Kit and method for detecting sensitivity of carbapenem-resistant enterobacteriaceae bacteria

Abstract

The invention discloses a carbapenem-resistant enterobacteriaceae bacterium sensitivity detection kit and a detection method, the detection kit comprises a reaction liquid, a drug sensitivity detection plate, a first group of antibiotics, a second group of antibiotics and a third group of antibiotics, the drug sensitivity detection plate is provided with a positive hole, a negative hole, a first experiment group hole, a second experiment group hole and a third experiment group hole; the first group of antibiotics is embedded in the first experimental group hole in a manner that the mass is in gradient change, and the first group of antibiotics is epracycline; the second group of antibiotics is embedded in the second experimental group holes in a mass gradient change manner, and the second group of antibiotics is ceftazidime; the third group of antibiotics is embedded in the holes of the third experimental group in a mass gradient change manner, and the third group of antibiotics is a combination of ceftazidime and aztreonam; the reaction liquid comprises glucose and a pH indicator. The detection kit provided by the invention can accurately and rapidly detect the sensitivity of CRE to CZA, CZA + ATM and ERV.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial detection, and in particular relates to a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit and a detection method. Background Art

[0002] Bacterial resistance has become a global concern, and infections caused by resistant bacteria have become a new challenge for clinical anti-infection treatment. Enterobacteriaceae are widely distributed and closely related to humans. Enterobacteriaceae are important nosocomial infections. Carbapenem antibiotics are the main antibiotics for the treatment of Enterobacteriaceae infections. However, in recent years, with the extensive use of carbapenem antibiotics, carbapenem-resistant Enterobacteriaceae (CRE) have appeared clinically. CRE is usually divided into two types: CRE that produces carbapenemase (CP-CRE) and CRE that does not produce carbapenemase (non-CP-CRE). Specifically, the common CRE that produces carbapenemase in clinical practice are mainly KPC type, NDM type, etc.

[0003] At present, tigecycline is often used in the clinic to treat infections of carbapenem-resistant Enterobacteriaceae (CRE). However, due to the toxic side effects of tigecycline, it is often not used in large doses for a long time in the clinic. Moreover, as one of the last lines of defense for anti-infection treatment, tigecycline is also used with caution in the clinic. In addition to tigecycline, there are also ceftazidime / avibactam (CZA), ceftazidime / avibactam + aztreonam (CZA + ATM), and eracycline (ERV). Since ceftazidime / avibactam only has an antibacterial effect on common KPC type (such as KPC-2 type) CRE, when KPC gene mutant strains appear, bacteria will be resistant to ceftazidime / avibactam, which will lead to erroneous results in some detection methods, such as detecting the KPC gene by PCR to determine the sensitivity of bacteria to ceftazidime / avibactam. When ceftazidime / avibactam-resistant strains appear, ceftazidime / avibactam + aztreonam can often be used for combined treatment in clinical practice. However, studies have found that some NDM-type CRE strains are resistant to ceftazidime / avibactam + aztreonam, and the resistance mechanism is not yet clear. This has led to the fact that the method of determining the sensitivity of bacteria to ceftazidime / avibactam + aztreonam by detecting NDM genes is not very accurate. Eracycline is considered to be one of the effective methods for treating CRE and carbapenem-resistant Acinetobacter baumannii. With its widespread clinical use, bacteria resistant to eracycline are a threat that cannot be ignored. However, in addition to bacteria expressing Tet(X) family proteins that are resistant to eracycline, other resistance mechanisms are still unclear.

[0004] Due to the limitations of methods such as PCR and mass spectrometry, existing methods cannot detect all drug-resistant phenotypes. For example, some KPC gene mutant strains, NDM-type strains, and strains with efflux pump-mediated resistance are resistant to ceftazidime / avibactam, ceftazidime / avibactam + aztreonam, and even elacycline. At present, the sensitivity of carbapenem-resistant Enterobacteriaceae to ceftazidime / avibactam, ceftazidime / avibactam + aztreonam, and elacycline is mainly detected by minimum inhibitory concentration (MICs), disk diffusion method (KB method) and E-test method. However, traditional MICs and KB methods require 12-24 hours to interpret the results, which will delay the time for feedback to clinical drug sensitivity results. Despite this, laboratories still generally use these three methods to detect the sensitivity of carbapenem-resistant Enterobacteriaceae to antibiotics, because MICs, KB method and E-test method are reliable methods to ensure the accuracy of the results.

