A bacterial drug resistance detection device and method thereof

Abstract

The application discloses a bacterial drug resistance detection device, which comprises a culture container, the culture container comprises at least two separable first parts and second parts, the first parts are used for containing aqueous solutions of microorganisms, the second parts are used for containing biological or chemical substances, the first parts and the second parts are changed in physical position to make the aqueous solutions of the microorganisms and the biological or chemical substances contact and interact with each other. A microscopic imaging system is configured to image the substances in the culture container multiple times, and the survival state of the microorganisms is obtained by analyzing the apparent morphology of the microorganisms. The application also provides a bacterial drug resistance detection method based on the device. The application provides a rapid and simple MIC quantitative detection device and method, and realizes high-throughput screening of the MIC of microorganisms in the case of combination of various antibiotics.

Description

Technical Field

[0001] This application relates to the fields of biochips and bacterial detection technology, and in particular to a bacterial drug resistance detection device and method. Background Technology

[0002] The discovery of antibiotics has saved many lives, but their inappropriate use and abuse have led to the emergence of drug-resistant bacteria and viruses. Multidrug-resistant microorganisms are becoming a significant threat to human health and public health security. Rapid identification of drug-resistant bacterial species and obtaining antibiotic susceptibility test results can guide clinicians to optimize antibiotic use, thereby reducing antibiotic overuse.

[0003] Currently, there are two main categories of methods for antibiotic susceptibility testing: phenotypic detection and genotypic detection. Phenotypic detection includes broth microdilution, disk diffusion (KB method), and concentration gradient agar diffusion. These traditional methods are cumbersome and take one to two days to obtain results. While some companies have developed semi-automatic or automated instruments, these are bulky and expensive. In recent years, molecular biology-based antibiotic susceptibility testing methods have become a promising approach. Genotypic detection methods can rapidly detect specific resistance genes or gene mutations through PCR, sequencing, etc. For example, methicillin-resistant Staphylococcus aureus can be identified within minutes by specifically amplifying the mecA and mecC genes using PCR. However, genotypic detection is highly dependent on understanding resistance mechanisms and resistance genes. Current research on bacterial resistance mechanisms is insufficient, resulting in only a limited number of resistant bacteria being identifiable using this method. Furthermore, the resistance mechanisms of Gram-negative bacteria are more complex than those of Gram-positive bacteria, making genotypic detection unsuitable for Gram-negative bacteria.

[0004] Microfluidic chips can manipulate solutions at nanoliter levels and have proven promising for antibiotic susceptibility testing. Microfluidic chips can generate large numbers of microdroplets for high-throughput screening; the small size and confinement effect accelerate the reaction and yield results more quickly. While microfluidic chips have been applied to bacterial antibiotic susceptibility phenotypic and genotypic analysis, current instruments still require complex control equipment and cannot achieve high throughput. Therefore, there is a need to develop more user-friendly and convenient microfluidic devices for antibiotic susceptibility testing. Summary of the Invention

[0005] The purpose of this application is to provide a bacterial resistance detection device and method to solve the problems of long detection time, low sensitivity, single detection results, and complicated operation in existing bacterial resistance detection methods.

[0006] One aspect of this application discloses a bacterial resistance detection device, comprising:

[0007] - A culture container, said culture container comprising at least two separable first parts and second parts.

[0008] The first portion is at least an aqueous solution for containing microorganisms.

[0009] The second part is used to contain biological or chemical substances.

[0010] The first part and the second part, through the relative change of their physical positions, allow the aqueous solution of the microorganism to come into contact with and interact with the biological or chemical substance;

[0011] - A microscopic imaging system configured to perform multiple imaging of interacting substances within the culture container and to obtain the survival status of microorganisms by analyzing their morphological appearance.

[0012] In a preferred embodiment, the culture vessel is a microfluidic chip for generating droplet arrays.

[0013] In a preferred embodiment, the microfluidic chip includes an upper chip and a lower chip, wherein

[0014] The upper chip includes at least a liquid inlet; the upper chip is used to form an array of droplets of microbial solution;

[0015] The lower chip includes at least an array of micropits for pre-embedding biological or chemical substances;

[0016] When the upper and lower chips are assembled into their initial positions, the droplet array of the upper chip does not overlap with the micro-pit array of the lower chip, and the aqueous solution of the microorganisms cannot come into contact with the biological or chemical substances.

