CN112014372A

CN112014372A - Method for quickly, qualitatively and quantitatively evaluating action effects of different antibacterial drugs

Info

Publication number: CN112014372A
Application number: CN202010815660.0A
Authority: CN
Inventors: 徐健; 陶一帆; 王允; 黄适; 凌均棨
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2017-02-20
Filing date: 2017-02-20
Publication date: 2020-12-01
Also published as: CN108459001B; CN108459001A

Abstract

The invention relates to the technical field of drug screening, in particular to a method for screening a drug by using heavy water (D)₂O) labeling and combining with living single cell Raman spectroscopy technology to quickly and quantitatively evaluate different antibacterial drugs. The method comprises the steps of culturing anti-bacteria by using heavy water markers and a drug to be detected, respectively obtaining single-cell Raman spectra at different time points in a culture period, generating Raman spectra of C-D peaks with different heights due to different metabolic activities of cells under different drugs to be detected and different concentrations of the drugs to be detected, and judging the sensitivity of the drug to be detected according to the Raman spectra, thereby identifying the action effect of the drug. The living single cell Raman spectrum technology adopted by the invention can be used for evaluating and screening the drugs causing different metabolic inhibition degrees to the target cells by combining the heavy water marking technology, and can evaluate the drug action level on the time axis span, thereby having universality in the fields of bacteria, mammalian cells and the like and having wide application prospect.

Description

Method for quickly, qualitatively and quantitatively evaluating action effects of different antibacterial drugs

Technical Field

The invention relates to the technical field of drug screening, in particular to a method for screening a drug by using heavy water (D)₂O) labeling and combining with living single cell Raman spectroscopy technology to quickly and quantitatively evaluate the action effects of different antibacterial drugs.

Background

With the increasing popularity of antibacterial drugs including antibiotics and the like, the problems of antibiotic abuse and bacterial resistance have become increasingly apparent, and the emergence of bacteria resistant to most commonly used antibiotics in severe infections has made the situation of no drug available a major global health safety issue faced in the 21 st century. Conventional evaluation criteria for antimicrobial agents are divided into cell growth-based means and non-cell growth-based means. Among the drug evaluation systems based on cell growth include agar diffusion method, thin-layer chromatography biological self-development, gradient dilution method, etc., which depend on cell division and proliferation, and therefore, usually take a long time (usually more than 16h) to complete. Meanwhile, the Minimum Inhibitory Concentration (MIC) is taken as a judgment index, which is defined as the concentration of the drug when the cells are not obviously grown after being exposed to the drug for 18-24h, but the cells do not grow normally and do not represent cell death, so that the method can only judge the inhibitory effect of the cells but not the bactericidal effect of the drug.

Since environmental bacteria exist in a non-growing but still metabolically active (NGMA) state, bacteria in this state cannot be identified by the above cell growth-based methods, but they play a critical role in the later disease recurrence process and are the subject of major concern. Therefore, methods relying solely on growth are still largely lacking in the evaluation of the effect of drugs.

On the other hand, the drug evaluation system not based on cell growth mainly comprises technologies of determining the integrity of cell membranes by ATP bioluminescence detection and dyeing with fluorochromes such as Propidium Iodide (PI), and the like, and saves time compared with the former technology because cell proliferation is not relied on, but because the action mechanisms of drugs are different, the change trend of internal ATP is caused, the destructiveness of cell membranes is obviously different, and even the opposite conclusion is produced, so the judgment of the method sometimes has errors. In addition, the method is based on the research of cells at the population level, so that the heterogeneity among the cells is ignored, and the problems of cell drug resistance and the like cannot be comprehensively evaluated. In view of the above, the development of novel antibacterial agents, including novel antibiotics, and means and criteria for rapidly evaluating the effects of drugs are becoming of great importance.

