Method for detecting bacterial drug resistance heterogeneity based on single-cell microfluidic system and application thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a single-cell microfluidic system for bacteria, which comprises a plurality of microchambers, at least two liquid inlet channels and at least one liquid outlet, the two liquid inlet channels are oppositely arranged on two sides of the microchambers, a stopper is arranged between the microchamber and a liquid inlet channel, the stopper is used for preventing the bacteria from flowing into the corresponding liquid inlet channel from the microchamber, and the height of the microchamber is equivalent to the diameter of a single bacterium to be detected. When the bacteria are cultured, the bacteria are independently distributed in the microchambers one by one, and the situation of multiple bacteria overlapping does not occur, so that single-cell capture and tracking are facilitated, and an ideal platform is provided for tracking single-bacterium division and observing the change of the bacterium morphology under different antibiotic pressures in real time.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for detecting bacterial drug resistance heterogeneity based on a single-cell microfluidic system and its application. Background Technology

[0002] Besides multidrug-resistant strains leading to antibiotic treatment failure in clinical practice, a growing body of research indicates that bacterial flora heterogeneity may also be a contributing factor. For example, clinically observed bacterial flora exhibiting drug resistance heterogeneity, persistent bacteria, or non-dividing bacteria may evade antibiotic treatment and, under suitable conditions, resume proliferation, causing secondary infections and requiring further treatment.

[0003] Therefore, detecting bacterial heterogeneity is particularly important when designing antibiotic dosing regimens. Currently, most traditional detection methods treat the bacterial community as the entire research object, often ignoring the differences between individual bacteria and lacking specific information about individual bacteria to guide medication regimens. Summary of the Invention

[0004] This invention provides a single-cell microfluidic system comprising a plurality of microchambers, at least two inlet channels and at least one outlet. The two inlet channels are disposed opposite to each other on both sides of the microchambers. A stop is provided between the microchamber and one inlet channel to prevent bacteria from flowing from the microchamber into the corresponding inlet channel. The height of the microchamber is approximately equal to the diameter of the single bacterium to be detected.

[0005] Preferably, the height of the microchamber is submicron.

[0006] According to an embodiment of the present invention, the interior of the microchamber is provided with a plurality of support columns, which are used to support the microchamber to prevent it from collapsing during the experiment.

[0007] According to an embodiment of the present invention, the liquid inlet channel is connected to an external liquid via a microfluidic pump. Under the action of the microfluidic pump, the external liquid is pumped into the liquid inlet channel and flows into the microchamber.

[0008] Preferably, the external liquid includes culture medium, bacterial culture, antibiotic solution, pure water, and buffer solution.

[0009] According to an embodiment of the present invention, the stop is a stepped structure disposed between the microchamber and the corresponding liquid inlet channel, the height of the stepped structure being less than the height of the microchamber, preferably, the height of the stepped structure being 0.4 μm; the stop can also be a baffle disposed at any position between the microchamber and the corresponding liquid inlet channel, more preferably, the baffle is disposed at the end of the microchamber, and the distance between the bottom of the baffle and the bottom of the chip is 0.2 to 0.6 μm.

[0010] Preferably, the number of micro-chamber structures is set according to actual needs, and the number can be any integer. Preferably, the number of micro-chamber structures is even and arranged in an array structure. Preferably, the number of micro-chamber structures is ≥6, and preferably, the number of micro-chamber structures is ≥8.

[0011] For example, the single-cell microfluidic system consists of two parallel channels (50μm×10μm; width×height) and eight identical microchambers (200μm×80μm×0.74μm; length×width×height). A stepped structure with a height of 0.4μm is provided on one side of each microchamber to prevent bacteria from flowing directly out of the microchamber during loading.

[0012] Preferably, the single-cell microfluidic system is further provided with a heating plate, which is used to provide constant temperature conditions for the microchamber.

[0013] The submicron-height chamber in this invention can easily achieve single-cell capture and tracking, providing an ideal platform for tracking single bacterial division and observing changes in bacterial morphology under different antibiotic pressures in real time.

[0014] Microchambers and liquid inlet channels are obtained by photolithography on a silicon substrate to obtain a template. PDMS (polydimethylsiloxane) silicone is mixed with a curing agent and poured into the template. After debubbling, drying and curing, the template is sliced ​​to obtain a PDMS chip. The PDMS chip is then bonded to a glass slide to obtain a single-cell microfluidic system.

[0015] Applications of a single-cell microfluidic system, including cell culture, cell morphology observation, bacterial resistance testing, drug screening, and drug delivery method screening.

[0016] A method for detecting bacterial drug resistance heterogeneity includes the following steps: first, introducing bacterial culture into the above-mentioned single-cell microfluidic system and culturing it until stable; then, introducing different concentrations of antibacterial drugs into the single-cell microfluidic system for co-incubation; and finally detecting the morphology and mortality rate of the bacteria to determine the drug resistance heterogeneity of the bacteria.

[0017] A method for screening antimicrobial drugs includes the following steps: introducing bacterial culture into the above-mentioned single-cell microfluidic system and culturing it until stable; introducing different antimicrobial drugs into the single-cell microfluidic system for co-incubation; and detecting bacterial mortality.

[0018] A method for testing the drug administration time of drug-resistant heterogeneous bacteria includes the following steps: introducing bacterial culture into the above-mentioned single-cell microfluidic system and culturing it until stable; introducing an antibacterial drug into the single-cell microfluidic system for co-incubation; separating undivided cells and culturing them again; and determining the time required for the undivided cells to divide again, wherein the time is the drug administration interval.

