阅读说明：本技术 一种检测促进抗生素杀菌效率提高的物质的方法和试剂盒 (Method and kit for detecting substance promoting antibiotic sterilization efficiency improvement ) 是由 李惠 彭博 彭宣宪 蒋明 陶建军 项娟娟 于 2021-07-01 设计创作，主要内容包括：本发明属于生物医药检测技术领域,具体涉及一种针对性的检测促进抗生素杀菌效率提高的物质的方法和试剂盒。该方法基于细菌在M9基本培养基中不能生长的特性,使细菌利用待测物质,结合不同浓度抗生素对细菌杀灭情况不同,细菌残存情况呈现相应梯度变化,因而可以用来检测化合物提高抗生素对细菌的杀菌效率,相比较于现有的最小抑菌浓度(MIC)方法,可以准确、有效评价促进抗生素杀菌效率提高的化合物的作用；并且操作简单,结果易观察,比利用试管法来检测化合物提高抗生素对细菌的杀菌效率,具有更好的操作性。(The invention belongs to the technical field of biomedical detection, and particularly relates to a method and a kit for pertinently detecting substances for promoting the improvement of antibiotic sterilization efficiency. The method is based on the characteristic that bacteria can not grow in an M9 minimal medium, so that bacteria utilize substances to be detected, different antibiotic killing conditions of different concentrations are combined, and the residual condition of the bacteria presents corresponding gradient change, so that the method can be used for detecting compounds to improve the bacterial sterilization efficiency of the antibiotics; and the operation is simple, the result is easy to observe, the sterilization efficiency of the antibiotic to the bacteria is improved compared with the method of detecting the compound by using a test tube method, and the operability is better.)

1. A method for detecting substances promoting the improvement of antibiotic sterilization efficiency is characterized in that macroscopic bacteria, substances to be detected and antibiotics are added into an M9 culture medium to be cultured to serve as an experimental group, and the substances to be detected are not added under the same condition to serve as a control group; the lowest antibiotic concentration of bacteria cannot be observed by naked eyes as the lowest bactericidal concentration, and the lowest bactericidal concentration of a control group/the lowest bactericidal concentration of an experimental group is more than or equal to 2, so that the substance to be detected promotes the improvement of the antibiotic sterilization efficiency and is positive.

2. The method according to claim 1, wherein the culturing is carried out under conditions selected to optimize the growth temperature of the bacteria and incubated for 8 to 24 hours.

3. The method of claim 1, wherein the macroscopic bacterial count is 5 x 10⁶CFU～4×10⁷CFU。

4. The method of claim 1, wherein the bacteria comprise Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella, Staphylococcus, enterococcus, and drug-resistant bacteria of the bacteria.

5. The method of claim 1, wherein the antibiotic is a bactericidal antibiotic that lyses bacteria.

6. The method of claim 5, wherein the antibiotic comprises a penicillium antibiotic, a cephalosporium antibiotic, or a carbapenem antibiotic.

7. The method of claim 1, wherein the test substance is a bioactive small molecule having a molecular weight of less than 1000.

8. The method of claim 1, wherein the M9 medium is Na-selective₂HPO₄、KH₂PO₄、NH₄Cl、NaCl、K₂HPO₄、KH₂PO₄And (NH)₄)₂SO₄More than two kinds of salt and water.

9. A kit constructed by the method for detecting substances promoting the improvement of antibiotic sterilization efficiency according to claims 1-8.

10. Use of the kit according to claim 9 for detecting and screening substances that promote an increase in the bactericidal efficiency of antibiotics.

Technical Field

The invention belongs to the technical field of biological medicine detection. And more particularly, to a method and kit for detecting a substance that promotes an increase in the sterilization efficiency of antibiotics.

Background

The Minimum Inhibitory Concentration (MIC) is an index for measuring the antibacterial activity of an antibacterial agent, refers to the minimum drug concentration that can inhibit the growth of pathogenic bacteria in a culture medium after bacteria are cultured in vitro for 18 to 24 hours, and is widely used for evaluating the drug resistance of bacteria and the sterilization efficiency of antibiotics. The MIC detection principle is that the minimum inhibitory concentration of the antibiotic is determined by comparing the number of bacteria by using the fact that the antibiotic can inhibit the bacteria growing in a specific culture medium. For the experiment, the number of bacteria invisible to the naked eye was added: if the antibiotic inhibits its growth, the bacteria are difficult to grow and no bacteria can be observed; if the antibiotic is unable to inhibit its growth, the bacteria can grow and the bacteria can be observed; the minimum antibiotic concentration for inhibiting bacteria until bacteria can not be observed is the minimum inhibitory concentration.

