Antimicrobial drug screening method based on bacterial resistance gene promoter strength detection

Abstract

The present invention belongs to the technical field of bacterial drug resistance analysis, and specifically relates to an antimicrobial drug screening method based on bacterial drug resistance gene promoter strength detection, which includes collecting and isolating drug-resistant strains; determining the drug resistance gene of the strain and its upstream promoter sequence; performing minimum inhibitory concentration value detection based on drug sensitivity experiments; establishing a mathematical analysis model for bacterial drug resistance analysis, performing boundary mathematical representation of the minimum inhibitory concentrations of drug resistance and non-drug resistance; performing a three-dimensional matrix representation of the strain drug sensitivity test results; performing an indicator function representation of the strain drug sensitivity test results; performing dimensionality reduction processing on the three-dimensional matrix of the strain drug sensitivity test results; screening for non-drug-resistant antimicrobial drugs for strains carrying any one drug resistance gene; screening for non-drug-resistant antimicrobial drugs for strains carrying any multiple drug resistance genes, and completing bacterial resistance analysis and antimicrobial drug screening. The present invention more conveniently and effectively guides clinical selection of antimicrobial drugs for drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial drug resistance analysis, and in particular relates to an antimicrobial drug screening method based on bacterial drug resistance gene promoter strength detection. Background Art

[0002] Severe infections caused by drug-resistant bacteria have a poor clinical prognosis and can easily lead to refractory infections and treatment failure. Among them, there are many types of drug-resistant genes that cause bacterial resistance, and there are also different types of upstream promoter sequences of drug-resistant genes. On the one hand, different drug-resistant genes will show different drug resistance strength characteristics due to mutations in gene sequence sites. On the other hand, the upstream promoter sequences of drug-resistant genes will also show different regulatory effects on the expression of drug-resistant genes due to site mutations and sequence structure changes, further affecting the expression level of drug-resistant genes, and thus affecting the hydrolysis activity of strains carrying these drug-resistant genes to antimicrobial drugs and whether the drug-resistant phenotype is expressed. However, the current existing bacterial resistance detection methods still have defects, such as:

[0003] (1) Only testing whether bacteria carry drug-resistant genes, but lacking analysis of promoter strength. When bacteria only carry drug-resistant genes but the promoter regulation strength is weak and does not show drug resistance, the test results will guide the clinical use of stronger antimicrobial drugs based solely on the results of drug-resistant gene testing, which will lead to excessive use of antimicrobial drugs;

[0004] (2) Using the drug sensitivity method to statically detect bacterial resistance in vitro is not only time-consuming, but also lacks dynamic analysis of the strength of bacterial resistance gene promoters, which may cause inconsistencies between static in vitro detection and dynamic evolution of bacterial resistance in vivo. Due to the defects of these bacterial resistance detection methods, the improper use of clinical antimicrobial drugs guided by resistance test results may even promote the evolution of bacterial resistance in vivo, enhance resistance, and other adverse consequences;

[0005] Although the introduction of resistance gene promoter strength analysis can more effectively monitor bacterial resistance, the strength of bacterial resistance depends not only on whether the bacteria carry resistance genes and the type of resistance genes, but also on the expression level of the resistance genes under promoter regulation. However, there are many types of resistance genes in clinical strains, and there are also many different types of promoter sequences upstream of resistance genes. If these are tested one by one, it may be time-consuming and laborious, increase time and testing costs, and encounter difficulties in detecting new promoter sequences. Therefore, the present invention proposes an antimicrobial drug screening method based on the detection of bacterial resistance gene promoter strength. Summary of the Invention

[0006] The purpose of the present invention is to provide an antimicrobial drug screening method based on bacterial resistance gene promoter strength detection. By combining bacterial resistance gene detection, resistance gene promoter strength analysis and bacterial MIC detection, it can better reflect the resistance characteristics of clinical drug-resistant bacteria and their possible potential changes in resistance. Therefore, through methods such as indicator function representation and matrix dimensionality reduction, it can more conveniently and effectively guide the clinical selection of antimicrobial drugs for drug-resistant bacteria.

[0007] The technical solutions adopted by the present invention are as follows:

[0008] The antimicrobial drug screening method based on bacterial resistance gene promoter strength detection includes the following steps:

[0009] Step 1: Collect and isolate drug-resistant strains;

[0010] Preferably, in step 1: isolating, culturing and identifying the strain from the clinical specimen, streaking the strain on a culture dish, culturing in a 37°C incubator, picking a single clone for passage, and then placing it in an Eppendorf tube containing 0.5 mL of bacterial freezing solution, sealing it with a sealing film, and freezing it in a -80°C refrigerator for later use.