[0005] Therefore, there is an urgent need for a carbapenem-resistant Enterobacteriaceae sensitivity detection kit and detection method to solve the deficiencies of the existing technical problems. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a carbapenem-resistant Enterobacteriaceae sensitivity detection kit and detection method, which can quickly and accurately detect the sensitivity of carbapenem-resistant Enterobacteriaceae to ceftazidime / avibactam, ceftazidime / avibactam+aztreonam, and elacycline.

[0007] To achieve the above objectives, the first aspect of the present invention provides a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit, comprising a reaction solution, a drug sensitivity detection plate, a first group of antibiotics, a second group of antibiotics, and a third group of antibiotics, the drug sensitivity detection plate having positive wells, negative wells, first experimental group wells, second experimental group wells, and third experimental group wells; the first group of antibiotics are embedded in the first experimental group wells in a gradient mass change manner, and the first group of antibiotics are elacycline; the second group of antibiotics are embedded in the second experimental group wells in a gradient mass change manner, the second group of antibiotics are ceftazidime, and avibactam is also embedded in the second experimental group wells; the third group of antibiotics are embedded in the third experimental group wells in a gradient mass change manner, the third group of antibiotics are a combination of ceftazidime and aztreonam, and avibactam is also embedded in the third experimental group wells; the reaction solution comprises peptone, beef extract, sodium chloride, glucose, pH indicator, and sterile water.

[0008] Further, the present invention mixes the suspension of carbapenem-resistant Enterobacteriaceae to be detected and the reaction solution to obtain the liquid to be detected, and then adds the liquid to be detected to the first experimental group well, the second experimental group well, and the third experimental group well, respectively. Each group of antibiotics includes a sensitivity breakpoint concentration and a resistance breakpoint concentration. The sensitivity breakpoint concentration is the sensitivity breakpoint of the antibiotic established by the Clinical Laboratory Standards Institute (CLSI) of the United States and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the resistance breakpoint concentration is the resistance breakpoint of the antibiotic established by the Clinical Laboratory Standards Institute (CLSI) of the United States and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Specifically, the latest CLSI and EUCAST guidelines should be referred to to ensure the use of the most accurate breakpoint data.

[0009] Further, according to the 2024 version of the CLSI and EUCAST guidelines, the first group of antibiotics of the present invention includes a first sensitivity breakpoint concentration of 0.5 mg / L, a second sensitivity breakpoint concentration of 1 mg / L, and a resistance breakpoint concentration of 2 mg / L. Specifically, for the first group of antibiotics (eracycline), the sensitivity breakpoint of Escherichia coli is 0.5 mg / L; the breakpoint of Klebsiella pneumoniae is 1 mg / L.

[0010] Further, according to the 2024 edition of the CLSI and EUCAST guidelines, the second group of antibiotics of the present invention includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L.

[0011] Further, according to the 2024 edition of the CLSI and EUCAST guidelines, the third group of antibiotics of the present invention includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L.

[0012] Further, the concentration of avibactam of the present invention is 4 mg / L. Avibactam itself is not an independent antibacterial drug, but a β-lactamase inhibitor, which is usually used in combination with ceftazidime antibiotics to form a compound drug ceftazidime / avibactam (abbreviated as CZA), which is designed to fight some drug-resistant bacteria that produce β-lactamases, which can decompose and render β-lactam antibiotics (such as penicillin and cephalosporin) ineffective.

[0013] Furthermore, the reaction solution of the present invention includes, by weight, 10 to 15 parts of peptone, 3 to 5 parts of beef extract, 5 to 7 parts of sodium chloride, 10 to 15 parts of glucose, 0.01 parts of pH indicator, and 1000 parts of sterile water.

[0014] Furthermore, the reaction solution of the present invention includes, by mass, 10 parts of peptone, 3 parts of beef extract, 5 parts of sodium chloride, 10 parts of glucose, 0.01 parts of pH indicator, and 1000 parts of sterilized water.

[0015] Further, the pH indicator of the present invention is selected from any one of methyl red, purple litmus, bromothymol blue and bromocresol purple. Preferably, the pH indicator is selected from bromocresol purple.

[0016] Furthermore, the carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit of the present invention also includes a container for storing the reaction solution.