[0017] When the solution is injected into the chip, the upper and lower chips are moved relative to each other, and the droplet array of the upper chip coincides with the micro-pit array of the lower chip, thereby mixing the aqueous solution of the microorganism with the biological or chemical substance to form a mixed droplet array.

[0018] In a preferred embodiment, the lower chip further includes one or more discontinuous microchannels.

[0019] In a preferred embodiment, the upper chip further includes one or more discontinuous microchannels and / or micropit arrays.

[0020] In a preferred embodiment, the micropit array density of the lower chip is 4-100,000 holes / cm², and / or

[0021] The droplet array density of the chip is 4-100,000 droplets / cm².

[0022] In a preferred embodiment, the volume of each micropit is 0.001-1000 nanoliters.

[0023] In a preferred embodiment, the microchannels of the upper and / or lower chip have the following dimensions: width of 1μm to 10cm, length of 100μm to 100cm, and depth of 1μm to 1cm.

[0024] In a preferred embodiment, the biological or chemical substance is used to promote, kill, or inhibit the growth or division of cells or microorganisms.

[0025] Another aspect of this application discloses a method for detecting bacterial resistance, comprising the following steps:

[0026] (In a preferred embodiment, biological or chemical substances are pre-embedded in the micro-pit array of the lower chip;)

[0027] (S2) Assemble the upper and lower chips into their initial positions and inject an aqueous solution of microorganisms into the upper chip through the liquid inlet to form a droplet array on the upper chip;

[0028] (S3) The upper chip and the lower chip are moved relative to each other, and the droplet array of the upper chip coincides with the micro-pit array of the lower chip, so that the aqueous solution of the microorganism is mixed with the biological or chemical substance to form a mixed droplet array;

[0029] (S4) The bacterial resistance detection device is placed in an incubator at a set temperature and incubated for a set time. During this time, a microscopic imaging system is used to capture continuous images of the mixed droplet array at fixed time intervals.

[0030] (S5) By analyzing the morphology of microorganisms, the survival status of microorganisms can be obtained, and the minimum inhibitory concentration can be determined.

[0031] In a preferred embodiment, the analysis of the apparent morphology includes at least one of the following: microbial quantity analysis, shape analysis, aggregation state analysis, and movement trajectory analysis.

[0032] In a preferred embodiment, the aqueous solution of the microorganism is an Escherichia coli culture medium.

[0033] In a preferred embodiment, the biological or chemical substance is one or more of the following: β-lactams, penicillins, cephalosporins, aminoglycosides, quinolones, macrolides, polypeptides, tetracyclines, chloramphenicol, sulfonamides, rifamycins, and bacteriophages.

[0034] In a preferred embodiment, the biological or chemical substance is pre-embedded in the micropits of the lower chip at a certain concentration gradient.

[0035] In a preferred embodiment, the concentration gradient ranges from 1 ng / mL to 1 mg / mL.

[0036] In a preferred embodiment, the concentration gradient is 0.125 × 2 x The concentration increases in μg / mL, where x ranges from -5 to x ≤ 12 and x is an integer.

[0037] This application has at least the following technical effects:

[0038] 1. The device uses a sliding chip as a culture device for microorganisms. The relative movement of the sliding chip device is used to manipulate droplets and form droplet arrays, without the need for a complex fluid control system, making it simple to operate.

[0039] 2. Achieving antibiotic susceptibility testing: Existing technologies require at least 48 hours for microbial culture, while this invention only requires 2-3 hours from microbial culture to completion of drug resistance determination.

[0040] 3. The device's microfluidic chip has a high-throughput micro-pit array, which can simultaneously screen multiple antibiotics in high throughput. The antibiotics are arranged and combined in different concentrations and types, and cultured and tested at the same time. There is no need to repeatedly control variables and test them one by one, which greatly improves the detection efficiency.

[0041] 4. The chip can be stored dry.

[0042] 5. Compared with other droplet generation methods, the present invention does not require a complex control system and can effectively control the size and shape of the generated droplets.

