spectrum, dynamic phenotypic rapid drug susceptibility testing method. Using this method, the drug resistance of 30 clinical bacterial strains (15 strains each of Staphylococcus aureus and Acinetobacter baumannii) to six antibiotics was detected with 100% accuracy. The detection time was only 1.0 to 5.5 hours, 3-18 times shorter than traditional dilution methods and commercial systems such as VITEK2. It also has a significant cost advantage, with the cost per sample being only about RMB 1. Currently, commercially available drug susceptibility analysis systems (such as VITEK2) all rely on turbidimetric methods to detect bacterial growth kinetic curves and determine drug susceptibility results by measuring the minimum inhibitory concentration (MIC). The method of this invention eliminates the lengthy process of traditional methods that require culturing to a point where turbidity or fluorescence signal is stable and visible to the naked eye. By capturing the differences in the metabolic or growth response of bacteria in the early stages (within a few hours) under antibiotic stress, it enables rapid determination and provides an innovative technical path for the development of next-generation drug resistance detection equipment. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 This is the growth curve of 20 strains of Acinetobacter baumannii in an amikacin-containing culture system in Example 5 of the present invention. Detailed Implementation

[0034] This invention provides a method for detecting microbial drug resistance, and the construction process of this method mainly includes the following steps.

[0035] 1. Establishment of core judgment criteria This invention breaks through the traditional method of interpreting the minimum inhibitory concentration (MIC) by relying on the culture endpoint, and proposes an "Application Threshold" (AT) based on early growth turbidity changes as a criterion for determining rapid drug resistance.

[0036] The principle for determining the application threshold (AT) is as follows: For the target pathogen-antibiotic combination, the AT is determined by analyzing the absorbance (OD value) changes of a large number of standard strains and clinical strains with known drug susceptibility spectra at specific time points, according to the following statistical principles: Define the formula for calculating the percentage of bacteria consumed by drugs: Percentage of bacteria consumed by drugs = (OD value excluding drug wells - OD value including drug wells) / OD value excluding drug wells × 100%, and calculate the percentage of bacteria consumed by drugs.

[0037] Let Rmax be the maximum percentage of drug consumption by a drug-resistant strain at a specific time point, and Smin be the minimum percentage of drug consumption by a susceptible strain at the same time point.

[0038] An effective application threshold (AT) must satisfy the condition that Smin - Rmax > 10%~15% at the same time point; take the percentage value of (Smin + Rmax) / 2 as the application threshold (AT) at that time point.

[0039] Determination of Application Measurement Time (AMT): The detection time corresponding to the application threshold is the final selected AMT. This time point is determined through system experiment optimization and is usually much shorter than the incubation time of the traditional MIC method.

[0040] The principle for determining the working concentration (WC) of antibiotics is as follows: Referencing the breakpoint standards for antimicrobial susceptibility testing specified in the Clinical and Laboratory Standards Institute (CLSI) M100 document (32nd edition), the working concentration of each antibiotic in the detection system is determined. For the target strain and the selected antibiotic, the resistance breakpoint concentration (R-BP) and susceptibility breakpoint concentration (S-BP) are determined according to CLSI standards. These two breakpoint concentrations are directly used as the working concentration (WC) of the corresponding antibiotic in the detection system of this invention.

[0041] 2. Database Construction Based on the above principles, through systematic experiments, this invention established a "strain-antibiotic-application threshold" database for four clinically important pathogens: Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. The application thresholds for Acinetobacter baumannii and Staphylococcus aureus include both ATR and ATS, while those for Escherichia coli and Pseudomonas aeruginosa only include ATR. The database covers the following: Strain range: Includes standard reference strains and a wide range of clinical isolates.

[0042] Antibiotic types: 15 antibiotics were selected based on drugs monitored by the China Antimicrobial Resistance Surveillance Network (CARSS) and clinical medications for 4 types of bacteria.

[0043] Core data: For each pair of "bacteria - antibiotic" combinations, the application measurement time AMT and the corresponding application thresholds AT at the working concentration are defined.

