Antimicrobial susceptibility analysis method based on direct detection of living bacteria in water

Abstract

The present disclosure provides a drug sensitivity analysis method based on direct detection of live bacteria in water, including: culturing bacteria in a culture medium; dividing the culture medium containing bacteria into two parts, adding antibiotics to one part and not adding antibiotics to the other part, and incubating them separately; after centrifugation and washing, resuspending the bacteria in water respectively; injecting the bacteria resuspended in water directly into a capillary; applying voltage to the capillary, detecting the metabolites of bacteria in water by mass spectrometry; judging the drug sensitivity of bacteria according to the changes in bacterial metabolites. The method provided by the present disclosure does not require pre-treatment processes such as lysis and organic solvent extraction, and can realize direct detection of the metabolism of live bacteria in water, meet the needs of rapid bacterial drug sensitivity analysis within 0.5-3 hours, and has the advantages of fast, simple operation, non-targeted analysis, wide application range, etc., and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a drug sensitivity analysis method based on direct detection of living bacteria in water. Background Art

[0002] Relatively fast and accurate antibiotic sensitivity testing is essential for guiding correct clinical intervention and appropriate antibiotic treatment. The current gold standard for clinical antibiotic sensitivity testing is based on culture, that is, co-incubating bacteria and antibiotics using broth dilution or agarose plate method, and judging the sensitivity of antibiotics to bacteria by whether the bacteria grow under antibiotics. This process generally takes 12 hours to 5 days, which is extremely time-consuming and cannot obtain accurate drug sensitivity information in a timely manner, missing the golden opportunity for patients to receive early treatment and increasing the patient's mortality rate.

[0003] In order to shorten the time of drug sensitivity judgment, guide doctors to use drugs in time, and reduce the mortality rate of patients, researchers have developed many other ways to speed up the judgment of drug sensitivity. For example, based on fluorescence colorimetry and mass spectrometry to analyze metabolites of biological samples, however, these analytical techniques currently require pre-treatment processes such as fluorescence colorimetry, lysis, and organic solvent extraction when analyzing bacterial metabolites, and these pre-treatment processes may cause changes in the composition of metabolites, making it impossible to obtain the true metabolic state and composition of living bacteria, which leads to the inability to accurately judge the drug sensitivity of bacteria.

[0004] Therefore, there is an urgent need for a technology that can quickly and accurately analyze the drug sensitivity information of living bacteria without the need for pre-treatment processes such as lysis and organic solution extraction. Summary of the invention

[0005] In view of the above-mentioned technical problems, the present disclosure can provide a drug sensitivity analysis method based on direct detection of living bacteria in water, in order to at least partially solve the above-mentioned technical problems.

[0006] In order to solve the above technical problems, the technical solutions provided by the present disclosure are as follows:

[0007] As one aspect of the present disclosure, the present disclosure provides a method for analyzing drug sensitivity of live bacteria in water, comprising:

[0008] Step (1): culturing bacteria in a culture medium;

[0009] Step (2): dividing the culture medium containing bacteria in step (1) into two parts, one part is added with antibiotics, and the other part is not added with antibiotics, and incubating them separately;

[0010] Step (3): centrifuging and washing the bacteria in step (2);

[0011] Step (4): resuspending the bacteria in step (3) in water;

[0012] Step (5): injecting the bacteria resuspended in water directly into the capillary tube;

[0013] Step (6): applying voltage to the capillary in step (5) to detect bacterial metabolites in the water by mass spectrometry;

[0014] Steps (3) to (6) are performed on the two parts of bacteria in step (2) respectively.

[0015] Step (7): Determine the drug sensitivity of bacteria based on changes in bacterial metabolites.

[0016] In one embodiment, the culture medium in step (1) comprises at least one of the following:

[0017] Peptone medium, broth medium, tryptic soytone medium.

[0018] In one embodiment, the bacteria in step (1) include at least one of the following:

[0019] Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa.

[0020] In one embodiment, the antibiotic in step (2) comprises at least one of the following:

[0021] Kanamycin, tobramycin, ampicillin.

[0022] In one embodiment, the concentration of the antibiotic is 0.5-10 times the critical inhibitory concentration of the antibiotic.

[0023] In one embodiment, the incubation in step (2) is performed at 16-45° C. for 0.5-3 hours.

[0024] In one embodiment, the centrifugal speed in step (3) is 4000-8000 rpm, and the centrifugal time is 3-10 min;

[0025] The number of centrifugal washings is 2-3 times, and the washing solvent is water.