[0043] 6. Compared with traditional micro-pit array microfluidic chips, the present invention can achieve better physical isolation, so that there is no cross-contamination between micro-pits. Attached Figure Description

[0044] Figure 1 The microfluidic chip according to the first embodiment of this application includes an upper chip and a lower chip;

[0045] Figure 2 It is a lower chip containing pre-embedded material according to the first embodiment of this application;

[0046] Figure 3 According to the first embodiment of this application, the microfluidic chip is in the liquid injection position, and an aqueous solution containing microorganisms is injected into the microfluidic chip;

[0047] Figure 4According to the first embodiment of this application, the microfluidic chip is in a segmented position, and the aqueous solution containing microorganisms is segmented into microdroplets and overlaps with the micropits of the lower chip.

[0048] Figure 5 According to the first embodiment of this application, the microfluidic chip is in a segmented position, and after a period of cultivation, the morphology of microorganisms in the microdroplets changes;

[0049] Figure 6a , 6b It is the upper chip of the microfluidic chip according to the second embodiment of this application;

[0050] Figure 7a , 7b It is the lower chip of the microfluidic chip according to the second embodiment of this application;

[0051] Figure 8 It is a lower chip containing pre-embedded material according to the second embodiment of this application;

[0052] Figure 9 It is a microfluidic chip assembled according to the second embodiment of this application;

[0053] Figure 10 The microfluidic chip assembled according to the second embodiment of this application is located at the liquid injection position, and an aqueous solution containing microorganisms is injected into the microfluidic chip;

[0054] Figure 11 The microfluidic chip assembled according to the second embodiment of this application is in a segmented position, wherein an aqueous solution containing microorganisms is segmented into microdroplets and overlaps with the micropits of the lower chip;

[0055] Figure 12 The microfluidic chip assembled according to the second embodiment of this application is in a segmented position. After a period of cultivation, the morphology of microorganisms in the microdroplets changes.

[0056] Figure 13 It is the upper chip of the microfluidic chip according to the third embodiment of this application;

[0057] Figure 14 It is the lower chip of the microfluidic chip according to the third embodiment of this application;

[0058] Figure 15 It is a microfluidic chip assembled according to the third embodiment of this application, located at the liquid injection position;

[0059] Figure 16 It is a microfluidic chip according to the third embodiment of this application, located in the segmented position;

[0060] Figure 17It is the upper chip of the microfluidic chip according to the fourth embodiment of this application;

[0061] Figure 18 It is the lower chip of the microfluidic chip according to the fourth embodiment of this application;

[0062] Figure 19 It is a microfluidic chip assembled according to the fourth embodiment of this application, located at the liquid injection position;

[0063] Figure 20 It is a microfluidic chip according to the fourth embodiment of this application, located at the segmentation position;

[0064] Figure 21 This is a schematic diagram of the bacterial resistance detection method according to this application;

[0065] Figure 22 This is a schematic diagram of the detection results of the sensitivity of Escherichia coli to nitrofurantoin and ampicillin in the bacterial resistance detection method according to this application;

[0066] Figure 23 This is a schematic diagram of the detection results of the sensitivity of Escherichia coli to ceftazidime and ciprofloxacin in the bacterial resistance detection method of this application;

[0067] Figure 24 This is a schematic diagram of four antibiotics in different combinations of types and dosages, applied to a microfluidic chip in a 2×2 dot matrix according to the bacterial resistance detection method of this application.

[0068] Figure 25 This is a statistical chart of bacterial counts after testing a combination of four antibiotics according to the bacterial resistance detection method of this application. Detailed Implementation

[0069] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0070] Explanation of some concepts:

[0071] Minimum Inhibitory Concentration (MIC)

[0072] The minimum inhibitory concentration (MIC) is generally considered the most basic laboratory measurement of the activity of antimicrobial agents. For a specific antimicrobial drug, the lowest concentration of the drug that can inhibit bacterial growth during bacterial culture is called the MIC. The MIC is generally regarded as the most fundamental indicator of the antimicrobial activity of an antimicrobial agent. Laboratories primarily use MIC values ​​to determine bacterial resistance. The MIC is also used to evaluate the activity of new antimicrobial agents.