[0044] 3. Drug resistance judgment rules Using the above database, the drug resistance of the test strain is judged according to the following rules: Resistant (R): In the R - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATR, or in the S - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATS.

[0045] Susceptible (S): In the R - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATR, or in the S - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATS.

[0046] Intermediate (I): In the R - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATR and in the S - BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATS.

[0047] For the bacteria - drug combinations with a clear non - intermediate range, the judgment can be simplified to only rely on a single concentration of R - BP or S - BP.

[0048] In the above judgment rules, the proportion of bacteria consumed by the drug = (OD value of the drug - free well - OD value of the drug - containing well) / OD value of the drug - free well × 100%.

[0049] 4. Drug resistance detection method Culture medium preparation: Weigh an appropriate amount of cation - adjusted M - H broth powder (Cation - Adjusted Mueller - Hinton Broth Powder, CAMHB) (e.g., 8.8 g), dissolve it in a certain volume (e.g., 400 mL) of distilled water, heat it to boiling or stir it magnetically until it is completely dissolved, and sterilize it at 121 °C for 20 minutes, then cool it to room temperature for standby.

[0050] Bacterial suspension preparation: Pick the test colonies, prepare a bacterial suspension with CAMHB medium, and adjust the concentration of the bacterial suspension to about 1.0×10 8 ~ 2.0×10 8 CFU / mL within the standard range (the typical value is 1.5×10 8 CFU / mL) by turbidimetry.

[0051] Sample addition and incubation: Add an equal volume (e.g., 150 μL) of the prepared bacterial suspension to each test well and control well of the test plate. Each plate can simultaneously test 6 test strains. After adding the samples, cover the test plate and place it in a constant temperature incubator at 37°C for incubation.

[0052] Measurement and Interpretation: At the corresponding Application Measurement Time (AMT) in the database, measure the OD value of each well and calculate the percentage of bacterial count consumed by the drug using the following formula: Percentage of bacterial count consumed by the drug = (OD value of wells without drug - OD value of wells with drug) / OD value of wells without drug × 100%. Compare the percentage of bacterial count consumed by the drug with the corresponding ATR and ATS standard thresholds in the database. Based on the above resistance judgment rules, determine the sensitivity result of the tested strain to each antibiotic (resistant (R), sensitive (S), or intermediate (I)).

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] The strains used in the following examples include 20 strains of Escherichia coli: ATCC 25922 (antimicrobial susceptibility control standard strain), BL21 (standard strain), MG1655 (standard strain), CHPC1.16891 to CHPC1.16907; 30 strains of Acinetobacter baumannii: ATCC 19606 (antimicrobial susceptibility control standard strain), CHPC1.16918 to CHPC1.16946; 20 strains of Staphylococcus aureus: ATCC29213 (antimicrobial susceptibility control standard strain, all sensitive), ATCC65382, CHPC1.16948 to CHPC1.16965; and 20 strains of Pseudomonas aeruginosa: ATCC27853 (antimicrobial susceptibility control standard strain), CHPC1.16971 to CHPC1.16989. All strains other than the standard strain were obtained from the Chinese Center for Disease Control and Prevention's Pathogenic Microorganism Culture Collection (CHPC). Among them, CHPC 1.16920, CHPC 1.16922, and CHPC 1.16924 have been published in the literature (Wang P, Li RQ, Wang L, et al. Proteomic Analyses of Acinetobacter baumannii Clinical Isolates to Identify Drug Resistant Mechanism[J]. Frontiers in Cellular and Infection Microbiology, 2021, 11:625430.), corresponding to the strains in that literature. Figure 4 The strains A7, A21, and A155 are among them.

