阅读说明：本技术 一种快速检测细菌耐药性的方法 (Method for rapidly detecting drug resistance of bacteria ) 是由 罗艳君 衣晓飞 彭迪 于 2019-10-31 设计创作，主要内容包括：本发明涉及一种快速检测细菌耐药性的方法,设置不同浓度的抗生素实验组及阳性对照组与阴性对照组,不同浓度的抗生素实验组是指将不同浓度的抗生素添加到培养基中,且后续会添加重水的实验组,阳性对照组是指培养基中加入抗生素浓度为0,并且后续添加重水的实验组,阴性对照组是指培养基中加入抗生素浓度为0,且后续不添加重水的实验组,孵育后离心清洗,对样品进行拉曼检测,根据得到的拉曼图谱分别计算C-D/(C-D+C-H),将不同浓度的抗生素实验组相对于对照组C-D/(C-D+C-H)比值下降的转折点作为抗生素对该菌的最低抑菌浓度,将最低抑菌浓度与CLSI药敏试验标准中给出的折点比较,判断细菌敏感、中介或耐药。本发明引入折点比较,更准确给出细菌对抗生素的敏感性。(The invention relates to a method for rapidly detecting bacterial drug resistance, which comprises the steps of setting antibiotic experimental groups with different concentrations, a positive control group and a negative control group, wherein the antibiotic experimental groups with different concentrations are experimental groups which are added into a culture medium and are added with heavy water subsequently, the positive control group is an experimental group which is added into the culture medium and is added with the antibiotic concentration of 0 and is added with the heavy water subsequently, the negative control group is an experimental group which is added into the culture medium and is added with the antibiotic concentration of 0 and is not added with the heavy water subsequently, carrying out centrifugal cleaning after incubation, carrying out Raman detection on samples, respectively calculating C-D/(C-D + C-H) according to the obtained Raman spectra, taking the turning point of the ratio reduction of the antibiotic experimental groups with different concentrations relative to the control group C-D/(C-D + C-H) as the minimum concentration of the antibiotic to the bacteria, and comparing the minimum inhibitory concentration with a break point given in a CLSI drug sensitivity test standard to judge the sensitivity, mediation or drug resistance of bacteria. The invention introduces breakpoint comparison, and more accurately gives the sensitivity of bacteria to antibiotics.)

1. A method for rapidly detecting bacterial drug resistance is characterized by comprising the following steps:

setting antibiotic experimental groups with different concentrations, a positive control group and a negative control group, wherein the antibiotic experimental groups with different concentrations are obtained by adding antibiotics with different concentrations into a culture medium, the antibiotic concentration of the positive control group is 0, the antibiotic concentration of the negative control group is 0,

incubating bacteria in an experimental group, a positive control group and a negative control group, then respectively adding heavy water into the bacteria liquid of the experimental group and the positive control group, not adding the heavy water into the bacteria liquid of the negative control group, continuously incubating and then centrifugally cleaning, carrying out Raman detection on the samples of the experimental group, the positive control group and the negative control group, respectively calculating C-D/(C-D + C-H) of the experimental group, the positive control group and the negative control group according to the obtained Raman spectra, taking the turning point of the antibiotic experimental group with different concentrations, which is reduced relative to the C-D/(C-D + C-H) of the control group, as the minimum inhibitory concentration of the antibiotic to the bacteria, comparing the minimum inhibitory concentration with the breaking point given in the CLSI drug sensitivity test standard (2019), and judging the sensitivity, intermediation or drug resistance of the bacteria.

2. The method of claim 1, wherein the antibiotic concentration setting is across 3 break points defined by the CLSI of the bacteria, i.e. sensitive (S), intermediate (I), and drug-resistant (R) concentrations, when performing antibiotic test group setting with different concentrations.

3. The method of claim 1, wherein the bacteria is sensitive when the minimum inhibitory concentration < the sensitivity value (S) specified by CLSI, the bacteria is intermediate when the sensitivity value (S) specified by CLSI < the resistance (R) specified by CLSI, and the bacteria is drug-resistant when the minimum inhibitory concentration > the resistance (R) specified by CLSI.

4. The method for rapidly detecting bacterial drug resistance according to claim 1, wherein the culture medium is MH culture medium.

5. The method for rapidly detecting bacterial drug resistance according to claim 1, wherein the heavy water is added in an amount of 40-60% of the total volume of the culture medium.

In one embodiment of the invention, the first incubation time is 0.5-3h and the incubation temperature is 37 ℃, the second incubation time is 0.5-3h and the incubation temperature is 37 ℃.