For example, chinese patent application CN105358982A discloses a method for the fast-reading determination of bacterial susceptibility or resistance to antibiotics based on the principle of MIC detection based on the evaluation of the increase in cells corresponding to the dose of antibiotic that inhibits synthesis of the cell wall. However, substances that promote increased antibiotic sterilization efficiency are difficult to determine based on current conventional or improved MIC detection methods. For example, in recent research, some biological cell metabolites (often referred to as compounds with molecular weight less than 1000) can significantly increase the bactericidal effect of antibiotics, and experiments have proved that the biological cell metabolites have the effect of promoting the sterilization of antibiotics, but when the biological cell metabolites are added, the MIC of the antibiotics is not significantly influenced by the addition of the biological cell metabolites when the biological cell metabolites are detected by a conventional MIC detection method. Therefore, there is an urgent need to provide a method for detecting a substance that promotes an increase in the sterilization efficiency of antibiotics.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect and the defect that the prior art lacks a method for determining substances for promoting the improvement of the antibiotic sterilization efficiency, and provides a method for detecting the substances for promoting the improvement of the antibiotic sterilization efficiency.

The invention aims to provide a method for detecting a substance which promotes the improvement of the sterilization efficiency of antibiotics.

The invention also aims to provide a kit constructed by the method for detecting the substance promoting the improvement of the antibiotic sterilization efficiency.

The invention also aims to provide application of the kit in detecting substances for promoting the improvement of the antibiotic sterilization efficiency.

The above purpose of the invention is realized by the following technical scheme:

the inventor finds that a plurality of small molecule cell metabolites (compounds with molecular weight less than 1000) can obviously increase the bactericidal effect of antibiotics, and the effect of synergistically improving the bactericidal efficiency is achieved. But when the sterilization efficiency of the comparison small-molecule cell metabolite and the antibiotic and the sterilization efficiency of the single antibiotic are measured by adopting the conventional MIC method, the effect is not obviously different. The inventor finds that the conventional MIC method adopts a nutrient-rich LB culture medium, bacteria obtain sufficient nutrition from the LB culture medium and cannot use small molecular cell metabolites to be detected, so that whether the small molecular cell metabolites can promote the improvement of the antibiotic sterilization efficiency cannot be detected by the conventional MIC method. Therefore, the present invention provides the following method for specifically detecting a substance that promotes an improvement in the sterilization efficiency of antibiotics.

A method for detecting substances promoting antibiotic sterilization efficiency improvement comprises adding macroscopic bacteria, substances to be detected and antibiotics into M9 culture medium for culture as experimental group, and adding no substances to be detected under the same conditions as control group; the lowest antibiotic concentration of the bacteria cannot be observed by naked eyes as the lowest bactericidal concentration (MKC), and the lowest bactericidal concentration of the control group/the lowest bactericidal concentration of the experimental group is more than or equal to 2, so that the substance to be detected promotes the improvement of the antibiotic sterilization efficiency and is positive.

The invention relates to a method for detecting substances for promoting the improvement of antibiotic sterilization efficiency, which takes an M9 culture medium which can only maintain the normal survival of bacteria and can not grow, has definite components and does not contain nutrient components as a culture medium during the determination, adds a large amount of bacteria visible to the naked eye, and determines the minimum sterilization concentration of the substance to be determined and the antibiotic (instead of adding a small amount of bacteria which can not be observed by the naked eye in the conventional MIC method to determine the minimum inhibitory concentration, the determination principle of the two has obvious difference). Under the culture condition, the bacteria can not absorb other nutrient components, and can only depend on the added substance to be detected, and the bacteria can use the substance to be detected, so that whether the bacteria can promote the improvement of the antibiotic sterilization efficiency can be reflected. During detection, if the antibiotic kills all or part of bacteria, the bacteria cannot be observed by naked eyes; if the antibiotic is unable to kill all of the bacteria, the bacteria can be observed by the naked eye.