[0017] Accordingly, the second aspect of the present invention also provides a detection method of a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit, comprising the steps of:

[0018] (1) Add the reaction solution to the negative wells;

[0019] (2) mixing the reaction solution and the suspension of carbapenem-resistant Enterobacteriaceae to be detected to obtain a solution to be detected, and then adding the solution to be detected to the positive wells, the wells of the first experimental group, the wells of the second experimental group, and the wells of the third experimental group, respectively;

[0020] (3) The drug sensitivity test plate was incubated at 35-37°C for 4.0-4.5 hours, and the color changes in each well were observed to determine the sensitivity of the carbapenem-resistant Enterobacteriaceae to each group of antibiotics.

[0021] Furthermore, in step (2) of the present invention, the concentration of carbapenem-resistant Enterobacteriaceae in the test solution is 1.5×10 6 CFU / mL.

[0022] The mechanism of the detection kit of the present invention for detecting the sensitivity of carbapenem-resistant Enterobacteriaceae to ceftazidime / avibactam, ceftazidime / avibactam+aztreonam, and elacycline is as follows: carbapenem-resistant Enterobacteriaceae (CRE) decompose glucose to produce acidic substances during growth, thereby causing the pH value of the reaction solution to decrease, and this change can be intuitively displayed by the color change of the pH indicator in the reaction solution. Specifically, when CRE shows resistance to a certain antibiotic, it can proliferate normally in the well containing the antibiotic, and the acidic environment generated by its metabolic activity will cause the color of the pH indicator to change significantly. On the contrary, if CRE is sensitive to a certain antibiotic, its growth will be effectively inhibited, and the pH value in the well and the color of the pH indicator will remain unchanged.

[0023] The beneficial effects of the present invention are:

[0024] (1) As the number of people infected with carbapenem-resistant bacteria continues to increase, the importance of CZA, CZA+ATM, and ERV becomes increasingly prominent. The detection kit of the present invention can quickly detect the sensitivity of CRE to CZA, CZA+ATM, and ERV, thereby improving clinical outcomes and accelerating microbial diagnosis.

[0025] (2) The detection kit of the present invention can determine the test result only based on the color change, does not require special instruments and equipment, and is simple to operate and easy to use.

[0026] (3) The detection kit of the present invention can greatly shorten the detection time. At the same time, as long as the accuracy of the antibiotic concentration is ensured, the detection kit of the present invention can provide highly reliable detection results, which buys valuable time for the correct clinical selection of antibacterial drugs for prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic structural diagram of a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to an embodiment of the present invention is shown.

[0028] Figure 2 for Figure 1 A schematic top view of a carbapenem-resistant Enterobacteriaceae susceptibility detection kit is shown. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be described clearly and completely below in conjunction with the drawings in the specification. It is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0030] Please see Figure 1-2 The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit 100 of the present invention comprises a reaction solution, a drug sensitivity detection plate 11, a first group of antibiotics, a second group of antibiotics, and a third group of antibiotics, wherein the drug sensitivity detection plate has a positive well 111, a negative well 112, a first experimental group well 113, a second experimental group well 114, and a third experimental group well 115; the first group of antibiotics are embedded in the first experimental group well 113 in a gradient manner, and the first group of antibiotics are elacycline; the second group of antibiotics are embedded in the second experimental group well 114 in a gradient manner, and the second group of antibiotics are ceftazidime, and avibactam is also embedded in the second experimental group well; the third group of antibiotics are embedded in the third experimental group well 115 in a gradient manner, and the third group of antibiotics are a combination of ceftazidime and aztreonam, and avibactam is also embedded in the third experimental group well; the reaction solution comprises peptone, beef extract, sodium chloride, glucose, pH indicator, and sterile water. Specifically, the pH value of sterile water is 7.2±0.4.

