Strain identification and drug sensitivity detection kit as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of strain identification and drug sensitivity detection, and discloses a strain identification and drug sensitivity detection kit and a preparation method and application thereof, the kit comprises a drug sensitivity disc, a pre-enrichment chromogenic culture medium, a chromogenic culture medium, sterile water and a diluent; the drug sensitive disc body comprises an enterobacteriaceae area, an enterococcus area, a staphylococcus area, a pseudomonas aeruginosa area and an acinetobacter baumannii area; the enterobacteriaceae area is provided with enterobacteriaceae identification culture medium micropores and drug micropores, the enterococcus area is provided with enterococcus identification culture medium micropores and drug micropores, the staphylococcus area is provided with staphylococcus identification culture medium micropores and drug micropores, and the pseudomonas aeruginosa area is provided with pseudomonas aeruginosa identification culture medium micropores and drug micropores. An acinetobacter baumannii identification culture medium micropore and a medicine micropore are matched in the acinetobacter baumannii area; the problems that an existing drug sensitivity test mode is complex in operation, high in cost and low in accuracy of identification and detection results are solved.

Description

Technical Field

[0001] This invention relates to the field of bacterial strain identification and antimicrobial susceptibility testing technology, specifically to a bacterial strain identification and antimicrobial susceptibility testing kit, its preparation method, and its application. Background Technology

[0002] With the frequent use of veterinary antibiotics, bacterial resistance has become an increasingly serious problem, and the widespread dissemination of drug-resistant strains has brought great difficulties to clinical treatment. Drug susceptibility testing is of great significance for the accurate use of antibiotics and the prevention of antibiotic abuse. Traditional drug susceptibility tests (such as the disk diffusion method and dilution method) have drawbacks such as complex operation, low detection efficiency, and strong subjectivity in interpretation, making them unsuitable for primary veterinary units and small pet hospitals. Automated instrument methods are convenient to operate, but are expensive and still unsuitable for primary veterinary units and small pet hospitals.

[0003] Chromogenic reagents in drug susceptibility testing can increase the sensitivity of the test and improve the speed of result interpretation. Patent CN108866152 A uses resazurin as a chromogenic reagent. This method requires co-incubating the bacterial culture and drug for 16 hours, then adding the resazurin solution and incubating for another 1.5 hours to check the results. This method is complex, cannot be completed in one step, and requires manual visual identification of the results. Patent CN 111304278 A uses EZMTT as a chromogenic reagent. This method requires an ELISA reader to detect the OD value, making it unsuitable for use in primary veterinary units. Patent CN 106701892 A uses TTC as a chromogenic reagent, but there are reports that TTC has some toxicity to Staphylococcus aureus, so TTC is not suitable as a Staphylococcus aureus drug susceptibility indicator and has certain limitations. Currently, commercially available drug susceptibility kits, such as the Beta-Bacterium and Yuge drug susceptibility kits, can be used in animal clinical testing, but neither of these kits can identify bacterial strains, and the drug susceptibility results they provide are inaccurate. Summary of the Invention

[0004] The present invention aims to provide a bacterial strain identification and drug susceptibility testing kit, its preparation method and application, to solve the problems of complex operation, high cost and low accuracy of identification and detection results in existing drug susceptibility testing methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A bacterial species identification and antimicrobial susceptibility testing kit, the kit comprising an antimicrobial susceptibility plate, pre-enrichment chromogenic medium, chromogenic medium, sterile water, and diluent; the antimicrobial susceptibility plate comprises a plate body, the plate body including an Enterobacteriaceae region, an Enterococcus spp. region, a Staphylococcus spp. region, a Pseudomonas aeruginosa region, and an Acinetobacter baumannii region; the Enterobacteriaceae region is configured with Enterobacteriaceae identification medium microwells and drug microwells, the Enterococcus spp. region is configured with Enterococcus spp. identification medium microwells and drug microwells, the Staphylococcus spp. region is configured with Staphylococcus spp. identification medium microwells and drug microwells, the Pseudomonas aeruginosa region is configured with Pseudomonas aeruginosa identification medium microwells and drug microwells, and the Acinetobacter baumannii region is configured with Acinetobacter baumannii identification medium microwells and drug microwells.

