A partitioned drug sensitivity plate and its application

Abstract

This invention relates to the field of microbial detection technology and discloses a partitioned antimicrobial susceptibility testing plate and its application. The plate comprises a plate body and a plurality of microwell reaction units disposed on the plate body. The plate body is spatially divided into at least two independent detection zones, including a first detection zone and a second detection zone. A first selective culture system is pre-installed within the microwell reaction units of the first detection zone. The first selective culture system includes neomycin and at least one antimicrobial drug concentration gradient targeting Gram-negative bacilli. The partitioned antimicrobial susceptibility testing plate provided by this invention enables direct and simultaneous antimicrobial susceptibility testing of mixed samples of Gram-negative bacilli and Gram-positive cocci.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and more specifically, to a zoned antimicrobial susceptibility testing plate and its application. Background Technology

[0002] In animal clinical infection detection, some samples, after pre-culture, pre-screening, or microscopic examination, may be identified as containing Gram-negative bacilli and / or Gram-positive cocci. For these samples, clinical practice usually requires further testing to obtain the susceptibility of the corresponding pathogens to antimicrobial agents, in order to assist in the selection of subsequent medications.

[0003] Currently, the antimicrobial susceptibility testing of animal-derived bacterial samples typically employs a technical approach of first isolating and culturing the bacteria, followed by susceptibility testing. Specifically, after collecting the sample, it is usually necessary to culture the bacteria in the sample first. When multiple bacteria are present in the sample, it is also necessary to obtain single colonies through methods such as streak plating or pure culture. The isolated strains are then inoculated into the antimicrobial susceptibility testing system, and the minimum inhibitory concentration (MIC) and corresponding susceptibility results are determined based on the growth after culture.

[0004] The aforementioned technical approach is suitable for drug susceptibility testing of single bacterial strains. However, when dealing with samples containing both Gram-negative bacilli and Gram-positive cocci, it suffers from a lengthy testing process and numerous operational steps. Especially when both types of bacteria coexist, direct cultivation and interpretation within the same detection system can lead to the co-growth and mutual interference of different bacterial communities, affecting the identification of growth signals from the target bacteria and the determination of MIC results. Therefore, current technologies typically still rely on first isolating different bacterial communities and then performing separate subsequent detections.

[0005] Furthermore, most existing antimicrobial susceptibility testing plates or procedures are designed around a single strain after pure culture, and their detection wells, culture systems, and interpretation methods are mainly suitable for detecting a single bacterial species. For animal-derived clinical bacterial samples that have been identified as containing Gram-negative bacilli and / or Gram-positive cocci, especially samples containing both types of bacteria, current technology lacks a solution that can establish corresponding detection environments for different bacterial groups within the same plate and achieve parallel detection.

[0006] Therefore, it is still necessary to provide a partitioned detection vector and its application scheme to establish a detection system that distinguishes between Gram-negative bacilli and Gram-positive cocci in at least animal-derived clinical bacterial samples, thereby reducing the mutual interference of different bacterial groups in the same detection process and providing a basis for obtaining the corresponding antimicrobial susceptibility results. Summary of the Invention

[0007] The purpose of this invention is to provide a drug susceptibility testing method and its application, which aims to use a partitioned plate structure as the core, target animal-derived clinical bacterial samples containing Gram-negative bacilli and / or Gram-positive cocci, and connect with the three types of detection modes already mentioned in your manuscript: first detection zone, second detection zone, positive / negative control well, turbidity time-series monitoring, single G-, single G+, and mixed sample.

[0008] To achieve the above objectives, the present invention provides the following solution: On one hand, the present invention provides a partitioned antimicrobial susceptibility plate for detecting the antimicrobial susceptibility of Gram-negative bacilli and / or Gram-positive cocci in animal-derived clinical bacterial samples, comprising a plate body and a plurality of microwell reaction units disposed on the plate body, wherein the plate body is spatially divided into at least two mutually independent detection zones, including a first detection zone and a second detection zone; The microwell reaction unit of the first detection area is pre-set with a first selective culture system, which includes neomycin and at least one antibacterial drug concentration gradient against Gram-negative bacilli. The neomycin is used to inhibit the growth of Gram-positive cocci in the first detection area, and the mass concentration of the neomycin in the first selective culture system is 5 μg / mL. The microwell reaction unit of the second detection area is pre-installed with a second selective culture system, which includes polymyxin B and at least one antimicrobial drug concentration gradient against Gram-positive cocci. The polymyxin B is used to inhibit the growth of Gram-negative bacilli in the second detection area, and the mass concentration of polymyxin B in the second selective culture system is 2 μg / mL. The first detection area and the second detection area are respectively provided with negative control wells and positive control wells. The negative control wells are kept clear when no antibacterial drugs are pre-placed in them and no samples are inoculated. The positive control wells are not pre-placed with antibacterial drugs and form a measurable growth curve after sample inoculation, which serves as the control baseline for determining the minimum inhibitory concentration (MIC).

[0009] Furthermore, the plate is a 96-well polystyrene microporous plate.

[0010] Furthermore, the first detection area is used to detect the susceptibility of Gram-negative bacilli in a single Gram-negative bacillus sample and a mixed sample containing both Gram-negative bacilli and Gram-positive cocci to antimicrobial agents, and the second detection area is used to detect the susceptibility of Gram-positive cocci in a single Gram-positive cocci sample and a mixed sample containing both Gram-negative bacilli and Gram-positive cocci to antimicrobial agents.

