Kit for rapidly identifying carbapenemase type of carbapenemase of enterobacter sp. Bacteremia and application of kit

Abstract

The invention relates to a kit for rapidly identifying the enzyme type of carbapenemase of bacteremia of enterobacter sp. Capable of producing carbapenemase and application of the kit, and relates to the technical field of biological detection. The kit comprises imipenem and any one of avibactam sodium, revobactam and 2, 6-dipicolinic acid, and is characterized in that the kit comprises imipenem and any one of avibactam sodium, revobactam and 2, 6-dipicolinic acid. Compared with the prior art, the method has the advantages of high speed, high accuracy, few interference factors, low cost (less than 1 dollar) and the like when being used for detecting the carbapenemase enzyme type of the carbapenemase-producing enterobacter bacteremia, the survival rate of a patient suffering from the carbapenemase-producing enterobacter bacteremia can be remarkably increased, the hospitalization time is shortened, and the method is suitable for popularization and application. Hospitalization cost is reduced, and great economic and social benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a kit for rapidly identifying the carbapenemase type of bacteremia of Enterobacteriales bacteria producing carbapenemase and its application. Background Art

[0002] Regarding the detection of carbapenemase-producing Enterobacteriales (CPE) enzyme types isolated from patients with blood infection, the existing methods are mainly molecular biology methods, colloidal gold method, modified carbapenemase inactivation test (mCIM), combined ethylenediaminetetraacetic acid carbapenemase inactivation test (eCIM) and carbapenemase enzyme inhibitor enhancement (APB-EDTA method). However, although the existing molecular biology 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 from human cells, and only being able to detect genes but not judging the survival status of bacteria, resulting in high false negative and false positive results; the colloidal gold method requires bacteria to be cultured before detection, which takes 18 to 24 hours, is time-consuming and expensive; the modified carbapenemase inactivation test (mCIM), combined ethylenediaminetetraacetic acid carbapenemase inactivation test (eCIM) and carbapenemase enzyme inhibitor enhancement (APB-EDTA method) are widely used, but they are time-consuming (>24 hours). It can be seen that the existing methods all have problems that seriously affect the early intervention of clinical anti-infection.

[0003] Studies have shown that early initiation of appropriate antibiotics is the key to successfully controlling bacteremia, and it is recommended that antibiotics be used rationally within 1 hour after the pathogen is identified. The importance of rational use of antibiotics in the first 6 hours is particularly emphasized, and the mortality rate of patients increases by 6% for every hour of extension. In view of this, the present invention provides a kit for rapidly identifying bacteremia of carbapenemase-producing Enterobacteriales (CPE) bacteria and their carbapenemase enzyme types and their application. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a kit and application for rapidly identifying the carbapenemase enzyme type of bacteremia in carbapenemase-producing Enterobacteriaceae. The purpose is to detect the carbapenemase enzyme type of carbapenemase-producing Enterobacteriaceae (CPE) quickly, with high accuracy, few interference factors and low cost (less than $1).

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

[0006] In a first aspect, a kit for rapidly identifying the carbapenemase type of bacteremia in carbapenemase-producing Enterobacteriales bacteria, the kit comprising imipenem, and any one of avibactam sodium, relebactam and 2,6-pyridinedicarboxylic acid.

[0007] The present invention combines imipenem with three other agents to form three combination modes. Among them, carbapenemase is a specific type of β-lactamase, which can significantly hydrolyze carbapenem antibiotics such as imipenem or meropenem. The following is a description of imipenem, avibactam sodium, relebactam and 2,6-pyridinedicarboxylic acid:

[0008] (1) Imipenem (IPM): molecular formula is C 12 H 17 N 3 O 4 S.H. 2 O; CAS number is 64221-86-9; belongs to the broad-spectrum β-lactam antibiotics; can be hydrolyzed by carbapenemase;

[0009] (2) Avibactam sodium (AVI): molecular formula is C 7 H 12 N 3 NaO 6 S; CAS number is 1192491-61-4; it is a new type of β-lactamase inhibitor, which forms a covalent bond through the amide bond and the nucleophilic attack of β-lactamase serine ring opening to obtain a stable enzyme-inhibitor complex, which can effectively prevent β-lactam antibiotics from being hydrolyzed and inactivated by β-lactamase (carbapenemase), and protect the antibacterial activity of β-lactam antibiotics against a variety of CPE-producing strains. It can mainly inhibit Class A (including ESBL and KPC) β-lactamase, Class C β-lactamase and Class D β-lactamase OXA-48-like;

