Organic metabolic marker for detecting bacteremia of extended-spectrum beta-lactamase-producing escherichia coli and application of organic metabolic marker

Abstract

The invention relates to an organic metabolic marker for detecting bacteremia of Escherichia coli producing extended-spectrum beta-lactamase and application of the organic metabolic marker, and relates to the field of clinical detection and biological detection, and the organic metabolic marker is obtained by hydrolyzing cefotaxime with the extended-spectrum beta-lactamase produced by Escherichia coli. Compared with an existing traditional ESBL-EC bacteremia detection method, overnight culture is needed to obtain pure bacterial colonies, and the detection process consumes too long time; a physical detection method is adopted, a molecular marker found from gas released in the process of hydrolyzing cefotaxime by ESBL generated by ESBL-EC serves as a target, and then ESBL-EC is rapidly identified. Therefore, blood flow infection caused by ESBL-EC can be detected early, efficiently and accurately, the survival rate of ESBL-EC blood flow infection patients is further increased, the hospitalization time is shortened, the hospitalization cost is reduced, and great economic and social benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the field of clinical detection and biological detection, and in particular to an organic metabolic marker for detecting bacteremia of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) and application thereof. Background Art

[0002] At present, the main method for detecting extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) and drug sensitivity in clinical bacteria is to perform double-disc synergy test and drug sensitivity test on Escherichia coli strains, but this method is time-consuming (18h-24h). Studies have shown that the early use of appropriate antibiotics is the key to successfully controlling bacteremia, and it is recommended to use antibiotics rationally within 1 hour after the pathogen is identified. It also emphasizes the importance of rational use of antibiotics in the first 6 hours. The mortality rate of patients increases by 6% for every hour of extension. However, the existing detection methods seriously affect the early intervention of clinical anti-infection. Although the existing molecular biological methods for detecting drug-resistant genes can greatly reduce the detection time, they have the disadvantages of complex operation, high price, easy mutation of drug-resistant genes, interference of human cells, etc., resulting in high false negative and false positive results.

[0003] In view of this, the present invention provides an organic metabolic marker for detecting bacteremia of extended-spectrum β-lactamase-producing Escherichia coli and application thereof. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an organic metabolic marker for detecting bacteremia of extended-spectrum β-lactamase-producing Escherichia coli and its application, so as to achieve rapid identification (12 minutes) of extended-spectrum β-lactamase-producing Escherichia coli.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, an organic metabolic marker for detecting ESBL-EC bacteremia is provided, wherein the organic metabolic marker is obtained by hydrolyzing ceftriaxone by extended-spectrum β-lactamase produced by Escherichia coli.

[0007] Furthermore, the mass-to-charge ratios m / z of the organic metabolic markers include: m / z 79.9, m / z 95.9, m / z 96.9, m / z 111.9, m / z 112.9, m / z 116.0, m / z 123.9 and m / z 126.9; the mass-to-charge ratios after rounding to integers are m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z 116, m / z 124 and m / z 127.

[0008] Furthermore, EESI-MS was coupled with LTQ-XL instrument (linear ion trap liquid-mass spectrometer) to detect the organic metabolic markers.

[0009] Furthermore, when detecting the organic metabolic markers, the ion mode and scanning range conditions of the EESI-MS ion source are set as follows: laboratory temperature 20°C, humidity 30%, electrospray N2 pressure control at 1.2MPa, extract silver nitrate injection rate 2μl / min, electrospray ionization discharge needle voltage 3.50kV, ion transfer tube temperature 150°C, pressure 5.0V, detection in positive and negative ion modes, and the scanning detection range is m / z 20-200.

[0010] Furthermore, the organic metabolic markers include pyridine, aldehydes, ethanol, dihydrogalactoside, chlorobenzene, indole, phenols, and alcohols.

[0011] In a second aspect, the organic metabolic marker for detecting ESBL-EC bacteremia is used to prepare a product for detecting ESBL-EC bacteremia.

[0012] Further, the product includes a reagent or kit for detecting ESBL-EC bacteremia.

[0013] Furthermore, the reagent includes cefotaxime.

[0014] Furthermore, the reagent also includes pharmaceutically acceptable excipients.

[0015] The beneficial effects of the present invention are as follows: compared with the existing traditional ESBL-EC bacteremia detection method, which is based on culture or chemical reaction after culture, overnight culture is required to obtain pure colonies, and the detection process takes too long (18-24h); the present invention adopts a physical detection method, and a class of molecular markers (m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z 116, m / z 124, m / z 127) found in the gas released during the hydrolysis of cefotaxime by ESBL produced by ESBL-EC are used as targets, thereby realizing rapid identification (12min) of ESBL-EC. Studies have found that the early use of appropriate antimicrobial drugs is the key to successfully controlling bacteremia, and the identification results of bacteria and drug-resistant phenotypes are the prerequisite for clinical selection of appropriate antimicrobial drugs. Therefore, the technology of rapid identification of ESBL-EC (12 min) of the present invention can guide the clinic to select appropriate antimicrobial drugs to treat ESBL-EC bacteremia patients as early as possible, improve the survival rate of ESBL-EC bloodstream infection patients, shorten the hospital stay, and reduce hospitalization costs, which has great economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A flow chart for the detection of the present invention;