The single cell Raman technology is increasingly becoming an important means for measuring cell phenotype due to its characteristics of simplicity, rapidness, no need of marking, capability of providing rich cell component information and the like. By observing the change of macromolecular substances in cells, the Single Cell Raman Spectrum (SCRS) can be used for distinguishing cell types and growth states, identifying product accumulation and the like. Recently, a new approach based on raman spectroscopy (raman) has been proposed and applied to determine the effect of external stimuli including drugs on cell phenotype at the single cell level (Teng, l.; Wang, x.; Gou, h.; Ren, l.; Wang, t.; Wang, y.; Ji, y.; Huang, w.e.; Xu, j., Label-free, rapid and qualitative phenotyping of stress response in e.coli viman raman 2016,6, 34359). The method is based on the statistical analysis of the full spectrum of the cell fingerprint area, and judges the response of the cell to the external stimulus by observing the change of the special Raman peak position, thereby evaluating the action mechanism of the stimulus to a certain extent. However, due to the weak change of the components in the cells and the sensitivity of the raman spectrum to other factors such as cell types, cell growth states, and test environments, the analysis process becomes complicated and is not suitable for all microorganisms, and even not applicable to non-culturable microorganisms. Therefore, a clearer universal mark needs to be found for evaluating the drug action, so as to obtain a method for rapidly and quantitatively evaluating the action effects of different antibacterial drugs and screening drug-resistant bacteria.

Disclosure of Invention

The invention aims to provide a method for quickly, qualitatively and quantitatively evaluating the action effects of different antibacterial drugs.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for quickly, qualitatively and quantitatively evaluating the action effect of different antibacterial drugs includes such steps as using heavy water label and the drug to be tested to culture antibacterial bacteria, respectively obtaining the Raman spectra of single cells at different time points in culture period, generating the Raman spectra of C-D peaks with different heights by the metabolic activities of cells under different drugs and concentrations of drugs, and judging the sensitivity of the drug to be tested according to the Raman spectra.

Adding the anti-bacteria into different drugs to be detected, comparing the generation speed of the C-D peak of the cell under different drugs to be detected with a control without the drugs, and judging the sensitivity to the drugs.

The heavy water mark and the drug to be detected are utilized to culture the anti-germ, and the C-D peak (1800 ion 2500 cm) of the single cell in the culture is detected by Raman spectrum under a period of time^-1) Changed and then passed through the C-D peak (1800-^-1) And (3) obtaining the C-D-ratio of a single cell by change calculation, obtaining the delta C-D-ratios at different time points by the C-D-ratio, and further obtaining the minimum inhibitory metabolic concentration MIC-MA of the drug to be detected, namely quantitatively judging the sensitivity of the drug to be detected.

The C-D-ratio is C-D/(C-D + C-H), wherein C-D is a C-D peak (1800 ion 2500 cm)^-1) Area, C-H is C-H peak (2600-3300 cm)^-1) Area;

the Δ C-D-ratio is subtracted from the average of 0h of C-D-ratio over each time point;

the minimum inhibitory metabolic concentration MIC-MA is the minimum drug concentration at which the median Δ C-D-ratio of the cells is below 0 after several hours of drug exposure.

And respectively acquiring the SCRS of the culture solution single cells at different time points in the culture period, and generating Raman spectra containing C-D peaks with different heights by utilizing different metabolic activities of the cells under different drugs to be detected and different concentrations of the drugs to be detected so as to judge the sensitivity of the drugs to be detected.

Culturing the disease-resistant bacteria in the stage of culturing to a culture medium containing the heavy water marker and the drug to be detected under different concentrations, respectively taking culture solution cells at different time points in the culture period and collecting the single-cell Raman spectrum, wherein the time for collecting the Raman spectrum is 0.01-100s under the laser with the energy of 1-100 mW.

The drug to be detected at the different concentrations is a gradient from below the minimum inhibitory concentration to a concentration above the minimum inhibitory concentration.