[0007] Furthermore, the pre-enrichment chromogenic medium comprises 1-5 g / 100 ml beef extract powder, 10-20 g / 100 ml acid-hydrolyzed casein, 5-10 g / 100 ml trypsin, 2-5 g / 100 ml sodium pyruvate, and 0.02-0.1 g / 100 ml chromogenic reagent WST-8.

[0008] Furthermore, the identification media for Enterobacteriaceae includes tryptone, yeast extract, sodium chloride, glucose, ferric ammonium citrate, ox bile salts, vancomycin, and bromocresol purple; the identification media for Enterococcus includes tryptone, yeast extract, beef extract, sodium pyruvate, magnesium sulfate, sodium citrate, aztreonam, nalidixic acid, 5-bromo-6-chloro-3-indole phosphate p-toluidine, and 5-bromo-6-chloro-3-indole-glucopyranoside; the identification media for Staphylococcus includes Mueller Hinton broth, sodium pyruvate, magnesium sulfate, ferric ammonium citrate, potassium tellurite, aztreonam, bacitracin, 5-bromo-4-chloro-3-indole-β-D-glucopyranoside, and 5-bromo-4-chloro-3-indole phosphate p-toluidine; and the identification media for Pseudomonas aeruginosa includes tryptone, yeast extract, sodium chloride, glucose, and 1M... HCl, ox bile salts, hexadecyltrimethylammonium bromide, cefazolin, bromocresol purple; Acinetobacter baumannii identification medium includes tryptone, casein peptone, sodium chloride, glucose, magnesium sulfate, sodium deoxycholate, ox bile salts, vancomycin, trimethoprim, and p-toluidine 5-bromo-6-chloro-3-indole phosphate.

[0009] Furthermore, the chromogenic culture medium comprises 5-10 g / 100 ml beef extract, 15-20 g / 100 ml acid-hydrolyzed casein, 2-5 g / 100 ml tryptone, 2-5 g / 100 ml sodium pyruvate, 0.01-0.03 g / 100 ml calcium ions, 0.01-0.03 g / 100 ml magnesium ions, 0.1-0.6 g / 100 ml 50% egg yolk emulsion, and 0.02-0.1 g / 100 ml WST-8 chromogenic reagent.

[0010] Furthermore, drugs for Enterobacteriaceae include ampicillin, amoxicillin / clavulanic acid, cefoperazone, cephalexin, tetracycline, trimethoprim / sulfamethoxazole, enrofloxacin, ciprofloxacin, and ofloxacin; drugs for Enterococcus include penicillin, ampicillin, amoxicillin / clavulanic acid, tetracycline, doxycycline, erythromycin, vancomycin, ciprofloxacin, and levofloxacin; and drugs for Staphylococcus include penicillin, azithromycin, and gentamicin. Drugs for *Pseudomonas aeruginosa* include cefepime, ceftazidime, amikacin, gentamicin, ciprofloxacin, levofloxacin, imipenem, and meropenem; drugs for *Acinetobacter baumannii* include cefepime, ceftazidime, amikacin, gentamicin, tetracycline, doxycycline, trimethoprim / sulfamethoxazole, ciprofloxacin, and levofloxacin.

[0011] The preparation method of the above-described bacterial strain identification and antimicrobial susceptibility testing kit includes the following steps:

[0012] S1. Preparation of pre-enrichment chromogenic medium: Prepare pre-enrichment chromogenic medium, dispense into dropper bottles, and freeze-dry using a freeze dryer;

[0013] S2. Preparation of the drug susceptibility test disc: Preparation of each drug microwell in the drug susceptibility test disc: Prepare each drug using chromogenic culture medium, and dispense each drug into the corresponding microwell in the main body of the drug susceptibility test disc; Preparation of microwells for strain identification areas: Prepare culture medium for each identification area, and dispense it into the corresponding microwell in the main body of the drug susceptibility test disc; After preparation, freeze dry using a freeze dryer and vacuum pack in aluminum foil bags.