[0017] Figure 2 This is a comparison diagram of mVOCs signals in the upper layer of gas in blood culture bottles of Escherichia coli with different drug resistance detected by EESI-MS of the present invention;

[0018] Figure 3 The figure is a flow chart of the experimental method of the present invention. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0020] Example

[0021] 1. Experimental methods

[0022] Three groups of experiments were set up: the ESBL-EC group, in which cefotaxime was added to the extended-spectrum β-lactamase-producing Escherichia coli; the EC group, in which cefotaxime was not added to the Escherichia coli that did not produce extended-spectrum β-lactamases; and the ESBL-EC+ group, in which cefotaxime and enzyme inhibitors were added to the blood culture bottles of extended-spectrum β-lactamase-producing Escherichia coli at the same time.

[0023] The specific experimental process is as follows: Figure 1 ):

[0024] The ESBL-EC strain confirmed by sequencing was cultured to obtain a pure culture, and a bacterial suspension was prepared with sterile physiological saline. The colony count was diluted to 100 CFU / ml, and 1 ml of the prepared bacterial suspension was added to a blood culture bottle for culture. After the blood culture instrument gave a positive alarm, cefotaxime (C16H17N5O7S2-1.0 g, Shanghai Xinya Pharmaceutical Co., Ltd.) was added to the blood culture bottle with a sterile syringe to make the final concentration of cefotaxime in the blood culture bottle 8 ug / ml, which was used as the ESBL-EC group; no cefotaxime was added to form the EC group; cefotaxime and tazobactam (T843979-1.0 g, Shanghai MacLean Biochemical Technology Co., Ltd.), an inhibitor of extended-spectrum β-lactamases, were added at the same time, so that the final concentrations of cefotaxime and tazobactam were 8 ug / ml and 32 ug / ml, respectively, which was used as the ESBL-EC+ group.

[0025] Continue to incubate at 37°C. After 10 minutes of incubation, inject clean air at a rate of 20 ml / min with a syringe. Draw the upper gas of the blood culture bottle from the sample outlet tube into the mass spectrometer. When the airflow is stable and the peak value is stable, the continuous injection time of each test sample is 30-60 seconds. Repeat the parallel test for each sample 3 times to obtain a sufficient number of mass spectrum peaks.

[0026] The specific EESI-MS mass spectrometry detection conditions are as follows: couple the EESI-MS source with the LTQ-XL instrument (linear ion trap liquid-mass spectrometer), set the ion mode and scan range of the EESI-MS ion source; the optimal conditions for experimental optimization: laboratory temperature 20°C, humidity 30%, electrospray N2 pressure controlled at 1.2MPa, extract silver nitrate injected at a rate of 2μl / min, electrospray ionization discharge needle voltage 3.50kV, ion transfer tube temperature 150°C, pressure 5.0V, detection in positive and negative ion modes, and a scan detection range of m / z 20-200.

[0027] 2. Experimental results

[0028] EESI-MS mass spectrometry detects volatile organic metabolites (mVOCs) produced by bacterial decomposition of substances in blood culture bottles and mVOCs released by ESBL-EC hydrolysis of ceftriaxone. The mVOCs produced by bacterial decomposition of substances in blood culture bottles are the basis for identifying bacteria, and "mVOCs molecular markers" or "mVOCs fingerprints" are obtained. They are compared with the established mVOCs molecular markers or mVOCs fingerprints, and ESBL-EC bacteremia is finally confirmed. By detecting mVOCs in the upper layer of Escherichia coli blood culture bottles in the negative ion mode of EESI-MS, it was found that "mVOCs molecular markers" or "mVOCs fingerprints" are m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z116, m / z 124 and m / z 127, which are special signal molecules ( Figure 2 ).

[0029] After adding cefotaxime and an inhibitor of extended-spectrum β-lactamase (tazobactam) at the same time, except for the characteristic peak of m / z 116 (identification peak of Escherichia coli), the other characteristic peaks disappeared, indicating that these characteristic peaks (m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z 124 and m / z 127) are the characteristic peaks of ESBL hydrolysis of cefotaxime ( Figure 2), confirming that the bacteria contain extended-spectrum β-lactamases. These characteristic peaks (m / z 80, m / z 96, m / z 97, m / z 112, m / z113, m / z 124 and m / z 127) combined with the characteristic peak for identification of Escherichia coli (m / z 116) can confirm that the bloodstream infection is caused by ESBL-EC.

[0030] Further comparison of the characteristic signal molecules with the National Institute of Standards and Technology data network showed that m / z 80 was pyridine, m / z 96 was aldehyde, m / z 97 was ethanol, m / z 112 was dihydrogalactoside, m / z 113 was chlorobenzene, m / z 116 was indole, m / z 124 was phenolic, and m / z 127 was alcohol. Our experiments showed that the detection rate of ESBL-EC in simulated blood culture detected by EESI-MS mass spectrometry was 100% ( Figure 3 ).