[0026] In one embodiment, the opening of the capillary in step (4) is 5-10 μm.

[0027] In one embodiment, the voltage applied to the capillary is an AC voltage or a DC voltage;

[0028] The voltage applied to the capillary was 2-5 kV.

[0029] In one embodiment, in step (7), the sensitivity of bacteria to antibiotics is determined by the increase or decrease of the detected metabolites.

[0030] Based on the above technical solution, the present disclosure provides a method for drug sensitivity analysis based on direct detection of living bacteria in water, which has at least one of the following beneficial effects:

[0031] (1) In the embodiments of the present disclosure, antibiotics are added to a culture medium in which bacteria are cultured for incubation, and after centrifugation and water washing, the culture medium is placed in a capillary tube. The live bacteria in the water are directly introduced into a mass spectrometer under the drive of a voltage for bacterial metabolite detection. Compared with the influence of pre-treatment processes such as lysis and organic solvent extraction on bacterial metabolites in the prior art, the present disclosure directly performs mass spectrometry detection on the metabolites of live bacteria in water to obtain more accurate bacterial metabolite information and achieve accurate drug sensitivity judgment; and based on the real metabolic information, the mechanism of action of antibiotics can be understood more accurately, guiding the combined use of metabolites and antibiotics.

[0032] (2) The method provided by the present disclosure is not affected by the growth rate and growth state of bacteria and is widely applicable to the determination of drug sensitivity of different bacteria with different action mechanisms.

[0033] (3) The analytical method provided by the present disclosure can detect and analyze metabolites of bacteria after incubation with antibiotics by simple centrifugation and water washing, without the need for pre-treatment processes such as bacterial lysis and amplification that are heavy and have an impact on bacterial metabolism in the prior art.

[0034] (4) The analysis method provided by the present invention is relatively fast. After the antibiotics and bacteria are incubated for 0.5 hours, the drug sensitivity of the bacteria can be determined based on the changes in the bacterial metabolites, and the medication can be guided. Compared with the existing technology, the speed of bacterial drug sensitivity analysis is significantly improved and the time for determining bacterial drug sensitivity is shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic flow chart of a drug sensitivity analysis method based on direct detection of living bacteria in water in an embodiment of the present disclosure;

[0036] Figure 2 is the mass spectrum of Escherichia coli metabolism in water of Example 1 of the present disclosure;

[0037] Figure 3 is a volcano plot of metabolite changes after ampicillin stimulates Escherichia coli in Example 2 of the present disclosure;

[0038] Figure 4 is a response diagram of hypoxanthine metabolism changes after Escherichia coli is stimulated by ampicillin in Example 2 of the present disclosure;

[0039] Figure 5 is a mass spectrum of changes in metabolites after different antibiotics stimulate Escherichia coli in Example 3 of the present disclosure;

[0040] Figure 6 This is a response diagram of the changes in glutamate metabolites after Escherichia coli is stimulated by different types of antibiotics in Example 3 of the present disclosure;

[0041] Figure 7 This is a response diagram of the changes in guanine metabolism after Escherichia coli was stimulated by different concentrations of ampicillin in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0043] In the prior art, in order to obtain the drug sensitivity information of bacteria, the fluorescent colorimetric method is usually used to judge the drug sensitivity of bacteria. Although this method has high sensitivity and can judge the sensitivity of antibiotics based on the fluorescence difference caused by the difference in activity of bacteria under antibiotics, it requires targeted labeling of bacteria, and thus cannot analyze the drug sensitivity of unknown bacteria.

[0044] Mass spectrometry is a technology used to measure and analyze the mass-to-charge ratio and to perform metabolite analysis based on the mass-to-charge ratio. Due to its high sensitivity and non-targeted analysis, mass spectrometry has been widely used in metabolic analysis of biological samples. However, there are also some problems with the current mass spectrometry-based analysis technology. For example, high-performance liquid chromatography-mass spectrometry requires pretreatment steps such as lysis and quenching of bacteria before analysis, and a long chromatographic separation process is required during the analysis. Other mass spectrometry analysis techniques, such as matrix-assisted laser desorption ionization mass spectrometry, desorption electrospray ionization mass spectrometry, and laser desorption ionization mass spectrometry, do not require time-consuming chromatographic separation steps and can achieve rapid bacterial metabolite analysis. However, these mass spectrometry analysis techniques also require pretreatment of bacteria with organic solutions such as methanol, acetonitrile, and formic acid before analysis. These pretreatment methods may stimulate changes in bacterial metabolites, and the true metabolic state of living bacteria cannot be obtained, resulting in an inability to accurately judge the drug sensitivity of bacteria.