[0011] On the other hand, the present invention also provides a method for detecting antimicrobial susceptibility using the above-mentioned partitioned antimicrobial susceptibility plate, comprising: Obtain clinical bacterial samples from animals; The animal-derived clinical bacterial samples were subjected to Gram staining and microscopic examination. Based on the staining results and bacterial morphology, the samples were identified as single Gram-negative bacilli, single Gram-positive cocci, or mixed samples containing both Gram-negative bacilli and Gram-positive cocci. The animal-derived clinical bacterial samples were pretreated and diluted according to the sample type to obtain the test sample solution; The test sample solution corresponding to a single Gram-negative bacillus sample is inoculated into the first detection area, the test sample solution corresponding to a single Gram-positive coccus sample is inoculated into the second detection area, and the test sample solution corresponding to the mixed sample is simultaneously inoculated into the first and second detection areas. The inoculated partitioned drug sensitivity plates were placed into the detection instrument for constant temperature incubation, and the turbidity of each well was monitored over time. Growth curves for each well were generated based on turbidity time-series monitoring data, and the growth curves for each well were compared with the corresponding control well growth curves to determine the minimum inhibitory concentration (MIC). Output sensitivity classification results based on MIC and corresponding interpretation rules.

[0012] Furthermore, when the sample type is blood, the liquid in the positive blood culture bottle is used as the test sample and diluted 100 times; when the sample type is urine, the sample is directly aspirated without dilution or diluted 10 times; when the sample type is skin swab or pus, it is washed with physiological saline and then diluted 100 times; when the sample type is ear canal secretions, it is emulsified with physiological saline and 0.1% Tween-80 and then diluted 100 times; when the sample type is sputum, it is washed with physiological saline and then diluted 100 times.

[0013] Furthermore, the 0h background OD value of each detection well was measured before constant temperature incubation; when determining the growth status, the net OD value after subtracting the corresponding 0h background OD value from the endpoint OD value was used as the basis for growth determination.

[0014] Furthermore, the temperature of the isothermal incubation is controlled at 38±1℃.

[0015] In another aspect, the present invention also provides an antimicrobial drug susceptibility testing system, including the above-mentioned partitioned drug susceptibility plate and a testing instrument. The testing instrument is used to perform isothermal incubation on the partitioned drug susceptibility plate, perform turbidity time-series monitoring on each well, automatically determine the minimum inhibitory concentration (MIC) based on the turbidity time-series monitoring data, and output the susceptibility grading results.

[0016] Furthermore, the detection instrument includes a temperature control component, an optical detection component, and an analysis and processing component; the temperature control component is used to control the temperature of the culture chamber at 38±1℃; the optical detection component is used to collect turbidity data at multiple times from each well and form a turbidity time series; the analysis and processing component is used to generate a growth curve based on the turbidity time series, perform MIC determination, and output a detection report containing the detection results of Gram-negative bacilli corresponding to the first detection area and the detection results of Gram-positive cocci corresponding to the second detection area.

[0017] The principle of spatial sorting and detection of mixed bacteria in this invention is as follows: by pre-placing different species-specific inhibitors in different zones of the antimicrobial susceptibility plate, different selective growth environments are created in physical space. Inhibitors that only inhibit Gram-positive cocci (such as neomycin) are added to the "bacterial detection zone," while inhibitors that only inhibit Gram-positive cocci (such as polymyxin B) are added to the "coccal detection zone," thus ensuring that this area only reflects the antimicrobial susceptibility results of Gram-negative bacilli; and vice versa. Based on this, mixed bacterial samples can be directly inoculated, enabling parallel detection. The bacterial detection zone and the coccal detection zone are either the first detection zone or the second detection zone.

[0018] As mentioned in the background section, existing technologies have the following problems: 1) Long detection cycle: The pure culture step is indispensable, making it impossible to shorten the total cycle to within 48 hours, thus failing to meet the clinical needs for rapid medication in acute infections. 2) Poor adaptability to different scenarios: The pure culture process needs to be carried out in a biosafety cabinet, requiring specialized operating space and equipment, which limits its application at the grassroots level. 3) Cumbersome operation and high labor costs: Steps such as isolation, transfection, and interpretation all require manual operation by professional personnel, resulting in low efficiency. 4) Incompatible standards: The drug sensitivity test system is based on human standards, which are out of sync with actual animal clinical medication use, resulting in weak guidance of the results.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: Firstly, by setting up a first detection area and a second detection area within the same plate, and pre-setting corresponding species-specific inhibitors and antibacterial drug concentration gradients in different detection areas, the present invention establishes a mutually distinguishable detection environment for different target bacterial groups within the same detection carrier, which helps to reduce the mutual interference between Gram-negative bacilli and Gram-positive cocci in the same detection process.

[0020] Secondly, the partitioned drug sensitivity plate of the present invention can be used for the detection of single Gram-negative bacillus samples, as well as single Gram-positive cocci samples, and can also be used for the parallel detection of mixed samples containing both Gram-negative bacillus and Gram-positive cocci, thereby improving the applicability of the detection plate.

[0021] Thirdly, by setting the positive control well and the negative control well in the corresponding detection area, and by determining the MIC based on the turbidity time-series monitoring data and the control baseline, the present invention helps to improve the consistency of the detection result determination.