[0031] For details, please continue to refer to Figure 1The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit 100 of the present invention further includes a container 12 for storing the reaction solution, such as a plastic bottle with a cover. The pH indicator is selected from any one of methyl red, purple litmus, bromothymol blue, and bromocresol purple; preferably, the pH indicator is selected from bromocresol purple, and the color of bromocresol purple gradually changes from yellow to purple as the pH value increases from 5.2 to 6.8. More specifically, the reaction solution includes 10-15 parts of peptone, 3-5 parts of beef extract, 5-7 parts of sodium chloride, 10-15 parts of glucose, 0.01 parts of pH indicator, and 1000 parts of sterile water; for example, the peptone content may be but not limited to 10 parts, 12 parts, 14 parts, and 15 parts, the beef extract content may be but not limited to 3 parts, 4 parts, and 5 parts; the sodium chloride content may be but not limited to 5 parts, 6 parts, and 7 parts; the glucose content may be but not limited to 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, and 15 parts. Preferably, the reaction solution includes 10 parts of peptone, 3 parts of beef extract, 5 parts of sodium chloride, 10 parts of glucose, 0.01 parts of pH indicator, and 1000 parts of sterile water. It is necessary to explain that yeast cannot be added to the reaction solution of the present invention, because yeast will decompose glucose to produce acid during growth and metabolism, resulting in a decrease in the pH of the solution, causing the solution to change from purple to yellow, resulting in a false positive experimental result.

[0032] Furthermore, during use, the suspension of carbapenem-resistant Enterobacteriaceae to be tested and the reaction solution are mixed to obtain a liquid to be tested, and then the liquid to be tested is added to the first experimental group hole 113, the second experimental group hole 114, and the third experimental group hole 115, respectively. Each group of antibiotics includes a sensitivity breakpoint concentration and a resistance breakpoint concentration. The sensitivity breakpoint concentration and the resistance breakpoint concentration are both the sensitivity breakpoint and resistance breakpoint of the antibiotic established by the Clinical Laboratory Standards Institute (CLSI) of the United States and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). More specifically, according to the 2024 version of the CLSI and EUCAST guidelines, the first group of antibiotics includes a first sensitivity breakpoint concentration of 0.5 mg / L, a second sensitivity breakpoint concentration of 1 mg / L, and a resistance breakpoint concentration of 2 mg / L; the second group of antibiotics includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L; the third group of antibiotics includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L. More specifically, the concentration of avibactam is 4 mg / L. As an example, Figure 2 As shown, the first experimental group of holes 113 of the present invention includes B2 hole, A3 hole, and B3 hole, the second experimental group of holes 114 includes A4 hole and B4 hole, and the third experimental group of holes 115 includes A5 hole and B5 hole; the positive hole 111 includes A2 hole, and the negative hole 112 includes A1 hole and B1 hole.

[0033] Please continue to refer to Figure 2 The steps of the detection method of the carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit of the present invention include: (1) adding the reaction solution to the negative well 112; (2) mixing the reaction solution and the suspension of carbapenem-resistant Enterobacteriaceae bacteria to be detected to obtain the test solution, and then adding the test solution to the positive well 111, the first experimental group well 113, the second experimental group well 114, and the third experimental group well 115 respectively; (3) placing the drug sensitivity detection plate 11 at 36-45°C for 3.5-4.5h, observing the color changes in each well, and judging the sensitivity of carbapenem-resistant Enterobacteriaceae bacteria to each group of antibiotics. Furthermore, in step (2) of the present invention, the concentration of carbapenem-resistant Enterobacteriaceae bacteria in the test solution is 1.5×10 6 CFU / mL.

[0034] The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit and detection method thereof of the present invention are described in more detail below based on specific Example 1.

[0035] Example 1

[0036] The present embodiment provides a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit, including a plastic bottle, a drug sensitivity detection plate, a first group of antibiotics, a second group of antibiotics, and a third group of antibiotics, the plastic bottle contains 3.37 ml of reaction solution, and the drug sensitivity detection plate has A2 well, A1 well, B1 well, a first experimental group well, a second experimental group well, and a third experimental group well;

[0037] The first experimental group of wells includes well B2, well A3, and well B3, well B2 is embedded with 0.1 μg of eracycline, well A3 is embedded with 0.2 μg of eracycline, and well B3 is embedded with 0.4 μg of eracycline;

[0038] The wells in the second experimental group include well A4 and well B4. Well A4 is embedded with 1.6 μg ceftazidime and 0.8 μg avibactam, and well B4 is embedded with 3.2 μg ceftazidime and 0.8 μg avibactam.

[0039] The third experimental group of wells includes well A5 and well B5. Well A5 is embedded with 1.6μg ceftazidime, 0.8μg avibactam and 1.6μg aztreonam, and well B5 is embedded with 3.2μg ceftazidime, 0.8μg avibactam and 3.2μg aztreonam.