[0055] Example 1: Determination of Working Concentration of Antibiotic This invention refers to the antimicrobial susceptibility testing (AST) criteria (breakpoint values) specified in the 32nd edition of the Performance Standards for Antimicrobial Susceptibility Testing (M100) published by the Clinical and Laboratory Standards Institute (CLSI) to determine the working concentration (WC) of the antibiotic in the culture system of the test strain. Two specific concentrations, the resistance breakpoint (R-BP) and the susceptibility breakpoint (S-BP), are selected as the working concentrations, i.e., the double breakpoint method. This allows for the simultaneous establishment of two clear "judges" or "thresholds" in a single experiment, enabling a clear and accurate classification of the test strain into "susceptible (S)," "intermediate (I)," or "resistant (R)" in one step.

[0056] The specific basis and logic for selecting these two inflection point concentrations are as follows: 1. Select the sensitive breakpoint (S-BP) as the basis for the working concentration (WC): S-BP is the highest antibiotic concentration required to inhibit the growth of 99.9% of wild-type susceptible bacterial strains (i.e., strains without acquired resistance mechanisms). It represents the expected effective therapeutic concentration of an antibiotic against a particular bacterium.

[0057] In a culture system containing antibiotics at an S-BP concentration, if the test strain can grow, it directly indicates that the MIC value of the strain is higher than S-BP. This means that even with conventional doses, the drug cannot reach an effective concentration at the site of infection, and treatment is likely to fail. Therefore, the first WC (S-BP) can preliminarily screen out "non-susceptible" strains (i.e., I or R), which can serve as a threshold for interpreting "susceptibility." If the strain cannot grow at an S-BP concentration, it indicates that its MIC value is lower than or equal to S-BP. This meets the definition of "susceptibility," that is, the strain can be inhibited by the drug concentration achieved after conventional doses, which can be used as confirmation of "susceptibility."

[0058] 2. Select the resistance breakpoint (R-BP) as the basis for the working concentration (WC): R-BP is a concentration set based on factors such as clinical efficacy, pharmacokinetic / pharmacodynamic data, and resistance mechanisms. It is not only higher than S-BP, but also typically higher than the highest blood concentration a drug can safely achieve in the human body. When a strain's MIC value is higher than this concentration, it indicates the presence of a clear, clinically detectable resistance mechanism, and treatment is likely to fail even with high doses or high concentrations at the site of infection. If a test strain can still grow in a culture system containing antibiotics at an R-BP concentration, it proves that its MIC value is higher than R-BP. This indicates a high level of resistance and must be classified as "resistant (R)," serving as a threshold for "resistance." Distinguishing between "intermediate" and "resistant" is one of the most crucial roles of R-BP as a whole-cell culture (WC). If a strain grows at an S-BP concentration (i.e., is not sensitive) but cannot grow at an R-BP concentration, its MIC value is between S-BP and R-BP. According to CLSI standards, strains in this range are classified as "intermediate (I)."

[0059] In summary, the core basis for this invention to use both the sensitivity breakpoint (S-BP) and resistance breakpoint (R-BP) specified in the CLSI M100 standard as the working concentration (WC) in the antimicrobial susceptibility test (AST) is that, through these two internationally recognized critical concentrations validated by a large amount of scientific and clinical data, a highly efficient dichotomous detection system can be constructed. This system can accurately and reliably classify any test strain into the categories of "sensitive (S)," "intermediate (I)," or "resistant (R)" in one go, and can ensure the consistency and comparability of results between different laboratories, providing the most critical basis for clinical anti-infective diagnosis and treatment and drug resistance monitoring.

[0060] Example 2: Application of threshold concept and rule optimization for determination The "Application Threshold (AT)" is a quantitative criterion proposed in this invention based on the rate of change of bacterial absorbance (OD value) at a specific time point. Its determination is derived from the summary of a large amount of research and development experimental data and is a value applicable to bacterial-drug combinations. This invention analyzes early growth kinetic data of a large number of strains with known drug susceptibility results after exposure to a specific antibiotic, statistically deriving the critical value of OD change rate that most significantly distinguishes between sensitive and resistant strains, and uses this as the Application Threshold (AT).