[0022] Fourth, the present invention adopts a method of combining a partitioned detection plate with a detection instrument, which can complete the determination of MIC and output of sensitivity results under constant temperature incubation and automatic turbidity monitoring conditions, thereby reducing manual well-by-well interpretation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a functional layout diagram of a partitioned antimicrobial susceptibility testing plate provided in an embodiment of the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] It should be noted that the "mixed bacteria" in this invention specifically refers to a mixed sample of Gram-negative bacilli and Gram-positive cocci, excluding fungi, anaerobic bacteria, mycoplasma, and other non-bacterial microorganisms. The "regional antimicrobial susceptibility testing plate" of this invention is only applicable to the detection of the aforementioned Gram-negative bacilli and Gram-positive cocci samples; the detection of other types of microorganisms is not within the scope of protection of this invention.

[0027] To facilitate understanding of the present invention, all terms used herein are defined in this section. Furthermore, unless otherwise defined, all technical or scientific terms used herein have the same general meaning as understood or commonly used by one of ordinary skill in the art to which this invention pertains.

[0028] EUCAST standard: refers to the antimicrobial susceptibility testing standard issued by the European Committee for Antimicrobial Susceptibility Testing. It has clear provisions on the detection methods and drug selection for animal-derived drug-resistant bacteria (such as ESBL-producing Escherichia coli and carbapenem-resistant bacteria). The drug formulation of this invention refers to its drug resistance testing requirements to improve the detection rate of drug-resistant bacteria.

[0029] Animal CLSI Standards: These refer to the animal pathogen susceptibility testing standards (such as VET01-A4) published by the Clinical Laboratory Standards Institute (CLSI). These standards specify the susceptibility testing methods, drug selection, and concentration gradients for common animal pathogens (such as Staphylococcus canis and Mycoplasma bovis). The concentration gradient design of this invention fully complies with these standards, ensuring that the MIC value matches the animal drug dosage.

[0030] Human CLSI Standards: These refer to the human pathogen susceptibility testing standards published by CLSI (such as M100). Traditional animal susceptibility testing boards often refer to these standards, leading to a mismatch between drugs and concentrations in animal clinical practice. This invention optimizes drug formulation and solves the "standard mismatch" problem by comparing EUCAST, animal CLSI, and human CLSI.

[0031] Strain-specific inhibitors: These are substances that inhibit the growth of only a certain type of pathogenic bacteria without affecting the target bacteria. In this invention, they are used to eliminate interference from mixed bacteria—such as polymyxin B (which only inhibits Gram-negative bacilli and has no effect on cocci) and neomycin (which, at the selected concentration, mainly inhibits Gram-positive cocci, while having no substantial effect on the growth and drug sensitivity of Gram-negative bacilli), ensuring that mixed bacteria can be directly detected without pure culture.

[0032] MIC (Minimum Inhibitory Concentration): refers to the lowest drug concentration that can inhibit the visible growth of bacteria. It is an important indicator for evaluating the antibacterial activity of antimicrobial drugs in drug susceptibility testing.

[0033] Broth dilution method: A method of drug sensitivity testing in which bacteria are inoculated into liquid culture medium containing different concentrations of antibacterial drugs, and the bacterial growth is observed after incubation to determine the MIC value.

[0034] Agar diffusion method: a method of drug sensitivity testing, in which an antimicrobial drug is placed on an agar plate inoculated with test bacteria, and after incubation, the sensitivity of bacteria to the drug is determined by measuring the diameter of the inhibition zone.

[0035] Gram-negative bacilli: a large group of bacteria that turn red after staining, such as Escherichia coli and Pseudomonas aeruginosa.

[0036] Gram-positive cocci: a large group of bacteria that stain purple after staining, such as Staphylococcus aureus and Streptococcus.

[0037] The following will explain the present invention in conjunction with embodiments. It should be noted that the strain-specific inhibitors can be replaced. For example, after the sample is diluted and inoculated onto the drug sensitivity plate, the inhibitor in the "bacterial zone" can be replaced from neomycin to nalidixic acid (at the selected concentration, it mainly inhibits Gram-positive cocci, but has no substantial effect on the growth and drug sensitivity of Gram-negative bacilli); the inhibitor in the "coccal zone" can be replaced from polymyxin B to cefotaxime (at the selected concentration, it mainly inhibits Gram-negative bacilli, but has no substantial effect on the growth and drug sensitivity of Gram-positive cocci). Only the drug concentration analysis threshold of the instrument needs to be adjusted (because the inhibition efficiency of nalidixic acid and cefotaxime is slightly lower than that of neomycin and polymyxin B, the turbidity determination threshold needs to be adjusted from 10% to 15%).

[0038] The structure of the partitioned drug sensitivity plate in the embodiments of the present invention is shown in the figure. Figure 1 As shown, this partitioned antimicrobial susceptibility testing plate uses a standard 96-well polystyrene microplate (8 rows × 12 columns). The plate is constructed to contain at least two functionally distinct detection zones: a first detection zone containing a first species-specific inhibitor in its culture medium for selective detection of Gram-negative bacilli in the presence of mixed bacteria; and a second detection zone containing a second species-specific inhibitor in its culture medium for selective detection of Gram-positive cocci in the presence of mixed bacteria.