Specifically, cells of a pathogen (such as common bacteria in the oral cavity, specifically Streptococcus mutans UA159) cultured overnight to a stationary phase are diluted to a volume ratio of 1:10 in a fresh culture medium (such as brain heart infusion Broth (BHI) culture medium) containing different concentrations of a drug to be detected and 30% of heavy water, and are cultured for 8 hours, and samples are respectively taken at 0,0.5,1,1.5,2,3,4,6 and 8 hours for raman measurement and SCRS are collected. The variation trend of the C-D peak, including the rising start time, the saturation height and the like of the C-D peak can be used for analyzing the response of cells to the medicine, so that the action effect and the mechanism of the medicine can be judged. Specifically, under the condition that a control group has no drug stimulation, the C-D peak rapidly rises and then enters a logarithmic growth period, and after reaching a certain height, the height of the C-D peak becomes stable and saturated and enters a plateau period; under the stimulation condition of the low-concentration medicament, the C-D peak does not appear in a period of time at the beginning of stimulation, then the C-D peak slowly rises, and finally the C-D peak is stabilized after reaching a relatively low saturation level; under the condition of high-concentration drug stimulation, no C-D peak or only extremely low C-D peak formation is observed in the observation time range.

The Streptococcus mutans UA159 is a common strain in an oral cavity, and is detected to have obvious C-D peak in the SCRS under the condition of existence of heavy water according to the mode, and the saturated C-D ratio is different along with different concentrations of the heavy water, and the Streptococcus mutans UA and the heavy water are in linear correlation relationship.

The drugs for detecting common bacteria in the oral cavity include, but are not limited to, sodium fluoride (NaF), Chlorhexidine (CHX), and ampicillin (Amp).

(some procedures for screening drug-resistant bacteria are given here)

The invention has the following function principle: in the presence of heavy water, the cells with metabolic activity can take up the heavy water for synthesizing internal macromolecules to carry out life activities, so that the C-H peak (2600-^-1) Drift, new C-D peak (1800 + 2500 cm)^-1). Drift speed can reflect the synthesis speed of cell macromolecules for the same speciesThe cells can then characterize changes in their metabolic activity.

Therefore, the target cells are exposed in different drug environments, and the sensitivity of the target cells to the drugs can be judged by comparing the generation speed of the C-D peak with that of a control without the drugs, so that the effect of the drugs is identified, and the action mechanism of the drugs is analyzed to a certain extent.

The invention has the advantages that: simple, rapid, low cost, based on metabolic activity and can reflect cellular heterogeneity at the single cell level. Firstly, the method is independent of cell proliferation, so that the time required is shorter than that of the traditional growth-based method, and the difference can be seen within 0.5 h; secondly, the method reflects the activity of cells, so that bacteria which do not grow but have activity can be identified, thereby giving more appropriate drug concentration and having important significance for subsequent disease recurrence control; in addition, the method can comprehensively detect the drug sensitivity of the cells at the single cell level, thereby discovering the heterogeneity phenomenon which cannot be observed at the population level and providing more comprehensive basis for understanding the drug resistance mechanism of the cells and controlling diseases.

Drawings

FIG. 1 is a graph of the C-D-ratio of S.mutans UA159 over time in the presence of different concentrations of NaF according to an embodiment of the invention;

FIG. 2 is a graph showing the Δ C-D-ratio distribution of S.mutans UA159 at various concentrations of NaF exposed for 8 hours according to an embodiment of the present invention;

FIG. 3 is a graph showing the variation of C-D-ratio of S.mutans UA159 over time in the presence of different concentrations of CHX according to an embodiment of the present invention;

FIG. 4 is a graph showing the Δ C-D-ratio distribution of S.mutans UA159 at different concentrations of CHX for 8h according to an embodiment of the present invention;

FIG. 5 is a graph of the C-D-ratio of S.mutans UA159 over time in the presence of various concentrations of Amp, according to an embodiment of the invention;

FIG. 6 is a distribution of Δ C-D-ratios of S.mutans UA159 at different concentrations of Amp exposed for 8h according to an embodiment of the invention;

FIG. 7 shows that S.mutans UA159 and S.mutans C180-2FR provided for embodiments of the invention respond differently in the presence of different concentrations of NaF.