[0061] This invention involves detecting 20 strains of each of four bacteria (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa) and 15 antibiotics at 11,000 time points. The experimental data were systematically analyzed, and two methods were used: turbidity difference and the percentage of bacteria consumed by the drug, to determine the application threshold judgment rules and application measurement time. The optimal rules were determined through optimization.

[0062] 1. Turbidity difference method For the tested strains, the maximum turbidity difference ΔOD value (ΔOD value is the difference between the OD values ​​of liquid culture without antibiotics and liquid culture with antibiotics) at the corresponding time point for all drug-resistant strains of the same antibiotic is Rmax, and the minimum turbidity difference ΔOD value (ΔOD value is the difference between the OD values ​​of liquid culture without antibiotics and liquid culture with antibiotics) at the corresponding time point for all sensitive strains is Smin. The application threshold (AT) meets the following two requirements: (1) at the same time point, Smin-Rmax>0.1; (2) at the same time point, (Smin+Rmax) / 2>0.1, and (Smin+Rmax) / 2 is used as the application threshold AT. The minimum measurement time point that meets both of the above requirements is the final application measurement time (AMT).

[0063] 2. Method based on the percentage of bacterial count consumed by the drug For the target pathogen-antibiotic combination, the absorbance (OD value) changes of a large number of standard strains and clinical strains with known drug susceptibility profiles at specific time points were analyzed. The application threshold (AT) was determined according to the following statistical principles: The formula for calculating the percentage of bacteria consumed by the drug was defined as: Percentage of bacteria consumed by the drug = (OD value of wells without drug - OD value of wells with drug) / OD value of wells without drug × 100%. The maximum percentage of drug consumption for resistant strains at a specific time point was set as Rmax, and the minimum percentage of drug consumption for susceptible strains at the same time point was set as Smin. The effective application threshold (AT) required that at the same time point, Smin - Rmax > 10~15%. The percentage of (Smin + Rmax) / 2 was taken as the application threshold (AT) at that time point. This time point is the final application measurement time (AMT). The AMT was determined through systematic experimental optimization and is usually much shorter than the culture time of the traditional MIC method.

[0064] Table 2 shows a comparison of the corresponding dataset information for "bacterium-antibiotic-application threshold (ATR)-application detection time (AMT)" for Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa, determined by the two methods described above. It is evident that the detection time using the drug-consumption bacterial count ratio method can be effectively shortened; therefore, this invention determines the application threshold using the drug-consumption bacterial count ratio method.

[0065] Table 2. Key parameters of 4 bacteria to 15 antibiotics determined by turbidity difference method and drug consumption ratio method Note: “—” in Table 2 indicates that no test was conducted.

[0066] Example 3 Determination Rules for Drug Resistance For each bacterium, each antibiotic, and each bacterium-antibiotic combination, drug resistance tests are respectively carried out using the working concentrations corresponding to the R-BP and S-BP breakpoints. The drug resistance of the test strain to the antibiotic is judged according to the following criteria: Resistant (R): In the R-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATR, or, in the S-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATS.

[0067] Susceptible (S): In the R-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATR, or, in the S-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATS.

[0068] Intermediate (I): In the R-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point > ATR and in the S-BP working concentration system, the proportion of bacteria consumed by the drug at the AMT time point < ATS.

[0069] If there are no intermediate strains, drug resistance can be judged only based on R-BP or S-BP.

[0070] For clinical diagnosis and treatment, only the R-BP working concentration is needed to determine bacterial drug resistance. If it is susceptible (S), the antibiotic can be used for infection treatment; for precise drug resistance detection and monitoring analysis that requires a detailed drug resistance spectrum, the R-BP working concentration analysis can be carried out first, and for susceptible strains, the S-BP working concentration analysis can be carried out to determine whether they are intermediate drug-resistant strains.