[0014] S3. Preparation of diluent: Dispense physiological saline into dropper bottles;

[0015] S4. Preparation of sterile water: After high-pressure sterilization, pure water is dispensed into dropper bottles.

[0016] The above-described bacterial identification and drug susceptibility testing kit is used for bacterial identification and drug susceptibility testing of skin samples, urine samples, pleural and peritoneal effusion samples, uterine effusion samples, ear canal samples, eye secretion samples, and porcine pleural and peritoneal effusion samples.

[0017] The beneficial effects of the technical solution are:

[0018] 1) Adding a colorimetric reagent to the pre-enrichment broth can indicate the bacterial content, ensuring that the bacterial inoculation amount in the drug sensitivity tray meets the standard and guarantees the accuracy of the results;

[0019] 2) A colorimetric reagent is added to the drug sensitivity test panel, which facilitates manual interpretation and interpretation via a mobile app, without requiring special equipment;

[0020] 3) It includes 5 strain identification regions (Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonas aeruginosa, Acinetobacter baumannii), which can directly determine drug sensitivity;

[0021] 4) Bacteria identified by the region can guide clinical medication and enable more precise treatment;

[0022] 5) The kit is easy to operate, has high efficiency in result interpretation, accurate experimental results, low cost, and quick results output, making it suitable for grassroots veterinary units and pet hospitals, thereby enabling precise use of antimicrobial drugs and preventing the abuse of antimicrobial drugs. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the chassis structure of the drug sensitivity tester of the present invention;

[0024] Figure 2 The above are color charts for the pre-enrichment culture and drug susceptibility of this invention. When comparing the pre-enrichment color charts, A in the chart represents the need for 10-fold dilution before use; B represents normal use; C represents low bacterial count requiring further culture; and D represents sterility. When comparing drug susceptibility, color charts A, B, and C all represent bacterial growth, and the result is recorded as drug resistant; color chart D represents sterile growth, and the result is recorded as sensitive.

[0025] Figure 3 For the identification of strains in this invention, the micropores in the Enterobacteriaceae identification area turn yellow, indicating that the bacteria are Enterobacteriaceae; the micropores in the Enterococcus spp. identification area turn purple, indicating that the bacteria are Enterococcus spp.; the micropores in the Staphylococcus spp. identification area turn green, indicating that the bacteria are Staphylococcus spp.; the micropores in the Pseudomonas aeruginosa identification area turn purple, indicating that the bacteria are Pseudomonas aeruginosa; and the micropores in the Acinetobacter baumannii identification area turn purple, indicating that the bacteria are Acinetobacter baumannii.

[0026] The names of the corresponding labels in the attached diagram are:

[0027] 1. Sample loading well; 2. Micropores; 3. Absorbent cotton. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments:

[0029] The antimicrobial susceptibility test kit of this invention includes strain identification and antimicrobial susceptibility testing. Based on clinical characteristics, the strain identification area of ​​this invention includes five identification areas: Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonas aeruginosa, and Acinetobacter baumannii. The antimicrobial susceptibility test includes a chromogenic reagent. For the antimicrobial susceptibility chromogenic reagent, this invention selects WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), which has low toxicity and high sensitivity. WST-8 is a novel tetrazolium dye. Its principle is that succinate dehydrogenase in living cells reacts with WST-8 to generate a water-soluble yellow formazan product, which is proportional to the number of living cells, thus reflecting the number of living cells.

[0030] Specifically, the bacterial strain identification and antimicrobial susceptibility testing kit includes an antimicrobial susceptibility test dish, pre-enrichment chromogenic medium, chromogenic medium, sterile water, and diluent. The antimicrobial susceptibility test dish includes the dish body, which is made of one of polymethyl methacrylate, polycarbonate, PET, and polystyrene. The dish body includes a base, a lid, a stopper, and a colorimetric card, such as... Figure 1 As shown, the chassis contains absorbent cotton 3, micropores 2 and sample application holes 1; after the drug micropores and strain identification area micropores are prepared, the lid and chassis are ultrasonically welded together, and the sample application holes are sealed with plugs.