[0039] It should be noted that this functional layout of the partitions is only an example layout, and the complete layout includes all drugs required to meet EUCAST and CLSI animal standards.

[0040] The process of this invention embodiment is as follows: Sampling: Veterinarians directly collect animal lesion samples (such as pus, urine, blood, skin swabs, ear canal secretions, sputum, etc.).

[0041] Gram staining determination: Take a small amount of sample smear, perform Gram staining and microscopic examination, and determine the sample type as a single Gram-negative bacillus, a single Gram-positive coccus, or a mixed bacillus based on the staining results (purple for G+, red for G-) and bacterial morphology. This takes about 5 minutes.

[0042] Sample processing: Pre-process according to the sample type (e.g., positive blood culture solution, direct urine aspiration, swab saline elution, etc.), and dilute 100 times with saline (or adjust according to sample characteristics). No sterile operating table is required; it can be completed on a regular examination table and takes about 3-5 minutes.

[0043] Inoculation and testing: Select the detection mode based on the Gram staining results—inoculate a single Gram-negative bacterium into the bacillus area, inoculate a single Gram-positive bacterium into the coccus area, or simultaneously cover both areas with mixed bacteria, and then place the instrument in place for testing.

[0044] Sampling: Veterinarians directly collect animal lesion samples (such as pus and urine).

[0045] Simple dilution: If the sample concentration is too high, perform a simple dilution using physiological saline (e.g., 10-fold dilution). No sterile operating table is required; this can be done on a regular examination table and takes approximately 3-5 minutes. It should be noted that the pre-test processing steps, including sample type, simple dilution, and direct inoculation, are all omitted. The procedure is changed to first obtaining the sample and then using a pet testing device (microbial identification and drug sensitivity analyzer, model: VetMicro-X) manufactured by Tianjin Ruide Biomedical Technology Co., Ltd. for incubation and positive result judgment. If the device judges the result as negative, the experiment is not started; if the result is positive, the dilution test is performed according to the pre-defined dilution concentration in the software. The pre-defined dilution concentration refers to the turbidity value obtained by the pet device when testing the sample turbidity, such as a turbidity of 0.5 mcf (1.5 mcg). With a culture volume of 5 ml (10⁸ CFU / ml), the concentration is fixed at 10⁴ CFU / ml during the instrumentation process, allowing you to calculate the corresponding volume to be added.

[0046] Direct inoculation: Use a pipette to take an appropriate amount of the diluted sample and add it directly to the partitioned antimicrobial susceptibility plate of this invention, ensuring that the sample solution covers both the bacillus and cocci zones. This process takes approximately 5 minutes.

[0047] Instrumental testing: Place the inoculated antimicrobial susceptibility plate into the matching antimicrobial susceptibility testing instrument and close the door. The instrument will automatically start the preset program (38℃ constant temperature incubation, real-time turbidity monitoring).

[0048] Automatic report generation: After the test is completed (usually 4-6 hours), the instrument’s built-in software automatically analyzes the growth data, determines the MIC value and sensitivity (S / I / R) according to the animal CLSI standard, and automatically generates a complete drug sensitivity report containing medication recommendations for veterinarians to use directly in clinical decision-making.

[0049] The detection system in this embodiment of the invention includes: a) The partitioned drug sensitivity test plate as described above; b) A detection instrument is used to receive the partitioned drug sensitivity plate, incubate it at 38±1℃, and monitor the turbidity of each well until the MIC value is automatically determined based on the turbidity data.

[0050] Example 1: Screening and Concentration Optimization of Strain-Specific Inhibitors The study aims to verify the selective inhibitory effects of different species-specific inhibitors on target and non-target bacterial populations, and to determine the minimum effective concentration (MIC) required for optimal inhibitory effect. This provides data support for the selection of inhibitors for drug sensitivity testing and further demonstrates the scientific rigor of the technical solution.

[0051] Materials: Candidate inhibitors (such as neomycin, polymyxin B, nalidixic acid, cefotaxime), standard strains of Gram-negative bacilli (such as Escherichia coli ATCC 25922), standard strains of Gram-positive cocci (such as Staphylococcus aureus ATCC 25923), MH broth, and matching drug sensitivity instruments (see Table 1 for details).

[0052] Table 1. List of Supporting Drug Sensitivity Instruments

[0053] method: Inhibitor type screening: Four inhibitor combinations were selected, and their inhibition rates against non-target bacteria and their effects on the growth of target bacteria were tested (calculated as the OD600 reduction rate after 6 hours).

[0054] Inhibition rate calculation: Inhibition rate = (1 - OD600 of well containing inhibitor / OD600 of control well) × 100% Concentration gradient optimization: Select the best inhibitors (neomyomycin and polymyxin B) from the screening and set multiple concentration gradients.

[0055] Non-target bacteria were inoculated separately, and the OD600 value was monitored after 6 hours to determine the minimum concentration required to achieve an inhibition rate of >99%.

[0056] Test results and data: Table 2 Comparison of Strain-Specific Inhibitor Types

[0057] Table 3. Optimization results of neomycin and polymyxin B concentrations

[0058] Data Analysis: Inhibitor type selection: As shown in Table 2, the neomycin / polymyxin B combination is significantly better than the nalidixic acid / cefotaxime combination in terms of inhibitory effect on non-target bacteria (both >99%), while having the least impact on the growth of target bacteria (<5%), making it the best choice for achieving efficient spatial sorting.