[0010] (3) Relebactam (REL): molecular formula is C 12 H 20 N 4 O 6 S; CAS number is 1174018-99-5; a new generation of β-lactamase inhibitors, as a potent and selective β-lactamase inhibitor, can tightly bind to β-lactamase and prevent its hydrolysis, thereby restoring the bactericidal effect of antibiotics on drug-resistant bacteria. It can protect the activity of antibiotics against Enterobacteriaceae that produce class A β-lactamase (KPC enzyme, etc.) and class C β-lactamase;

[0011] (4) 2,6-pyridinedicarboxylic acid (DPA): molecular formula is C 7 H 3 NO 4 , CAS No. 499-83-2, is a potent metal chelator that inhibits class B carbapenemases (metalloenzymes).

[0012] Based on the above technical solution, the present invention can also be improved as follows.

[0013] Furthermore, the kit comprises imipenem, avibactam sodium, relebactam and 2,6-pyridinedicarboxylic acid.

[0014] Furthermore, the mass ratio of the imipenem, the avibactam sodium, the relebactam and the 2,6-pyridinedicarboxylic acid is: 1.8-7.2: 3.6-14.4: 3.2-12.8: 87.5-350.

[0015] Further, the kit includes LB broth, and the amount of the LB broth is not less than 12 ml.

[0016] In a second aspect, a kit for rapidly identifying the carbapenemase type of bacteremia in Enterobacteriaces bacteria producing carbapenemase is provided, characterized in that each reagent in the kit for identifying the carbapenemase type of Enterobacteriaces bacteria is used to prepare a detection reagent for identifying the carbapenemase type of Enterobacteriaces bacteria.

[0017] Furthermore, each reagent in the kit for rapid identification of carbapenemase enzyme types of bacteremia in carbapenemase-producing Enterobacteriales (CPE) is used to prepare a detection reagent for identifying carbapenemase enzyme types of Enterobacteriales bacteria through blood culture bacterial suspension.

[0018] Further, when the carbapenemase type of bacteremia in carbapenemase-producing Enterobacteriales (CPE) is rapidly identified, the imipenem and avibactam sodium in the kit for identifying the carbapenemase type of Enterobacteriales bacteria are added to the same LB broth, and the mass ratio of the imipenem to the avibactam sodium is 1.8-7.2:3.6-14.4; the LB broth containing imipenem and avibactam sodium is mixed with a blood culture bacterial suspension (the bacterial inoculum is about 1.5×10 8 The volume ratio of 1:1 was 3:1.

[0019] Further, when the carbapenemase type of bacteremia in carbapenemase-producing Enterobacteriales (CPE) is rapidly identified, the imipenem and relebactam in the kit for identifying the carbapenemase type phenotype of Enterobacteriales bacteria are added to the same LB broth, and the mass ratio of the imipenem to the relebactam is 1.8-7.2:3.2-12.8; the LB broth containing imipenem and relebactam is mixed with a blood culture suspension (the bacterial inoculum is about 1.5×10 8 The volume ratio of 1:1 was 3:1.

[0020] Further, when the carbapenemase phenotype of Enterobacteriaceae bacteria is identified, the imipenem and 2,6-pyridinedicarboxylic acid in the kit for identifying the carbapenemase type of Enterobacteriaceae bacteria are added to the same LB broth, and the mass ratio of the imipenem to the 2,6-pyridinedicarboxylic acid is 1.8-7.2:87.5-350; the LB broth containing imipenem and 2,6-pyridinedicarboxylic acid is mixed with a blood culture bacterial suspension (the bacterial inoculum is about 1.5×10 8 The volume ratio of 1:1 was 3:1.

[0021] Furthermore, the Enterobacteriales bacterial carbapenemase type includes class A carbapenemase, class B carbapenemase (metalloenzyme), class C carbapenemase and class D carbapenemase OXA-48-like.

[0022] The present invention adopts a detection method in which different substances (relebactam, avibactam, 2,6-pyridinedicarboxylic acid and imipenem, etc.) are added to the bacterial suspension after the blood culture is positive, and the absorbance of the bacterial suspension in the culture is measured after culturing for 1 hour (200 μl per well, 630 nm). If the absorbance is ≥0.115, the centrifuge tube is judged to be "+", and if the absorbance is <0.115, the centrifuge tube is judged to be "-". If the incubation time is 1.5 hours, the turbidity of each centrifuge tube is observed with the naked eye, turbidity is "+", and clarity is "-". When the naked eye cannot determine turbidity / clarity, the absorbance can also be measured to determine, and then the rapid detection (1 to 1.5 hours) is identified as CPE bacteremia.