[0031] The microwells of the drug sensitivity disc are divided into Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonas aeruginosa identification, and Acinetobacter baumannii identification regions. The Enterobacteriaceae region is equipped with microwells for Enterobacteriaceae identification culture medium and drug microwells; the Enterococcus region is equipped with microwells for Enterococcus identification culture medium and drug microwells; the Staphylococcus region is equipped with microwells for Staphylococcus identification culture medium and drug microwells; the Pseudomonas aeruginosa region is equipped with microwells for Pseudomonas aeruginosa identification culture medium and drug microwells; and the Acinetobacter baumannii region is equipped with microwells for Acinetobacter baumannii identification culture medium and drug microwells.

[0032] The pre-enrichment chromogenic medium contains the following nutrients: beef extract powder 1-5g / 100ml, acid-hydrolyzed casein 10-20g / 100ml, trypsin 5-10g / 100ml, sodium pyruvate 2-5g / 100ml, and chromogenic agent: WST-8, full name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt, 0.02-0.1 g / 100ml; the solvent is water.

[0033] The formulations of identification media for Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonas aeruginosa, and Acinetobacter baumannii are shown in Table 1 below.

[0034] Table 1. Culture medium formulation for identification zone (g / 100ml)

[0035]

[0036] Nutrients in the chromogenic medium: Beef extract powder 5-10g / 100ml, acid-hydrolyzed casein 15-20g / 100ml, tryptone 2-5g / 100ml, sodium pyruvate 2-5g / 100ml, calcium ions 0.01-0.03g / 100ml, magnesium ions 0.01-0.03g / 100ml, 50% egg yolk emulsion 0.1-0.6g / 100ml. Chromogenic reagent: WST-8, full name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt, 0.02-0.1 g / 100ml; the solvent is water.

[0037] Drug options for Enterobacteriaceae: Ampicillin, Amoxicillin / Clavulanic acid, Cefoperazone, Cephalexin, Tetracycline, Trimethoprim / Sulfamethoxazole, Enrofloxacin, Ciprofloxacin, Ofloxacin.

[0038] Drugs for Enterococcus spp. include: penicillin, ampicillin, amoxicillin / clavulanic acid, tetracycline, doxycycline, erythromycin, vancomycin, ciprofloxacin, and levofloxacin.

[0039] Drugs for Staphylococcus spp. include: penicillin, azithromycin, gentamicin, tetracycline, doxycycline, erythromycin, clindamycin, ciprofloxacin, and levofloxacin.

[0040] Drug options for Pseudomonas aeruginosa: cefepime, ceftazidime, amikacin, gentamicin, ciprofloxacin, levofloxacin, ofloxacin, imipenem, meropenem.

[0041] Drug options for Acinetobacter baumannii: cefepime, ceftazidime, amikacin, gentamicin, tetracycline, doxycycline, trimethoprim / sulfamethoxazole, ciprofloxacin, levofloxacin.

[0042] The preparation method of the bacterial strain identification and antimicrobial susceptibility testing kit includes the following steps:

[0043] S1. Preparation of pre-enrichment chromogenic medium: Prepare pre-enrichment chromogenic medium according to Table 1, dispense 200ul / well into 5ml dropper bottles, and freeze dry using a freeze dryer;

[0044] S2. Preparation of the drug sensitivity test panel:

[0045] Preparation of each drug microwell in the drug sensitivity test plate: The drug was prepared using the above-mentioned chromogenic culture medium, and 10 μL / well was dispensed into the corresponding microwell of the drug sensitivity test plate. The specific drug layout is shown in Table 2, and the drug concentration was 10 times that in Table 2.

[0046] Preparation of microwells for strain identification: Prepare culture medium for each identification zone using the formula in Table 1, and dispense 10 μL / well into the corresponding microwells of the identification zone on the antimicrobial susceptibility test plate; the layout of microwells for the identification zone is shown in Table 2 below;

[0047] Table 2. Location of drugs and identification areas in the drug susceptibility testing panel.

[0048]

[0049] Note: The "32" in "Ampicillin 32" represents an ampicillin concentration of 32 ug / ml. Other drugs follow the same principle.