[0059] Concentration optimization results: As shown in Table 3, the minimum effective concentration of neomycin is 5 μg / mL, and the minimum effective concentration of polymyxin B is 2 μg / mL. Although higher concentrations of polymyxin B (e.g., 4 μg / mL) have the same inhibitory effect, they have too large an impact on the OD600 of the target bacteria (>15%), which may affect the accuracy of the drug sensitivity results.

[0060] To further verify the universality of inhibitor concentrations, the following common animal-derived pathogens were tested at the optimized concentrations mentioned above (neomyomycin 5 μg / mL, polymyxin B 2 μg / mL): Gram-negative bacilli (n=10): Canine Escherichia coli (n=3), Feline Klebsiella pneumoniae (n=3), Avian Salmonella (n=2), Porcine Pasteurella (n=2) Gram-positive cocci (n=10): Canine Staphylococcus aureus (n=3), Feline Streptococcus (n=3), Bovine Streptococcus mammologica (n=2), Porcine Streptococcus suis (n=2) Table 4

[0061] Conclusion: This embodiment determined the optimal configuration of neomycin (5 μg / mL) and polymyxin B (2 μg / mL) in the drug sensitivity test plate through experimental data, as shown in Table 4. Stable selective inhibitory effects were maintained across different animal sources and bacterial species, and the accuracy of the target bacterial MIC determination met the requirements, confirming the universality of this technical solution in animal clinical settings. It demonstrates the non-empirical nature and technical difficulty of the core technical parameters, providing a solid experimental basis for limiting the types and concentrations of inhibitors.

[0062] Example 2: Verification of the selective inhibitory effect of the partitioning inhibitor on mixed bacteria This embodiment verifies whether the core principle of the present invention—the partitioning design of the drug sensitivity plate and the strain-specific inhibitors—can effectively achieve the selective growth of target bacteria and the effective inhibition of non-target bacteria in mixed bacteria, and verifies the applicability of the three detection modes.

[0063] Materials: Antimicrobial susceptibility testing plates of this invention (containing 5 μg / mL neomycin in the bacillus zone and 2 μg / mL polymyxin B in the cocci zone), ordinary antimicrobial susceptibility testing plates (without inhibitors, used as controls), *Escherichia coli* (ATCC 25922, Gram-negative bacillus), *Staphylococcus aureus* (ATCC 25923, Gram-positive cocci), canine *Escherichia coli* (C-001), canine *Staphylococcus aureus* (C-011), MH broth, turbidimeter, physiological saline, and matching antimicrobial susceptibility testing instruments (see Table 1 for details).

[0064] Methods: Single-strain validation group: Control group 2: Purified Escherichia coli ATCC 25922 bacterial suspension was inoculated into the cocci zone (containing polymyxin B) of the drug sensitivity plate of this invention. Control group 3: Purified Staphylococcus aureus ATCC 25923 bacterial suspension was inoculated into the bacillus zone of the antimicrobial susceptibility plate of this invention (containing neomycin). Control group 4: Purified canine Staphylococcus aureus C-011 bacterial suspension was inoculated into the bacillus zone of the antimicrobial susceptibility plate of this invention (containing neomycin). Mixed bacteria validation group (1:1 ratio): Experimental group: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were mixed at a 1:1 ratio (0.5 McFarland units) and inoculated into the antimicrobial susceptibility plate of this invention. Control group 1: The above 1:1 mixed bacterial solution was inoculated onto a regular antimicrobial susceptibility plate (without inhibitors). Mixed bacterial validation groups (different proportions): Control group 5: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were mixed at a ratio of 1:10 (G+ dominant) and inoculated into the antimicrobial susceptibility plate of this invention. Control group 6: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were mixed at a ratio of 10:1 (G-dominant) and inoculated into the antimicrobial susceptibility plate of this invention. Mixed bacterial validation group (animal-derived strains): Control group 7: Canine Escherichia coli C-001 and canine Staphylococcus aureus C-011 were mixed at a 1:1 ratio and inoculated into the drug sensitivity plate of this invention. All plates were placed in a matching antimicrobial susceptibility testing instrument for incubation, and the turbidity changes in each well were monitored in real time.

[0065] The test results and data are shown in Table 5: Table 5. Turbidity (OD600) Record of Each Well After 6 Hours of Incubation

[0066] Table 6. Verification of the accuracy of drug sensitivity results in experimental and control groups 5-7

[0067] Data analysis (as shown in Table 6): Validation of single-species detection mode: The results of control groups 2, 3, and 4 showed that purified Escherichia coli could not grow in the coccal region containing polymyxin B (OD600<0.1), and purified Staphylococcus aureus (standard strain and animal-derived strain) could not grow in the bacillus region containing neomycin (OD600<0.1), confirming that single-species samples were completely inhibited in non-target areas and would not produce false positives.

[0068] Validation of the mixed bacterial detection mode (1:1): The experimental group results showed that, although a 1:1 mixed bacterial culture was used, both the bacillus region (containing neomycin) and the coccus region (containing polymyxin B) achieved good growth of the single target bacteria (OD600>0.2), while the growth of non-target bacteria was effectively inhibited. The MIC values ​​were completely consistent with the gold standard method.