[0073] The following is a brief summary of some of the innovative aspects of the embodiments of this application:

[0074] Microfluidic biochips can be used as containers for culturing cells and microorganisms, altering the internal fluid connections through relative changes in physical position. A microfluidic chip comprises two wafers: an upper wafer and a lower wafer. At least one wafer includes micropits into which biological or chemical substances can be added; these substances can be in solid, liquid, or colloidal form. Specifically, these substances can be compounds that promote, kill, or inhibit the (active) growth / division of cells and microorganisms (e.g., antibiotics or bacteriophages). Different types, concentrations, and combinations of substances can also be placed in the micropits to screen for cellular and microbial responses.

[0075] The upper chip includes a portion of the flow channel. The assembly of the upper and lower chips forms a complete flow channel. The flow channel does not overlap with the aforementioned micropits. An aqueous solution containing microorganisms can be injected into the chip through this flow channel. Specifically, the microorganisms can be bacteria or fungi.

[0076] By changing the relative physical positions of the two chips, the liquid in the original flow channel is divided into independent volumes and mixed with the material in the micro-pits.

[0077] Specifically, changes in the number of microorganisms during culture can be used to determine their sensitivity to a particular type or combination of antibiotics. For example, bacteria are photographed at 0 hours and at 1, 2, and 3 hours. If the number of bacteria in the 1, 2, and 3-hour photographs is significantly higher than that in the 0-hour photographs, then the bacteria are not sensitive to the antibiotic or the combination of antibiotics.

[0078] Specifically, changes in the morphology of microorganisms can be used to determine their sensitivity to a particular type or combination of antibiotics. For example, under the influence of ceftazidime, the division of *E. coli* is affected, resulting in *E. coli* becoming longer. This indicates that *E. coli* is sensitive to ceftazidime.

[0079] Specifically, changes in microbial movement patterns can be used to determine whether a microorganism is sensitive to a particular type or combination of antibiotics. This can be achieved by scanning the culture vessel at intervals, or by continuously photographing or capturing images of the same location. The movement patterns of *E. coli* under the influence of ciprofloxacin are compared to those of *E. coli* without antibiotics (e.g., length of movement, frequency and manner of twisting). A reduction in these patterns indicates sensitivity to ciprofloxacin.

[0080] Microbial resistance can be determined by using one, two, or three of the following methods: quantity, shape, and movement trajectory.

[0081] In particular, software can be used to analyze the number, shape, and movement trajectory of cells and microorganisms.

[0082] The microscopic imaging system includes an imaging submodule. When the upper and lower chips are combined and their respective contents are mixed, the imaging submodule continuously images the micro-pits on the chips at fixed time intervals. The imaging submodule can include, for example, a research-grade inverted microscope.

[0083] The microscopic imaging system further includes a quantitative analysis submodule, which statistically analyzes the number, length variation, and community size of microorganisms in the images recorded by the imaging submodule, and then quantifies their MIC (microbial microstructure). The quantitative analysis submodule can be, for example, ImageJ image software.

[0084] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0085] The first embodiment of this application relates to a microfluidic chip, such as Figures 1 to 4 As shown.

[0086] like Figure 1 The diagram shows the upper and lower chips of this embodiment. The upper chip includes a portion of the flow channels.

[0087] like Figure 2 As shown, Substance-1, Substance-2, Substance-3… can be pre-embedded in the micro-pits of the lower chip. The substance can be a chemical or biological substance, a single substance, or a mixture of two or more substances. The substance can be in a solid, liquid, or colloidal state. In one embodiment, the substance is a compound (e.g., an antibiotic or bacteriophage) that promotes, kills, or inhibits the growth / division of cells and microorganisms (active).

[0088] like Figure 3 As shown, a solution containing cells or microorganisms is injected into the interconnected flow channels. The solution may also contain culture components.

[0089] like Figure 4 As shown, microdroplets are formed by changing the physical position of the upper and lower chips. Substances-1,-2, and-3, which were pre-embedded, dissolve into the microdroplets. Microscopic observation is then performed, and the phenotypes (shape, number, movement, etc.) of the cells or microorganisms within the microdroplets are recorded.