[0045] To this end, the present disclosure aims at the problems existing in the above-mentioned prior art in judging the drug sensitivity of bacteria. The present disclosure provides a drug sensitivity analysis method based on direct detection of living bacteria in water, which does not require pre-treatment such as lysis and organic solvent extraction. The method is carried out by co-incubating antibiotics with bacteria, and then simply centrifuging and washing with water, and then directly injecting the bacteria in the water into a capillary for mass spectrometry analysis, thereby achieving direct detection of metabolites of living bacteria in water and obtaining drug sensitivity information of bacteria.

[0046] Figure 1 It is a schematic flow chart of the drug sensitivity analysis method based on direct detection of living bacteria in water in the embodiment of the present disclosure.

[0047] like Figure 1 As shown, the present disclosure provides a method for drug sensitivity analysis based on direct detection of living bacteria in water, comprising the following steps:

[0048] Growing bacteria in culture medium;

[0049] The cultured bacteria were centrifuged and washed;

[0050] Resuspend the live bacteria in water;

[0051] Direct mass spectrometric analysis of live bacteria in water.

[0052] In order to determine the drug sensitivity of bacteria based on the changes in bacterial metabolites, the bacteria were divided into two parts for culture, one part was added with antibiotics, and the other part was not added with antibiotics. Figure 1 The steps shown are used to determine the drug sensitivity of bacteria based on the effect of adding antibiotics on bacterial metabolites.

[0053] As a specific embodiment, the drug sensitivity analysis method based on direct detection of living bacteria in water may include:

[0054] Step (1): Cultivate bacteria in a culture medium.

[0055] Step (2): the culture medium containing bacteria in step (1) is divided into two parts, one part is added with antibiotics, and the other part is not added with antibiotics, and they are incubated separately.

[0056] Step (3): Centrifuge and wash the bacteria in step (2).

[0057] Step (4): Resuspend the bacteria in step (3) in water.

[0058] Step (5): Inject the bacteria resuspended in water directly into the capillary tube.

[0059] Step (6): applying voltage to the capillary in step (5) and detecting metabolites of bacteria in water by mass spectrometry.

[0060] Steps (3) to (6) are performed on the two parts of bacteria in step (2) respectively.

[0061] Step (7): Determine the drug sensitivity of bacteria based on changes in bacterial metabolites.

[0062] According to an embodiment of the present disclosure, in step (1), the culture medium includes at least one of the following: peptone culture medium, broth culture medium, tryptic soy culture medium; the bacteria include at least one of the following: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa. It should be noted that the types of culture medium are not limited to the types listed in the present disclosure, and any matrix that can provide nutrients for bacteria can be used as a culture medium; the types of bacteria are not limited to the bacterial categories provided in the present disclosure, and can be other common or uncommon bacterial infection species, or even unknown bacteria; the water used in the present disclosure is deionized water.

[0063] According to an embodiment of the present disclosure, the antibiotic in step (2) includes at least one of the following: kanamycin, tobramycin, ampicillin, and the concentration of the antibiotic is 0.5-10 times the critical inhibitory concentration of the antibiotic, wherein the critical inhibitory concentration represents the minimum concentration value for bacterial death. The antibiotic can be an antibiotic used to treat bacterial infection, and its type is not limited to the antibiotics listed in the present disclosure.

[0064] According to an embodiment of the present disclosure, in step (2), the antibiotic and the bacteria are incubated at 16-45° C. for 0.5-3 hours, wherein after the antibiotic and the bacteria are incubated for 0.5 hours, the drug sensitivity of the antibiotic to the bacteria can be determined based on the changes in the bacterial metabolites, thereby significantly shortening the time for drug sensitivity determination.

[0065] According to the embodiments of the present disclosure, in order to ensure the activity of bacteria, during the centrifugation and water washing of the bacteria after incubation with antibiotics, the centrifugal speed needs to be less than 8000rpm, preferably 4000-8000rpm, more preferably 6000-8000rpm, such as the centrifugal speed can be 4000rpm, 5000rpm, 6000rpm, 7000rpm, 8000rpm, etc. At this speed, the culture matrix can be removed without lysing the bacteria, thereby not affecting the activity of the bacteria. The centrifugal time can be selected to be 3-10 minutes, such as 3, 5, 7, 10 minutes, etc.