[0069] Verification using mixed bacterial strains at different ratios: Control group 5 (1:10, Gram-positive dominance) and control group 6 (10:1, Gram-negative dominance) showed that even when one bacterial species was absolutely dominant (10:1), the other species could still grow normally in the target area, with OD600 > 0.2 in both cases, and accurate MIC values. This confirms that different mixing ratios do not affect the selective inhibition effect.

[0070] Animal-derived strain validation: Control group 7 used canine clinical isolates, and the results showed the same effect as the standard strain, with accurate MIC values, confirming the applicability of this method to animal-derived strains.

[0071] Verification of interference elimination from mixed growth: The turbid growth (OD600=0.30) of control group 1 (without inhibitor) demonstrates that mixed bacteria will co-grow on conventional plates, making it impossible to interpret drug sensitivity results. This invention, however, completely eliminates this interference through a partitioned inhibitor design.

[0072] Conclusion: The partitioned antimicrobial susceptibility testing plate of this invention is applicable to three detection scenarios: detection of single Gram-negative bacilli, single Gram-positive cocci, and mixed bacteria. In mixed bacterial samples, regardless of the mixing ratio (1:1 to 10:1 or 1:10) or the source of the strain (standard strain or animal-derived strain), both partitions can achieve selective growth of target bacteria while effectively inhibiting non-target bacteria, and the obtained MIC values ​​are completely consistent with the gold standard method. In contrast, traditional antimicrobial susceptibility testing plates without inhibitors cannot distinguish the growth of mixed bacteria, resulting in uninterpretable antimicrobial susceptibility results. This embodiment confirms the core principle of the "partitioning + inhibitor" technical approach.

[0073] Example 3: On-site testing of clinical samples with different matrices This embodiment verifies the application effect of the method of the present invention in different types of animal-derived clinical samples, covering various matrices such as blood, urine, skin swabs / pus, ear canal secretions, and sputum, and clarifies the sample processing procedure and matrix interference elimination method.

[0074] Materials: The drug sensitivity test strip for dogs and cats, the matching drug sensitivity instrument (see Table 1 for details), VETAST pet testing equipment (Tianjin VETAST Biomedical Technology Co., Ltd.), and clinical samples from pet hospitals and farms.

[0075] Sample inclusion criteria: Samples that were pre-screened positive by the RIDIA pet testing equipment and confirmed to contain Gram staining containing Gram-negative bacilli and / or Gram-positive cocci.

[0076] The sample processing flow is shown in Table 7: Table 7

[0077] method: The sample processing procedure is shown in Table 6. After processing, the 0-hour background OD value was measured for all samples. After inoculation, samples were incubated at 38℃, and turbidity was monitored in real time. The endpoint OD value was subtracted from the background value to determine growth. The detection time is shown in Table 8. For each sample, a traditional pure culture followed by drug sensitivity testing was used as a control.

[0078] Table 8

[0079] Table 9 Comparison of the accuracy of drug sensitivity results

[0080] As shown in Table 9, the matrix interference elimination effect is verified as follows: Ten samples were randomly selected to compare the detection accuracy of the background subtraction method and the method without subtraction. The results are shown in Table 10. Table 10

[0081] The results showed that the background subtraction method can avoid false positives caused by matrix interference.

[0082] Conclusion: The average detection time of this invention is 6.2 hours, which is 88% shorter than traditional methods. It performs well for common sample types such as blood, urine, and skin swabs / pus. The background subtraction method effectively eliminates matrix interference, ensuring detection accuracy. The system performs excellently in veterinary hospitals and farms, providing rapid and accurate medication guidance.

[0083] Example 4: Detection of drug-resistant mixed bacteria This embodiment is used to verify the detection capability of drug-resistant mixed bacterial infections with significant clinical importance, and to demonstrate that the detection results meet the requirements of the EUCAST standard and can accurately identify key drug resistance phenotypes.

[0084] Materials: The drug susceptibility plate of this invention, and a mixed bacterial suspension of known drug-resistant strains (ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus MRSA).

[0085] method: ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) were cultured to 0.5 McFarland units and mixed bacterial suspensions were prepared in a 1:1 ratio.

[0086] Experimental group: The mixed bacterial solution was directly inoculated onto the drug sensitivity plate of the present invention according to the operating procedure of the present invention (after dilution) and then tested on the instrument.

[0087] Reference method group: Purified ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) bacterial suspensions were subjected to drug susceptibility testing on standard plates according to the micro-broth dilution method established by EUCAST.

[0088] Compare the MIC values ​​and sensitivity determination results of the two methods for key drugs (especially those used to determine drug resistance phenotypes).

[0089] The test results and data are shown in Table 11-13: Table 11 Comparison of Antimicrobial Susceptibility Results of ESBL-Producing Escherichia coli (ESBL-EC)

[0090] Table 12 Comparison of methicillin-resistant Staphylococcus aureus (MRSA) susceptibility test results

[0091] Table 13 Consistency in Identification of Key Drug Resistance Phenotypes

[0092] Validation of clinically resistant strains from animal sources: The results of testing on 20 clinically resistant animal isolates showed that the MIC values ​​obtained by the method of this invention were completely consistent with the traditional drug susceptibility testing method after pure culture, and the identification of drug resistance phenotypes of all strains met expectations. Results for some representative strains are shown in the table above.

[0093] The results of validation of clinically resistant strains from animal sources are shown in Table 14.