6. The method for rapidly detecting bacterial drug resistance according to claim 1, wherein the conditions of centrifugal washing are as follows: setting the rotating speed of a centrifuge to 3000-10000 rpm for 1-3min, adding sterile water each time, blowing and beating for 5-6 times by using a gun head, and uniformly mixing, wherein the sterile water is sterilized deionized water.

7. The method of claim 1, wherein when performing Raman detection on the samples of the experimental group, the positive control group and the negative control group, the samples of the experimental group, the positive control group and the negative control group are dropped on a low-Raman background chip to perform Raman detection, and the low-background Raman chip adopts a glass slide glass containing a metal coating; the low raman background chip was purchased from shanghai deuterium peak medical instruments ltd, Cat No 1001.

8. The method for rapidly detecting bacterial drug resistance according to claim 1, wherein 532nm laser is used for collecting Raman spectrum, grating is 600g/mm, spectrum center is set to 2300, and spectrum range is 188-3950 cm^-1(ii) a The C-D peak and the C-H peak are respectively positioned at 2000-2300cm^-1And 2800-^-1。

9. The method of claim 1, wherein C-D/(C-D + C-H) is C-D peak area/(C-D peak area + C-H peak area),

at a wavelength X at the maximum of the C-D peak_dCentering on, setting range A_dRange A_dHas a wavelength of X at the boundary₁And X₂Wherein X is₁<X_d<X₂And X₂-X₁>7nm, set range B_dRange B_dHas a wavelength of X at the boundary₀And X₁Wherein X is₁-X₀＝X₂-X₁Setting a range C_dRange C_dHas a boundary wavelength of X₂And X₃Wherein X is₃-X₂＝X₂-X₁Calculating the area of C-D peak: s_d＝S_ad-(S_bd+S_cd) /2 wherein S_ad，S_bdAnd S_cdRespectively is the range A_d，B_dAnd C_dArea under the internal raman spectrum;

at a wavelength Y at the maximum of the C-H peak_hCentering on, setting range A_hRange A_hIs bounded by the wavelength Y₁And Y₂Wherein Y is₁<Y_h<Y₂And Y is₂-Y₁>9nm, set range B_hRange B_hIs bounded by the wavelength Y₀And Y₁Wherein Y is₁-Y₀＝Y₂-Y₁Setting a range C_hRange C_hHas a boundary wavelength of Y₂And Y₃Wherein Y is₃-Y₂＝Y₂-Y₁Calculating the area of C-H peak: s_h＝S_ah-(S_bh+S_ch) /2 wherein S_ah，S_bhAnd S_chRespectively is the range A_h，B_hAnd C_hArea under the internal raman spectrum.

10. The method of claim 1 for rapidly detecting bacterial drug resistanceThe method is characterized in that 1770-2400 cm is intercepted during data processing^-1Mapping, and then performing background removal and normalization processing on the data.

Technical Field

The invention relates to a bacteria detection method, in particular to a method for rapidly detecting bacterial drug resistance.

Background

The traditional methods for detecting the drug resistance of the bacteria mainly comprise a paper diffusion method, a broth dilution method and an agar dilution method (Melvin P, Performance Standards for Antimicrobial surgery testing. CLSIM100.28,30(2018)), wherein before the drug sensitivity test is carried out by using the methods, the culture and purification steps of the bacteria are required, the culture takes 24-48 hours, and the sensitivity of the bacteria to the drugs is judged by observing whether the bacteria grow and proliferate, such as the turbidity of the bacteria or the size of bacteriostatic spots, and the time is 16-20 hours. Clinically, a drug sensitivity report obtained by a traditional drug sensitivity detection method needs 3-5 days, and the requirements of doctors and patients in an acute infectious department on rapid diagnosis cannot be met.

In recent years, researchers have proposed rapid drug-sensitive detection of bacteria using Single-Cell Raman technology in combination with heavy water labeling (Tao, Y., et al, Metabolic-Activity-Based Assessment of Antimicrobial Effect D2O-laboratory Single-Cell Raman microscopy. Anal. chem.89,4108-4115 (2017); Song, Y., et al, Raman-Deuterium Isotope binding for in-situ identification of Antimicrobial bacteria in diameter river experiments. scientific reports 7(1):16648 (2017)).

In the existing method for carrying out drug sensitive detection on bacteria by combining Raman with heavy water labeling, heavy water and antibiotics are generally added into a culture medium at the same time, then the bacteria are incubated, and the C-D intensity of single cells is detected by a Raman spectrometer after a period of time. It is believed that the C-D intensity of resistant bacteria is not affected by antibiotics, while the C-D intensity of susceptible bacteria is reduced by inhibition by antibiotics.