[0019] The concentration of the antibacterial drug is such that it can induce antibiotic pressure in the bacteria, and the concentration of the antibacterial drug is greater than 300 mg / L, preferably greater than 400 mg / L, and more preferably greater than 500 mg / L.

[0020] A method for determining the dosing time of multidrug-resistant Acinetobacter baumannii includes the following steps: introducing a bacterial culture of multidrug-resistant Acinetobacter baumannii into the above-mentioned single-cell microfluidic system and culturing it until stable; introducing a PBP-targeting drug into the single-cell microfluidic system for co-incubation; separating undivided cells and culturing them again; and determining the time required for the undivided cells to divide again, wherein the time is the dosing interval.

[0021] The targeted PBP drugs include targeted PBP1 drugs, targeted PBP2 drugs, and / or targeted PBP3 drugs, preferably berberine hydrochloride, meropenem, mecillin, sulbactam, ceftazidime, and / or aztreonam; preferably, berberine hydrochloride / meropenem, berberine hydrochloride / meropenem / mecillin, berberine hydrochloride / meropenem / sulbactam, or berberine hydrochloride / meropenem / mecillin / sulbactam.

[0022] A drug for combating multidrug-resistant Acinetobacter baumannii, comprising berberine hydrochloride and at least one antibiotic that targets the PBP-binding protein.

[0023] Preferably, it includes berberine hydrochloride and at least one antibiotic that targets the PBP2 binding protein.

[0024] Preferably, it includes berberine hydrochloride, at least one antibiotic targeting PBP2 binding protein, at least one antibiotic targeting PBP1 binding protein, and / or at least one antibiotic targeting PBP3 binding protein.

[0025] Preferably, it includes berberine hydrochloride, an antibiotic targeting PBP2 binding protein, at least one antibiotic targeting PBP1 binding protein, and / or at least one antibiotic targeting PBP3 binding protein.

[0026] Preferably, it includes berberine hydrochloride, an antibiotic targeting PBP2 binding protein, and an antibiotic targeting PBP1 binding protein.

[0027] Preferably, it includes berberine hydrochloride, an antibiotic targeting PBP2 binding protein, and an antibiotic targeting PBP3 binding protein.

[0028] Preferably, it includes berberine hydrochloride, an antibiotic targeting PBP2 binding protein, an antibiotic targeting PBP1 binding protein, and an antibiotic targeting PBP3 binding protein.

[0029] Preferably, it includes berberine hydrochloride and meropenem; preferably, it includes berberine hydrochloride, meropenem, and sulbactam; preferably, it includes berberine hydrochloride, meropenem, and aztreonam.

[0030] Preferably, it includes berberine hydrochloride and mecillin; preferably, it includes berberine hydrochloride, mecillin, and sulbactam; preferably, it includes berberine hydrochloride, mecillin, and aztreonam.

[0031] Preferably, when berberine hydrochloride and meropenem are used, preferably, the concentration of berberine hydrochloride is less than 800 mg / L and the concentration of meropenem is less than 64 mg / L; preferably, the concentration of berberine hydrochloride is less than 500 mg / L and the concentration of meropenem is less than 50 mg / L; most preferably, the concentration of berberine hydrochloride is 256 mg / L and the concentration of meropenem is 16 mg / L.

[0032] Preferably, the mixture comprises berberine hydrochloride and mecillin; preferably, the concentration of berberine hydrochloride is less than 800 mg / L and the concentration of mecillin is less than 500 mg / L; preferably, the concentration of berberine hydrochloride is less than 500 mg / L and the concentration of mecillin is less than 300 mg / L; most preferably, the concentration of berberine hydrochloride is 256 mg / L and the concentration of mecillin is 256 mg / L.

[0033] Preferably, the mixture includes berberine hydrochloride, mecillin, and sulbactam. Preferably, the concentration of berberine hydrochloride is less than 800 mg / L, the concentration of mecillin is less than 64 mg / L, and the concentration of sulbactam is less than 64 mg / L. Preferably, the concentration of berberine hydrochloride is less than 500 mg / L, the concentration of mecillin is less than 50 mg / L, and the concentration of sulbactam is less than 50 mg / L. Most preferably, the concentration of berberine hydrochloride is 256 mg / L, the concentration of mecillin is 4 mg / L, and the concentration of sulbactam is 4 mg / L.

[0034] Beneficial effects

[0035] (1) The single-cell microfluidic system of the present invention includes several microchambers, at least two inlet channels and at least one outlet. The two inlet channels are arranged opposite to each other on both sides of the microchamber. A stop is provided between the microchamber and the inlet channel. The stop is used to prevent bacteria from flowing from the microchamber into the corresponding inlet channel. The height of the microchamber is equivalent to the diameter of the single bacterium to be detected. During bacterial culture, the bacteria are distributed independently in the microchamber, and multiple bacteria will not overlap. This facilitates single-cell capture and tracking, and provides an ideal platform for tracking the division of single bacteria and observing the changes in bacterial morphology under different antibiotic pressures in real time.

[0036] (2) This invention utilizes a single-cell microfluidic system with single-cell capture technology to monitor the changes of individual bacteria under antibiotic stress in real time. Using a combination of berberine hydrochloride and antibiotics targeting PBP binding proteins as a dosing model, the growth status of Acinetobacter baumannii under independent / combined drug administration was studied. The drug resistance mechanism of bacteria was revealed at the single-bacterial level, namely, the bacteria respond to the attack of antibiotics targeting PBP binding proteins through morphological changes. The observations in this invention include bacteria maintaining a non-dividing state and forming "spherical" or "filamentous" morphologies during growth to cope with the predicament of cell wall synthesis.