[0090] like Figure 5 As shown, after a period of cultivation (e.g., 37 degrees Celsius for 1 hour), microscopic observations were performed again, and the phenotypes (shape, number, motility, etc.) of the cells or microorganisms in the microdroplets were recorded. By comparing the phenotypes with the previous ones, it was determined whether substances-1,-2,-3, etc., had any effect on the growth of cells or microorganisms (inhibition or promotion).

[0091] The second embodiment of this application relates to another microfluidic chip, as shown in 6a, 6b, 7a, and 7b. The upper chip has a series of micropits and microchannels, the micropits being T1, T2, and T3. The lower chip also has a series of micropits and microchannels. The micropits of the lower chip are B1, B2, and B3.

[0092] like Figure 8 As shown, substances-1,-2,-3, etc. can be pre-embedded in the micro-pits of the lower chip. The substances can be chemical or biological substances, and can be a single substance or a mixture of two or more substances.

[0093] like Figure 9 As shown, the upper and lower chips are assembled, and the micro-pits of the upper chip and the micro-channels of the lower chip partially overlap to form a connected flow channel;

[0094] like Figure 10 As shown, a solution containing cells or microorganisms will be injected into the connected flow channels. The solution may also contain culture components.

[0095] like Figure 11 As shown, microdroplets are formed by changing the physical position of the upper and lower chips. Substances-1,-2, and-3, which were pre-embedded, dissolve into the microdroplets. Microscopic observation is then performed, and the phenotypes (shape, number, movement, etc.) of the cells or microorganisms within the microdroplets are recorded.

[0096] like Figure 12 As shown, after a period of cultivation (e.g., 37 degrees Celsius for 1 hour), microscopic observations were performed again, and the phenotypes (shape, number, motility, etc.) of the cells or microorganisms in the microdroplets were recorded. By comparing the phenotypes with the previous ones, it was determined whether substances-1,-2,-3, etc., had any effect on the growth of cells or microorganisms (inhibition or promotion).

[0097] The third embodiment of this application relates to a high-throughput microfluidic chip, such as... Figures 13 to 16 As shown.

[0098] On chip such as Figure 13 As shown;

[0099] Next chip Figure 14 As shown, each micro-pit can be pre-buried with Material-1, Material-2, Material-3…:

[0100] like Figure 15 As shown, the upper and lower chips are assembled into their initial positions (by injecting a solution containing cells or microorganisms);

[0101] like Figure 16 As shown, the upper chip moves relative to the lower chip, generating droplets of solution and mixing them with the pre-embedded material.

[0102] The fourth embodiment of the application relates to a high-throughput microfluidic chip, such as Figures 17 to 20 As shown.

[0103] On chip such as Figure 17 As shown;

[0104] Next chip Figure 18 As shown, each micro-pit can be pre-buried with substance-1, substance-2, substance-3, etc.;

[0105] like Figure 19 As shown, the upper and lower chips are assembled into their initial positions (by injecting a solution containing cells or microorganisms);

[0106] like Figure 20 As shown, the upper chip moves relative to the lower chip, generating droplets of solution and mixing them with the pre-embedded material.

[0107] To better understand the technical solution of this application, a specific example is provided below. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of this application.

[0108] The microfluidic chip in this embodiment is made of glass, measuring 5.5 cm in length and 3.5 cm in width, and consists of two layers. The upper chip includes micropits with interconnected channels, each with a radius of 500 micrometers and a depth of 50 micrometers. The lower chip includes 192 micropits with a radius of 400 micrometers and a depth of 30 micrometers. The combined volume of the upper and lower chips is only 50 nL.

[0109] First, based on the minimum inhibitory concentration (MIC) of the antibiotics, 12 different concentration solutions were prepared according to a 2x concentration gradient. Using a manual spotting instrument, 200 nmol of each antibiotic solution was spotted into the corresponding well of the lower chip. The spotting pattern for each antibiotic was a 2×2 matrix. Figure 21 (A, B, F in the text). After the antibiotics have dried, the upper and lower chips are assembled together in PMX-200 dimethyl silicone oil. Figure 21 (C in the text). Next, the concentration was 5-10 × 10⁻⁶. 5 CFU / mL of E. coli culture medium was injected into the chip using a pipette. Figure 21 (D) The bacterial solution is blended with the antibiotic through a simple sliding motion. Figure 21 (E in the image). The chip was then incubated in a 37°C incubator for three hours. Every hour, a Nikon Ti2 microscope was used to photograph each well to count the number of bacteria and observe changes in bacterial morphology and motility.