In addition, it should be noted that the method established by the present invention is not the same as the current method for detecting Minimum Bacterial Concentration (MBC). MBC refers to the lowest concentration that can kill bacteria in the medium (i.e., kill 99.9% of the test microorganisms), and it is often performed by viable count on well plates below the lowest inhibitory concentration based on MIC determination using broth dilution microplate assay, and the number of bacteria added and the medium used are consistent with MIC determination, and therefore, is different from the principles and methods of the present application.

Further, the culture condition is to select the optimal growth temperature of the bacteria and incubate for 8-24 hours.

Further, the macroscopic bacterial count was 5 × 10⁶CFU～4×10⁷CFU。

Further, the bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella, Staphylococcus, enterococcus, and drug-resistant bacteria of the bacteria.

Further, the antibiotic is a bactericidal antibiotic that lyses bacteria, rather than a bacterial growth-inhibiting antibiotic.

Preferably, the antibiotics include penicillins, cephalosporins, carbapenems; the antibiotic can lyse to kill the bacteria.

Preferably, the penicillium antibiotic includes, but is not limited to, ampicillin, amoxicillin or temocillin.

Preferably, the cephalosporin antibiotics include, but are not limited to, ceftizoline, ceftriaxone, cefoperazone, ceftazidime, cefepime, cefpirome, cefotaxime or cefixime.

Preferably, the carbapenem antibiotic includes, but is not limited to, meropenem.

Further, the substance to be detected is a bioactive small molecule, and the molecular weight is less than 1000. Such as amino acids, glucose, fructose, and pyruvic acid, etc.

Further, the M9 medium is selected from Na₂HPO₄、KH₂PO₄、NH₄Cl、NaCl、K₂HPO₄、KH₂PO₄And (NH)₄)₂SO₄More than two kinds of salt and water.

In addition, the invention also provides a kit constructed by the method for detecting the substance promoting the improvement of the antibiotic sterilization efficiency.

In addition, the invention also provides application of the kit in detecting and screening substances for promoting the improvement of the antibiotic sterilization efficiency.

The invention has the following beneficial effects:

the invention provides a method for pertinently detecting substances promoting the improvement of the antibiotic sterilization efficiency, which is characterized in that based on the characteristic that bacteria can not grow in an M9 minimal medium, the bacteria utilize the substances to be detected, different antibiotic killing conditions of different concentrations are combined, and the residual condition of the bacteria presents corresponding gradient change, so that the method can be used for detecting the compounds to improve the sterilization efficiency of the antibiotics to the bacteria, and compared with the existing Minimum Inhibitory Concentration (MIC) method, the method can accurately and effectively evaluate the effect of the compounds promoting the improvement of the antibiotic sterilization efficiency; and the operation is simple, the result is easy to observe, the sterilization efficiency of the antibiotic to the bacteria is improved compared with the method of detecting the compound by using a test tube method, and the operability is better.

Drawings

FIG. 1 is a statistical chart of data of experiments in which glutamine can improve the sensitivity of clinically resistant Escherichia coli to ampicillin in example 1.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 establishment of a method for detecting a substance that promotes improvement in antibiotic sterilization efficiency

1.1 Glutamine can improve the sensitivity of clinical drug-resistant Escherichia coli to ampicillin

1. Experimental methods

Preparing a bacterial sample: selecting 20 clinical drug-resistant Escherichia coli monoclonals to be cultured in 100 ml LB liquid medium at 37 ℃ and 200rpm for 16 hours to reach a saturated state; collecting 20 ml of bacterial liquid, centrifuging at 8,000rpm for 5min, and then removing a supernatant; the cells were washed with an equal volume of 0.85% physiological saline and finally with M9 containing 10mM acetate (17.1g Na)₂HPO₄·12H₂O，3g KH₂PO₄、1g NH₄Cl and 0.5g NaCl are dissolved in 1L of ultrapure water for sterilization), suspended bacteria are obtained in the minimal medium, the OD value of the bacteria liquid is adjusted to 0.5, and then 5ml of the bacteria liquid is respectively subpackaged in test tubes for later use.

Each prepared bacterial sample was divided into 2 groups, one group was added with 200. mu.g/ml Ampicillin (AMP), and the other group was added with 20mM Glutamine (Glutamine, Gln) simultaneously with ampicillin. After incubation at 37 ℃ for 4 hours at 200rpm, 100. mu.l of the broth was diluted and counted for viable plate count.