[0071] Example 4 Establishment of a Drug Resistance Detection Method This example provides a drug resistance detection method for microorganisms, which includes the following steps: Preparation of culture medium: Weigh an appropriate amount of cation-adjusted M-H broth powder (Cation-Adjusted Mueller-Hinton Broth Powder, CAMHB) (for example, 8.8 g), dissolve it in a certain volume (for example, 400 mL) of distilled water, heat it to boiling or stir it magnetically to completely dissolve it, and sterilize it at 121 °C for 20 minutes, then cool it to room temperature for standby.

[0072] Preparation of bacterial suspension: Pick the test colonies, prepare a bacterial suspension with CAMHB medium, and adjust the concentration of the bacterial suspension to about 1.0×10 8 ~ 2.0×10 8 CFU / mL within the standard range (the typical value is 1.5×10 8 CFU / mL) by turbidimetry.

[0073] Sample addition and incubation: Add an equal volume (e.g., 150 μL) of the prepared bacterial suspension to each test well and control well of the test plate. Each plate can simultaneously test 6 test strains. After adding the samples, cover the test plate and place it in a constant temperature incubator at 37°C for incubation.

[0074] Measurement and Interpretation: At the application measurement time (AMT) predetermined according to the known strain, the OD value of each well was measured, and the percentage of bacterial count consumed by the drug was calculated according to the following formula: Percentage of bacterial count consumed by the drug = (OD value of wells without drug - OD value of wells with drug) / OD value of wells without drug × 100%. The percentage of bacterial count consumed by the drug was compared with the corresponding ATR and ATS predetermined according to the known strain. According to the resistance judgment rules in Example 3, the sensitivity result (resistance R, sensitivity S, or intermediate I) of the test strain to each antibiotic was determined.

[0075] Example 5: Establishment of a Bacterium-Antibiotic-Application Threshold (ATR)-Application Detection Time (AMT) Database Taking Acinetobacter baumannii and Staphylococcus aureus as examples, a database of bacteria-antibiotic-application threshold (ATR)-application detection time (AMT) was established.

[0076] Twenty clinical isolates each of Acinetobacter baumannii and Staphylococcus aureus were selected. Based on the drugs monitored by the China Antimicrobial Resistance Surveillance Network (CARSS) and clinical medications, the antibiotics to be tested for Acinetobacter baumannii included amikacin, gentamicin, ceftazidime, tobramycin, imipenem, and levofloxacin (a total of six antibiotics); the antibiotics to be tested for Staphylococcus aureus included azithromycin, erythromycin, clarithromycin, clindamycin, penicillin, and tetracycline (a total of six antibiotics).

[0077] Pick an appropriate amount of colonies from the solid culture medium and add them to 6 mL of CAMHB liquid medium. Mix well and incubate overnight at 37°C using a shaker. Prepare an antibiotic stock solution using CAMHB as the solute. Add an appropriate amount of the liquid culture to the CAMHB liquid medium to achieve a bacterial concentration of 1.5 × 10⁻⁶. 8 CFU / mL. Dispense the prepared bacterial culture into two tubes: one with antibiotics and one without. Add the calculated amount of antibiotic stock solution (calculated according to the working concentrations corresponding to the R-BP and S-BP breakpoints) to the tube with antibiotics. Add an equal volume of CAMHB liquid medium to the tube without antibiotics. Mix the two bacterial cultures by shaking and then dispense 1 mL into each reaction tube according to the desired time points. Place all reaction tubes in a shaker and incubate at 37°C. At each set time point, remove the corresponding two sets of reaction tubes, measure the OD value, plot the growth curve, and calculate the ΔOD value.