[0037] The applicant unexpectedly discovered that the combination of berberine hydrochloride and PBP2-binding protein-targeting antibiotics was the most effective, achieving a good bactericidal effect even at relatively low concentrations of both berberine hydrochloride and PBP2-binding protein-targeting antibiotics.

[0038] (3) The present invention has observed and studied bacteria and found that there are individual differences among drug-resistant bacteria, that is, there are drug-resistant heterogeneous bacteria. For example, some bacteria can remain in a non-dividing state under high doses of antibiotic pressure, thereby escaping the bactericidal effect of antibiotics. After the antibiotic pressure disappears, they resume division and proliferation. This provides a reference for antibiotic use, that is, to select appropriate antibiotic doses and appropriate dosing intervals to attack "dormant bacteria" that have escaped antibiotic treatment, so as to avoid reinfection. This provides a research platform for the development of new antibiotics and the study of antibacterial mechanisms, and provides corresponding reference and analysis for clinical antibiotic use. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of the single-cell microfluidic system for bacteria in this invention;

[0040] Figure 2 This is a flowchart illustrating the preparation process of the single-cell microfluidic system for bacterial resistance in this invention.

[0041] Figure 3 The images show the bacterial morphology at different time points when MDR-TJ was cultured using a single-cell microfluidic system.

[0042] Figure 4 This is a scatter plot of the lag time λ and growth rate μ of a single bacterium in MDR-TJ under different temperature conditions in this invention.

[0043] Figure 5 Delayed recordings of MDR-TJ under different drug conditions;

[0044] Figure 6 The left side shows the population growth curves of MDR-TJ under different drug effects;

[0045] Figure 6 The value in μ represents the growth rate of MDR-TJ under different drug treatments.

[0046] Figure 6 The right side represents the sum and delay time λ of MDR-TJ under different drug effects;

[0047] Figure 7 Delayed recording images of MDR-TJ under combined drug administration conditions;

[0048] Figure 8 c represents the population growth curve of MDR-TJ under combined drug administration;

[0049] Figure 8 d represents the growth rate μ of MDR-TJ under combined drug administration;

[0050] Figure 8 e represents the sum and delay time λ of MDR-TJ under combined drug administration;

[0051] Figure 9 Scatter plot of μ-λ for MDR-TJ under different drug conditions;

[0052] Figure 10 This is a diagram showing the cell structure under the influence of different drugs.

[0053] Figure 11 The graph shows the results of treatment with 512 mg / L meropenem for different durations. Detailed Implementation

[0054] The following detailed description, in conjunction with specific embodiments, illustrates the general formula compounds of the present invention, their preparation methods, and applications in further detail. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0056] The following examples demonstrate the use of the following methods and reagents for staining live / dead bacteria:

[0057] The live / dead bacteria staining kit (L13152, Invitrogen) was used to stain bacteria in the chip after drug administration to verify the survival or death of bacteria after final culture. Two hours after bacterial administration, the solution in the channel was replaced with staining solution (PBS solution containing 10 μM propidium iodide and 2 μM SYTO 9, where SYTO 9 is a green fluorescent nucleic acid dye). The flow rate was maintained at 0.15 μL / min, and the staining process lasted approximately 1 hour. After staining, the infusion of staining solution was stopped, and the fluorescence signals of SYTO 9 (green) and propidium iodide (red) in the stained chip were detected using excitation wavelengths of 455–495 nm / 505–555 nm and excitation wavelengths of 533–558 nm / 570–640 nm, respectively. SYTO 9 stained both live and dead bacteria green, while PI stained only dead bacteria red.

[0058] The following equipment and methods were used to acquire optical images:

[0059] The equipment used was either a fluorescence inverted microscope (Zeiss Axio Observer 7, Axiocam 506 color camera) equipped with a long working distance plan-neofluar 40X / 0.6 objective or an automated inverted fluorescence microscope (Olympus IX83, fully automated ultrasonic stage, DP74 color camera) equipped with a long working distance plansemiapochromat 60X / 0.7 objective, which was used to record bright-field time-lapse images and live / dead bacterial staining fluorescence images.

[0060] The bright-field image acquisition method is as follows: the incubation time on the chip lasts for about 2 hours. During the incubation process, photos are taken at intervals of 20 minutes or 60 minutes. The specific incubation time and interval depend on the growth of bacteria. However, a longer incubation time will cause the microchamber of the chip to be filled with bacteria, making it difficult to distinguish the boundaries of colonies produced by individual bacteria. Therefore, the incubation time is usually controlled within 2 hours.

[0061] Fluorescence images can be obtained directly after staining.

[0062] The growth parameters μ and λ are calculated in the following examples:

[0063] In the chip, bacteria grow in a monolayer, and the area of ​​the colony is directly proportional to the mass of the cell. Therefore, the growth rate of bacteria can be analyzed using Formula 1:

[0064] μ=ln(S t / S0) / (t-λ)……Formula 1

[0065] Where S0 and S t λ represents the area of ​​a single colony at time t=0 and time t, where λ is the lag time.

[0066] All images were processed using ImageJ software (a Java-based public image processing software). By inputting the images into ImageJ, the growth rate μ and lag time λ of a single bacterium can be calculated.