[0023] The beneficial effects of the present invention are as follows: compared with the existing traditional technology, the present invention has the advantages of fast speed (1-1.5h), high accuracy, few interference factors and low cost (<1$) for detecting CPE bacteremia carbapenemase enzyme types, and overcomes the disadvantages of traditional methods (colloidal gold method, mCIM, eCIM and APB-EDTA method) such as long time consumption (18-24h), complex molecular biology method operation, high price, easy mutation of drug-resistant genes, interference of human cells and only being able to detect genes but not judge the survival status of bacteria; the present invention can significantly improve the survival rate of patients infected with CPE bloodstream infection, shorten the hospitalization time, reduce the hospitalization expenses, and has great economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the experimental flow chart of the present invention;

[0025] Figure 2 This is a graph showing the experimental results of incubating 4 standard bacteria of the present invention for 1.5 hours;

[0026] Figure 3 The absorbance distribution diagram of 1h and 1.5h incubation was statistically analyzed for the present invention. In the results, "+" represents bacterial growth and "-" represents bacterial inhibition;

[0027] Figure 4 The absorbance determination graph after 1 hour of incubation + / - ROC AUC: 0.999 (95% CI: 0.997-1.000) and the absorbance determination graph after 1.5 hours of incubation + / - ROC AUC: 0.998 (95% CI: 0.995-1.000) of the present invention are shown. DETAILED DESCRIPTION

[0028] 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.

[0029] Example

[0030] 1. Experimental Materials and Reagents

[0031] 5ml centrifuge tube, 10ml centrifuge tube, 10ml syringe, disposable sterile swab, glass tube, turbidimeter, microplate reader, 96-well plate, pipette, shaking bed, microbalance, test tube rack;

[0032] Imipenem (Solebau), avibactam sodium (Solebau), relebactam (McLean), 2,6-pyridinedicarboxylic acid (Aladdin), LB broth (Solebau), positive blood culture (Thermo Fisher blood culture bottle).

[0033] 2. Experimental methods

[0034] 2.1 Identification methods

[0035] A method for identifying the carbapenemase phenotype of Enterobacteriales bacteria, comprising the following steps: Figure 1 ):

[0036] (1) Use a microbalance to weigh and prepare 5 mg / ml 2,6-pyridinedicarboxylic acid solution (stored at room temperature for a long time), 10 mg / ml avibactam sodium solution (stored at 4°C for a long time), 10 mg / ml relebactam solution (stored at 4°C for a long time), and 5 mg / ml imipenem solution (prepare and use immediately, store at 4°C and use within 24 hours).

[0037] (2) Take out the blood culture bottle that is positive after incubation, use a 10 ml syringe to extract 10 ml of positive blood culture into a sterile 10 ml centrifuge tube, and centrifuge at 1500 rpm (403×g) for 5 minutes.

[0038] (3) Use a pipette to aspirate the supernatant after centrifugation (do not aspirate the blood cell pellet at the bottom) into another 10 ml centrifuge tube and centrifuge at 2500 rpm (1118 × g) for 10 minutes.

[0039] (4) Discard the supernatant and add 0.5 ml to 1 ml of LB broth to the centrifuge tube (adjust according to the amount of white bacterial sediment at the bottom of the centrifuge tube). Use a pipette to gently blow the bacterial sediment at the bottom (try to avoid blowing the red blood clots) until the bacterial suspension is obtained.

[0040] (5) Add 4-5 ml of LB broth to the glass tube, add an appropriate amount of the bacterial suspension in the centrifuge tube, and adjust the turbidity to 2 McFarland (when adjusting the turbidity to 2 McFarland, be careful to eliminate the interference of the LB broth background color).

[0041] (6) Take out four 5 ml sterile centrifuge tubes and label them as 1, 2, 3, and 4. Add 1 ml of 2 McFarland turbidity LB broth bacterial suspension to each centrifuge tube. Then add 3 ml of LB broth to each centrifuge tube to dilute the LB broth bacterial suspension to 0.5 McFarland turbidity.

[0042] (7) Add 3.2 μl of 10 mg / ml relebactam solution to centrifuge tube No. 2; add 175 μl of 2,6-pyridinedicarboxylic acid solution to centrifuge tube No. 3; add 3.6 μl of 10 mg / ml avibactam sodium solution to test tube No. 4. Finally, add 3.6 μl of 5 mg / ml imipenem solution to centrifuge tubes No. 2, 3, and 4.