However, this method has disadvantages: after the antibiotic is added into the cell culture solution, the bacteriostatic or bactericidal action on the bacteria needs a certain time to take effect. Before the antibiotic becomes effective, the bacteria still have strong metabolic activity in the culture medium and combine heavy water during the metabolic process. Therefore, even if bacteria are sensitive to the applied medicine, C-D peaks can be detected to exist during Raman detection, so that the judgment of medicine sensitivity results is inaccurate.

In interpreting the results, the methods disclosed so far are generally judged to be sensitive if the ratio of C-D/(C-D + C-H) of the treated group to the control group is 0.75 or less and to be resistant if it is 0.75 or more.

The sensitivity or drug resistance is judged by simply using the ratio of C-D/(C-D + C-H) of a treatment group and a control group with a certain antibiotic concentration, and the bacteria sensitivity, drug resistance or mediation is obtained by comparing the MIC of the detected drug to the bacteria with the breakpoint when the treatment group and the control group do not meet the clinical judgment standard.

Disclosure of Invention

The present invention aims at overcoming the defects of the prior art and providing a method for rapidly detecting bacterial drug resistance.

The purpose of the invention can be realized by the following technical scheme:

a method for rapidly detecting bacterial drug resistance comprises the following steps:

setting antibiotic experimental groups with different concentrations, a positive control group and a negative control group, wherein the antibiotic experimental groups with different concentrations are obtained by adding antibiotics with different concentrations into a culture medium, the antibiotic concentration of the positive control group is 0, the antibiotic concentration of the negative control group is 0,

incubating bacteria in an experimental group, a positive control group and a negative control group, then respectively adding heavy water into the bacteria liquid of the experimental group and the positive control group, but not adding heavy water into the bacteria liquid of the negative control group, namely the antibiotic experimental group with different concentrations is an experimental group which adds antibiotics with different concentrations into a culture medium and can be added with heavy water subsequently, the positive control group is an experimental group which is added with 0 antibiotic concentration into the culture medium and is added with heavy water subsequently, the negative control group is an experimental group which is added with 0 antibiotic concentration into the culture medium and is not added with heavy water subsequently, carrying out centrifugal cleaning after continuous incubation, carrying out Raman detection on samples of the experimental group, the positive control group and the negative control group, respectively calculating C-D/(C-D + C-H) of the experimental group, the positive control group and the negative control group according to the obtained Raman spectrums, and taking the turning point of the antibiotic experimental group with different concentrations, which is reduced relative to the C-D/(C-D + C-H) ratio of the control group, as the Minimum Inhibitory Concentration (MIC) of the antibiotic to the bacteria, comparing the minimum inhibitory concentration with the turning point given in the CLSI drug sensitivity test standard (2019), and rapidly judging the sensitivity, mediation or drug resistance of the bacteria within 3-5 hours.

Further, when the antibiotic experimental group setting of different concentrations is carried out, the antibiotic concentration setting should span 3 break points defined by the CLSI of the bacteria, namely sensitive (S), intermediate (I) and drug-resistant (R) concentrations.

Further, when the Minimum Inhibitory Concentration (MIC) is less than the sensitivity value (S) specified by CLSI, the patient is judged to be sensitive, when the sensitivity value (S) specified by CLSI is less than the Minimum Inhibitory Concentration (MIC) is less than the drug resistance (R) specified by CLSI, the patient is judged to be intermediate, and when the Minimum Inhibitory Concentration (MIC) is greater than the drug resistance (R) specified by CLSI, the patient is judged to be drug resistant.

In one embodiment of the present invention, the culture medium uses MH medium.

In one embodiment of the invention, the heavy water is added in an amount of 40% to 60% based on the total volume of the medium.

In one embodiment of the invention, the first incubation time is 0.5-3h and the incubation temperature is 37 ℃, the second incubation time is 0.5-3h and the incubation temperature is 37 ℃.

In one embodiment of the present invention, the conditions for centrifugal washing are: setting the rotating speed of a centrifuge to 3000rpm-6000rpm (preferably 5000rpm) for 1-3min (preferably 2min), adding sterile water each time, blowing and beating by using a gun head for 5-6 times, and uniformly mixing, wherein the sterile water is sterilized deionized water.

In one embodiment of the invention, when performing raman detection on samples of an experimental group, a positive control group and a negative control group, the samples of the experimental group, the positive control group and the negative control group are dripped on a low-raman background chip to perform raman detection, and the low-background raman chip adopts a glass slide glass containing a metal coating; the low raman background chip was purchased from shanghai deuterium peak medical instruments ltd, Cat No 1001.