[0031] in, Figure 2 ESBL-EC (NO.14006574): It is a clinically isolated strain of Escherichia coli producing extended-spectrum β-lactamases (the strain was isolated from a patient with bloodstream infection in the Second Affiliated Hospital of Nanchang University. The pathogen with the highest detection rate of bacteremia in my country is Escherichia coli, accounting for 34.7%, of which the detection rate of ESBL-EC is as high as 55.7%, which is very easy to obtain. [1] Chen Yunbo, Ji Jinru, Ying Chaoqun, Wang Peipei, Yang Qing, Kong Haishen, Liu Yongyun, Huang Ying, Dai Yuanyuan, Zhang Liping, Ding Hui, Guo Liang, Zhang Baohua, Zhu Lisha, Mao Haifeng, Liao Zhixiang, Li Yanhong, Wang Lu, Hu Shuyan, Yang Zhenghai, Gu Beiqing, Dong Haixin, Du Fei, Zheng Lin, Quan Bo, Zhu Wencheng, Wang Jianzhong, Ma Lan, Xu Rong, Sun Li, Li Aiyun, Cao Junmin, Liang Jinhua, Xu Hongyun, Liang Kunpeng, Qiao Deng Yan, Qi Xiaoyan, Xia Xiusan, Li Lanjuan, Xiao Yonghong. Report on the National Antimicrobial Resistance Surveillance (BRICS) of Bloodstream Infections from 2016 to 2017 [J]. Chinese Journal of Clinical Infectious Diseases, 2020(01):42-54.》); ESBL-EC (NO.14005001): standard strain of Escherichia coli producing extended-spectrum β-lactamases ATCC35218 (purchased from Wenzhou Kangtai Biological Co., Ltd.); EC: standard strain of Escherichia coli not producing extended-spectrum β-lactamases ATCC25922 (from Jiangxi Provincial Clinical Laboratory Center); ESBL-EC+: ceftriaxone and enzyme inhibitor (tazobactam) were added to the blood culture bottle of extended-spectrum β-lactamase-producing Escherichia coli ATCC35218 at the same time.

[0032] In summary, compared with the existing traditional ESBL-EC bacteremia detection method, based on culture or post-culture chemical reaction, overnight culture is required to obtain pure colonies, and the detection process takes too long (18h-24h); the present invention adopts a physical detection method, and a class of molecular markers (m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z 116, m / z 124, m / z 127) found in the gas released during the hydrolysis of cefotaxime by ESBL produced by ESBL-EC are used as targets, thereby realizing rapid identification (12min) of ESBL-EC. Therefore, the present invention can improve the survival rate of patients infected with ESBL-EC, shorten the length of hospital stay, and reduce hospitalization costs, and has great economic and social benefits.

[0033] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An organic metabolic marker for detecting ESBL-EC bacteremia, characterized in that: The organic metabolic marker is obtained by hydrolyzing ceftriaxone with extended-spectrum beta-lactamase produced by Escherichia coli.

2. An organic metabolic marker for detecting ESBL-EC bacteremia according to claim 1, characterized in that: The mass-to-charge ratios m / z of the organic metabolic markers include: m / z 79.9, m / z 95.9, m / z 96.9, m / z 111.9, m / z 112.9, m / z 116.0, m / z 123.9 and m / z 126.

9.

3. An organic metabolic marker for detecting ESBL-EC bacteremia according to claim 1, characterized in that: The mass-to-charge ratios m / z of the organic metabolic markers include: m / z 80, m / z 96, m / z 97, m / z 112, m / z 113, m / z 116, m / z 124 and m / z 127.

4. An organic metabolic marker for detecting ESBL-EC bacteremia according to claim 1, characterized in that: The organic metabolic markers were detected using EESI-MS coupled to a LTQ-XL instrument.

5. An organic metabolic marker for detecting ESBL-EC bacteremia according to claim 4, characterized in that: When detecting the organic metabolic markers, the ion mode and scanning range conditions of the EESI-MS ion source are set as follows: laboratory temperature 20°C, humidity 30%, electrospray N2 pressure control at 1.2MPa, extract silver nitrate injection rate 2μl / min, electrospray ionization discharge needle voltage 3.50kV, ion transfer tube temperature 150°C, pressure 5.0V, detection in positive and negative ion modes, and the scanning detection range is m / z 20-200.

6. An organic metabolic marker for detecting ESBL-EC bacteremia according to any one of claims 2 to 5, characterized in that: The organic metabolic markers include pyridine, aldehydes, ethanol, dihydrogalactoside, chlorobenzene, indole, phenols and alcohols.

7. Use of an organic metabolic marker for detecting ESBL-EC bacteremia according to any one of claims 1 to 6 in the preparation of a product for detecting ESBL-EC bacteremia.

8. The use according to claim 7, characterized in that: The products include reagents or kits for detecting ESBL-EC bacteremia.

9. The use according to claim 8, characterized in that: The agent includes cefotaxime.

10. The use according to claim 8, characterized in that: The reagent also includes pharmaceutically acceptable excipients.

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