[0043] (8) Place four centrifuge tubes in a shaking bed and incubate at 200 rpm.

[0044] 2.2 Interpretation of results

[0045] If the incubation time is 1h, the absorbance of the bacterial suspension in each centrifuge tube can be measured (200μl per well, 630nm). If the absorbance is ≥0.115, the centrifuge tube is judged as "+", and if the absorbance is <0.115, the centrifuge tube is judged as "-". If the incubation time is 1.5h, the turbidity of each centrifuge tube is observed with the naked eye, turbid is "+" and clear is "-". When the turbidity / clarity cannot be determined by the naked eye, the absorbance can also be measured to make a judgment.

[0046] 2.3 Interpretation principles

[0047] Phenotypic interpretation principles for identification of carbapenemase types in Enterobacteriales bacteria:

[0048] ① The results of centrifuge tubes 1 and 3 are “+”, and those of tubes 2 and 4 are “-”, indicating that the isolate may produce class A carbapenemase (such as KPC, GES, IMI, etc.);

[0049] ② No. 1, No. 2, and No. 4 centrifuge tubes are "+", and No. 3 test tube is "-", indicating that the isolate may produce Class B carbapenemase (such as NDM, IMP, VIM, etc.);

[0050] ③ Test tubes 1, 2, and 3 are “+”, and test tube 4 is “-”, indicating that the isolate may produce class D carbapenemase OXA-48-like (such as OXA-48, OXA-181, OXA-232);

[0051] ④ The number 1 centrifuge tube is “+”, and the number 2 centrifuge tube is “-”, indicating that the isolate may be carbapenemase negative;

[0052] ⑤ When all four test tubes are "+", the isolate may have other resistance mechanisms, such as overexpression of AmpC enzyme combined with loss of membrane pore protein or co-production of multiple different types of carbapenemases;

[0053] ⑥ When test tube No. 1 is judged as “-”, no interpretation can be made on the result.

[0054] 3. Experimental strains

[0055] The experimental strains included: 105 strains of Klebsiella pneumoniae, 54 strains of Escherichia coli, 28 strains of Enterobacter cloacae complex (Enterobacter agglomerans, Enterobacter hallii, Enterobacter kobe or Enterobacter cloacae), 9 strains of Klebsiella aerogenes, 8 strains of Klebsiella oxytoca, 4 strains of Citrobacter freudii, 2 strains of Pantoea agglomerans, 1 strain of Pantoea dispersae and 1 strain of Citrobacter braunii.

[0056] The strains contained the following genotypes: 41 strains of class A carbapenemases (KPC-2, KPC-33); 81 strains of class B carbapenemases (NDM-1, NDM-3, NDM-4, ​​NDM-5, NDM-16b, NDM-51, IMP-4); 6 strains of class D carbapenemases OXA-48-like (OXA-48, OXA-181, OXA-232); 7 strains that produced both class A and class B carbapenemases (KPC-2+IMP-4, KPC-2+NDM-1). 77 strains were carbapenemase negative;

[0057] Note: Except for the standard strains, all other strains were isolated from infected patients in the Second Affiliated Hospital of Nanchang University. The detection rate of clinical Enterobacteriaceae in my country ranks first, accounting for >50%. Among them, carbapenem-resistant Enterobacteriaceae (CRE) has been increasing year by year, especially carbapenem-resistant Klebsiella pneumoniae (CRKP), with a detection rate as high as 23.6%. Such strains are very easy to obtain "China Bacterial Resistance Monitoring Network (CHINET 2023), https: / / www.chinets.com / Data / AntibioticDrugFast".

[0058] It includes 4 standard strains of Klebsiella pneumoniae: ATCC 1705 (producing KPC), ATCC 1706 (carbapenemase negative), ATCC 2146 (producing NDM) and ACTT 2524 (producing OXA-48) strains purchased from the American Type Culture Collection (ATCC).

[0059] 4. Experimental Results

[0060] The results of incubation for 1 hour were as follows: the detection results of class A carbapenemase were 97.56% (40 / 41), the detection results of class B carbapenemase were 100% (81 / 81), and the detection results of class D carbapenemase OXA-48-like were 83.33% (5 / 6) ( Figures 2 to 4 ).

[0061] The results of incubation for 1.5 hours were as follows: the detection results of class A carbapenemase were 97.56% (40 / 41), the detection results of class B carbapenemase were 100% (81 / 81), and the detection results of class D carbapenemase OXA-48-like were 100% (6 / 6) ( Figures 2 to 4 ).