In one embodiment of the invention, 532nm laser is used for collecting Raman spectrum, the grating is 600g/mm, the spectrum center is set to 2300, and the spectrum range is 188-3950 cm^-1(ii) a The C-D peak and the C-H peak are respectively positioned at 2000-2300cm^-1And 2800-^-1。

In one embodiment of the present invention, C-D/(C-D + C-H) means C-D peak area/(C-D peak area + C-H peak area),

the calculation mode of the C-D peak area is as follows: at the wavelength at the maximum of the C-D peakX_dCentering on, setting range A_dRange A_dHas a wavelength of X at the boundary₁And X₂Wherein X is₁<X_d<X₂And X₂-X₁>7nm, set range B_dRange B_dHas a wavelength of X at the boundary₀And X₁Wherein X is₁-X₀＝X₂-X₁Setting a range C_dRange C_dHas a boundary wavelength of X₂And X₃Wherein X is₃-X₂＝X₂-X₁Calculating the area of C-D peak: s_d＝S_ad-(S_bd+S_cd) /2 wherein S_ad，S_bdAnd S_cdRespectively is the range A_d，B_dAnd C_dArea under the internal raman spectrum;

the C-H peak area calculation mode is as follows: at a wavelength Y at the maximum of the C-H peak_hCentering on, setting range A_hRange A_hIs bounded by the wavelength Y₁And Y₂Wherein Y is₁<Y_h<Y₂And Y is₂-Y₁>9nm, set range B_hRange B_hIs bounded by the wavelength Y₀And Y₁Wherein Y is₁-Y₀＝Y₂-Y₁Setting a range C_hRange C_hHas a boundary wavelength of Y₂And Y₃Wherein Y is₃-Y₂＝Y₂-Y₁Calculating the area of C-H peak: s_h＝S_ah-(S_bh+S_ch) /2 wherein S_ah，S_bhAnd S_chRespectively is the range A_h，B_hAnd C_hArea under the internal raman spectrum.

In one embodiment of the invention, the interception is 1770-2400 cm during data processing^-1Atlas, data were then processed for background (linearity) and normalization (/ area).

The invention realizes the sensitivity detection of bacteria to antibiotics and guides reasonable medication by utilizing different metabolic activities of bacteria under the action of different antibiotics. Working of the inventionThe principle is as follows: during the reduction of NAD/NADP, the bacteria convert hydrogen in water into biomass, especially biomacromolecules such as lipids and proteins. The C-H bond in these macromolecules has a characteristic peak in the Raman spectrum (2800-^-1In between). Deuterium (D) in heavy water is used to synthesize important biological macromolecules as bacteria metabolize when heavy water is present, thereby enabling the C-H peak (2800-3000 cm) in Raman spectra^-1) The shift occurs, and the C-D peak (2000-^-1) The intensity of the C-D peak reflects the metabolic activity of the bacteria. After antibiotics with different concentrations act for a period of time, bacteria are killed or inhibited, then heavy water is added, the minimum inhibitory concentration MIC of the antibiotics to the bacteria can be easily obtained according to the existence or nonexistence of C-D peaks and the strength difference, the bacteria sensitivity or drug resistance can be judged by comparing the MIC with the break point of the genus of the bacteria to which the bacteria belong, and then effective antibiotics can be rapidly screened out.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the adding time of the heavy water is adjusted, and the heavy water is added after the antibiotics act for a period of time, so that the situation that the bacteria still have strong metabolic activity and have strong deuterium peak at the early stage of the antibiotics act can be avoided.

2. The break point comparison is introduced while the MIC of the bacteria is detected by combining Raman and heavy water, and the sensitivity of the bacteria to antibiotics is more accurately given by comparing the MIC detected by Raman with the break point specified in the CLSI used clinically.

Drawings

FIG. 1 shows the mean values of the Raman spectra of single cells with different concentrations of Ciprofloxacin (Ciprofloxacin), before the modification (addition of deuterium oxide and antibiotics simultaneously) on the left and after the modification (addition of deuterium oxide after 2h of antibiotics).

In FIG. 1, each of cip0.25, cip0.12, cip0.06, cip0.03, cip0.015, cip0.008, cip0.004, and cip0.002 indicates an experimental group of Ciprofloxacin (Ciprofloxacin) at different concentrations (in ug/mL), pos indicates a positive control group, and neg indicates a negative control group.

FIG. 2 shows the ratio of the C-D peak area to the sum of the C-H and C-D areas of a single-cell Raman spectrum under the action of Ciprofloxacin (Ciprofloxacin) with different concentrations, wherein the left side shows the method before the improvement (heavy water and antibiotics are added simultaneously), and the right side shows the method after the improvement (heavy water is added after the antibiotics act for 2H).

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.