[0067] Example 1

[0068] Preparation of single-cell microfluidic systems

[0069] Template preparation: The silicon wafer is sequentially immersed in acetone, methanol and isopropanol for ultrasonic cleaning for 5 minutes each, then dried with nitrogen. Surface treatments such as adding correction marks are then performed. First, a layer of SU-8 photoresist is spread on the silicon wafer, then a photomask is added on top of the photoresist. After ultraviolet exposure, it is placed in a developing solution. The SU-8 photoresist covered by the photomask is cured, while other areas are dissolved. This process is repeated 3 times to obtain micro-chamber channels. Finally, the processed silicon wafer is surface treated with trichlorosilane to improve its lifespan, thus obtaining the template.

[0070] PDMS mold preparation: Weigh PDMS silicone and curing agent in a weight ratio of 10:1, stir to mix them thoroughly, pour the mixed adhesive into the mold to a suitable height, place it in a vacuum chamber to evacuate the vacuum, and then slowly connect the vacuum chamber to the atmosphere to make all the air bubbles in the adhesive burst and disappear.

[0071] Place the defoamed PDMS template in an oven and bake at 80°C for more than 2 hours until the PDMS is cured. Use a scalpel to cut out the PDMS chip containing the channel. Use a 0.5mm diameter punch to drill holes at the channel inlet and outlet of the PDMS chip. Then, immerse the PDMS chip and glass slide (glass slide size 25×75mm, no.3) in acetone, methanol, and isopropanol for ultrasonic cleaning for 5 minutes each. After drying with nitrogen, place the PDMS chip and glass slide in an oven and bake at 80°C for 15 minutes to further remove surface solvent residue.

[0072] PDMS bonding to glass slides: The PDMS chip and glass slide were surface treated using an oxygen plasma surface treatment instrument. The oxygen vacuum was set to 0.4 mbar and the treatment time was set to 30 s. The treated PDMS chip and glass slide were then bonded together. After the bubbles disappeared automatically, the chips were placed in an oven and dried at 80 ℃ for more than 8 hours to obtain the crude single-cell microfluidic system.

[0073] Under a microscope, observe whether the channels of the crude single-cell microfluidic system are intact and whether the microchambers have collapsed. Select the crude single-cell microfluidic system with intact structure for further processing.

[0074] Remove the front and back of a 0.6 mm diameter needle, smooth it with a file, insert it into the inlet and outlet of the crude single-cell microfluidic system, and glue the inlet and outlet needles together to prevent leakage, thus obtaining the single-cell microfluidic system.

[0075] Example 2: The culture method of bacteria (in this example, the multidrug-resistant Acinetobacter baumannii strain MDR-TJ is used as an example) in the above-mentioned single-cell microfluidic system (hereinafter referred to as chip) is as follows:

[0076] S1. After culturing the multidrug-resistant Acinetobacter baumannii strain MDR-TJ overnight in CAMHB medium (cation-adjusted Mueller-Hinton broth), it was then inoculated at a 1% inoculum and recultured in fresh CAMHB medium until the logarithmic phase (OD600nm = 0.6-0.8). The bacteria were then washed twice with PBS (pH = 7.2), resuspended in PBS, and diluted to a bacterial density of approximately 0.02 (OD600nm). This density was used to control the appropriate number of single bacteria loaded onto the subsequent chip.

[0077] S2. Before loading the bacteria into the microfluidic device, CAMHB culture medium is introduced into the two channels of the chip at a rate of 2 μL / min. When the culture medium flows out of the chip outlet, the flow rate of the culture medium is changed to 0.15 μL / min.

[0078] S3. Loading bacteria into the chip: Load bacteria into the chip through the loading channel, controlling the flow rate at 2 μL / min. Then, replace one of the two channels with culture medium and adjust the flow rate of the syringe pump (e.g., the flow rate of the culture medium channel is 2 μL / min, and the flow rate of the bacterial loading channel is 0.5 μL / min) to control the cell density in the microchamber. At the same time, the PBS buffer used for loading bacteria can be drained from the microchamber.

[0079] S4. Once the number of bacteria in the microchamber is appropriate, adjust both channels to culture medium and the flow rate to 0.15 μL / min to begin bacterial culture on the chip.

[0080] This continuous low flow rate not only provides a constant supply of nutrients for the bacteria, but also removes their metabolic products, similar to a miniature constant flow culture. Therefore, it can be assumed that the bacteria are always in the logarithmic growth phase in the chip (except for a short delay in adapting to the new environment).

[0081] To bring the experiment closer to the optimal growth temperature of Acinetobacter baumannii, a heating plate (TPi-SQX, Tokai Hit Co., Ltd., Japan) was installed on the microscope stage to control the bacterial culture temperature at 37°C (this can also be achieved using a heating plate on the chip). Meanwhile, a culture temperature of 25°C was used as a control.

[0082] See Figure 3 The image shown is a typical time-lapse observation of the MDR Acinetobacter baumannii strain MDR-TJ cultured on a chip. As can be seen from the image, Acinetobacter baumannii is short rod-shaped and mostly arranged in pairs. Through continuous culture, individual cells begin to divide and then continuously produce the next generation.

[0083] However, a few individual cells remain in a special non-growth state, without dividing or changing morphology during the culture phase. These cells are referred to in the art as dormant cells (the cells indicated by the black arrows at 0, 60h, and 100h in the figure, which always remain in a non-growth state). The dormant state is generally considered to be an effective survival strategy for bacteria to cope with environmental pressures: by reducing metabolic activity, they are in a non-dividing phase and return to the normal dividing phase when environmental conditions improve.