[0040] The reaction solution includes, by mass, 10 parts of peptone, 3 parts of beef extract, 5 parts of sodium chloride, 10 parts of glucose, 0.01 parts of bromocresol purple, and 1000 parts of sterile water.

[0041] This embodiment provides a detection method for a carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit, the steps comprising:

[0042] (1) Pipette the reaction solution from the plastic bottle, and then add 200 μL of the reaction solution to well A1 and well B1 respectively;

[0043] (2) 30 μL of the suspension of carbapenem-resistant Enterobacteriaceae to be tested (0.5 McFarland turbidity) was added to a plastic bottle and mixed evenly with 2.97 mL of the reaction solution to obtain the test solution (the concentration of carbapenem-resistant Enterobacteriaceae was 1.5×10 6 CFU / mL), and then add 200 μL of the test solution to wells A2, B2, A3, B3, A4, B4, A5, and B5 respectively;

[0044] (3) The drug sensitivity test plate was incubated at 37°C for 4 h, and the color changes in each well were observed to determine the sensitivity of the carbapenem-resistant Enterobacteriaceae to each group of antibiotics;

[0045] The criteria for interpreting the test results are:

[0046] The negative wells remain purple, and the positive wells change from purple to yellow; that is, wells A1 and B1 are both purple, and well A2 is yellow, otherwise the test results are invalid.

[0047] The breakpoint for Escherichia coli is 0.5 mg / L. When testing the sensitivity of carbapenem-resistant Escherichia coli to ERV, when wells B2, A3, and B3 do not change color, or when well B2 changes color, wells A3 and B3 do not change color, it is judged to be sensitive to ERV; when wells B2 and A3 change color, well B3 does not change color, or wells B2, A3, and B3 all change color, it should be judged to be resistant;

[0048] The breakpoint for Klebsiella pneumoniae is 1 mg / L. When testing the sensitivity of Klebsiella pneumoniae to ERV of carbapenems, when wells B2, A3, and B3 do not change color, or well B2 changes color, wells A3 and B3 do not change color, or wells B2 and A3 change color, and well B3 does not change color, it is judged as sensitive; when wells B2, A3, and B3 all change color, it should be judged as resistant.

[0049] When both wells A4 and B4 do not change color; or when well A4 changes color but well B4 does not change color, it should be judged as sensitive to CZA; when both wells A4 and B4 change color, it should be judged as resistant to CZA;

[0050] When both A5 and B5 do not change color, they should be judged as sensitive to CZA+ATM; when A4 changes color but B4 does not, they should be judged as intermediate to CZA+ATM; when both A4 and B4 change color, they should be judged as resistant to CZA+ATM.

[0051] Furthermore, the inventors of the present invention collected 160 carbapenem-resistant Enterobacteriaceae (CRE) samples from January 2021 to June 2024. Among them, the KPN142 strain carrying blaKPC-2 is sensitive to ceftazidime (CZA), elacycline (ERV) and ceftazidime / avibactam + aztreonam (CZA + ATM), and is used as a negative control strain; an NDM-type CRE strain resistant to CZA and CZA + ATM is used as a positive control strain; the ECO67 strain resistant to ERV constructed by the laboratory is used as a positive control strain for ERV sensitivity detection.

[0052] The sensitivity detection kit and detection method for carbapenem-resistant Enterobacteriaceae of Example 1 (referred to as MultiRapid CAE detection method) were used to rapidly detect the sensitivity of the above 160 CRE strains to CZA, CZA+ATM and ERV, so as to evaluate and verify the accuracy and reliability of the sensitivity detection kit and detection method for carbapenem-resistant Enterobacteriaceae of the present invention.

[0053] In addition, in order to further confirm the accuracy and reliability of the detection kit and the detection method of the present invention, the present invention also uses the extended site confirmation test method (eCIM), the Kirby-Bauer disk diffusion method (KB method) and the E-test method for comparison according to the 2024 version of the Clinical and Laboratory Standards Institute (CLSI) and the 2024 version of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Expert Guidelines, and the comparison results are shown in Table 1; all experimental steps are carried out in accordance with the requirements of the minimum inhibitory concentration (MIC) determination method, as follows:

[0054] Select a single clone of the strain and prepare it into a 0.5 McFarland turbidity bacterial suspension (1.5×10 8 CFU / mL).