[0050] After preparation, the product was freeze-dried in a freeze dryer and then vacuum-packed in aluminum foil bags.

[0051] S3. Preparation of diluent: Dispense 5 ml / bottle of physiological saline into dropper bottles;

[0052] S4. Preparation of sterile water: After high-pressure sterilization of pure water, dispense 2ml / bottle into dropper bottles.

[0053] The method of using the antimicrobial susceptibility test kit is as follows:

[0054] 1) Preparation of pre-enrichment broth: Add all the sterile water from the kit to the pre-enrichment (lyophilized) culture medium, invert it 5 times, and set aside.

[0055] 2) Sample preparation:

[0056] Swab and pus samples: Use the sampling swab provided in the kit to collect an appropriate amount of sample, rinse several times with the pre-enrichment medium, discard the swab, and mix thoroughly by inverting the swab 5 times. Incubate at 37°C for 3-8 hours (refer to...). Figure 2 (Refer to the color chart shown) to obtain the pre-enrichment broth.

[0057] Urine sample: Take 100 μl of urine and add it to the pre-enrichment medium. Mix thoroughly by inverting the container 5 times. Incubate at 37°C for 3-8 hours (refer to the reference). Figure 2 (Refer to the color chart shown) to obtain the pre-enrichment broth.

[0058] Figure 2 In the diagram, A indicates that the product needs to be diluted 10 times before use; B indicates normal use; C indicates that the bacterial count is low and further culturing is required; and D indicates sterility.

[0059] 3) Antimicrobial sensitivity testing

[0060] ① Mix the pre-enriched bacterial culture by inverting it 5 times, then add 2 drops to the diluent and mix by inverting it 5 times to prepare the bacterial culture diluent; ② Place the antimicrobial susceptibility plate at a 30-degree angle and add the bacterial culture diluent dropwise into the sample wells until the bacterial culture diluent enters the last well, then stop adding samples; ③ Place the antimicrobial susceptibility plate in a 37°C incubator and incubate for 16 hours.

[0061] 4) Result Interpretation

[0062] There are two methods: automatic interpretation via mobile app and manual interpretation.

[0063] ① Automatic interpretation via mobile app: Open the mobile app (Nabai Micro-Mintong), click "Interpretation", and the result report will be generated automatically.

[0064] ② Manual interpretation

[0065] For reference Figure 3 Interpretation of the results of artificial strain identification shown on the color chart:

[0066] Enterobacteriaceae: The micropores in the Enterobacteriaceae identification area turn yellow.

[0067] Enterococcus: The micropores in the Enterococcus identification area turn purple.

[0068] Staphylococcus: The micropores in the identification area of ​​Staphylococcus turn green.

[0069] Pseudomonas aeruginosa: The micropores in the identification area of ​​Pseudomonas aeruginosa turn purple.

[0070] Acinetobacter baumannii: The micropores in the identification area of ​​Acinetobacter baumannii turn purple.

[0071] Interpretation of artificial drug sensitivity results:

[0072] For reference Figure 2 The color charts shown indicate that colors A, B, and C represent bacterial growth, and the result is recorded as drug resistant; color chart D represents sterile growth, and the result is recorded as sensitive.

[0073] The above-mentioned bacterial identification and drug susceptibility testing kits were applied to the identification of bacterial strains and drug susceptibility testing of skin samples, urine samples, pleural and peritoneal effusion samples, uterine effusion samples, ear canal samples, eye secretion samples, and porcine pleural and peritoneal effusion samples.

[0074] 1. Pre-enrichment chromogenic medium

[0075] Beef extract powder 1g / 100ml, acid-hydrolyzed casein 10g / 100ml, tryptone 5g / 100ml, sodium pyruvate 2g / 100ml, color developer: WST-8 0.02 g / 100ml.

[0076] 2. Identify regional formulas

[0077] The culture medium formulations for strain identification are shown in Table 4 below.