[0084] The area occupied by a single colony was measured to plot a growth curve. A linear fit was then performed on this curve to calculate the growth rate μ and lag time λ of a single cell, as shown in Table 1. The growth rate of strain MDR-TJ under both conventional broth culture (shake flask) and microarray culture conditions was 1.46 h. -1 and 1.61h -1 The values ​​of the two are basically close, and the growth rates at different temperatures (25℃, 37℃) in the chip are 0.70h respectively. -1 and 1.61h -1 The difference is significant.

[0085] Table 1. Growth rate and lag time of MDR-TJ strain under different culture conditions

[0086]

[0087] Therefore, all culturing and related tests in all chips of this invention are performed at 37°C, because at this temperature, not only is it closer to the growth temperature at which bacteria infect the human body, but the bacterial population is also more uniformly distributed than at room temperature (25°C) (see...). Figure 4 ), with a greater growth rate.

[0088] Example 3: Chip Antibacterial Test

[0089] Each class of antibiotics possesses a specific antibacterial mechanism, and multidrug-resistant bacteria have evolved corresponding strategies to evade antibiotic attacks. Typically, some bacteria may undergo various morphological changes to survive under antibiotic stress. However, during suspension bacterial culture, conventional microscopes not only make it difficult to observe and record bacterial morphological changes but also prevent real-time image recording. The single-cell microfluidic system of this invention enables single-cell capture and tracking using a conventional microscope while culturing cells.

[0090] This embodiment experimentally recorded the morphological changes of MDR-TJ under the action of the test antibiotics meropenem (MEM), berberine hydrochloride (BBH), and the MEM / BBH drug complex.

[0091] The concentrations of MEM were set to 16 mg / L, 64 mg / L, and 320 mg / L, the concentrations of BBH were set to 256 mg / L and 800 mg / L, and the concentration of the MEM / BBH drug complex was 320 mg / L. The bacterial culture steps in this embodiment were the same as steps S1 to S4 in Example 1.

[0092] After 2 hours of incubation, the antibiotic to be tested was added to the culture medium and introduced into the microchamber through the flow channel for co-incubation with the bacteria.

[0093] Meropenem (MEM), a carbapenem antibiotic, has been shown to have broad-spectrum antibacterial activity against Gram-negative bacteria, including Acinetobacter baumannii. (See also...) Figure 5 As shown, when treated with a low concentration (16 mg / L) of MEM, MDR-TJ bacteria slowly become round and maintain this shape over time. Figure 5 (represented by a pentagram in the image), when the MEM concentration increases to 64 mg / L, most bacteria undergo a series of shape transformations: first becoming round, then the bacteria lyse entirely. Figure 5 (Indicated by the circle in the middle). However, some bacteria retain their original shape (rod-shaped), and even at a MEM concentration of 320 mg / L, some bacteria still retain their original rod shape.

[0094] The presence of live and dead bacteria can be indicated by staining with SYTO 9 (which stains both live and dead bacteria green, while PI stains only dead bacteria red) to show that all bacteria are dead in this state.

[0095] The mechanism of action of MEM can be analyzed through bacterial morphology during antibiotic action: Current research shows that the rounding of bacteria is consistent with the inhibitory effect related to penicillin-binding protein 2 (PBP2). Penicillin-binding protein 2 has been confirmed as a key protein in bacterial cell wall synthesis. However, the overall size of the rounded bacteria is smaller than the specific PBP2 inhibition. A reasonable explanation is that MEM may inhibit not only PBP2, but also PBP1.

[0096] Combination Figure 6 Growth curve analysis showed that the MIC (minimum inhibitory concentration) of MEM against MDR-TJ was 64 mg / L, which was consistent with the results of the susceptibility testing of the plate suspension (see Table 2).

[0097] See Figure 6 As shown in the left figure, MDR-TJ cells continued to divide under a series of different concentrations of BBH (BBH concentrations were set at 256 mg / L and 800 mg / L) with almost no morphological changes, although it inhibited cell growth at a concentration of 1024 mg / L in the well plate suspension drug sensitivity test (see Table 2).

[0098] Table 2. MIC values ​​of antimicrobial agents against Acinetobacter baumannii MDR-TJ strain

[0099]

[0100] Note: R, drug resistance; S, sensitivity; I, intermediate.

[0101] However, due to the poor solubility of berberine hydrochloride, it is difficult to conduct high-concentration berberine hydrochloride inhibition experiments in single-cell microfluidic systems. The figure only shows a small number of bacteria turning into filaments under the action of berberine hydrochloride at a concentration of 256 mg / L. Figure 5 (Represented by a triangle in the middle) Most other bacteria showed little change in morphology under the action of berberine hydrochloride. In addition, more dormant cells could be observed after treatment with higher concentrations of berberine hydrochloride (such as 800 mg / L). Therefore, it is not recommended to use berberine hydrochloride alone to combat MDR-TJ.

[0102] Depend on Figure 5 It was found that neither 16 mg / L MEM nor 256 mg / L BBH alone could effectively inhibit the growth of Acinetobacter baumannii. However, the combination of the two low-concentration antibiotics showed a significant antibacterial effect. With the combined treatment, cells rapidly became rounded before 1 hour of drug action, and at 2 hours, the bacterial morphology was larger and rounder than when treated with 16 mg / L MEM alone, resembling more of a "protoplasmic sphere." Figure 5 (as shown by the pentagram in the image); according to Figure 5 and 6Live / dead bacteria staining diagram, growth curve diagram ( Figure 6 (Left) and growth rate diagram ( Figure 6 The study showed that combination therapy can effectively combat MDR-TJ.