[0066] According to an embodiment of the present disclosure, the washed bacteria are resuspended in water to maintain the activity of the bacteria.

[0067] According to an embodiment of the present disclosure, bacteria resuspended in water are directly injected into a capillary to facilitate mass spectrometry detection of bacteria. The opening of the capillary is set to be larger than 5 μm, preferably 5-10 μm. Capillaries within this opening size range will not squeeze the bacteria or destroy the structure of the bacteria to affect bacterial detection.

[0068] According to the embodiments of the present disclosure, the voltage applied to the capillary is an AC voltage or a DC voltage; the voltage applied to the capillary is 2-5 kV, wherein the applied voltage can be selected as 2 kV, 3 kV, 4 kV, 5 kV, etc. Applying voltage to the capillary can drive the bacteria in the capillary to eject the bacteria into the mass spectrometer. It should be noted that the voltage applied to the capillary is related to the size of the capillary opening. A larger capillary opening may require a larger voltage to allow the bacteria to enter the mass spectrometer.

[0069] According to an embodiment of the present disclosure, bacteria incubated without antibiotics are compared with bacteria incubated with antibiotics, and the sensitivity of bacteria to antibiotics is determined based on the increase or decrease in detected bacterial metabolites, thereby obtaining bacterial susceptibility information.

[0070] In order to facilitate a better understanding of the present disclosure, the present disclosure is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments. However, it should be noted that the specific embodiments cited are only for further explanation of the present disclosure, and the present disclosure is not limited to the following embodiments.

[0071] Example 1

[0072] The detection of metabolites of living Escherichia coli in water includes the following steps:

[0073] (1) Preparation of samples of live Escherichia coli in water

[0074] Take 1 mL of E. coli in LB medium (lysing broth medium), dilute the E. coli concentration with LB medium to an optical density (OD) value of about 0.3, centrifuge the diluted E. coli solution at 6000 rpm for 3 minutes, discard the supernatant, resuspend the bacteria in water, repeat the above centrifugation operation twice to wash the bacteria. After washing, resuspend the E. coli in 1 mL of water to keep the E. coli active.

[0075] (2) Capillary preparation:

[0076] Using a P1000 needle puller, capillaries with a tip diameter of 5 μm were pulled.

[0077] (3) Mass spectrometry analysis of metabolites of living Escherichia coli in water

[0078] The prepared water containing E. coli was injected into the capillary, and the capillary containing bacteria was placed at the front end of the mass spectrometer. A 3kV AC voltage was applied to collect the mass spectrum. The metabolic spectrum of the living E. coli in the water was obtained as shown in the figure. Figure 2 shown.

[0079] Example 2

[0080] The sensitivity test of Escherichia coli to ampicillin includes the following steps:

[0081] (1) Incubate E. coli with ampicillin

[0082] Take 1mL of E. coli in LB medium, dilute the E. coli concentration with LB medium to an OD value of about 0.3, add 1μL of ampicillin to make the final concentration of ampicillin 4μg / mL. Place the mixed E. coli and ampicillin solution in a constant temperature shaking box at 37℃ and 220rpm for 0.5h, and the E. coli without ampicillin is used as the control group.

[0083] (2) Centrifugation and washing of bacteria

[0084] The E. coli incubated with ampicillin and the E. coli without ampicillin were collected and centrifuged at 6000 rpm for 3 minutes. After discarding the supernatant, the E. coli were resuspended in water, and the centrifugation operation was repeated twice to wash the E. coli. After washing, the E. coli were resuspended in 1 mL of water to keep the E. coli active.

[0085] (3) Capillary preparation:

[0086] Using a P1000 needle puller, capillaries with a tip diameter of 5 μm were pulled.

[0087] (4) Mass spectrometry analysis of metabolism of live bacteria in water

[0088] The prepared water incubated with E. coli with or without antibiotics was injected into the capillary tubes respectively, the capillary tubes containing bacteria were placed at the front end of the mass spectrometer port, 3 kV AC voltage was applied, and mass spectra were collected respectively.

[0089] (5) Determination of drug sensitivity of Escherichia coli

[0090] Compare the changes in bacterial metabolism after incubation with ampicillin, and the change folds and significant differences of metabolites after treatment with ampicillin.

[0091] Figure 3 It is a volcano plot of metabolite changes after Escherichia coli is stimulated by ampicillin in Example 2 of the present disclosure.