2. Results of the experiment

The results are shown in fig. 1, and it can be seen from the figure that after glutamine is added, the sensitivities of the 20 clinical drug-resistant escherichia coli to ampicillin are remarkably improved, the times of the improvements are 13-205 times, and the survival rate of bacteria is remarkably reduced.

1.2 measurement of the efficiency of ampicillin-added bacteria to sterilize bacteria by the conventional MIC method

1. Experimental methods

Selecting a clinical drug-resistant Escherichia coli monoclonal to be cultured in 5mL LB culture medium overnight at 37 ℃, then inoculating the Escherichia coli monoclonal to a new 5mL LB culture medium at a ratio of 1:100(v/v), culturing until OD600 is 0.5, and diluting with LB culture medium at a ratio of 1: 100; mu.L of LB medium containing 20mM glutamine diluted in two-fold was added to a 96-well microplate in advance, and 10. mu.L (about 10. mu.L) of LB medium containing no glutamine diluted in two-fold was used as a control⁵CFU) bacteria into each well, three biological replicates of each strain. After culturing at 37 ℃ for about 16h, observing the growth condition of bacteria by naked eyes, wherein the minimum concentration at which the bacteria can not grow is the Minimum Inhibitory Concentration (MIC); the antibiotic concentration (MIC) of bacteria that did not grow long after antibiotic addition alone was recorded separately_Antibiotic) Antibiotic concentration (MIC) of bacteria that do not grow long after addition of glutamine and antibiotics_{Antibiotic + Gln}) Then using MIC_AntibioticDivided by MIC_{Antibiotic + Gln}Ampicillin was added to the cells as a bactericidal factor against bacteria. The sterilization times of the test results of the experiments are 1-2, the inventor dilutes the antibiotics with 1/3 concentration on the basis of the corresponding MIC concentration, and the MIC determination is carried out again by the method.

2. Results of the experiment

The results are shown in Table 1, where the MIC of each clinically resistant Escherichia coli strain is shown after glutamine addition_{Antibiotic + Gln}Divided by MIC_AntibioticThe multiple of (A) is still between 1 and 2.

TABLE 1 measurement of the efficiency of ampicillin addition to bacteria for killing bacteria by the conventional MIC method

Note: AMP concentration was in mg/mL and glutamine concentration was 20 mM.

According to the MIC judgment standard: equal to or more than 4 times of positive, equal to 2 times of positive candidate and less than 2 times of negative candidate; the glutamine positive rate was 0%, and the positive candidate rate was 25%. However, the results of example 1.1 show that 20 clinically resistant E.coli strains with significantly improved ampicillin sensitivity were obtained after glutamine addition, whereas the MIC was determined from the results of 1.2_AntibioticDivided by MIC_{Antibiotic + Gln}The fold of (A) is less than 2 times for 15 strains and 2 times for 5 strains, and the experimental results are contradictory. These results demonstrate that it was not possible to determine whether adding glutamine could improve the efficiency of ampicillin sterilization of bacteria using conventional MIC.

1.3 detecting the efficiency of promoting antibiotic sterilization by glutamine by using an enzyme-linked immunosorbent assay (ELISA) plate method diluted by M9 culture medium

1. Experimental methods

The bacteria were cultured overnight in LB medium, centrifuged at 8,000rpm for 5min to collect the bacteria, washed three times with sterile saline, and M9(17.1g Na) containing 10mM acetate was added₂HPO₄·12H₂O，3g KH₂PO₄，1g NH₄Cl, 0.5g NaCl dissolved in 1L ultrapure water for sterilization) the medium was diluted to OD600 ═ 1.0 for use; mu.L of M9 medium containing 20mM glutamine diluted in two fold was added to a 96-well microplate as an experimental group, and M9 medium containing no glutamine diluted in two fold was used as a control group, and then 100. mu.L (containing 1X 10 ampicillin) was added to each well⁷CFU) bacteria, mixing uniformly, incubating for 8 hours at 37 ℃, and observing the retention condition of the bacteria in the micropores by naked eyes; three biological replicates were performed.