[0011] Step 2: Determine the drug-resistant gene of the strain and its upstream promoter sequence;

[0012] In step 2: using a commercial kit to extract the bacterial genome and plasmid, and designing primers for the drug-resistant gene and its upstream promoter and the spacer sequence between the downstream gene, using the extracted genome and plasmid as templates, amplifying the corresponding sequence by PCR and sequencing to determine the type of drug-resistant gene and the upstream promoter sequence structure;

[0013] Step 3: Based on the drug sensitivity test, the minimum inhibitory concentration (MIC) value is detected; in the present invention, the minimum inhibitory concentration is abbreviated as MIC;

[0014] In step 3: according to the drug sensitivity test, the MIC values of the resistant strains to different antimicrobial drugs are detected respectively, that is, the MIC values of the resistant strains to the drugs at different antimicrobial drugs and their different concentrations are monitored, and the MIC values are recorded and used as the input values of the mathematical analysis model for bacterial resistance analysis.

[0015] Step 4: Establish a mathematical analysis model for bacterial resistance analysis and mathematically represent the boundary between the minimum inhibitory concentration (MIC) of drug-resistant and non-resistant bacteria;

[0016] In step 4, according to the drug resistance standards specified by clinical and microbiological standardization laboratories, combined with the MIC value obtained by strain testing, the drug sensitivity test results of the strain to the antimicrobial drug at different resistance genes and different upstream promoters, i.e., the degree of drug resistance, are determined; the antimicrobial drug is defined as According to the drug sensitivity test standards, the drug sensitivity MIC results of the strains were recorded as the strains’ resistance to the corresponding antimicrobial drugs. Resistance level: sensitive ,intermediary or drug resistance ;

[0017] The results of the drug sensitivity test of the strains were ,but: Indicates that the drug sensitivity test result is sensitive, the superscript Refers to sensitivity;

[0018] Indicates that the drug sensitivity test result is intermediate, the superscript Refers to the intermediary;

[0019] Indicates that the result of drug sensitivity test is drug resistant, Refers to drug resistance.

[0020] According to the degree of drug resistance, .

[0021] Then, define the MIC boundary value between resistance and non-resistance to any antimicrobial drug when the strain carries any resistance gene and any upstream promoter;

[0022] Indicates strain resistance to drugs The drug sensitivity test is the sensitive MIC boundary value, that is, the lowest MIC value that the drug sensitivity test can achieve is sensitive;

[0023] Indicates strain resistance to drugs The MIC boundary value with the drug sensitivity test as the medium; Since the intermediate state is a state in which the sensitivity of the strain to a certain antimicrobial drug is between sensitive and resistant, the strain MIC is intermediate when it is in a specific range; the MIC boundary value with the drug sensitivity test as the medium includes two boundary values, high and low; furthermore, For strains to treat drugs The drug susceptibility test is close to the intermediate MIC boundary value of sensitivity. For strains to treat drugs The drug susceptibility test was close to the intermediate MIC boundary value of drug resistance;

[0024] Indicates strain resistance to drugs The drug sensitivity test is the MIC boundary value of drug resistance, that is, the highest MIC value of drug resistance that can be achieved in the drug sensitivity test;

[0025] Step 5: Perform a three-dimensional matrix representation of the strain drug sensitivity test results;

[0026] In step 5, define For bacterial resistance genes, is the upstream promoter sequence of the strain's drug-resistant gene; Represents any promoter sequence , carry any drug resistance gene strains are resistant to any drug MIC value of drug susceptibility test;

[0027] For a strain carrying any drug-resistant gene and any promoter sequence at the same time, the MIC value of its drug susceptibility test for any drug must be between the sensitive boundary value and the drug-resistant boundary value of the drug susceptibility test of the strain;

[0028] Then, any promoter sequence Any drug resistance gene strains, resistant to any antimicrobial drugs ,have ;

[0029] Then, based on the original MIC value of the strain, a three-dimensional matrix of different promoter sequences, different resistance genes and different types of antimicrobial drugs is formed.

[0030] Step 6: Use indicator function to represent the results of the strain drug sensitivity test;

[0031] In step 6, any promoter sequence is carried Any drug resistance gene strains, to any drug The indicator function for judging drug susceptibility test results can be expressed as:

[0032] ,

[0033] if ,otherwise ; Then, under any drug-resistant gene and any upstream promoter, the drug sensitivity test results of the strain will be indicated by the indicator function Represented as an integer value of 0 or 1.