[0078] Taking the Acinetobacter baumannii R-BP system for amikacin resistance testing as an example, Table 3 shows the percentage of bacterial counts consuming the drug at different time points in culture systems with and without amikacin for 20 Acinetobacter baumannii strains with known resistance profiles. Growth curves are shown in [Figure 3]. Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 The application threshold (AT) and application measurement time (AMT) of this invention are determined using the following principles: The formula for calculating the percentage of bacteria consumed by the drug is defined as: Percentage of bacteria consumed by the drug = (OD value of wells without drug - OD value of wells with drug) / OD value of wells without drug × 100%. The percentage of bacteria consumed by the drug is calculated. Let Rmax be the maximum percentage of drug consumption for all drug-resistant strains at a specific time point, and Smin be the minimum percentage of drug consumption for all susceptible strains at the same time point. The application threshold (AT) satisfies the condition that Smin - Rmax > 10-15% at the same time point. With a measurement time of 1 hour, Smin = 49.73%, Rmax = 12.85%, and Smin - Rmax = 36.88%, satisfying the above conditions. Therefore, the ATR is (Smin + Rmax) / 2 = 31.29%, and the AMT is 1 hour. Similarly, the application threshold (ATR) and application measurement time (AMT) for Acinetobacter baumannii against all antibiotics are determined, and the results are shown in Table 4. The ATR and AMT of Staphylococcus aureus against six antibiotics were obtained using the same method (Table 5). Based on the determined ATR and AMT, the resistance of the tested strains to the six antibiotics can be determined.

[0079] Table 3. Percentage of bacterial count consumed by Acinetobacter baumannii-amikacin at different culture time points

[0080] Table 4. Key parameters for detecting Acinetobacter baumannii resistance.

[0081] Table 5. Key parameters for detecting Staphylococcus aureus drug resistance

[0082] Example 6: Evaluation of the effectiveness of the drug resistance detection method The drug resistance detection method of this invention (see Examples 4 and 5) was used to detect the drug resistance of 10 strains of Acinetobacter baumannii. These strains were isolates independent of the strains used in the database construction in Example 5. The commercial drug resistance analysis system VITEK2 (bioMérieux, France) and the E-Test detection method were used as control methods. The detection results are shown in Tables 6, 7, 8, 9, 10, and 11, and the method comparison summary is shown in Table 12. The drug resistance detection method of this invention has significant advantages over the two most commonly used methods: the detection time is reduced by 3-18 times compared to conventional methods, and the detection can be completed within 2 hours, with drug resistance results obtained in as little as 1 hour, and the cost is significantly reduced. Therefore, the detection device based on the drug resistance detection method of this invention can effectively help clinical infection diagnosis and treatment move from empirical medication to precise treatment within hours.

[0083] Table 6. Results of Amikacin Resistance Test for Acinetobacter baumannii

[0084] Table 7. Results of levofloxacin resistance testing for Acinetobacter baumannii

[0085] Table 8. Results of imipenem resistance testing for Acinetobacter baumannii

[0086] Table 9. Results of Tobramycin Resistance Test for Acinetobacter baumannii

[0087] Table 10. Results of ceftazidime resistance test for Acinetobacter baumannii

[0088] Table 11. Results of gentamicin resistance testing for Acinetobacter baumannii

[0089] Table 12 Evaluation of the effectiveness of drug resistance testing methods