[0110] By statistically analyzing changes in bacterial counts in different micropits, combined with changes in bacterial morphology and motility, the sensitivity of bacteria to a particular antibiotic can be determined, and the MIC (microinjection threshold) can be quantified. Figure 21 (G in the middle).

[0111] To verify that our chip can rapidly obtain antibiotic susceptibility results, the inventors selected the standard strain of Escherichia coli, E. coli ATCC 25922, for testing. We selected four commonly used antibiotics: ampicillin (Amp), ciprofloxacin (Cip), ceftazidime (Cef), and nitrofurantoin (Nit) (Table 1).

[0112] Table 1

[0113]

[0114] Four antibiotics at twelve concentration gradients were applied to the wells of the lower chip using a spotting method. After the antibiotics dried, the upper and lower chip layers were assembled. Then, *E. coli* bacteria were injected into the chip through the injection wells, and the antibiotics were mixed with the bacterial culture by sliding the injection tube. The chip was then incubated at 37°C. Every hour, each micro-pit of the chip was photographed to analyze the dynamic changes in the bacteria. Figure 22As shown in Figures A and B, a concentration gradient from 0.5 μg / mL to 512 μg / mL was established in the nitrofurantoin group. When the concentration was below 2 μg / mL, the proliferation rate of *E. coli* was basically the same as that of the control group; when the nitrofurantoin concentration was between 4-16 μg / mL, the length of *E. coli* increased to some extent, and the number still increased to some extent; when the nitrofurantoin concentration was greater than or equal to 32 μg / mL, the growth of *E. coli* was completely inhibited, and the number of bacteria no longer changed. Through statistical analysis of the changes in the number of bacteria, we were able to obtain the MIC of *E. coli* ATCC 25922 as approximately 32 μg / mL, which is basically consistent with the MIC results obtained by conventional bacterial culture methods in the American Classification of Microbial Susceptibility Testing (CLSI 2021).

[0115] In the ampicillin group, a concentration gradient ranging from 0.125 μg / mL to 128 μg / mL was established. For example... Figure 22 As shown in Figure C, *E. coli* can proliferate when the concentration of ampicillin is below 1 μg / mL; *E. coli* will lengthen when the concentration is between 2 μg / mL and 8 μg / mL; and when the concentration is greater than or equal to 8 μg / mL, *E. coli* will not divide at all and will directly shrink into protoplasmic spheroids. Therefore, the sensitivity of bacteria to ampicillin can be determined and the MIC can be quantified by observing changes in bacterial number and morphology.

[0116] β-lactam antibiotics, such as cephalosporins, can bind to penicillin-binding proteins (PBPs) and inhibit cell wall peptidoglycan synthase, thereby hindering cell wall peptidoglycan synthesis and achieving a bactericidal effect. For example... Figure 23 In the ceftazidime group, a concentration gradient ranging from 0.0625 μg / mL to 64 μg / mL was established. When the ceftazidime concentration was below 0.5 μg / mL, the growth of *E. coli* was completely unaffected. When the cephalosporin concentration was between 1 and 4 μg / mL, bacterial division was inhibited, and the bacteria gradually became longer. When the concentration was greater than or equal to 4 μg / mL, the bacterial condition began to deteriorate, and the bacteria gradually died. The minimum inhibitory concentration (MIC) could be quantified by statistically analyzing the changes in bacterial number and length.

[0117] Urinary tract infections are among the most common infections worldwide. The emergence of multidrug-resistant strains exacerbates this public health problem. To explore the clinical application of this chip, the inventors obtained five strains of multidrug-resistant Escherichia coli from urine samples obtained from a hospital (Table 2).

[0118] Table 2

[0119]

[0120] Ciprofloxacin is a third-generation fluoroquinolone antibiotic widely used to treat bacterial infections, such as urinary tract infections. It exerts its antibacterial effect by inhibiting bacterial DNA replication through the inhibition of bacterial DNA topoisomerase and DNA cyclase. At ciprofloxacin concentrations below 0.0625 μg / mL, *E. coli* divides slowly and becomes longer. At concentrations between 0.0625 and 0.25 μg / mL, *E. coli* no longer proliferates, but its length increases slightly. At concentrations above 0.25 μg / mL, bacterial activity is completely inhibited.