Detailed Description

The method comprises the steps of firstly adding activated microbial cells into a culture medium containing heavy water with a certain concentration and corresponding drug concentration for culturing for a period of time, then collecting the Raman spectrum of the single cell, and evaluating the action effect of the drug at the single cell level according to the action principle of different drugs on bacterial cells and the difference of Raman heavy water peak morphological areas generated by different effects. The method has the advantages of simple operation, less sample consumption, relatively short time and low cost, and the living single-cell Raman spectrum technology adopted by the method can be combined with the heavy water labeling technology to evaluate and screen the drugs which cause different metabolic inhibition degrees on the target cells, and can evaluate the drug action level on a time axis span, so the method has universality in the fields of bacteria, mammalian cells and the like and has wide application prospect.

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

Example 1 evaluation of the Effect of sodium fluoride (NaF) on S.mutans UA159 by the deuterium-Raman technique

NaF is a common mouthwash and toothpaste additive, and has a remarkable effect on preventing oral diseases such as dental caries. S. mutans UA159 has a MIC of 0.4g/L for NaF.

1) Cell culture

S. mutans UA159 strain was streaked from-80 ℃ and anaerobically cultured at 37 ℃ for about 36 hours; then selecting a single clone to be transferred into 5-10ml of BHI liquid culture medium, standing and activating at 37 ℃ overnight;

inoculating 20mL of strain containing D into the strain after the activated strain is centrifuged according to the inoculation amount of 1:10 in volume ratio₂O and BHI liquid culture medium with different NaF concentrations, and anaerobic static culture at 37 ℃. The different concentrations of NaF are 0,0.2,0.4,0.6,1.2g/L NaF, D₂The O concentration was 30%.

2) Raman measurement

Respectively taking 500 μ L of the above D-containing solution at different time periods of 0,0.5,1,1.5,2,3,4,6 and 8h₂O and NaF at various concentrations, with ddH₂O washing for 3 times, centrifugal speedAt 2500 Xg, centrifuge for 3min, use ddH of the same volume₂O heavy suspension and corresponding dilution are carried out to make the final concentration of the cells reach 10⁶and/mL. Spotting 1.5. mu.L of the dilution onto CaF₂And (5) drying on a glass slide.

Observing the air-dried sample under a Raman microscope, positioning a laser point to a cell to be detected, and then carrying out Raman spectrum collection. The laser wavelength is 532nm, the laser intensity on the sample is about 1-100mW, and the collection time of the Raman spectrum is 0.01-100 s. Each concentration was set up with 3 biological replicates, each replicate taking 20 cell profiles at each time point (see figure 1).

3) Data processing

And carrying out background removal, baseline normalization and maximum value standardization processing on the collected Raman spectrum by using LabSpec software.

After treatment, the obvious C-D peak appears in the SCRS under the condition of the existence of heavy water, and the C-D peak (1800 + 2500 cm) is obtained at the same time^-1) Area, and then calculate C-D-ratio, and Δ C-D-ratio.

Wherein the C-D-ratio is defined by a C-D peak (1800-^-1) Area divided by C-D peak (1800-^-1) And C-H peak (2600-^-1) And calculating the sum of the areas. Δ C-D-ratio is obtained by subtracting the average value of C-D-ratio of 0h from C-D-ratio of each time point.

As can be seen from the above, the SCRS has obvious C-D peak, and the saturated C-D ratio is different with the heavy water concentration, and the two are in linear correlation relationship.

According to the above experimental results, the present invention proposes a new drug concentration use guide standard MIC-MA, i.e. "minimum inhibitory metabolic concentration". This new index is defined as the minimum drug concentration at which the median Δ C-D-ratio of the cells is below 0 after 8h drug exposure.

FIG. 1 shows the change in C-D-ratio of cells treated with different concentrations of NaF. All of the C-D peaks of cells exposed to NaF increased slowly compared to the control without NaF, and most of the saturated C-D-ratio was lower than the control (except for 0.2g/L NaF). Furthermore, as the NaF concentration increases, the increase in C-D-ratio is regularly delayed and the saturated C-D-ratio value gradually decreases. At 0.2g/L NaF below the MIC, the increase in C-D-ratio slowed, but the final saturated C-D-ratio value still rose to the same level as the control; at 0.4g/L NaF, which is equal to the MIC, a significant lag phase in the increase in C-D-ratio occurred, but there was still an increase in the late phase, demonstrating that under the conditions in which cell growth was inhibited, its activity was not completely inhibited; above MIC, the lag phase was longer (0.6g/L NaF) or the C-D-ratio did not increase (1.2g/L NaF) all the time, demonstrating that the cell activity was completely inhibited.