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting microbial drug resistance, characterized by, The application relates to a method for determining the drug resistance of a microorganism to a drug, comprising the following steps: inoculating a microorganism suspension of the microorganism to be tested into a culture medium containing the drug to be tested and a culture medium not containing the drug to be tested respectively, and culturing the culture medium to an application measurement time; determining the total number of microorganisms in the culture system, and calculating the proportion of the drug-consumed microorganism amount; comparing the proportion of the drug-consumed microorganism amount with an application threshold value, and determining the drug resistance of the microorganism to the drug according to the comparison result; wherein the application measurement time and the application threshold value are determined according to known drug-resistant strains and known sensitive strains of the microorganism to the drug; the application threshold value is a percentage value of (Smin + Rmax) / 2, wherein Smin and Rmax are the minimum drug-consumed microorganism amount proportion and the maximum drug-consumed microorganism amount proportion of the known sensitive strain and the known drug-resistant strain respectively at the same culture time point, and Smin - Rmax > 10%~15%; the calculation formula of the proportion of the drug-consumed microorganism amount is: drug-consumed microorganism amount proportion = (the total number of microorganisms in the culture medium not containing the drug to be tested - the total number of microorganisms in the culture medium containing the drug to be tested) / the total number of microorganisms in the culture medium not containing the drug to be tested * 100%; the total number of microorganisms in the culture medium not containing the drug to be tested and the total number of microorganisms in the culture medium containing the drug to be tested are derived from the same culture time point; the application measurement time is the culture time corresponding to the application threshold value; the application threshold value comprises a drug-resistant breakpoint application threshold value ATR with the drug-resistant breakpoint concentration as the drug working concentration and / or a sensitive breakpoint application threshold value ATS with the sensitive breakpoint concentration as the drug working concentration, wherein the drug-resistant breakpoint application threshold value is obtained by taking the culture medium containing the drug to be tested as the drug working concentration; and the sensitive breakpoint application threshold value is obtained by taking the culture medium containing the drug to be tested as the drug working concentration. The method for determining the drug resistance of the microorganism to the drug is as follows: if the proportion of the drug-consumed microorganism amount is less than the drug-resistant breakpoint application threshold value in the culture system with the drug-resistant breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, or the proportion of the drug-consumed microorganism amount is less than the sensitive breakpoint application threshold value in the culture system with the sensitive breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, the microorganism is determined to be drug-resistant; if the proportion of the drug-consumed microorganism amount is greater than the drug-resistant breakpoint application threshold value in the culture system with the drug-resistant breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, or the proportion of the drug-consumed microorganism amount is greater than the sensitive breakpoint application threshold value in the culture system with the sensitive breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, the microorganism is determined to be sensitive; and if the proportion of the drug-consumed microorganism amount is greater than the drug-resistant breakpoint application threshold value in the culture system with the drug-resistant breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, and the proportion of the drug-consumed microorganism amount is less than the sensitive breakpoint application threshold value in the culture system with the sensitive breakpoint concentration as the drug working concentration when the culture system is cultured to the application measurement time, the microorganism is determined to be intermediate. ​ ​ ​ ​ ​ ​ 2. The method of claim 1, wherein the microorganism is a bacterium. ​ ​ ​ ​ 3. The method of claim 1, wherein the microorganism is bacteria. The method for determining the application measurement time and the application threshold value according to known drug-resistant strains and known sensitive strains of the microorganism to be tested comprises: inoculating the bacterial suspension of the known drug-resistant strains and the known sensitive strains into culture media containing and not containing the drug to be tested respectively for culture, determining the total number of microorganisms in each culture system at different time points, and calculating the proportion of the drug-consuming bacteria of each known drug-resistant strain and known sensitive strain, determining the maximum proportion of drug-consuming bacteria of all detected known drug-resistant strains at the same culture time point, and the minimum proportion of drug-consuming bacteria of all detected known sensitive strains at the same culture time point.

4. The method according to any one of claims 1 to 3, wherein The total number of microorganisms is characterized by absorbance; and / or the concentration of the bacterial suspension is 0.5 x 10 8 ~ 5.0 x 10 8 CFU / mL.

5. The method according to any one of claims 1 to 3, wherein the microorganism is bacteria. The microorganism comprises bacteria or fungi.