[0121] The discovery of new antibiotics largely relies on the chemical modification and de novo synthesis of natural products. The process from research and development to clinical application of a new antibiotic is quite slow. Based on existing antibiotics, one approach to treating multidrug-resistant bacteria is combination therapy, including combinations of different antibiotics and combinations of antibiotics with non-antibiotic small molecules. Combination therapy has been successfully applied to treat Mycobacterium tuberculosis, HIV, and malaria, and it can also prevent pathogen resistance to single drugs. Furthermore, antibiotic combination therapy has shown good results in treating drug-resistant MRSA and Enterococcus faecalis. Regarding drug resistance in urinary tract infections, we created 15 combinations by permuting four antibiotics. Using the microfluidic chip described in this application, all 15 combinations can be detected simultaneously (each combination includes three different concentrations (0.5S, S, and R), with each concentration including four replicates). Figure 24 As shown, we can quickly obtain sensitivity profiles of bacteria against multiple antibiotics and combinations of antibiotics, thereby guiding clinicians in medication use.

[0122] The results are as follows Figure 25As shown, by statistically analyzing the absolute number of bacteria, we can determine the antibiotic susceptibility of the bacteria. The changes in bacterial count reveal that all five *E. coli* strains are resistant to ampicillin and ciprofloxacin. Specifically, b10046 is resistant to all four antibiotics; b09088 is sensitive to nitrofurantoin; and b08920, b08712, and b09314 are sensitive to both nitrofurantoin and ceftazidime. These results are consistent with clinically obtained antibiotic susceptibility data. For *E. coli* b10046, combinations of low concentrations (0.5S) and medium concentrations (S) had no inhibitory effect; however, combinations containing high concentrations (R) of nitrofurantoin inhibited the growth of *E. coli* b10046; and high concentrations of ampicillin and ciprofloxacin also inhibited the proliferation of b10046. Since b08920, b08712, and b09314 are sensitive to nitrofurantoin and ceftazidime, their growth can be inhibited as long as nitrofurantoin or ceftazidime is present in the combination, even at antibiotic concentrations only half of the MIC. A medium concentration (S) of ampicillin and ciprofloxacin in combination also showed good inhibitory effects on these three ampicillin and ciprofloxacin-resistant bacteria. b09088 is sensitive to nitrofurantoin; a medium concentration (S) of nitrofurantoin in the combination significantly inhibited its growth. In combinations of three and four antibiotics, even low concentrations (0.5S) of nitrofurantoin significantly inhibited the proliferation of b09088. Furthermore, high concentrations of antibiotics, besides inhibiting bacterial division, also lead to a decrease in bacterial motility. We can record bacterial motility over a period of time using a microscope, and the changes in motility can help determine the antibiotic resistance status of the bacteria.

[0123] The above results indicate that multidrug-resistant bacteria can be treated by using antibiotics in combination, and that this combination can effectively inhibit bacterial proliferation while reducing the concentration of antibiotics used. This provides valuable insights for clinicians in choosing antibiotics.

[0124] In addition, by changing the chip material, such as PDMS, and the chip structure, SlipChip can also be used for cell culture and screening of related drugs.

[0125] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0126] This specification includes combinations of various embodiments described herein. Individual references to “one embodiment” or a particular embodiment, etc., do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless indicated to be mutually exclusive or are readily apparent to those skilled in the art. It should be noted that the word “or” is used in a non-exclusive sense throughout this specification unless the context explicitly indicates or requires it.