[0103] The delay time varied considerably among individual bacteria throughout the experiment, resulting in no significant difference in delay time among the four groups of drugs (see [link]). Figure 6 (Right). However, compared with no drug effect (the graph corresponding to CAMHB) or single drug effect (MEM or BBH), combined drug treatment (MEM / BBH) can significantly reduce the growth rate of MDR-TJ.

[0104] Example 4

[0105] Berberine hydrochloride in combination with other antibiotics targeting PBP

[0106] Given that berberine hydrochloride can significantly enhance the sensitivity of MDR-TJ strains to MEM, the applicant infers that berberine hydrochloride may also have a synergistic effect with other antibiotics that target PBPs.

[0107] In this embodiment, two antibiotics targeting PBPs proteins, mecillinam (MEC) and sulbactam (SAM), a β-lactamase inhibitor, were used as examples for testing.

[0108] The antibiotic mecillin inhibits bacterial proliferation by specifically targeting the PBP2 protein. (See also:) Figure 7 As shown in a, when MDR-TJ was treated with 256 mg / L MEC, the bacteria became spherical ( Figure 7 (The circles in 'a' represent the spheres), but compared to those treated with MEM, the spheres were larger, consistent with reports of studies specifically targeting the PBP2 protein. However, after adding an additional 256 mg / L of berberine hydrochloride, most bacteria stopped dividing or became rounded, with only a few cells becoming smaller rounds. Staining with live and dead bacteria further confirmed that all bacteria were dead. Two hours after drug treatment, the antibiotic was replaced with fresh culture medium and cultured for another 4 hours; the bacteria remained non-dividing, indicating that all bacteria were dead.

[0109] The β-lactamase inhibitor sulbactam binds to the PBP1 and PBP3 proteins of Acinetobacter baumannii with a weak affinity, exhibiting inherent antibacterial activity against the baumannii. When 16 mg / L of SAM was added, different morphological changes were observed in the bacteria. Initially, the bacteria became elongated rod-shaped, but with prolonged exposure, their morphology changed to filamentous or "spaghetti-like" forms. Figure 4-7 (represented by a triangle in the diagram), but the bacterial area continues to expand.

[0110] When berberine hydrochloride was added, the transformation of these filamentous bacteria became more pronounced, but some colonies still failed to stain red (i.e., they were not dead). Furthermore, when the SAM / BBH medium was replaced with fresh medium, some filamentous bacteria began to divide into normal short rod-shaped forms after 2 hours. Figure 7 (As shown in the rectangle in b), this indicates that Acinetobacter baumannii strain MDR-TJ can escape antibiotic treatment by altering its morphology to remain non-dividing.

[0111] The antibacterial experimental results of the combination of two antibiotics targeting PBPs proteins with berberine are shown in [the table below]. Figure 7 b. The difference in morphological changes in Acinetobacter baumannii caused by MEC and SAM may be due to their different mechanisms of action. MEC mainly targets PBP2 binding proteins, while SAM targets PBP1 and PBP3 binding proteins with weaker binding. PBP binding proteins are essential enzymes in response to peptidoglycan (a major component of the bacterial cell wall). In addition to contributing to peptidoglycan synthesis, PBP2 can also catalyze transglycosylation. Therefore, we can infer that the elongated or spaghetti-like filamentous form is more likely to be related to the failure of PBP1 or PBP3 synthesis, while the disruption of PBP2 induces more spherical or unique "protoplast" shapes.

[0112] Figure 8 As shown in c and 8d, the combination therapy of 16SAM / 256BBH did not significantly reduce the bacterial growth rate compared to 16SAM alone. Conversely, the growth curve and growth rate of MDR-TJ decreased sharply after the combination therapy of 256MEC / 256BBH. Therefore, for MDR-TJ strains, the combination therapy of MEC / BBH is superior to SAM / BBH. In addition, based on the plate antibacterial assay, the antibacterial activity of berberine hydrochloride combined with other antibiotics targeting PBPs was also determined, and the results are shown in Table 3.

[0113] Table 3. Determination of antibacterial activity of berberine hydrochloride in combination with other antibiotics targeting PBPs.

[0114]

[0115]

[0116] Note: a: The final concentration of berberine hydrochloride (BBH) added is 256 mg / L; b: 256 mg / L BBH may or may not be added when using ceftazidime / aztreonam in combination.

[0117] As shown in Table 2, compared with antibiotics targeting PBP2, antibiotics targeting PBP1 or PBP3 have poor antibacterial effects when used in combination with berberine hydrochloride. For example, ceftazidime (CAZ) or aztreonam (ATM) have no synergistic effect when used in combination with berberine hydrochloride (FIC>0.5).

[0118] Therefore, BBH may enhance the efficacy of antibiotics targeting PBP2, while also demonstrating that PBP2 is crucial for cell wall synthesis. When MDR-TJ was treated with a combination of mecillin and sulbactam, it simultaneously targeted PBP1, PBP2, and PBP3. The combination significantly reduced the MICs of MDR-TJ against both (mecillin decreased by 8-fold, and sulbactam by 4-fold). When 256 mg / L of berberine hydrochloride was added, the MICs of both decreased even more dramatically, both to 4 mg / L, with inhibition rates exceeding 95%. According to the CLSI (Clinical and Laboratory Standards Institute) sensitivity cutoff determination, the MDR-TJ strain regained sensitivity to both antibiotics.