Because the method adopts the M9 culture medium, bacteria can not grow in the culture medium, and after high-concentration macroscopic bacteria are added, the bacteria killing capability of different micropores is different because the antibiotic concentration in the micropores is different, so that the residual condition of the bacteria in the micropores can show corresponding gradient change. If no bacteria remain in the micropores, the corresponding antibiotic concentration is the antibiotic concentration for killing all bacteria. The lowest antibiotic concentration not observable to the naked eye was designated as the minimum bactericidal concentration (MKC).

The antibiotic concentration (MKC) without residual bacteria after antibiotic addition alone was recorded_Antibiotic) Antibiotic concentration (MKC) without residual micropores after addition of glutamine and antibiotic_{Antibiotic + Gln}) Then using MKC_AntibioticDivided by MKC_{Antibiotic + Gln}Namely the bactericidal multiple of the ampicillin on the bacteria after the glutamine is added. Theoretically, if a compound increases the sensitivity of bacteria to ampicillin, the antibiotic concentration MKC at which there are no bacterial pores left after the compound is added_{Antibiotic + Gln}Should be less than the antibiotic concentration MKC in the antibiotic-free micropores with antibiotic alone_Antibiotic(ii) a And the larger the increase factor is, the more remarkable the substance improves the ampicillin sterilization efficiency of the bacteria.

2. Results of the experiment

The results are shown in Table 2, which shows that the clinically resistant Escherichia coli MKC is obtained after glutamine addition_AntibioticDivided by MKC_{Antibiotic + Gln}There were 16 strains with a multiple of 4, 1 strain with a multiple of 2, and 3 strains with a multiple of 1. If the drug resistance and the sterilization efficiency are changed into indexes according to 4 times, the strains with the changed drug resistance and sterilization efficiency after the glutamine is added account for 80 percent, namely the positive rate is 80 percent; in fact, the drug resistance and the sterilization efficiency of the strain with the multiple of 2 also change, and the strain with the drug resistance and the sterilization efficiency changed after the glutamine is added accounts for 85 percent, namely the positive rate is 85 percent. The results are substantially consistent with the results of the 1.1 experiment in which the bacteria showed increased ampicillin sensitivity after glutamine addition, indicating that this method can be used to determine whether a compound can promote the bactericidal efficacy of an antibiotic.

TABLE 2 detection of glutamine by dilution of enzyme-linked immunosorbent assay (ELISA) plate method using M9 culture medium

Note: AMP concentration was in mg/mL and glutamine concentration was 20 mM.

EXAMPLE 2 comparison of the Sterilization efficiencies of the conventional MIC method and the MKC method of the present invention for various antibiotics after glutamine addition

2.1 measurement of the efficiency of various antibiotics in sterilizing bacteria after addition of Glutamine by the conventional MIC method

1. Experimental methods

A sample of 20 clinically resistant E.coli strains was prepared according to the method 1.2 in example 1, and MIC was determined by adding various antibiotics (antibiotic names shown in Table 3) separately based on glutamine.

TABLE 3 antibiotic designations used in the experiments

2. Results of the experiment

The results are shown in Table 4, and it can be seen that the fold of resistance of glutamine added is between 1 and 2 for any antibiotic, that is, the sterilization efficiency of glutamine added antibiotic to bacteria cannot be correctly determined by the conventional MIC method.

TABLE 4 measurement of the efficiency of various antibiotics to sterilize bacteria after glutamine addition by the conventional MIC method

Note: antibiotic concentration units are mg/mL.

2.2 MKC method is adopted to measure the sterilization efficiency of various antibiotics to bacteria after glutamine addition

1. Test method

A sample of 20 clinically resistant E.coli strains was prepared by the method of 1.3 in example 1, and after glutamine was added, the antibiotics shown in Table 3 were added to the samples, respectively, to measure MKC.