[0034] Step 7: Dimensionality reduction processing is performed on the three-dimensional matrix of the strain drug sensitivity test results;

[0035] The upstream promoter sequence of the drug-resistant gene of the strain involved in the previous step Perform dimensionality reduction processing;

[0036] definition Promoter sequence The weight coefficient is used to represent the frequency of different types of upstream promoter sequences appearing upstream of related drug-resistant genes in the bacterial population; and antimicrobial drugs Based on the strength of bacterial resistance gene promoter, the promoter sequence can be Perform weighted summation according to weight considerations and convert the strain drug sensitivity test results from a three-dimensional matrix into a two-dimensional matrix. , for any antimicrobial drug The drug susceptibility test results can be calculated using the following formula: , then the drug resistance results can be reduced from a three-dimensional matrix to a two-dimensional matrix.

[0037] Step 8: Screening for non-resistant antimicrobial drugs for strains carrying any drug-resistant gene;

[0038] In step 8, the two-dimensional matrix calculated from step 7 can be obtained. When the value is 0, it means that the strain carries any drug-resistant gene. , any promoter sequence , strains are resistant to any antimicrobial drugs The drug sensitivity test results are all non-resistant; when the value is not 0, it means that the strain carries any drug resistance gene , there are different types of promoter sequences upstream of the drug-resistant gene In this case, any antimicrobial drug The drug sensitivity test results indicate that the strain has drug resistance. The larger the value, the more drug resistance genes the strain has. In a variety of different promoter sequences In case of this drug The greater the possibility that the drug sensitivity test result is drug resistance, the stronger the overall resistance to the drug; The different values of were analyzed to obtain the bacterial resistance analysis based on the detection of bacterial resistance gene promoter strength;

[0039] Screen out strains carrying any drug-resistant gene When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then drug-resistant bacteria can carry any drug-resistant gene Screening for non-resistant antimicrobial drugs.

[0040] Step 9: Screening for non-resistant antimicrobial drugs that carry any number of resistance genes;

[0041] In step 9, Sum the different values to get ,when A value of 0 indicates that the strain carries any number of drug-resistant genes. , strains are resistant to any antimicrobial drugs The drug sensitivity test result is non-resistant; when the value is not 0, it means that the strain carries any number of drug resistance genes ,according to The different values of are analyzed to obtain the bacterial resistance analysis of resistant bacteria carrying any multiple drug-resistant genes.

[0042] Screen out strains carrying any number of drug-resistant genes When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then drug-resistant bacteria can carry any number of drug-resistant genes Screening for non-resistant antimicrobial drugs.

[0043] Step 10: Output the final results based on the calculation results in step 8 and step 9 respectively, and complete the bacterial resistance analysis and antimicrobial drug screening.

[0044] Preferably, in step 10, according to the value calculated in step 8, the resistance analysis of bacteria to different antimicrobial drugs based on the promoter sequence weight consideration when the resistant bacteria carry any kind of resistance gene can be output, and the information of antimicrobial drugs whose drug sensitivity test results are non-resistant based on promoter strength detection can be output.

[0045] According to the value calculated in step 9, the resistance analysis of bacteria to different antimicrobial drugs based on promoter sequence weight considerations when resistant bacteria carry any multiple resistance genes can be output, and the information of antimicrobial drugs whose drug sensitivity test results are non-resistant based on promoter strength detection can be conveniently output.

[0046] The technical effects achieved by the present invention are:

[0047] This invention, based on the analysis of bacterial resistance gene promoter strength, establishes a relevant mathematical representation model and utilizes methods such as indicator function representation and matrix dimensionality reduction to develop a convenient bacterial resistance analysis and antimicrobial drug screening method. The method includes the following basic steps: collecting and isolating drug-resistant strains, determining the strain's resistance gene and its upstream promoter sequence, testing the minimum inhibitory concentration (MIC) value of the drug-resistant bacteria in each of the drug-resistant strains, inputting the MIC test result data, defining the MIC boundary between resistance and non-resistance to any antimicrobial drug under any resistance gene and any upstream promoter, and mathematically representing it; a three-dimensional matrix representation of the strain drug susceptibility test results; an indicator function representation of the strain drug susceptibility test results; dimensionality reduction processing of the three-dimensional matrix of the strain drug susceptibility test results; screening for non-resistance to antimicrobial drugs in strains carrying any one or more resistance genes; and outputting the results.

[0048] By combining bacterial resistance gene detection, resistance gene promoter strength analysis and bacterial MIC detection, the present invention can better reflect the resistance characteristics of clinical drug-resistant bacteria and their possible potential changes in resistance. Therefore, through methods such as indicator function representation and matrix dimensionality reduction, it can more conveniently and effectively guide the clinical selection of antimicrobial drugs for drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a flow chart of the antimicrobial drug screening method based on bacterial resistance gene promoter strength detection of the present invention;

[0050] Figure 2 It is the drug resistance analysis and sensitive antimicrobial drug screening of the strain of the present invention carrying a single drug resistance gene;

[0051] Figure 3 The invention relates to drug resistance analysis and sensitive antimicrobial drug screening of the bacterial strain carrying multiple drug resistance genes. DETAILED DESCRIPTION

[0052] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following examples. It should be understood that the following text is only used to describe one or more specific embodiments of the present invention and does not strictly limit the scope of protection of the present invention.