[0119] Analysis of bacterial community heterogeneity under drug influence

[0120] μ-λ scatter plots are a common research tool used to study the heterogeneous distribution of bacterial communities under different stresses. To further understand the growth dynamics of the entire bacterial community under different antibiotic stresses, μ-λ scatter plots of bacteria under different antibiotic stresses were plotted. See [link to relevant documentation]. Figure 9 As shown, the scatter plot reveals the heterogeneity of the population. If the growth states of the bacteria are similar, that is, they are homogeneous, then all data points will cluster due to the similarity of μ and λ values. On the contrary, cellular heterogeneity is characterized by an increase in the amount of scattering, which is information that cannot be obtained from ordinary shake-flask batch culture.

[0121] like Figure 9 As shown, the bacteria in CAMHB medium exhibit a relatively homogeneous bacterial community. The box in the μ-λ scatter plot represents the space where more than 95% of the individual bacteria reside. The horizontal axis in the scatter plot represents the bacterial growth rate μ (h). -1 The vertical axis represents the bacterial lag time λ (h). The value to the right of the red box indicates that the bacteria have a greater growth rate and a shorter doubling time; the value above the right of the red box indicates that the bacteria have a longer lag time.

[0122] When using antibiotics, the optimal bacterial flora is one that is on the left side of the box and homogeneous, meaning that the bacteria in the flora generally have a longer doubling time and a shorter lag time. The lag time λ refers to the time required for bacteria to adapt to the environment without dividing. An extended lag time is often associated with "resistant" bacteria, known as "antibiotic tolerance." These bacteria evade the effects of antibiotics by prolonging the time to their first division and remaining in a dormant state for a longer period. Subsequently, they resume replication after the antibiotic pressure disappears. These bacteria are similar to multidrug-resistant strains, both of which can lead to clinical treatment failure.

[0123] Depend on Figure 9 It can be seen that when berberine hydrochloride is used alone, the heterogeneity of the bacterial community is more obvious and the proliferation rate is higher. However, the combination of 256MEC / 256BBH or 16MEM / 256BBH can significantly reduce bacterial heterogeneity and effectively inhibit bacterial proliferation (greater growth rate μ). Based on this, the effect of using berberine hydrochloride alone against multidrug-resistant Acinetobacter baumannii is poor, while the combination of MEM / BBH can effectively combat multidrug-resistant Acinetobacter baumannii.

[0124] dormant bacteria detection

[0125] Dormant cells were observed under different culture conditions, meaning that the morphology of the bacteria did not change or divide during the culture time. Table 4 lists the proportion of dormant cells under different culture conditions.

[0126] Table 4. Proportion of dormant bacteria under different culture conditions

[0127]

[0128]

[0129] Note: a: The percentage of dormant cells was calculated through two independent tests. b: Under these conditions, cells were inhibited at the initial time.

[0130] Although dormant bacteria exhibit a state of non-division and non-replication, staining analysis of live and dead bacteria shows that not all dormant bacteria retain intact cell membrane structures. For example... Figure 10 As shown, under the action of different drugs, the cell membranes of some dormant bacteria have ruptured and been stained red, while some bacteria have maintained an intact cell structure but remain in a non-dividing state during the culture period (2h). Such bacteria may resume replication function after being replaced with fresh culture medium, so such dormant bacteria may also lead to the failure of antibiotic treatment.

[0131] Because bacterial division is inhibited under high doses of antibiotics, relying solely on bacterial morphology observation is insufficient to determine bacterial death under these conditions. Therefore, this embodiment utilizes a microfluidic experimental platform to treat single bacteria with different doses and durations of meropenem antibiotics, followed by reculturing in fresh culture medium to observe whether bacterial growth resumes.

[0132] like Figure 11 As shown, after treatment with 512 mg / L meropenem for 5 hours, the medium was replaced with fresh MH medium. After about 9.5 hours of incubation, bacteria began to divide (indicated by the black arrow at 14.5 hours in the figure). After another 1.5 hours of incubation, the bacteria began to divide and proliferate continuously, forming a colony.

[0133] In addition, the number of bacteria that regrow under other drug administration conditions is shown in Table 5. Only a very small percentage of bacteria (<0.2%) resumed growth after reculturing. After the regrown bacteria were cultured to a certain quantity, they were removed from the chip with fresh culture medium, and their MICs were re-measured. The results showed that the MIC of some of the regrowth bacteria was doubled. This indicates that even multidrug-resistant Acinetobacter baumannii can retain a very small percentage of bacteria in a dormant state under high concentrations of antibiotics. Once the environment is suitable, these bacteria will re-divide and proliferate. However, the reculturing time required for such bacteria is at least more than 6 hours, therefore, continuous intermittent administration of antibiotics is necessary during treatment.

[0134] Therefore, when receiving antibiotic treatment, the dosage of antibiotics must be sufficient and the appropriate interval between administrations must be selected in order to inhibit the re-division of dormant bacteria and prevent treatment failure.

[0135] Table 5. Number of persistent or resistant bacteria under high-dose meropenem treatment.

[0136]

[0137] In addition to multidrug-resistant strains leading to antibiotic treatment failure in clinical practice, an increasing number of studies have shown that bacterial flora heterogeneity may also be a factor in antibiotic treatment failure. For example, in clinical observations of bacterial flora resistance heterogeneity, persistent bacteria or non-dividing bacteria may evade antibiotic attack and, under suitable conditions, resume proliferation, causing secondary bacterial infections.

[0138] Therefore, detecting bacterial heterogeneity is particularly important when designing antibiotic dosing regimens. Traditional detection methods mostly treat the bacterial community as the entire research object, often ignoring the differences between individual bacteria and lacking specific information about individual bacteria to guide medication regimens.