From the results of the Δ C-D-ratio calculation at 8h, the MIC-MA of S.mutans UA159 to NaF was judged to be 1.2g/L (FIG. 2). This concentration is much higher than its MIC value (0.4 g/L). At MIC concentrations, although cells do not grow significantly, their metabolic activity is not completely inhibited, and thus the pathogenic bacteria will reactivate and cause disease recurrence after removal of the drug. The risk is greatly reduced by the use of drugs under MIC-MA conditions.

Example 2 evaluation of the Effect of Chlorhexidine (CHX) on S.mutans UA159 Using the deuterium-Raman technique

Chlorhexidine is a common mouthwash additive, is a spectral bactericide, and can interfere the formation of bacterial plaque and reduce cell adsorption, thereby achieving the purpose of preventing and reducing periodontal diseases and dental caries. S. mutans UA159 has a MIC of 2mg/L for CHX.

1) Cell culture

inoculating 20mL of strain containing D into the strain after the activated strain is centrifuged according to the inoculation amount of 1:10 in volume ratio₂O and BHI liquid culture medium with different CHX concentrations, and anaerobic static culture at 37 ℃. The different concentrations of CHX are 0,1.2,2,4mg/L CHX, D₂The O concentration was 30%.

2) The Raman measurement is carried out by taking 500 μ L of the above D-containing substance at different times of 0,0.5,1,1.5,2,3,4,6 and 8h₂O and CHX at various concentrations, with ddH₂Washing with O for 3 times at 2500 Xg for 3min, and washing with ddH of the same volume₂O heavy suspension and corresponding dilution are carried out to make the final concentration of the cells reach 10⁶and/mL. Taking 1.5. mu.L of diluent and adding into CaF₂And (5) drying on a glass slide.

3) Data processing

FIG. 3 shows the change in C-D-ratio of cells after different concentrations of CHX treatment. All of the cells exposed to CHX showed a slower increase in the C-D peak and a lower saturation C-D-ratio than the control, compared to the control without CHX. Furthermore, as CHX concentration increases, the increase in C-D-ratio is regularly delayed and the saturated C-D-ratio value gradually decreases. At 1.2mg/L CHX below the MIC, the C-D-ratio increased relatively slowly and the final saturated C-D-ratio value was about 84% of the control level; at 2mg/L CHX, which is equal to MIC, there was a significant lag phase in the increase in C-D-ratio, but there was still an increase in the later phase, demonstrating that under the conditions in which cell growth was inhibited, its activity was not completely inhibited; the C-D-ratio was never increased above the MIC condition, i.e., 4mg/L CHX, demonstrating that cellular activity was completely inhibited.

From the calculation of Δ C-D-ratio at 8h, the MIC-MA of S.mutans UA159 to CHX was judged to be 4mg/L (FIG. 4). This concentration is much higher than its MIC value (2 mg/L). At MIC concentrations, although cells do not grow significantly, their metabolic activity is not completely inhibited, and thus the pathogenic bacteria will reactivate and cause disease recurrence after removal of the drug. The risk is greatly reduced by the use of drugs under MIC-MA conditions.

Example 3 evaluation of the Effect of ampicillin (Amp) on S.mutans UA159 by the deuterium-Raman technique

Ampicillin is a commonly used antibiotic and can treat oral inflammation. S. mutans UA159 has a MIC of 0.8mg/L for Amp.