[0094] Table 14

[0095] Note: All strains were clinical isolates from veterinary hospitals, and their drug resistance phenotypes were confirmed by traditional pure culture identification and drug susceptibility testing.

[0096] Data Analysis: Precise detection of drug resistance: As shown in Tables 11 and 12, this embodiment accurately detected high levels of resistance in ESBL-EC to amoxicillin / clavulanic acid and ceftiofur, as well as resistance in penicillin-resistant Staphylococcus aureus to multiple drugs, including amoxicillin. The MIC values ​​of all key drugs were completely consistent with the EUCAST standard method.

[0097] Correct sensitivity retention: This example also accurately identified the sensitivity of ESBL-EC to enrofloxacin and florfenicol. This indicates that the regional inhibitor design does not affect drug efficacy assessment.

[0098] Perfect consistency in judgment: As shown in Table 13, the final phenotypic identification conclusions of ESBL generation and penicillin resistance derived from the MIC value in this embodiment are 100% consistent with the judgment criteria of the reference method.

[0099] Superior performance in mixed environments: Most importantly, all these accurate resistance and susceptibility results were obtained in a single, complex sample containing both ESBL-EC and penicillin-resistant Staphylococcus aureus. This demonstrates that the "partitioning + inhibitor" technique of this embodiment can effectively eliminate interference from mixed bacteria.

[0100] The detection results obtained by the method of this invention are 100% consistent with those of the traditional method. Specifically, the MICs of ESBL+ strains against ceftiofur are all >64 μg / mL, and the MICs of MRSA strains against oxacillin are all >8 μg / mL, meeting the criteria for drug resistance determination.

[0101] Validation of mixed drug-resistant bacteria: DR-001 (ESBL Escherichia coli) and DR-003 (MRSA) were mixed at a ratio of 1:1 to prepare a mixed bacterial sample. The samples were tested according to the method of this invention. The drug susceptibility results of the two bacteria were completely consistent with the individual test results (ceftiofur > 64, oxacillin > 8), proving that this invention is also applicable in mixed drug-resistant bacterial environments.

[0102] Conclusion: The data provided in this embodiment fully demonstrate that the present invention is not only applicable to common susceptible bacteria, but also provides accurate and rapid drug susceptibility results highly consistent with the international gold standard (EUCAST) for mixed infections of important multidrug-resistant bacteria such as ESBL-EC and MRSA. This makes it extremely valuable and authoritative in the field of monitoring and controlling antibiotic resistance in animal-derived bacteria.

[0103] Example 5: Comprehensive Performance Comparison Analysis This embodiment uses systematic and standardized parallel comparative tests to quantitatively evaluate the comprehensive performance of the invention in terms of detection cycle, operation process, manpower input, economic cost and result reliability.

[0104] Materials and Methods: Samples: Ten clinical animal samples (including pet pus / urine, avian intestinal contents, etc.) that were pre-diagnosed as mixed bacterial infections by microscopic examination were selected.

[0105] Grouping: Each sample was tested in parallel using the following two methods: Experimental group: The system of this invention (drug sensitivity plate + matching drug sensitivity instrument).

[0106] Control group: Traditional method (pure culture isolation + traditional antimicrobial susceptibility testing + manual interpretation). Method: Parallel evaluation indicators for each sample: Detailed recording and comparison of the following data from the two methods: Total testing time (from sample receipt to report generation); Active operation time (time when personnel directly participate in the operation); Number of operation steps; List of required large equipment and space required; Total cost per sample (including consumables, equipment depreciation, and labor costs); Sample contamination rate; Accuracy of results (calculated based on the results of traditional methods, with a compliance rate).

[0107] Test results and data are detailed in Table 15-17: Table 15 Comprehensive Performance Quantitative Comparison Table

[0108] Table 16. Detailed breakdown of testing time for 10 samples (unit: hours)

[0109] Table 17 Cost Composition Analysis (based on a single sample, unit: yuan)

[0110] Data Analysis: Significant efficiency advantages: This embodiment reduces the total testing cycle from over 2.5 days (60.5 hours) to less than half a day (11.2 hours), improving efficiency by over 80%. More importantly, the active operation time is reduced from nearly 4 hours (228 minutes) to less than 20 minutes, meaning that veterinarians can devote more of their valuable time to the diagnosis and treatment itself, greatly liberating productivity.

[0111] Significant process simplification and space savings: The operation steps have been reduced from 9 to 3, lowering operational complexity and the risk of errors. Equipment requirements have been reduced from biosafety cabinets and incubators requiring dedicated space to a single small desktop instrument, reducing space occupancy by 98%, making it possible to use in space-constrained veterinary hospitals or farms.

[0112] Economic benefits: Comprehensive cost analysis shows that the total cost per sample of this invention is reduced by nearly 60%. This is mainly due to: a) reduced consumable costs as no two cultures are required; b) significantly reduced labor costs due to extremely simplified operation; and c) avoidance of additional expenses from repeated testing due to the low contamination rate (10% vs 30%).

[0113] Detection quality: While achieving significant advantages in speed and cost, the system of this invention maintains an accuracy that is highly consistent with traditional methods (96.7%), proving the high reliability of its detection results.