6. A microorganism drug resistance detection device, characterized by, The device comprises: a calling module configured to call the application measurement time and the application threshold value corresponding to the microorganism to be tested and the drug to be tested; wherein the application measurement time and the application threshold value are determined according to known drug-resistant strains and known sensitive strains of the microorganism to be tested to the drug to be tested; The application threshold value is a percentage value of (Smin + Rmax) / 2, wherein Smin and Rmax are measured at the same culture time point and satisfy Smin - Rmax > 10%~15%; Rmax is the maximum proportion of drug-consuming bacteria of the known drug-resistant strain at a certain culture time point, and Smin is the minimum proportion of drug-consuming bacteria of the known sensitive strain at the same time point as Rmax; the calculation formula of the proportion of drug-consuming bacteria is: proportion of drug-consuming bacteria = (total number of microorganisms in the culture system without the drug to be tested - total number of microorganisms in the culture system with the drug to be tested) / total number of microorganisms in the culture system without the drug to be tested × 100%; in the calculation formula of the proportion of drug-consuming bacteria, the total number of microorganisms in the culture system without the drug to be tested and the total number of microorganisms in the culture system with the drug to be tested are derived from the same culture time point; The application measurement time is the culture time corresponding to the application threshold value; The application threshold value comprises a resistant breakpoint application threshold value ATR with a resistant breakpoint concentration as the working concentration of the drug and / or a sensitive breakpoint application threshold value ATS with a sensitive breakpoint concentration as the working concentration of the drug; wherein the resistant breakpoint application threshold value is obtained by taking the culture system with the drug to be tested as the working concentration of the drug at the resistant breakpoint concentration; and the sensitive breakpoint application threshold value is obtained by taking the culture system with the drug to be tested as the working concentration of the drug at the sensitive breakpoint concentration; a detection module configured to detect the growth of the microorganism to be tested based on the application measurement time corresponding to the microorganism to be tested and the drug to be tested called by the calling module; comprising: inoculating the bacterial suspension of the microorganism to be tested into culture media containing and not containing the drug to be tested respectively for culture to the application measurement time, determining the total number of microorganisms in the culture system, and calculating the proportion of drug-consuming bacteria. The analysis module is configured to determine the drug resistance of the test microorganism to the test drug based on the application threshold corresponding to the test microorganism and the test drug retrieved by the retrieval module. The analysis module includes: comparing the drug-consuming bacteria amount proportion determined by the detection module with the application threshold, and determining the drug resistance of the test microorganism to the test drug according to the comparison result. The output module is configured to output the result of the drug resistance of the test microorganism to the test drug.

7. The microbial drug resistance detection device according to claim 6, characterized in that, The method for determining the drug resistance of the test microorganism to the test drug includes: If, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is less than the drug application threshold when the culture is cultured to the application measurement time; or, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is less than the drug application threshold when the culture is cultured to the application measurement time, the drug resistance is determined. If, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is greater than the drug application threshold when the culture is cultured to the application measurement time; or, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is greater than the drug application threshold when the culture is cultured to the application measurement time, the drug sensitivity is determined. If, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is greater than the drug application threshold when the culture is cultured to the application measurement time, and, in a culture system with the drug working concentration being the drug breakpoint concentration, the drug-consuming bacteria amount proportion is less than the drug application threshold when the culture is cultured to the application measurement time, the drug intermediate is determined.

8. The microorganism drug resistance detection device according to claim 6 or 7, wherein The method for determining the application measurement time and the application threshold according to the known drug-resistant strains and the known sensitive strains of the test microorganism includes: inoculating the bacterial suspensions of the known drug-resistant strains and the known sensitive strains into culture media containing the test drug and culture media not containing the test drug respectively, determining the total number of microorganisms in each culture system at different time points, calculating the drug-consuming bacteria amount proportion of each known drug-resistant strain and each known sensitive strain, determining the maximum drug-consuming bacteria amount proportion of all the detected known drug-resistant strains at the same culture time point, and determining the minimum drug-consuming bacteria amount proportion of all the detected known sensitive strains at the same culture time point. The total number of microorganisms is characterized by absorbance. and / or the concentration of the bacterial suspension is 0.5 x 10 8 ~ 5.0 x 10 8 CFU / mL; The microorganisms include bacteria or fungi.

9. A computer device, comprising: The computer device includes a processor and a memory, and the memory stores a computer program. When the processor executes the computer program, the microorganism drug resistance detection method in any one of claims 1-5 is implemented.

10. A computer storage medium, characterized in that, The computer storage medium stores a computer program, and the computer program is executed to implement the microorganism drug resistance detection method in any one of claims 1-5.

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