2. Results of the experiment

The results are shown in Table 5, in which 16 strains with a fold of 4 or more and 1 bacterial fold of 4 strains are shown for AMX; according to the 4-time sterilization efficiency index, 80% of strains can be detected to have improved sterilization efficiency after glutamine addition, namely the positive rate is 80%. For CZO, 15 strains with a fold of 4 or more, 2 bacteria with a fold of 3, and 1 bacterial change fold of 2 strains; according to the 4-fold standard, the positive rate is 75 percent of strains, and the sterilization efficiency of the strains with the actual multiple of 2 is also changed, so that the positive rate is 95 percent together. For CRO, 19 strains with a fold of 4 or more and 2 bacteria with a fold of 1 strain; according to the 4-fold standard, the positive rate is 95%, and all strains can be detected by adding the strains with the fold of 2. For CFP, 13 strains with the fold of more than 4 are used, the fold of 4 strains is 2, and the fold of 3 strains is 1; according to the 4-fold standard, the positive rate is 65%, and the positive rate is 85% when the multiple of the strain is 2. For the CAZ, 19 strains with the fold of more than 4 are obtained, and 1 strain has the fold of 1 bacterium; according to 4-fold standard, the positive rate is 95% of the strain. In MEM, 19 strains with a fold of 4 or more were used, and 1 strain had a fold change of 1; according to 4-fold standard, the positive rate is 95% of the strain.

However, the MKC values of the other 4 antibiotics (FOX cefoxitin, GEN gentamicin, CIP ciprofloxacin and TET tetracycline) are all less than 2, and the reason probably is that the 4 antibiotics mainly inhibit the growth of bacteria but not crack and kill the bacteria, and the improvement of the bacterial killing rate of the antibiotics after the compound is added cannot be detected by the method.

TABLE 5 MKC method for determining bacterial sterilizing efficiency of multiple antibiotics after adding glutamine

Note: antibiotic concentration units are mg/mL.

The above results demonstrate that the method of the present invention can be used to detect substances that promote the enhancement of the bactericidal efficiency of antibiotics. Notably, the MKC method is only applicable to bactericidal antibiotics, whereas stationary phase bactericidal and non-bactericidal antibiotics (e.g. GEN, FOX, CIP, TET) are not suitable for this method.

Example 3 determination of the Sterilization efficiency of Cefoperazone-sulbactam sodium to clinically drug-resistant Escherichia coli after amino acid addition by MKC method

1. Experimental methods

Referring to the method of 1.3 in example 1, 8 clinically resistant E.coli (including a model E.coli K12) with improved sensitivity after addition of exogenous compound were randomly selected and cultured overnight in LB medium, centrifuged at 8,000rpm for 5min to collect the bacteria, washed three times with sterile physiological saline, and added with M9(17.1g Na) containing 10mM acetate₂HPO₄·12H₂O，3g KH₂PO₄、1g NH₄Cl, 0.5g NaCl dissolved in 1L ultrapure water for sterilization) the medium was diluted to OD600 ═ 1.0 for use. First, 100. mu.L of M9 medium containing 5mM amino acid diluted in two times in terms of cefoperazone-sulbactam Sodium (SCF) was added to a 96-well microplate to serve as an experimental group, and M9 medium containing no amino acid diluted in two times in terms of cefoperazone-sulbactam sodium was used as a control group, and then 100. mu.L (containing 1X 10 amino acid) was added to each well of the 96-well microplate⁷CFU) bacteria. After mixing and incubation at 37 ℃ for 8 hours, the bacterial persistence in the wells was visually observed and three biological replicates were performed.

2. Results of the experiment

The results are shown in Table 6, which shows that the strains with a fold of 4 or more for inosine (inosine) were 8 strains and that the fold of 1 strain was 2; the sensitivity change index is obtained according to 4 times, which indicates that the strain which improves the sensitivity of bacteria to antibiotics after adding inosine accounts for 89 percent, namely the positive rate is 89 percent, and the sensitivity of the strain with the multiple of 2 is changed actually, and the positive rate is 100 percent together. For Glycine (Glycine), the total fold of the strain was greater than 4, indicating a detectable positive rate of 100% after addition of Glycine. For alanine (alanine), 8 strains with a fold of 4 or more and 2 strains with a fold of 1 bacterium; according to the 4-fold index, the positive rate is 89%, and the sensitivity of the strain with the multiple of 2 is changed actually, and the positive rate is 100%. For Glutamine (Glutamine), 7 strains with a fold of 4 or more and 2 strains with a bacterial fold of 2; according to the 4-fold index, the positive rate is 78%, and the sensitivity of the strain with the multiple of 2 is changed actually, and the positive rate is 100%.