[0062] In summary, compared with the existing traditional technology, the detection of CPE bacteremia carbapenemase type by the present invention has the advantages of fast speed (1 to 1.5 hours), high accuracy, few interference factors and low cost (<1 US dollar), etc., which overcomes the disadvantages of traditional methods (colloidal gold method, mCIM, eCIM and APB-EDTA method) that are time-consuming (18 to 24 hours), complex molecular biology methods, high prices, easy mutation of drug-resistant genes, interference from human cells, and only being able to detect genes but not determine the survival status of bacteria. The present invention can significantly improve the survival rate of patients infected with CPE bacteremia, shorten the length of hospital stay, reduce hospitalization costs, and has great economic and social benefits.

[0063] 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. A kit for rapid identification of carbapenemase enzyme types in bacteremia of carbapenemase-producing Enterobacteriales, characterized in that: The kit comprises imipenem and any one of avibactam sodium, relebactam and 2,6-pyridinedicarboxylic acid.

2. A kit for rapidly identifying carbapenemase enzyme types of bacteremia in Enterobacteriales bacteria producing carbapenemase according to claim 1, characterized in that: The kit comprises imipenem, avibactam sodium, relebactam and 2,6-pyridinedicarboxylic acid.

3. A kit for rapidly identifying carbapenemase enzyme types of bacteremia in Enterobacteriales bacteria producing carbapenemase according to claim 2, characterized in that: The mass ratio of the imipenem, the avibactam sodium, the relebactam and the 2,6-pyridinedicarboxylic acid is: 1.8-7.2: 3.6-14.4: 3.2-12.8: 87.5-350.

4. A kit for rapidly identifying carbapenemase enzyme types of bacteremia in carbapenemase-producing Enterobacteriales according to claim 1, characterized in that: The kit comprises LB broth, and the amount of the LB broth is not less than 12 ml.

5. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia in Enterobacteriales bacteria producing carbapenemase, characterized in that: The reagents in the kit for rapidly identifying the carbapenemase type of bacteremia in Enterobacteriaces bacteria according to any one of claims 1 to 4 are used to prepare a detection reagent for identifying the carbapenemase type of Enterobacteriaces bacteria.

6. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia in Enterobacteriales bacteria producing carbapenemase according to claim 5, characterized in that: The reagents in the kit for rapidly identifying the carbapenemase type of bacteremia in Enterobacteriaceae bacteria producing carbapenemase are used to prepare a detection reagent for identifying the carbapenemase type of Enterobacteriaceae bacteria through blood culture bacterial suspension.

7. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia in Enterobacteriales bacteria producing carbapenemase according to claim 5, characterized in that: When identifying the carbapenemase type of Enterobacteriales bacteria, the imipenem and avibactam sodium in the kit for identifying the carbapenemase type of Enterobacteriales bacteria are added to the same LB broth, and the mass ratio of the imipenem to the avibactam sodium is 1.8-7.2:3.6-14.4; the volume ratio of the LB broth containing imipenem and avibactam sodium to the blood culture bacterial suspension is 3:

1.

8. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia in carbapenemase-producing Enterobacteriales according to claim 5, characterized in that: When identifying the carbapenemase phenotype of Enterobacteriales bacteria, the imipenem and relebactam in the kit for identifying the carbapenemase phenotype of Enterobacteriales bacteria are added to the same LB broth, and the mass ratio of the imipenem to the relebactam is 1.8-7.2:3.2-12.8; the volume ratio of the LB broth containing imipenem and relebactam to the blood culture bacterial suspension is 3:

1.

9. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia in carbapenemase-producing Enterobacteriales according to claim 5, characterized in that: When identifying the carbapenemase phenotype of Enterobacteriales bacteria, the imipenem and 2,6-pyridinedicarboxylic acid in the kit for identifying the carbapenemase phenotype of Enterobacteriales bacteria are added to the same LB broth, and the mass ratio of the imipenem to the 2,6-pyridinedicarboxylic acid is 1.8-7.2:87.5-350; the volume ratio of the LB broth containing imipenem and 2,6-pyridinedicarboxylic acid to the blood culture bacterial suspension is 3:

1.

10. Use of a kit for rapid identification of carbapenemase enzyme types of bacteremia of carbapenemase-producing Enterobacteriales according to any one of claims 5 to 9, characterized in that: The Enterobacteriales bacterial carbapenemase types include class A carbapenemase, class B carbapenemase (metalloenzyme), class C carbapenemase and class D carbapenemase OXA-48-like.

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