[0078] Table 4. Culture medium formulations for strain identification (g / 100ml)

[0079]

[0080] 3. Chromogenic culture medium

[0081] Beef extract powder 5g / 100ml, acid-hydrolyzed casein 15g / 100ml, tryptone 2g / 100ml, sodium pyruvate 2g / 100ml, calcium ions 0.01g / 100ml, magnesium ions 0.01g / 100ml, 50% egg yolk emulsion 0.1g / 100ml, WST-8 0.02g / 100ml.

[0082] 4. Materials for the drug sensitivity test panel

[0083] The material is: polymethyl methacrylate

[0084] 5. Application of Antimicrobial Susceptibility Testing Kits

[0085] Standard strains were validated and clinical samples were tested in accordance with CLSI and the "Technical Procedure for Testing Antimicrobial Susceptibility of Animal-Derived Bacteria - Microbroth Dilution Method".

[0086] 5.1 Validation of Standard Strains

[0087] The strains were validated using Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), and Acinetobacter baumannii (CICC 10980), and the disk partition method (KB) was used for comparison.

[0088] The results showed that the kit results were consistent with the KB results, as shown in Table 5 below.

[0089] Table 5. Results of Standard Strains Reagent Kit and KB Results

[0090]

[0091] Note: " / " indicates that the experiment was not conducted.

[0092] 5.2 Validation of clinical samples

[0093] A total of 35 clinical samples were collected, including 30 pet clinical samples (5 skin samples, 5 urine samples, 5 pleural and peritoneal effusion samples, 5 uterine effusion samples, 5 ear canal samples, and 5 eye discharge samples) and 5 pig pleural and peritoneal effusion samples.

[0094] Strain identification: The isolated single colonies were sent to a sequencing company for identification.

[0095] The results showed that the kit yielded results consistent with the KB method. See the table below.

[0096] (1) Skin sample results

[0097] The results of bacterial strain identification from skin samples are shown in Table 6: the results from the kit are consistent with the sequencing results.

[0098] The results of drug sensitivity testing on skin samples are shown in Table 7: The results from the kit are consistent with those from KB.

[0099] Table 6. Results of bacterial strain identification from skin samples

[0100]

[0101] Table 7. Drug sensitivity results of skin samples

[0102]

[0103] Note: " / " indicates that the experiment was not conducted.

[0104] (2) Results of urine sample

[0105] The results of strain identification in urine samples are shown in Table 8: the results from the kit are consistent with the results from sequencing.

[0106] The results of drug sensitivity testing of urine samples are shown in Table 9: The results of the kit are consistent with those of KB.

[0107] Table 8. Results of bacterial strain identification in urine samples

[0108]

[0109] Table 9. Antimicrobial susceptibility results of urine samples

[0110]

[0111] Note: " / " indicates that the experiment was not conducted.

[0112] (3) Results of pleural and peritoneal effusion samples

[0113] The results of strain identification in pleural and peritoneal effusion samples are shown in Table 10: the kit results are consistent with the sequencing identification results.

[0114] The drug sensitivity results of the pleural and peritoneal effusion samples are shown in Table 11: The results of the kit are consistent with the results of KB.

[0115] Table 10. Results of bacterial strain identification in pleural and peritoneal effusion samples.

[0116]

[0117] Table 11 Drug sensitivity results of pleural and peritoneal effusion samples

[0118]

[0119] Note: " / " indicates that the experiment was not conducted.

[0120] (4) Results of uterine fluid samples

[0121] The results of strain identification in the uterine fluid samples are shown in Table 12: the kit results are consistent with the sequencing identification results.

[0122] The drug sensitivity results of the uterine fluid samples are shown in Table 13: The results of the kit are consistent with those of KB.

[0123] Table 12 Results of bacterial strain identification in uterine fluid samples

[0124]

[0125] Table 13 Drug sensitivity results of uterine fluid samples

[0126]

[0127] Note: " / " indicates that the experiment was not conducted.

[0128] (5) Ear canal sample results

[0129] The results of strain identification in ear canal samples are shown in Table 14: the results from the kit are consistent with the results from sequencing.

[0130] The drug sensitivity results of the ear canal samples are shown in Table 15: the results of the kit are consistent with the results of KB.