[0053] like Figure 1 As shown, the antimicrobial drug screening method based on bacterial resistance gene promoter strength detection includes the following steps:

[0054] Step 1: Collect and isolate drug-resistant strains;

[0055] In step 1: Isolate, culture, and identify the strain from the clinical specimen. Streak the strain on a culture dish, culture it in a 37°C incubator, pick a single clone for subculture, and then place it in an Eppendorf tube containing 0.5 mL of bacterial freezing solution. Seal it with parafilm and freeze it in a -80°C freezer until use.

[0056] Step 2: Determine the drug-resistant gene of the strain and its upstream promoter sequence;

[0057] In step 2: The bacterial genome and plasmid are extracted using a commercial kit, and primers are designed targeting the resistance gene, its upstream promoter, and the spacer sequence between it and the downstream gene. Using the extracted genome and plasmid as templates, the corresponding sequences are amplified by PCR and then sequenced to determine the type of resistance gene and the structure of the upstream promoter sequence.

[0058] Step 3: Based on the drug sensitivity test, perform minimum inhibitory concentration (MIC) value detection;

[0059] In step 3: Based on the drug sensitivity test, the MIC values of the resistant strains to different antimicrobial drugs are detected respectively, that is, the MIC values of the resistant strains to the drugs at different antimicrobial drugs and their different concentrations are monitored, and the MIC values are recorded and used as the input value of the mathematical analysis model for bacterial resistance analysis.

[0060] Step 4: Establish a mathematical analysis model for bacterial resistance analysis and mathematically represent the boundary between the minimum inhibitory concentration (MIC) of drug-resistant and non-resistant bacteria;

[0061] In step 4: Based on the drug resistance standards specified by clinical and microbiological standardization laboratories, combined with the MIC values obtained from strain testing, the drug sensitivity test results of the strain to the antimicrobial drug at different resistance genes and different upstream promoters are determined, i.e., the degree of drug resistance; the antimicrobial drug is defined as According to the drug sensitivity test standards, the drug sensitivity MIC results of the strains were recorded as the strains’ resistance to the corresponding antimicrobial drugs. Resistance level: sensitive ,intermediary or drug resistance ;

[0062] The results of the drug sensitivity test of the strains were ,but: Indicates that the drug sensitivity test result is sensitive, the superscript Refers to sensitivity;

[0063] Indicates that the drug sensitivity test result is intermediate, the superscript Refers to the intermediary;

[0064] Indicates that the result of drug sensitivity test is drug resistant, Refers to drug resistance.

[0065] According to the degree of drug resistance, .

[0066] Then, define the MIC boundary value between resistance and non-resistance to any antimicrobial drug when the strain carries any resistance gene and any upstream promoter;

[0067] Indicates strain resistance to drugs The drug sensitivity test is the sensitive MIC boundary value, that is, the lowest MIC value that the drug sensitivity test can achieve is sensitive;

[0068] Indicates strain resistance to drugs The MIC boundary value with the drug sensitivity test as the medium; Since the intermediate state is a state in which the sensitivity of the strain to a certain antimicrobial drug is between sensitive and resistant, the strain MIC is intermediate when it is in a specific range; the MIC boundary value with the drug sensitivity test as the medium includes two boundary values, high and low; furthermore, For strains to treat drugs The drug susceptibility test is close to the intermediate MIC boundary value of sensitivity. For strains to treat drugs The drug susceptibility test was close to the intermediate MIC boundary value of drug resistance;

[0069] Indicates strain resistance to drugs The drug sensitivity test is the MIC boundary value of drug resistance, that is, the highest MIC value of drug resistance that can be achieved in the drug sensitivity test;

[0070] Step 5: Perform a three-dimensional matrix representation of the strain drug sensitivity test results;

[0071] Preferably, in step 5, define For bacterial resistance genes, is the upstream promoter sequence of the strain's drug-resistant gene; Represents any promoter sequence , carry any drug resistance gene strains are resistant to any drug MIC value of drug susceptibility test;

[0072] For a strain carrying any drug-resistant gene and any promoter sequence at the same time, the MIC value of its drug sensitivity test for any drug must be between the sensitive boundary value and the resistant boundary value of the drug sensitivity test of the strain; Any drug resistance gene strains, resistant to any antimicrobial drugs ,have ;

[0073] Then, based on the original MIC value of the strain, a three-dimensional matrix of different promoter sequences, different resistance genes and different types of antimicrobial drugs is formed.