[0092] like Figure 3 As shown in Figure 1, when E. coli is stimulated by ampicillin antibiotics, the signal intensity of E. coli metabolites such as guanine, hypoxanthine, arginine, lysine, putrescine, and spermidine will increase significantly. Specifically, Figure 3 The horizontal axis is the multiple of the change in bacterial metabolites between the treatment with ampicillin antibiotics and the treatment without ampicillin antibiotics (i.e., the ratio); the vertical axis is the significant difference (p value) in bacterial metabolites between the treatment with antibiotics and the treatment without antibiotics. The larger the value, the more significant the change in bacterial metabolites. Figure 3 The middle triangle represents the bacterial metabolites that have undergone significant changes (i.e., p-value is less than 0.5) and have an increasing trend after treatment with ampicillin antibiotics; the five-pointed star represents the bacterial metabolites that have undergone significant changes (i.e., p-value is less than 0.5) but have a decreasing trend after treatment with ampicillin antibiotics; the circle represents the bacterial metabolites that have not undergone significant changes (i.e., p-value is greater than 0.5) after treatment with ampicillin antibiotics.

[0093] Among them, the changes in hypoxanthine metabolites, which have extremely significant changes, are taken as an example for detailed explanation.

[0094] Figure 4 This is a response diagram of the changes in hypoxanthine metabolism after Escherichia coli is stimulated by ampicillin in Example 2 of the present disclosure.

[0095] like Figure 4 As shown in the figure, after incubation with ampicillin, the hypoxanthine signal decreased significantly. Therefore, the sensitivity of ampicillin to Escherichia coli can be judged by whether the hypoxanthine decreases significantly (**** represents that the metabolite signal is significantly different from the control group, that is, the p value is <0.0001).

[0096] Example 3

[0097] The sensitivity test of E. coli to different types of antibiotics includes the following steps:

[0098] (1) Incubate E. coli with different types of antibiotics

[0099] Take 1mL of E. coli in LB medium, dilute the E. coli concentration with LB medium to an OD value of about 0.3, add 1μL of kanamycin, ampicillin and tobramycin respectively, so that the final concentration of kanamycin is 4μg / mL, the final concentration of ampicillin is 8μg / mL, and the final concentration of tobramycin is 0.25μg / mL. The E. coli solutions mixed with different types of antibiotics are placed in a constant temperature shaking box at 37°C and 220rpm for 0.5h, and bacteria without antibiotics are used as the control group.

[0100] (2) Centrifugal washing of bacteria

[0101] The E. coli incubated with different types of antibiotics were collected, centrifuged at 6000 rpm for 3 minutes, the supernatant was discarded, and the bacteria were resuspended in water, and the above centrifugation operation was repeated twice to wash the bacteria. After washing, the E. coli were resuspended in 1 mL of water to keep the E. coli active.

[0102] (3) Capillary preparation:

[0103] Using a P1000 needle puller, capillaries with a tip diameter of 5 μm were pulled.

[0104] (4) Mass spectrometry analysis of metabolites of living Escherichia coli in water

[0105] The prepared bacterial solutions incubated with different antibiotics were injected into the capillaries respectively, the capillaries containing the bacterial solutions were placed at the front end of the mass spectrometer port, 3 kV AC voltage was applied, and the mass spectrum was collected.

[0106] (5) Determination of the drug sensitivity of Escherichia coli to different antibiotics

[0107] Compare the changes in E. coli metabolites after incubation with different types of antibiotics. When E. coli is stimulated by antibiotics, the metabolites of E. coli will change. The mass spectra of E. coli metabolic changes after three different antibiotics induce E. coli are shown in Figure 1. Figure 5 As shown, different antibiotics have both similar and different effects on bacterial metabolism.

[0108] Taking the changes in glutamate metabolism as an example, a detailed explanation is given.

[0109] Figure 6 This is a response diagram of the changes in glutamate metabolites after Escherichia coli is stimulated by different types of antibiotics in Example 3 of the present disclosure.

[0110] like Figure 6 As shown in the figure, after incubation with antibiotics, the glutamate signal was significantly reduced. Therefore, the sensitivity of different antibiotics to Escherichia coli can be judged by whether glutamate is significantly reduced.