[0131] Table 14 Results of strain identification from ear canal samples

[0132]

[0133] Table 15. Drug sensitivity results of ear canal samples

[0134]

[0135] Note: " / " indicates that the experiment was not conducted.

[0136] (6) Results of eye secretion samples

[0137] The results of strain identification from eye secretion samples are shown in Table 16: the kit results are consistent with the sequencing identification results.

[0138] The drug sensitivity results of the eye secretion samples are shown in Table 17: The results of the kit are consistent with the results of KB.

[0139] Table 16 Results of bacterial strain identification in ocular secretion samples

[0140]

[0141] Table 17 Drug sensitivity results of eye secretion samples

[0142]

[0143] Note: " / " indicates that the experiment was not conducted.

[0144] (7) Results of pleural and peritoneal effusion samples from pigs

[0145] The results of strain identification in porcine pleural and peritoneal effusion samples are shown in Table 18: the kit results are consistent with the sequencing identification results.

[0146] The drug sensitivity results of porcine pleural and peritoneal effusion samples are shown in Table 19: The results from the kit are consistent with those from KB.

[0147] Table 18. Results of bacterial strain identification in porcine pleural and peritoneal effusion samples.

[0148]

[0149] Table 19 Drug sensitivity results of porcine pleural and peritoneal effusion samples

[0150]

[0151] Note: " / " indicates that the experiment was not conducted.

[0152] In summary, adding a chromogenic agent to the pre-enrichment broth indicates the bacterial content, ensuring that the bacterial inoculation amount in the drug sensitivity test tray meets the standard and guarantees the accuracy of the results. The addition of a chromogenic agent to the drug sensitivity test tray facilitates manual interpretation and mobile app interpretation, requiring no special equipment. It includes five strain identification areas (Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonas aeruginosa, and Acinetobacter baumannii), which can directly determine drug sensitivity. The bacteria identified in the strain identification areas can guide clinical medication, leading to more precise treatment. The kit is simple to operate, has high result interpretation efficiency, accurate experimental results, low cost, and rapid result output, making it suitable for primary veterinary units and pet hospitals, thereby achieving precise use of antimicrobial drugs and preventing antimicrobial overuse.

[0153] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A kit for bacterial identification and antimicrobial susceptibility testing, characterized in that, The kit includes an antimicrobial susceptibility testing disc, pre-enrichment chromogenic medium, chromogenic medium, sterile water, and diluent. The antimicrobial susceptibility testing disc includes a disc body, which comprises an Enterobacteriaceae region, an Enterococcus spp. region, a Staphylococcus spp. region, a Pseudomonas aeruginosa region, and an Acinetobacter baumannii region. The Enterobacteriaceae region is equipped with microwells for Enterobacteriaceae identification medium and drug microwells; the Enterococcus spp. region is equipped with microwells for Enterococcus spp. identification medium and drug microwells; the Staphylococcus spp. region is equipped with microwells for Staphylococcus spp. identification medium and drug microwells; the Pseudomonas aeruginosa region is equipped with microwells for Pseudomonas aeruginosa identification medium and drug microwells; and the Acinetobacter baumannii region is equipped with microwells for Acinetobacter baumannii identification medium and drug microwells.

2. The bacterial strain identification and antimicrobial susceptibility testing kit according to claim 1, characterized in that: The pre-enrichment chromogenic medium comprises 1-5 g / 100 ml beef extract powder, 10-20 g / 100 ml acid-hydrolyzed casein, 5-10 g / 100 ml trypsin, 2-5 g / 100 ml sodium pyruvate, and 0.02-0.1 g / 100 ml chromogenic reagent WST-8.