[0114] Conclusion: This embodiment demonstrates through detailed quantitative data that this invention is not a simple improvement, but a revolutionary innovation. It comprehensively surpasses traditional methods in five dimensions: detection efficiency, ease of operation, scenario adaptability, economic cost, and reliability of control results. It is particularly suitable for large-scale application in grassroots animal clinics, veterinary hospitals, and farms that prioritize efficiency and cost-effectiveness, possessing extremely high market value and industrial driving force.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A zoned drug sensitivity plate, characterized in that, It includes a plate body and a plurality of microporous reaction units disposed on the plate body. The plate body is spatially divided into at least two independent detection zones, including a first detection zone and a second detection zone. The microwell reaction unit of the first detection area is pre-set with a first selective culture system, which includes neomycin and at least one antibacterial drug concentration gradient against Gram-negative bacilli. The neomycin is used to inhibit the growth of Gram-positive cocci in the first detection area, and the mass concentration of the neomycin in the first selective culture system is 5 μg / mL. The microwell reaction unit of the second detection area is pre-installed with a second selective culture system, which includes polymyxin B and at least one antimicrobial drug concentration gradient against Gram-positive cocci. The polymyxin B is used to inhibit the growth of Gram-negative bacilli in the second detection area, and the mass concentration of polymyxin B in the second selective culture system is 2 μg / mL. The first detection area and the second detection area are respectively provided with negative control wells and positive control wells. The negative control wells are kept clear when no antibacterial drugs are pre-placed in them and no samples are inoculated. The positive control wells are not pre-placed with antibacterial drugs and form a measurable growth curve after sample inoculation, which serves as the control baseline for determining the minimum inhibitory concentration (MIC).

2. The zoned drug sensitivity plate according to claim 1, characterized in that, The plate is a 96-well polystyrene microplate; and the partitioned antimicrobial susceptibility plate is used for the detection of antimicrobial susceptibility of Gram-negative bacilli and / or Gram-positive cocci in animal-derived clinical bacterial samples.

3. The zoned drug sensitivity plate according to claim 2, characterized in that, The first detection area is used to detect the susceptibility of Gram-negative bacilli in a single Gram-negative bacillus sample and a mixed sample containing both Gram-negative bacilli and Gram-positive cocci to antimicrobial agents. The second detection area is used to detect the susceptibility of Gram-positive cocci in a single Gram-positive cocci sample and a mixed sample containing both Gram-negative bacilli and Gram-positive cocci to antimicrobial agents.

4. A method for detecting antimicrobial susceptibility using a partitioned antimicrobial susceptibility plate according to any one of claims 1-3, characterized in that, include: Obtain clinical bacterial samples from animals; The animal-derived clinical bacterial samples were subjected to Gram staining and microscopic examination. Based on the staining results and bacterial morphology, the samples were identified as single Gram-negative bacilli, single Gram-positive cocci, or mixed samples containing both Gram-negative bacilli and Gram-positive cocci. The animal-derived clinical bacterial samples were pretreated and diluted according to the sample type to obtain the test sample solution; The test sample solution corresponding to a single Gram-negative bacillus sample is inoculated into the first detection area, the test sample solution corresponding to a single Gram-positive coccus sample is inoculated into the second detection area, and the test sample solution corresponding to the mixed sample is simultaneously inoculated into the first and second detection areas. The inoculated partitioned drug sensitivity plates were placed into the detection instrument for constant temperature incubation, and the turbidity of each well was monitored over time. Growth curves for each well were generated based on turbidity time-series monitoring data, and the growth curves for each well were compared with the corresponding control well growth curves to determine the minimum inhibitory concentration (MIC). Output sensitivity classification results based on MIC and corresponding interpretation rules.

5. The method according to claim 4, characterized in that, When the sample type is blood, use the liquid in the positive blood culture bottle as the test sample and dilute it 100 times; when the sample type is urine, directly aspirate the sample without dilution or dilute it 10 times; when the sample type is skin swab or pus, wash with physiological saline and then dilute it 100 times; when the sample type is ear canal secretions, emulsify with physiological saline and 0.1% Tween-80 and then dilute it 100 times; when the sample type is sputum, wash with physiological saline and then dilute it 100 times.

6. The method according to claim 5, characterized in that, Before constant temperature incubation, the 0h background OD value of each test well was measured; when determining the growth status, the net OD value after subtracting the corresponding 0h background OD value from the endpoint OD value was used as the basis for growth determination.

7. The method according to claim 6, characterized in that, The temperature for the isothermal incubation was controlled at 38±1℃.

8. An antimicrobial susceptibility testing system, characterized in that, The invention includes the partitioned antimicrobial susceptibility plate and the detection instrument as described in any one of claims 1-3. The detection instrument is used to perform isothermal incubation on the partitioned antimicrobial susceptibility plate, perform turbidity time-series monitoring on each well, automatically determine the minimum inhibitory concentration (MIC) based on the turbidity time-series monitoring data, and output the sensitivity grading results.

9. The antimicrobial susceptibility testing system according to claim 8, characterized in that, The detection instrument includes a temperature control component, an optical detection component, and an analysis and processing component. The temperature control component is used to control the temperature of the culture chamber at 38±1℃. The optical detection component is used to collect turbidity data from each well at multiple time points and form a turbidity time series. The analysis and processing component is used to generate a growth curve based on the turbidity time series, perform MIC determination, and output a detection report containing the detection results of Gram-negative bacilli corresponding to the first detection area and the detection results of Gram-positive cocci corresponding to the second detection area.

Images