[0055] Negative control group: 10 μL of 0.45% sterile saline was added to 990 μL of solution (glucose biochemical reaction solution).

[0056] Positive control group: add 10 μL of 0.5 McLaughlin suspension (final concentration of 1.5×10 6 CFU / mL).

[0057] Low-concentration CZA experimental group: 10 μL of 0.5 McLaughlin suspension, 5 μL of ceftazidime (1.6 mg / mL), and 5 μL of avibactam (0.8 mg / mL) were added to 980 μL of glucose solution.

[0058] High-concentration CZA experimental group: 10 μL of 0.5 McLaughlin suspension, 5 μL of ceftazidime (3.2 mg / mL), and 5 μL of avibactam (0.8 mg / mL) were added to 980 μL of glucose solution.

[0059] Low-concentration CZA+ATM experimental group: 10 μL of 0.5 McLaughlin suspension, 5 μL of ceftazidime (1.6 mg / mL), 5 μL of avibactam (0.8 mg / mL), and 10 μL of aztreonam (0.8 mg / mL) were added to 970 μL of glucose solution.

[0060] High concentration CZA+ATM experimental group: 10 μL of 0.5 McLaughlin suspension, 5 μL of ceftazidime (3.2 mg / mL), 5 μL of avibactam (0.8 mg / mL), and 20 μL of aztreonam (0.8 mg / mL) were added to 960 μL of glucose solution.

[0061] Low-concentration ERV experimental group: 10 μL of 0.5 McLaughlin suspension and 5 μL of eracycline (0.1 mg / mL) were added to 985 μL of glucose solution.

[0062] Medium concentration ERV experimental group: 10 μL of 0.5 McLaughlin suspension and 10 μL of eracycline (0.1 mg / mL) were added to 980 μL of glucose solution.

[0063] High-concentration ERV experimental group: 10 μL of 0.5 McLaughlin suspension and 20 μL of eracycline (0.1 mg / mL) were added to 970 μL of glucose solution.

[0064] The present invention detected a total of 160 CRE strains. The eCIM detection results showed that 48 CRE strains were NDM type, 95 were KPC type, and 17 were non-carbapenemase-producing strains.

[0065] The KB method results of CZA showed that 48 NDM-type, 24 KPC-type CRE strains and 9 non-carbapenemase-producing CRE strains were resistant to CZA (<21 mm), while 79 CRE strains (including 71 KPC-type CRE strains and 8 non-carbapenemase-producing CRE strains) were sensitive to CZA (>21 mm).

[0066] The KB results of CZA+ATM showed that 144 CRE strains were sensitive to CZA+ATM (the inhibition zone of aztreonam was >21mm); 16 NDM-type CRE strains were resistant to CZA+ATM (the inhibition zone of aztreonam was <17mm).

[0067] The results of the ERV E-test method showed that 143 CRE strains (23 Escherichia coli and 120 Klebsiella pneumoniae) were sensitive to ERV (Escherichia coli ≤ 0.5 mg / L; Klebsiella pneumoniae ≤ 1 mg / L).

[0068] In the CZA sensitivity test of the MultiRapid CAE method of the present invention, 48 NDM-type CRE strains, 24 KPC-type CRE strains and 9 non-carbapenemase-producing CRE strains (CZA inhibition zone less than 21 mm) showed positive results and were resistant; while 71 KPC-type CRE strains and 8 non-carbapenemase-producing CRE strains (CZA inhibition zone greater than 21 mm) showed negative results and were sensitive. As shown in Table 1 , the sensitivity, specificity, positive predictive value, negative predictive value and Cohen's Kappa reliability were 97.59% (95% CI: 90.76-99.58%), 97.41% (95% CI: 90.07-99.55%), 97.59% (95% CI: 90.76-99.58%), 97.41% (95% CI: 90.07-99.55%) and 0.95, respectively.

[0069] In the CZA+ATM sensitivity test of the MultiRapid CAE method of the present invention, 139 CRE strains were negative results, indicating sensitivity; 21 NDM-type CRE strains were positive results, indicating drug resistance. As shown in Table 1, the sensitivity, specificity, positive predictive value, negative predictive value, and Cohen's Kappa reliability were 100% (95% CI: 75.93-100%), 96.53% (95% CI: 91.07-98.62%), 76.19% (95% CI: 52.45-90.88%), 100% (95% CI: 96.65-1000%), and 0.85, respectively.