Meanwhile, the MKC multiple is 4 and the rest is 1 after only one clinical drug-resistant Escherichia coli strain is added with Leucine (Leucine); and after adding Isoleucine (Isoleucine), the MKC multiples of all strains are less than 1 except that 1 MKC is 2. The above results indicate that addition of exogenous isoleucine and leucine does not promote antibiotics to increase the efficiency of sterilization of bacteria.

TABLE 6 multiple amino acids to improve the bactericidal efficiency of cefoperazone-sulbactam sodium against clinical drug-resistant Escherichia coli

Note: antibiotic concentration units are mg/mL.

Example 4 determination of the Sterilization efficiency of Cefoperazone-sulbactam sodium to clinical Klebsiella pneumoniae by MKC method after amino acid addition

1. Test method

Referring to the method of 1.3 in example 1, 10 clinical Klebsiella pneumoniae (including the model strain ATCC700603) with increased sensitivity after addition of exogenous compounds were randomly selected and cultured overnight in LB medium, the bacteria were collected by centrifugation at 8,000rpm for 3min, washed three times with sterile physiological saline, and then treated with M9(17.1g Na) containing 10mM acetate, 2mM magnesium sulfate and 0.1mM calcium chloride₂HPO₄·12H₂O，3g KH₂PO₄、1g NH₄Cl, 0.5g NaCl dissolved in 1L ultrapure water for sterilization) the medium was diluted to OD600 ═ 1.0 for use. Firstly, 100 mu L of diluted cefoperazone containing 5mM amino acid in a double ratio is added into a 96-hole microplateM9 medium of sulbactam sodium as experimental group, M9 medium of cefoperazone-sulbactam sodium diluted at amino acid-free multiple ratio as control group, and then 100. mu.L (containing 1X 10) of each well of 96-well plate⁷CFU) bacteria. After mixing and incubation at 37 ℃ for 8 hours, the bacterial persistence in the wells was visually observed and three biological replicates were performed.

2. Results of the experiment

The results are shown in Table 7, which shows that the fold of all strains is greater than 4 after the exogenous amino acid is added, indicating that the positive rate is 100%. Meanwhile, after only one clinical klebsiella pneumoniae is added (Isoleucine), the MKC multiple is 4, 3 MKC strains are 2, and the rest is 1; after Leucine (Leucine) is added, the MKC of 2 strains is 2, and the MKC times of the other strains are less than 1, which indicates that the sterilization efficiency of antibiotics on bacteria cannot be promoted after exogenous isoleucine and Leucine are added.

TABLE 7 multiple amino acids to improve the sterilizing efficiency of cefoperazone-sulbactam sodium to clinical Klebsiella pneumoniae

Note: antibiotic concentration units are mg/mL.

Example 5 determination of the Sterilization efficiency of Cefoperazone-sulbactam sodium to clinical Acinetobacter baumannii after amino acid addition by MKC method

1. Experimental methods

Referring to 1.3 of example 1, 10 clinical A.baumannii strains with improved sensitivity after addition of exogenous compounds were selected, cultured overnight in LB medium, centrifuged at 8,000rpm for 3min to collect bacteria, washed three times with sterile physiological saline, and added with M9(17.1 gNa) containing 10mM acetate₂HPO₄·12H₂O，3g KH₂PO₄、1g NH₄Cl, 0.5g NaCl dissolved in 1L ultrapure water for sterilization) the medium was diluted to OD600 ═ 1.0 for use. Firstly, 100 mu L of M9 culture medium containing 5mM amino acid and diluted by times is added into a 96-well microplate to serve as an experimental group, and no amino group is addedAcid dilution of cefoperazone-sulbactam sodium in M9 medium at a fold ratio as a control, and then 100. mu.L (containing 1X 10 sodium acetate) was added to each well of a 96-well plate⁷CFU) bacteria. After mixing, incubating for about 20 hours at 37 ℃, observing the retention condition of bacteria in the micropores by naked eyes, and carrying out three times of biological repetition.

2. Results of the experiment

The results are shown in Table 8, in which 8 strains with a fold of 4 or more and 2 strains with a fold of 2 bacteria are shown for Inosine (Inosine); according to the 4-fold index, 80% of detectable strains are detected, namely 80% of positive strains which can be detected by the method, and actually, the sensitivity of strains with the multiple of 2 is changed, and the positive strains account for 100% together. For Glycine (Glycine), 9 strains with a fold of 4 or more and 1 strain with a bacterial fold of 2; according to the 4-fold index, the detectable positive strain accounts for 90 percent, and the 2-fold index accounts for 100 percent.