[0074] Step 6: Use indicator function to represent the results of the strain drug sensitivity test;

[0075] In step 6, any promoter sequence is carried Any drug resistance gene strains, to any drug The indicator function for judging drug susceptibility test results can be expressed as: ,

[0076] if ,otherwise ; Then, under any drug-resistant gene and any upstream promoter, the drug sensitivity test results of the strain will be indicated by the indicator function Represented as an integer value of 0 or 1.

[0077] Step 7: Dimensionality reduction processing is performed on the three-dimensional matrix of the strain drug sensitivity test results;

[0078] Preferably, in step 7, considering that different upstream promoter structure sequences can significantly affect the transcription and expression of downstream drug-resistant genes, clinical special detection of upstream promoter sequences is rarely used, unless the whole genome of the strain is sequenced and analyzed to clarify the upstream promoter structure sequence of the drug-resistant gene, but the time and detection cost of this operation are also high, so the upstream promoter sequence of the drug-resistant gene of the strain involved in the previous step will be Perform dimensionality reduction processing;

[0079] To achieve the above functions, define Promoter sequence The weight coefficient is used to represent the frequency of different types of upstream promoter sequences appearing upstream of related drug-resistant genes in the bacterial population; and antimicrobial drugs Based on the strength of bacterial resistance gene promoter, the promoter sequence can be The weighted summation is performed according to the weight considerations, and the results of the strain drug sensitivity test are converted from a three-dimensional matrix to a two-dimensional matrix, so as to achieve the purpose of reducing the dimension of the three-dimensional numerical matrix to a two-dimensional matrix, which is convenient for the analysis of bacterial resistance to antimicrobial drugs for any drug-resistant gene; then the strain carries any drug-resistant gene. , for any antimicrobial drug The drug susceptibility test results can be calculated using the following formula: , then the drug resistance results can be reduced from a three-dimensional matrix to a two-dimensional matrix.

[0080] Step 8: Screening for non-resistant antimicrobial drugs for strains carrying any drug-resistant gene;

[0081] Preferably, in step 8, the two-dimensional matrix calculated in step 7 can be obtained, when the value is 0, it means that the strain carries any drug resistance gene , any promoter sequence , strains are resistant to any antimicrobial drugs The drug sensitivity test results are all non-resistant; when the value is not 0, it means that the strain carries any drug resistance gene , there are different types of promoter sequences upstream of the drug-resistant gene In this case, any antimicrobial drug The drug sensitivity test results indicate that the strain has drug resistance. The larger the value, the more drug resistance genes the strain has. In a variety of different promoter sequences In case of this drug The greater the possibility that the drug sensitivity test result is drug resistance, the stronger the overall resistance to the drug; The different values of were analyzed to obtain the bacterial resistance analysis based on the detection of bacterial resistance gene promoter strength;

[0082] In order to further obtain and screen drug-resistant bacteria carrying any drug-resistant gene When the drug sensitivity test result is non-resistant antimicrobial drug information, it is necessary to screen out the strains carrying any drug resistance gene When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then drug-resistant bacteria can carry any drug-resistant gene Screening for non-resistant antimicrobial drugs.

[0083] Step 9: Screening for non-resistant antimicrobial drugs that carry any number of resistance genes;

[0084] Preferably, in step 9, it can be known from the calculation in step 8 that when When the value is 0, the drug-resistant bacteria carry any drug-resistant gene. Non-resistant antimicrobial drugs, when When the value is non-zero, it means that the resistant bacteria carry any drug-resistant gene When the drug The results of drug sensitivity test show drug resistance; if drug-resistant bacteria carry any number of drug-resistant genes, Sum the different values to get ,when A value of 0 indicates that the strain carries any number of drug-resistant genes. , strains are resistant to any antimicrobial drugs The drug sensitivity test result is non-resistant; when the value is not 0, it means that the strain carries any number of drug resistance genes , for any antimicrobial drug The drug sensitivity test results show that the strain is resistant to the drug. The larger the value, the more susceptible the strain is to the drug. The greater the possibility that the drug sensitivity test result is drug resistance, the stronger the overall resistance to the drug; The different values of are analyzed to obtain the bacterial resistance analysis of resistant bacteria carrying any multiple drug-resistant genes.

[0085] In order to further obtain and screen out drug-resistant bacteria carrying any number of drug-resistant genes When the drug sensitivity test result is non-resistant antimicrobial drug information, it is necessary to screen out the strains carrying any multiple drug resistance genes When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then drug-resistant bacteria can carry any number of drug-resistant genes Screening for non-resistant antimicrobial drugs.

[0086] Step 10: Output the final results based on the calculation results in step 8 and step 9 respectively, and complete the bacterial resistance analysis and antimicrobial drug screening.