[0127] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A bacterial resistance detection device, characterized in that, include: - A culture container, comprising at least two separable first parts and a second part, wherein the first part is used to contain an aqueous solution of microorganisms. The second part includes at least micropits for containing biological or chemical substances; the volume of the micropits is 0.001-1000 nanoliters. The first part and the second part, through the relative change of their physical positions, allow the aqueous solution of the microorganism to come into contact with and interact with the biological or chemical substance; - A microscopic imaging system configured to perform multiple imaging of interacting substances within the culture container and to obtain the survival status of microorganisms by analyzing their morphological appearance; the morphological analysis includes at least one of the following: microbial quantity analysis, shape analysis, aggregation state analysis, and movement trajectory analysis. The culture container is a microfluidic chip used to generate droplet arrays; The microfluidic chip includes an upper chip and a lower chip, wherein The upper chip includes at least a liquid inlet; the upper chip is used to form an array of droplets of microbial solution; The lower chip includes at least an array of micropits for pre-embedding biological or chemical substances; When the upper and lower chips are assembled into their initial positions, the droplet array of the upper chip does not overlap with the micro-pit array of the lower chip, and the aqueous solution of the microorganisms cannot come into contact with the biological or chemical substances. When the solution is injected into the chip, the upper and lower chips are moved relative to each other, and the droplet array of the upper chip coincides with the micro-pit array of the lower chip, thereby mixing the aqueous solution of the microorganism with the biological or chemical substance to form a mixed droplet array.

2. The bacterial resistance detection device as described in claim 1, characterized in that, The lower chip also includes one or more discontinuous microchannels.

3. The bacterial resistance detection device as described in claim 1, characterized in that, The chip also includes one or more discontinuous microchannels and / or micropit arrays.

4. The bacterial resistance detection device as described in claim 1, characterized in that, The micro-pit array density of the lower chip is 4-100,000 holes / cm². 2 , and / or The droplet array density of the upper chip is 4-100,000 droplets / cm². 2 .

5. The bacterial resistance detection device as described in claim 1, characterized in that, The volume of each micropit is 0.001-1000 nanoliters.

6. The bacterial resistance detection device as described in claim 2 or 3, characterized in that, The dimensions of the microchannels of the upper and / or lower chips are as follows: width 1μm to 10cm, length 100μm to 100cm, and depth 1μm to 1cm.

7. The bacterial resistance detection device according to any one of claims 1-5, characterized in that, The biological or chemical substance is used to promote, kill, or inhibit the growth or division of cells or microorganisms.

8. A method for detecting bacterial resistance, characterized in that, Includes the following steps: (S1) Using the bacterial resistance detection device as described in any one of claims 1-5, pre-embed biological or chemical substances in the micro-pit array of the lower chip; (S2) Assemble the upper and lower chips into their initial positions and inject an aqueous solution of microorganisms into the upper chip through the liquid inlet to form a droplet array on the upper chip; (S3) The upper chip and the lower chip are moved relative to each other, and the droplet array of the upper chip coincides with the micro-pit array of the lower chip, so that the aqueous solution of the microorganism is mixed with the biological or chemical substance to form a mixed droplet array; (S4) Place the bacterial resistance detection device in an incubator at a set temperature and incubate for a set time. During this period, a microscopic imaging system was used to capture continuous images of the mixed droplet array at fixed time intervals; (S5) By analyzing the morphology of microorganisms, the survival status of microorganisms can be obtained, and the minimum inhibitory concentration can be determined.

9. The bacterial resistance detection method as described in claim 8, characterized in that, The analysis of the apparent morphology includes at least one of the following: microbial quantity analysis, shape analysis, aggregation state analysis, and movement trajectory analysis.

10. The bacterial resistance detection method as described in claim 8, characterized in that, The aqueous solution of the microorganism is an Escherichia coli culture medium.

11. The bacterial resistance detection method according to any one of claims 8-10, characterized in that, The biological or chemical substance is one or more of the following: β-lactams, aminoglycosides, quinolones, macrolides, polypeptides, tetracyclines, chloramphenicols, sulfonamides, rifamycins, and bacteriophages.

12. The bacterial resistance detection method according to any one of claims 8-10, characterized in that, The biological or chemical substances are pre-embedded in the micropits of the lower chip at a certain concentration gradient.

13. The bacterial resistance detection method as described in claim 12, characterized in that, The concentration The gradient ranges from 1 ng / mL to 1 mg / mL.

14. The bacterial resistance detection method as described in claim 12, characterized in that, The concentration gradient is according to 0.125 × 2 x The concentration increases in μg / mL, where x ranges from -5 to x ≤ 12, and x is an integer.

Images