[0111] Example 4

[0112] The sensitivity test of Escherichia coli to different concentrations of ampicillin antibiotics includes the following steps:

[0113] (1) Incubate E. coli with different concentrations of ampicillin

[0114] Take 1mL of E. coli in LB medium, dilute the E. coli concentration with LB medium to an OD value of about 0.3, add 1μL of ampicillin with different initial concentrations, and make the final concentrations of ampicillin 0μg / mL, 2μg / mL, 4μg / mL, 8μg / mL, and 16μg / mL. The E. coli solution mixed with different concentrations of ampicillin was placed in a constant temperature shaking box at 37°C and 220rpm for 0.5h, and the bacterial solution without ampicillin was used as the control group.

[0115] (2) Centrifugal washing of bacteria

[0116] The E. coli solution incubated with different concentrations of ampicillin was collected, centrifuged at 6000 rpm for 3 minutes, the supernatant was discarded, the E. coli was resuspended in water, and the above centrifugation operation was repeated twice to wash the bacteria. After washing, the E. coli was resuspended in 1 mL of water to keep the E. coli active.

[0117] (3) Capillary preparation:

[0118] Using a P1000 needle puller, capillaries with a tip diameter of 5 μm were pulled.

[0119] (4) Mass spectrometry analysis of metabolism of living bacteria in water

[0120] Inject the prepared water containing bacteria incubated with different concentrations of ampicillin into the capillary tubes. Place the capillary tube containing bacteria at the front end of the mass spectrometer port, apply 3kV AC voltage, and collect the mass spectrum. (5) Determination of the drug sensitivity of Escherichia coli to different concentrations of ampicillin antibiotics

[0121] Compare the changes in bacterial metabolism after incubation with different concentrations of ampicillin. When bacteria are stimulated by different concentrations of ampicillin, bacterial metabolites will undergo different changes. Take the changes in guanine metabolism as an example to explain in detail.

[0122] Figure 7 This is a response diagram of the changes in guanine metabolism after Escherichia coli was stimulated by different concentrations of ampicillin in Example 4 of the present disclosure.

[0123] like Figure 7 As shown in the figure, the critical inhibitory concentration of ampicillin for Escherichia coli is 4μg / mL. When the concentration of ampicillin applied is less than the critical inhibitory concentration (2μg / mL), the signal of guanine is not significantly different from the control group (0μg / mL). As the concentration of ampicillin increases, the signal of glutamate gradually decreases, showing a significant difference from the control group. Therefore, the sensitivity of ampicillin to bacteria can be judged by the degree of change in the xanthine signal ( Figure 7 In the table, *** represents that the metabolite signal is significantly different from the control group, that is, p value < 0.001; ns represents that there is no significant difference from the control group (p value > 0.05).

[0124] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for drug sensitivity analysis based on direct detection of living bacteria in water, characterized in that: include: Step (1): culturing bacteria in a culture medium; Step (2): dividing the culture medium containing bacteria in step (1) into two parts, one part is added with antibiotics, and the other part is not added with antibiotics, and incubating them separately; Step (3): centrifuging and washing the bacteria in step (2); Step (4): resuspending the bacteria in step (3) in water; Step (5): injecting the bacteria resuspended in water directly into the capillary tube; Step (6): applying voltage to the capillary in step (5) to detect bacterial metabolites in the water by mass spectrometry; Steps (3) to (6) are performed on the two parts of bacteria in step (2) respectively. Step (7): Determine the drug sensitivity of bacteria based on changes in bacterial metabolites.

2. The method according to claim 1, characterized in that The culture medium in step (1) comprises at least one of the following: Peptone medium, broth medium, tryptic soytone medium.

3. The method according to claim 1, characterized in that The bacteria in step (1) include at least one of the following: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa.

4. The method according to claim 1, characterized in that: The antibiotic in step (2) includes at least one of the following: Kanamycin, tobramycin, ampicillin.

5. The method according to claim 4, characterized in that The concentration of the antibiotic is 0.5-10 times the critical inhibitory concentration value of the antibiotic.

6. The method according to claim 1, characterized in that The incubation in step (2) is performed at 16-45°C for 0.5-3 hours.

7. The method according to claim 1, characterized in that The centrifugal speed in step (3) is 4000-8000 rpm, and the centrifugal time is 3-10 min; The number of centrifugal washings is 2-3 times, and the washing solvent is water.

8. The method according to claim 1, characterized in that The opening of the capillary in step (4) is 5-10 μm.

9. The method according to claim 7, characterized in that: The voltage applied to the capillary is an AC voltage or a DC voltage; The voltage applied to the capillary was 2-5 kV.

10. The method according to claim 1, characterized in that In the step (7), the sensitivity of the bacteria to the antibiotic is determined by the increase or decrease of the detected metabolites.

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