[0087] Preferably, according to the value calculated in step 8, when the resistant bacteria carry any drug-resistant gene, the resistance analysis of the bacteria to different antimicrobial drugs based on the promoter sequence weight consideration can be output, and the information of antimicrobial drugs whose drug sensitivity test results are non-resistant based on promoter strength detection can be conveniently output.

[0088] According to the value calculated in step 9, the resistance analysis of bacteria to different antimicrobial drugs based on promoter sequence weight considerations when resistant bacteria carry any number of resistance genes can be output, and the information of antimicrobial drugs whose drug sensitivity test results are non-resistant based on promoter strength detection can be conveniently output.

[0089] This result combines bacterial resistance gene detection, resistance gene promoter strength analysis and bacterial MIC detection, so it can better reflect the resistance characteristics of clinical bacteria, and can more sensitively detect the evolution of bacterial resistance through promoter changes, and therefore can more effectively guide the clinical selection of antimicrobial drugs to control resistant bacteria.

[0090] This invention, based on the analysis of bacterial resistance gene promoter strength, establishes a relevant mathematical representation model and utilizes methods such as indicator function representation and matrix dimensionality reduction to develop a convenient bacterial resistance analysis and antimicrobial drug screening method. The method includes the following basic steps: collecting and isolating drug-resistant strains, determining the strain's resistance gene and its upstream promoter sequence, testing the minimum inhibitory concentration (MIC) values of the drug-resistant bacteria in each drug-resistant strain, inputting the MIC test result data, defining the MIC boundary between resistance and non-resistance to any antimicrobial drug under any resistance gene and any upstream promoter, and mathematically representing it; a three-dimensional matrix representation of the strain's drug susceptibility test results; an indicator function representation of the strain's drug susceptibility test results; dimensionality reduction processing of the three-dimensional matrix of the strain's drug susceptibility test results; screening for non-resistance to antimicrobial drugs in strains carrying any one or more resistance genes; and outputting the results.

[0091] By combining bacterial resistance gene detection, resistance gene promoter strength analysis and bacterial MIC detection, the present invention can better reflect the resistance characteristics of clinical drug-resistant bacteria and their possible potential changes in resistance. Therefore, through methods such as indicator function representation and matrix dimensionality reduction, it can more conveniently and effectively guide the clinical selection of antimicrobial drugs for drug-resistant bacteria.

[0092] In the actual experimental process of the present invention, the Escherichia coli that has been treated through step 1, step 2 and step 3 is used as an indicator strain to perform drug resistance analysis and effective antimicrobial drug screening.

[0093] A total of 12 strains of Escherichia coli were isolated and identified; four strains from each group were found to carry drug-resistant genes. bla SHV-5 、 bla SHV-27 、 bla SHV-28 There are four promoter-related sequence structures upstream of the resistance genes, labeled PW, PS, P-IS, and P-WPD. The four strains carried the same resistance gene, with four promoter-related sequence structures upstream. All strains were tested for MICs against the following drugs: ampicillin / sulbactam (SAM), piperacillin (PIP), piperacillin / tazobactam (TZP), cefazolin (CFZ), cefuroxime (CXM), cefuroxime axetil (CFA), ceftazidime (CAZ), ceftriaxone (CRO), aztreonam (ATM), cefotaxime (CTX), amoxicillin / clavulanate (AMC), cefoxitin (FOX), and amoxicillin (AMX). The results of drug susceptibility testing are shown in Table 1.

[0094]

[0095] Due to the limited number of strains in this experiment, it is assumed that the frequencies of the different upstream promoter sequences mentioned above in the examples above are the same in the upstream of the drug-resistant genes in the bacterial population, that is, the weight coefficients of the four promoters are A p Both are 1.

[0096] After processing and calculating each MIC value through steps 4 to 8, the following table 2 and Figure 2 , that is, the drug resistance analysis of strains carrying a single drug-resistant gene and the screening results of non-resistant antimicrobial drugs.

[0097]

[0098] Substitute each MIC value into the formula of step 4 to step 9 and calculate to obtain Table 3 and Figure 3 , that is, the drug resistance analysis of strains carrying multiple drug-resistant genes and the screening results of non-drug-resistant antimicrobial drugs.

[0099]

[0100] The larger the value in Table 2 and Table 3, the greater the possibility that the strain will be resistant to the drug in the drug sensitivity test when carrying any different upstream promoters and drug resistance genes, and the stronger the overall drug resistance to the drug; 0 means that the strain is not resistant to the drug sensitivity test results when carrying any different upstream promoters and drug resistance genes.