[0070] In the ERV sensitivity test of the MultiRapid CAE method of the present invention, 143 CRE strains showed negative results and were sensitive. As shown in Table 1, the specificity and negative predictive value were 100% (95% CI: 96.74-100%) and 100% (95% CI: 96.74-100%), respectively.

[0071] Table 1

[0072]

[0073] PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; K, Cohen's Kappa reliability; “-”, value cannot be calculated.

[0074] In summary, the MultiRapid CAE detection method of the present invention has good sensitivity, specificity and Cohen's Kappa reliability, and is a simple, efficient, sensitive and specific detection method, that is, the detection kit of the present invention can quickly and accurately detect the sensitivity of CRE to ceftazidime / avibactam, ceftazidime / avibactam+aztreonam, and eracycline.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit, characterized in that: It includes a reaction solution, a drug sensitivity test plate, a first group of antibiotics, a second group of antibiotics, and a third group of antibiotics, wherein the drug sensitivity test plate has positive wells, negative wells, first experimental group wells, second experimental group wells, and third experimental group wells; The first group of antibiotics is embedded in the first experimental group of wells in a gradient manner, and the first group of antibiotics is eracycline; The second group of antibiotics are embedded in the second experimental group wells in a manner of gradient mass change, the second group of antibiotics is ceftazidime, and the second experimental group wells also have avibactam embedded therein; The third group of antibiotics is embedded in the third experimental group wells in a manner of gradient mass change, the third group of antibiotics is a combination of ceftazidime and aztreonam, and avibactam is also embedded in the third experimental group wells; The reaction solution comprises peptone, beef extract, sodium chloride, glucose, a pH indicator and sterilized water.

2. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 1, characterized in that: A suspension of carbapenem-resistant Enterobacteriaceae bacteria to be detected and the reaction solution are mixed to obtain a liquid to be detected, and then the liquid to be detected is added to the first experimental group wells, the second experimental group wells, and the third experimental group wells, respectively. Each group of antibiotics includes a sensitivity breakpoint concentration and a resistance breakpoint concentration. The sensitivity breakpoint concentration is the sensitivity breakpoint of the antibiotic established by the American Clinical Laboratory Standards Association and the European Antimicrobial Susceptibility Testing Committee, and the resistance breakpoint concentration is the resistance breakpoint of the antibiotic established by the American Clinical Laboratory Standards Association and the European Antimicrobial Susceptibility Testing Committee.

3. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 2, characterized in that: The first group of antibiotics includes a first sensitivity breakpoint concentration of 0.5 mg / L, a second sensitivity breakpoint concentration of 1 mg / L, and a resistance breakpoint concentration of 2 mg / L.

4. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 2, characterized in that: The second group of antibiotics includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L.

5. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 2, characterized in that: The third group of antibiotics includes a sensitivity breakpoint concentration of 8 mg / L and a resistance breakpoint concentration of 16 mg / L.

6. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to any one of claims 4 to 5, characterized in that: The concentration of avibactam is 4 mg / L.

7. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 1, characterized in that: In parts by mass, the reaction solution includes 10 to 15 parts of peptone, 3 to 5 parts of beef extract, 5 to 7 parts of sodium chloride, 10 to 15 parts of glucose, 0.01 parts of pH indicator, and 1000 parts of sterile water.

8. The carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 1, characterized in that: The pH indicator is selected from any one of methyl red, purple litmus, bromothymol blue and bromocresol purple.

9. The detection method of the carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to any one of claims 1 to 8, characterized in that the steps include: (1) Add the reaction solution to the negative wells; (2) mixing the reaction solution and the suspension of carbapenem-resistant Enterobacteriaceae to be detected to obtain a solution to be detected, and then adding the solution to be detected to the positive wells, the wells of the first experimental group, the wells of the second experimental group, and the wells of the third experimental group, respectively; (3) The drug sensitivity test plate was incubated at 35-37°C for 4.0-4.5 hours, and the color changes in each well were observed to determine the sensitivity of the carbapenem-resistant Enterobacteriaceae to each group of antibiotics.

10. The detection method of the carbapenem-resistant Enterobacteriaceae bacteria sensitivity detection kit according to claim 9, characterized in that: The concentration of carbapenem-resistant Enterobacteriaceae in the test solution in step (2) is 1.5×10 6 CFU / mL.

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