1) Cell culture

S. mutans UA159 strain was streaked from-80 ℃ and anaerobically cultured at 37 ℃ for about 36 hours; then selecting a single clone to be transferred into 5-10ml of BHI liquid culture medium, standing overnight at 37 ℃ for activation;

inoculating 20mL of strain containing D into the strain after the activated strain is centrifuged according to the inoculation amount of 1:10 in volume ratio₂O and BHI liquid culture medium with different concentrations of Amp, and anaerobic static culture at 37 ℃. The different concentrations of Amp are 0,0.4,0.8,1.6,4,50mg/L Amp, D₂The O concentration was 30%.

2) Raman measurement

Respectively taking 500 μ L of the above D-containing solution at different time periods of 0,0.5,1,1.5,2,3,4,6 and 8h₂O and Amp at different concentrations, with ddH₂Washing with O for 3 times at 2500 Xg for 3min, and washing with ddH of the same volume₂O heavy suspension and corresponding dilution are carried out to make the final concentration of the cells reach 10⁶and/mL. Spotting 1.5. mu.L of the dilution onto CaF₂And (5) drying on a glass slide.

3) Data processing

FIG. 5 shows the change in C-D-ratio of cells treated with different concentrations of Amp. All cells exposed to ampicillin showed a slower increase in the C-D peak and a lower saturation of C-D-ratio than the control without Amp. Furthermore, as ampicillin concentration increased, the increase in C-D-ratio was substantially regularly delayed and the saturated C-D-ratio value gradually decreased (except for 0.4mg/L Amp). At 0.4mg/L Amp below the MIC, the C-D-ratio increases relatively slowly; equal to MIC, i.e., 0.8mg/L Amp, the final saturation C-D-ratio value was about 83% of the control level, demonstrating that the activity was not completely inhibited under conditions in which cell growth was inhibited; the C-D-ratio also increased significantly above the MIC, and even at 60-fold MIC (50mg/L Amp), the final saturated C-D-ratio reached 51% of the control level. At all concentrations, a significant increase in the C-D peak was observed at 0.5h, with no lag phase present, demonstrating that cell activity was not completely inhibited under all conditions tested. The reason for this is presumed to be that Amp acts by inhibiting the transpeptidase activity of cells, thereby affecting the synthesis of cell walls during division to make them unable to divide, but does not absolutely inhibit the activity of living cells.

From the Δ C-D-ratio calculation at 8h, it was judged that S.mutans UA159 could not find MIC-MA at the tested concentration for Amp (FIG. 6). At or above the MIC tested, the cells did not grow but retained a level of metabolic activity all the time, and therefore their bactericidal effect was left to be tested further.

Example 4 evaluation of different resistance of S.mutans UA159 and S.mutans C180-2FR to NaF Using the deuterium-Raman technique

S. mutans C180-2FR is a NaF-tolerant bacterium with a MIC for NaF of 1.6g/L, which is much higher than 0.4g/L for S.mutans UA 159. The existence of the bacteria can cause that the addition of NaF with low concentration has no obvious bactericidal and bacteriostatic effects. Therefore, the method for screening the drug-resistant bacteria in the environment also has important clinical significance.

1) Cell culture

Streaking S.mutans UA159 and S.mutans C180-2FR strains from-80 deg.C, and anaerobically culturing at 37 deg.C for about 36 h; then respectively selecting monoclonals to be transferred into 5-10ml of BHI liquid culture medium, and standing overnight at 37 ℃ for activation;

inoculating 20mL of activated S.mutans UA159 strain into the strain containing D according to the inoculation amount of 1:10 in volume ratio after centrifugation₂O and BHI liquid culture medium with different NaF concentrations, and anaerobic static culture at 37 ℃. The different concentrations of NaF are 0,0.4,1.2g/L NaF, D₂The O concentration was 30%.

Inoculating 20mL of the activated S.mutans C180-2FR strain into the strain containing D according to the inoculation amount of 1:10 in volume ratio after centrifugation₂O and BHI liquid culture medium with different NaF concentrations, and anaerobic static culture at 37 ℃. The different concentrations of NaF are 0,0.4,1.6g/L NaF, D₂The O concentration was 30%.