[0101] Finally, the results of the antimicrobial drugs to which the screened strains are not resistant are output:

[0102] If the strain only carries bla SHV-5 If the strain has drug-resistant genes, then the strain is non-resistant to TZP, AMC, and FOX, so these three drugs can be selected;

[0103] If the strain only carries bla SHV-27 If there are drug-resistant genes, the strain is non-resistant to TZP, CAZ, CRO, ATM, CTX, AMC, and FOX, so these drugs can be selected;

[0104] If the strain only carries bla SHV-28 If the strain contains drug-resistant genes, the strain is non-resistant to CXM, CFA, CAZ, CRO, ATM, CTX, AMC, and FOX, so these drugs can be selected;

[0105] If the strain carries bla SHV-5 and bla SHV-27 If the strain has drug-resistant genes, then the strain is non-resistant to TZP, AMC, and FOX, so these drugs can be selected;

[0106] If the strain carries bla SHV-5 and bla SHV-28 If the strain has drug-resistant genes, then it is non-resistant to AMC and FOX, so these two drugs can be selected;

[0107] If the strain carries bla SHV-27 and bla SHV-28 If the strain has drug-resistant genes, then it is non-resistant to CAZ, CRO, ATM, CTX, AMC, and FOX, so these two drugs can be selected;

[0108] If the strain carries bla SHV-5 、 blaSHV-27 and bla SHV-28 If the strain has drug-resistant genes, the strain is not resistant to both AMC and FOX, so these two drugs can be selected.

[0109] The foregoing is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained herein shall, unless otherwise specified or limited, be implemented in accordance with conventional means in the art.

Claims

1. An antimicrobial drug screening method based on bacterial resistance gene promoter strength detection, characterized in that: The following steps are involved: Step 1: Collect and isolate drug-resistant strains; Step 2: Determine the drug-resistant gene of the strain and its upstream promoter sequence; Step 3: Based on the drug sensitivity test, perform minimum inhibitory concentration (MIC) value detection; Step 4: Establish a mathematical analysis model for bacterial resistance analysis and perform a mathematical representation of the minimum inhibitory concentration (MIC) of drug-resistant and non-resistant bacteria; Step 5: Perform a three-dimensional matrix representation of the strain drug sensitivity test results; In step 5, define For bacterial resistance genes, is the upstream promoter sequence of the strain's drug-resistant gene; Represents any promoter sequence , carry any drug resistance gene strains are resistant to any drug MIC value of drug susceptibility test; For a strain carrying any drug-resistant gene and any promoter sequence at the same time, the MIC value of its drug susceptibility test for any drug must be between the sensitive boundary value and the drug-resistant boundary value of the drug susceptibility test of the strain; Then, any promoter sequence Any drug resistance gene strains, resistant to any antimicrobial drugs ,have ; Then, based on the original MIC value of the strain, a three-dimensional matrix of different promoter sequences, different drug resistance genes and different types of antimicrobial drugs was formed; Step 6: Use indicator function to represent the results of the strain drug sensitivity test; Step 7: Perform dimensionality reduction processing on the three-dimensional matrix of the strain drug sensitivity test results; Step 8: Screening for non-resistant antimicrobial drugs for strains carrying any drug-resistant gene; Step 9: Screening for non-resistant antimicrobial drugs that carry any number of resistance genes; Step 10: Output the final results based on the calculation results in step 8 and step 9 respectively, and complete the bacterial resistance analysis and antimicrobial drug screening.

2. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 1, characterized in that: In step 1, the strain is isolated, cultured, and identified from a clinical specimen, streaked on a culture dish, cultured in a 37°C incubator, and then a single clone is picked for passage. The tube is then placed in an Eppendorf tube containing 0.5 mL of bacterial freezing solution, sealed with a sealing film, and stored in a -80°C refrigerator for later use.

3. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 2, characterized in that: In step 2: using a commercial kit to extract the bacterial genome and plasmid, and designing primers for the drug-resistant gene and its upstream promoter and the spacer sequence between the downstream gene, using the extracted genome and plasmid as templates, amplifying the corresponding sequence by PCR and sequencing to determine the type of drug-resistant gene and the upstream promoter sequence structure; In step 3: according to the drug sensitivity test, the MIC values of the resistant strains to different antimicrobial drugs are detected respectively, that is, the MIC values of the resistant strains to the drugs at different antimicrobial drugs and their different concentrations are monitored, and the MIC values are recorded and used as the input values of the mathematical analysis model for bacterial resistance analysis.

4. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 3, characterized in that: In step 4, according to the drug resistance standards specified by clinical and microbiological standardization laboratories, combined with the MIC value obtained by strain testing, the drug sensitivity test results of the strain to the antimicrobial drug at different resistance genes and different upstream promoters, i.e., the degree of drug resistance, are determined; the antimicrobial drug is defined as According to the drug sensitivity test standards, the drug sensitivity MIC results of the strains were recorded as the strains’ resistance to the corresponding antimicrobial drugs. Resistance level: sensitive ,intermediary or drug resistance ; The results of the drug sensitivity test of the strains were ,but: Indicates that the drug sensitivity test result is sensitive, the superscript Refers to sensitivity; Indicates that the drug sensitivity test result is intermediate, the superscript Refers to the intermediary; Indicates that the result of drug sensitivity test is drug resistant, Refers to drug resistance; According to the degree of drug resistance, ; Then, define the MIC boundary value between resistance and non-resistance to any antimicrobial drug when the strain carries any resistance gene and any upstream promoter; Indicates strain resistance to drugs The drug sensitivity test is the sensitive MIC boundary value, that is, the lowest MIC value that the drug sensitivity test can achieve is sensitive; Indicates strain resistance to drugs The MIC boundary value with the drug sensitivity test as the medium; Since the intermediate state is a state in which the sensitivity of the strain to a certain antimicrobial drug is between sensitive and resistant, the strain MIC is intermediate when it is in a specific range; the MIC boundary value with the drug sensitivity test as the medium includes two boundary values, high and low; furthermore, For strains to treat drugs The drug susceptibility test is close to the intermediate MIC boundary value of sensitivity. For strains to treat drugs The drug susceptibility test was close to the intermediate MIC boundary value of drug resistance; Indicates strain resistance to drugs The drug sensitivity test is the MIC boundary value of drug resistance, that is, the highest MIC value of drug resistance that can be achieved in the drug sensitivity test.

5. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 4, characterized in that: In step 6, any promoter sequence is carried Any drug resistance gene strains, to any drug The indicator function for judging the results of drug susceptibility test is expressed as: ， if ,but ,otherwise ; Then, under any drug-resistant gene and any upstream promoter, the drug sensitivity test results of the strain will be indicated by the indicator function Represented as an integer value of 0 or 1.

6. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 5, characterized in that: In step 7, the upstream promoter sequence of the drug-resistant gene of the strain involved in the previous step is Perform dimensionality reduction processing; definition Promoter sequence The weight coefficient is used to represent the frequency of different types of upstream promoter sequences appearing upstream of related drug-resistant genes in the bacterial population; and antimicrobial drugs Based on the promoter strength of bacterial resistance genes, the promoter sequence Perform weighted summation according to weight considerations and convert the strain drug sensitivity test results from a three-dimensional matrix into a two-dimensional matrix. , for any antimicrobial drug The drug susceptibility test results were calculated using the following formula: , then the drug resistance results are reduced from a three-dimensional matrix to a two-dimensional matrix.

7. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 6, characterized in that: In step 8, the two-dimensional matrix calculated in step 7 is obtained. When the value is 0, it means that the strain carries any drug-resistant gene. , any promoter sequence , strains are resistant to any antimicrobial drugs The drug sensitivity test results are all non-resistant; when the value is not 0, it means that the strain carries any drug resistance gene , there are different types of promoter sequences upstream of the drug-resistant gene In this case, any antimicrobial drug The drug sensitivity test results indicate that the strain has drug resistance. The larger the value, the more drug resistance genes the strain has. In a variety of different promoter sequences In case of this drug The greater the possibility that the drug sensitivity test result is drug resistance, the stronger the overall resistance to the drug; The different values of were analyzed to obtain the bacterial resistance analysis based on the detection of bacterial resistance gene promoter strength; Screen out strains carrying any drug-resistant gene When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then the drug-resistant bacteria carry any drug-resistant gene Screening for non-resistant antimicrobial drugs.

8. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 7, characterized in that: In step 9, Sum the different values to get ,when A value of 0 indicates that the strain carries any number of drug-resistant genes. , strains are resistant to any antimicrobial drugs The drug sensitivity test result is non-resistant; when the value is not 0, it means that the strain carries any number of drug resistance genes ,according to Different values of are used to analyze the drug resistance of resistant bacteria carrying any number of drug resistance genes; Screen out strains carrying any number of drug-resistant genes When any antimicrobial drug of The antibacterial drugs corresponding to the value of 0 are: , then the drug-resistant bacteria carry any number of drug-resistant genes Screening for non-resistant antimicrobial drugs.

9. The method for screening antimicrobial drugs based on bacterial resistance gene promoter strength detection according to claim 1, characterized in that: In step 10, based on the value calculated in step 8, the drug resistance analysis of the bacteria to different antimicrobial drugs based on the promoter sequence weight consideration when the resistant bacteria carry any drug-resistant gene is output, and the antimicrobial drug information for which the drug sensitivity test result based on the promoter strength detection is non-resistant is output; According to the value calculated in step 9, when the resistant bacteria carry any multiple resistance genes, the resistance analysis of the bacteria to different antimicrobial drugs based on the promoter sequence weight consideration is output, and the antimicrobial drug information of the antimicrobial drug whose sensitivity test result is non-resistant based on the promoter strength detection is output.

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