Meanwhile, the result shows that the MKC multiple is 2 and the positive rate is only 20% after only 2 clinical strains are added with Leucine (Leucine) or Isoleucine (Isoleucine).

TABLE 8 multiple amino acids to increase the bactericidal efficiency of cefoperazone-sulbactam sodium on clinical acinetobacter baumannii

Note: antibiotic concentration units are mg/mL.

Example 6 determination of the Bactericidal efficacy of cefoperazone-sulbactam sodium to clinical Pseudomonas aeruginosa by MKC method after amino acid addition

1. Experimental methods

Referring to the method of 1.3 in example 1, 10 clinical P.aeruginosa strains with increased sensitivity after addition of exogenous compound were selected, cultured overnight in 5ml LB medium, centrifuged at 8,000rpm for 3min to collect bacteria, washed three times with sterile physiological saline, and added with M9(7g K) containing 2mM sodium citrate and 1.2mM magnesium sulfate₂HPO₄、3g KH₂PO₄、1g(NH₄)₂SO₄Sterilizing with ultrapure water dissolved in 1L) The medium was diluted to OD600 ═ 1.0 for use. First, 100. mu.L of M9 medium containing 5mM amino acid diluted in two times was added to a 96-well microplate as an experimental group, and M9 medium containing no amino acid diluted in two times was used as a control group, and then 100. mu.L (containing 1X 10 amino acid) was added to each well of the 96-well plate⁷CFU) bacteria. After mixing, incubating for about 20 hours at 37 ℃, observing the retention condition of bacteria in the micropores by naked eyes, and carrying out three times of biological repetition.

2. Results of the experiment

See table 9 for results, from which it can be seen that for Serine (Serine), the total strain fold is 4; detectable positive strains accounted for 100% according to the 4-fold index. For Glycine (Glycine), 7 strains with a fold of 4 or more, 2 bacteria with a fold of 1 strain, and 1 bacteria with a fold of 2 strains; according to 4-fold index, the detectable positive strain accounts for 70%, and the detectable positive strain accounts for 80% of 2-fold index.

Meanwhile, the result shows that after only one strain of clinical bacteria is added with Isoleucine (Isoleucine), the MKC multiple is 2, and the positive rate only accounts for 20%.

TABLE 9 multiple amino acids to increase the bactericidal efficiency of cefoperazone-sulbactam sodium against clinical pseudomonas aeruginosa

Note: antibiotic concentration units are mg/mL.

Example 7 optimal number of bacteria experiment in MKC method

1. Test method

Samples of E.coli K12 and 1 strain of clinically resistant E.coli were prepared according to the method of 1.3 in example 1, and the bacteria were diluted differently for use. First, 100. mu.L of M9 medium containing 5mM inosine diluted in two times in cefoperazone-sulbactam Sodium (SCF) was added to a 96-well microplate to prepare a test group, and M9 medium (17.1g of Na) containing cefoperazone-sulbactam sodium diluted in two times without amino acid₂HPO₄·12H₂O，3gKH₂PO₄、1g NH₄CI，0.5g NaCl dissolved in 1L ultrapure water for sterilization) was used as a control group. Then adding the mixture containing 5 × 10⁶CFU，1×10⁷CFU，2×10⁷CFU，4×10⁷CFU) bacteria 100 μ L. After mixing and incubation at 37 ℃ for 8 hours, the bacterial persistence in the wells was visually observed and three biological replicates were performed.

2. Results of the experiment

The results are shown in Table 10, from which it can be seen that the number of bacteria is 5X 10⁶CFU，1×10⁷CFU，2×10⁷CFU，4×10⁷In CFU, the MKC of the cefoperazone sulbactam sodium to the bacteria is more than 2 after inosine is added into K12 and the clinical drug-resistant Escherichia coli 17. This result indicates that the number of bacteria was 5X 10⁶CFU～4×10⁷In CFU, the bactericidal efficiency of the antibiotic to bacteria can be detected after the compound is added.

TABLE 10 results of the experiment of the number of bacteria in the MKC method of the present invention

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.