2) Raman measurement

Respectively taking 500 μ L of the above D-containing substances at different time periods of 0,0.5,1,1.5,2,3,4,6 and 8h₂Of O and NaF in different concentrationsCulture solution using ddH₂Washing with O for 3 times at 2500 Xg for 3min, and washing with ddH of the same volume₂O heavy suspension and corresponding dilution are carried out to make the final concentration of the cells reach 10⁶and/mL. Spotting 1.5. mu.L of the dilution onto CaF₂And (5) drying on a glass slide.

3) Data processing

FIG. 7 shows the different change patterns of C-D-ratio of two cells after treatment with NaF at different concentrations. In the absence of NaF, the C-D-ratio of C180-2FR was lower than UA159 for the first 3h and leveled off after 4 h. This phenomenon is mainly caused by the slightly slower growth rate of C180-2FR compared to UA 159. Whereas at 0.4g/L NaF (MIC of UA159), the increase in C-D-ratio of UA159 showed a significant lag phase, beginning to climb after 2h, with the saturated C-D-ratio value remaining at the level of 44% of the control; whereas the increase in C-D-ratio of C180-2FR was essentially unaffected. Under 1.2g/L NaF, the C-D-ratio of UA159 is not increased at all, and the metabolic activity is completely inhibited; on the other hand, even in the presence of 1.6g/L NaF (MIC of C180-2 FR), the increase in C-D-ratio of C180-2FR climbed slowly after a lag of 0.5h had occurred, eventually remaining at a level corresponding to 62% of the control. Therefore, drug-resistant bacteria and drug-sensitive bacteria can be rapidly distinguished and identified by using a deuterium-Raman technology and comparing the metabolic activity change trends of different bacteria under the same drug action.

The application embodiment of the invention adopts common oral microorganisms as a mode system, but the heavy water can be metabolized and utilized by most microorganisms and can be detected by a Raman technology, namely the heavy water-Raman technology has universality among different microorganisms, and the application of the technology can be extended to different human bodies and even environmental microorganisms, even human bodies and mammalian cells.

Claims

1. A method for rapidly qualitatively/quantitatively evaluating the action effects of different antibacterial drugs is characterized in that: and (3) culturing the anti-bacteria by using the heavy water marker and the drug to be detected, respectively acquiring single-cell Raman spectra at different time points in a culture period, generating Raman spectra of C-D peaks with different heights due to different metabolic activities of cells under different drugs to be detected and different concentrations of the drugs to be detected, and judging the sensitivity of the drug to be detected according to the Raman spectra.

2. The method of claim 1, wherein: adding the anti-bacteria into different drugs to be detected, comparing the generation speed of the C-D peak of the cell under different drugs to be detected with a control without the drugs, and judging the sensitivity to the drugs.

3. The method of claim 1, wherein: the method detects the C-D peak (1800-^-1) Change by the C-D peak (1800-^-1) Obtaining the C-D-ratio of the single cell by variation calculation; and obtaining the delta C-D-ratio at different time points by the C-D-ratio, further obtaining the minimum inhibitory metabolic concentration MIC-MA of the drug to be detected, namely quantitatively judging the sensitivity of the drug to be detected.

4. The method of claim 3, wherein: the C-D-ratio is C-D/(C-D + C-H), wherein C-D is a C-D peak (1800 ion 2500 cm)^-1) Area, C-H is C-H peak (2600-3300 cm)^-1) Area; the Δ C-D-ratio is subtracted from the average of 0h of C-D-ratio over each time point; the minimum inhibitory metabolic concentration MIC-MA is the minimum drug concentration at which the median Δ C-D-ratio of the cells is below 0 after several hours of drug exposure.

5. The method of claim 4, wherein: and (3) culturing the disease-resistant bacteria in the stage of culturing to a culture medium containing heavy water and the drug to be detected with different concentrations, and respectively acquiring the single-cell Raman spectra of the culture solution cells at different time points in the culture period, wherein the time for acquiring the Raman spectra is 0.01-100s under the laser with the energy of 1-100 mW.

6. The method of claim 5, wherein: the different concentrations of the drug to be detected are gradients from below the minimum inhibitory concentration to above the minimum inhibitory concentration.