3. The bacterial strain identification and antimicrobial susceptibility testing kit according to claim 1, characterized in that: The identification media for Enterobacteriaceae include tryptone, yeast extract, sodium chloride, glucose, ferric ammonium citrate, ox bile salts, vancomycin, and bromocresol purple; the identification media for Enterococcus includes tryptone, yeast extract, beef extract, sodium pyruvate, magnesium sulfate, sodium citrate, aztreonam, nalidixic acid, 5-bromo-6-chloro-3-indole phosphate p-toluidine, and 5-bromo-6-chloro-3-indole-glucopyranoside; the identification media for Staphylococcus includes Mueller Hinton broth, sodium pyruvate, magnesium sulfate, ferric ammonium citrate, potassium tellurite, aztreonam, bacitracin, 5-bromo-4-chloro-3-indole-β-D-glucopyranoside, and 5-bromo-4-chloro-3-indole phosphate p-toluidine; the identification media for Pseudomonas aeruginosa includes tryptone, yeast extract, sodium chloride, glucose, and 1M... HCl, ox bile salts, hexadecyltrimethylammonium bromide, cefazolin, bromocresol purple; Acinetobacter baumannii identification medium includes tryptone, casein peptone, sodium chloride, glucose, magnesium sulfate, sodium deoxycholate, ox bile salts, vancomycin, trimethoprim, and p-toluidine 5-bromo-6-chloro-3-indole phosphate.

4. The bacterial strain identification and antimicrobial susceptibility testing kit according to claim 1, characterized in that: The chromogenic culture medium consists of 5-10 g / 100 ml beef extract, 15-20 g / 100 ml acid-hydrolyzed casein, 2-5 g / 100 ml tryptone, 2-5 g / 100 ml sodium pyruvate, 0.01-0.03 g / 100 ml calcium ions, 0.01-0.03 g / 100 ml magnesium ions, 0.1-0.6 g / 100 ml 50% egg yolk emulsion, and 0.02-0.1 g / 100 ml WST-8 chromogenic reagent.

5. The bacterial strain identification and antimicrobial susceptibility testing kit according to claim 1, characterized in that: Drugs for Enterobacteriaceae include ampicillin, amoxicillin / clavulanic acid, cefoperazone, cephalexin, tetracycline, trimethoprim / sulfamethoxazole, enrofloxacin, ciprofloxacin, and ofloxacin; drugs for Enterococcus include penicillin, ampicillin, amoxicillin / clavulanic acid, tetracycline, doxycycline, erythromycin, vancomycin, ciprofloxacin, and levofloxacin; drugs for Staphylococcus include penicillin, azithromycin, gentamicin, and tetracycline. Drugs for Pseudomonas aeruginosa include cefepime, doxycycline, erythromycin, clindamycin, ciprofloxacin, and levofloxacin; drugs for Pseudomonas aeruginosa include cefepime, ceftazidime, amikacin, gentamicin, ciprofloxacin, levofloxacin, ofloxacin, imipenem, and meropenem; drugs for Acinetobacter baumannii include cefepime, ceftazidime, amikacin, gentamicin, tetracycline, doxycycline, trimethoprim / sulfamethoxazole, ciprofloxacin, and levofloxacin.

6. The method for preparing the bacterial strain identification and antimicrobial susceptibility testing kit according to any one of claims 1 to 5, characterized in that: Includes the following steps: S1. Preparation of pre-enrichment chromogenic medium: Prepare pre-enrichment chromogenic medium, dispense into dropper bottles, and freeze-dry using a freeze dryer; S2. Preparation of the drug sensitivity test tray: Preparation of each drug microwell in the drug sensitivity test tray: Each drug is prepared using a chromogenic culture medium, and each drug is dispensed into the corresponding microwell in the drug sensitivity test tray body; Preparation of microwells for strain identification: Prepare culture medium for each identification area and dispense it into the corresponding microwells of the antimicrobial susceptibility plate. After preparation, the product was freeze-dried in a freeze dryer and then vacuum-packed in aluminum foil bags. S3. Preparation of diluent: Dispense physiological saline into dropper bottles; S4. Preparation of sterile water: After high-pressure sterilization, pure water is dispensed into dropper bottles.

7. The application of the bacterial strain identification and antimicrobial susceptibility testing kit according to any one of claims 1 to 5, characterized in that: It is used for bacterial identification and drug susceptibility testing of skin samples, urine samples, pleural and peritoneal effusion samples, uterine effusion samples, ear canal samples, eye secretion samples, and porcine pleural and peritoneal effusion samples.

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