[0139] This invention utilizes a single-cell microfluidic system with single-cell capture technology to monitor changes in individual bacteria under antibiotic stress in real time. Using a combination of berberine hydrochloride and antibiotics targeting PBP-binding proteins as a dosing model, the optimal dosing regimen for combating multidrug-resistant Acinetobacter baumannii was explored. Results showed that the combination of berberine hydrochloride and antibiotics targeting PBP2-binding proteins was the most effective. Furthermore, the antibiotic resistance mechanism of bacteria was revealed at the single-bacterial level, namely, responding to antibiotic attack targeting PBP-binding proteins through morphological changes, such as maintaining a non-dividing state and continuously growing, forming "spherical" or "filamentous" morphologies to cope with the challenges of cell wall synthesis.

[0140] Furthermore, the study results indicate that individual bacterial differences also exist within drug-resistant bacterial communities. For example, some bacteria can remain in a non-dividing state under high-dose antibiotic (meropenem) pressure, and resume division and proliferation after the antibiotic pressure disappears. This provides a reference for antibiotic use strategies, namely, selecting appropriate antibiotic doses and dosing intervals to attack "dormant bacteria" that have escaped antibiotic treatment.

[0141] In summary, this platform technology may provide a research platform for developing novel antibiotics and studying their antibacterial mechanisms, and provide corresponding references and analyses for clinical antibiotic use protocols.

[0142] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for testing the administration time of drug-resistant heterogeneous bacteria, comprising the following steps: The bacterial culture is introduced into a single-cell microfluidic system for bacteria and cultured until stable. An antibacterial drug is introduced into the single-cell microfluidic system for co-incubation. Undivided cells are separated and cultured again. The time required for the undivided cells to divide again is determined, and the time is the dosing interval. The single-cell microfluidic system for bacteria includes several microchambers, at least two inlet channels, and at least one outlet. The two inlet channels are arranged opposite each other on both sides of the microchambers. A stop is provided between the microchamber and one inlet channel to prevent bacteria from flowing from the microchamber into the corresponding inlet channel. The height of the microchamber is 100 nm-1 μm. The stop is a stepped structure between the microchamber and the corresponding inlet channel. The height of the stepped structure is less than the height of the microchamber. The height of the microchamber is approximately equal to the diameter of the single bacterium to be tested. During bacterial culture, the bacteria are independently distributed in the microchambers and grow in a monolayer within the microchambers.

2. The method for testing drug-resistant heterogeneous bacteria according to claim 1, characterized in that, The microchamber is equipped with several support columns.

3. The method for testing drug-resistant heterogeneous bacteria according to claim 1, characterized in that, The liquid inlet channel is connected to the external liquid via a microfluidic pump. Under the action of the microfluidic pump, the external liquid is pumped into the liquid inlet channel and flows into the micro-chamber.

4. The method for testing drug-resistant heterogeneous bacteria according to claim 1, characterized in that, The stop is a baffle disposed between the microchamber and the corresponding liquid inlet channel. The baffle is disposed at the end of the microchamber, and the distance between the bottom of the baffle and the bottom of the chip is 0.2 to 0.6 μm.

5. The method for testing drug-resistant heterogeneous bacteria administration time according to any one of claims 1-4, characterized in that, The number of microchamber structures is even and they are arranged in an array structure, with a total number of ≥6 microchamber structures.

6. The method for testing drug-resistant heterogeneous bacteria according to any one of claims 1-4, characterized in that, The single-cell microfluidic system is also equipped with a heating plate, which is used to provide constant temperature conditions for the microchamber.

7. The method for testing drug-resistant heterogeneous bacteria administration time according to any one of claims 1-4, characterized in that, The concentration of the antibacterial drug is such that it can induce antibiotic pressure in the bacteria, and the concentration of the antibacterial drug is greater than 300 mg / L.

8. The method for testing drug-resistant heterogeneous bacteria administration time according to any one of claims 1-4, characterized in that, The preparation method of the microfluidic system includes the following steps: preparing a template with microchambers and liquid inlet channels, adding PDMS to the template to obtain a PDMS chip, and obtaining the above-mentioned single-cell microfluidic system.

9. The method for testing drug-resistant heterogeneous bacteria according to claim 8, characterized in that, The template is obtained by photolithography on a substrate, which may include a silicon plate, a glass plate, or a metal plate.

10. The method for testing drug-resistant heterogeneous bacteria according to claim 8, characterized in that, The addition of PDMS to the template also includes the steps of adding a curing agent and mixing. After the PDMS and curing agent are mixed and introduced into the template, the steps of defoaming, drying and curing, and then slicing are also included.

11. The method for testing drug-resistant heterogeneous bacteria administration time according to claim 8, characterized in that, After obtaining the PDMS chip, the process further includes the step of bonding the PDMS chip to a glass slide to obtain a single-cell microfluidic system.

12. A method for determining the administration time of multidrug-resistant Acinetobacter baumannii, comprising the following steps: The bacterial culture of multidrug-resistant Acinetobacter baumannii is introduced into the single-cell microfluidic system of the method for testing drug administration time of drug-resistant heterogeneous bacteria according to any one of claims 1 to 11 and cultured until stable. The targeted PBP drug is introduced into the single-cell microfluidic system for co-incubation. Undivided cells are separated and cultured again. The time required for the undivided cells to divide again is determined. The time is the drug administration interval. The targeted PBP drug includes at least one of the targeted PBP1 drug, targeted PBP2 drug, and targeted PBP3 drug.

13. The method for determining the administration time of multidrug-resistant Acinetobacter baumannii according to claim 12, characterized in that, The targeted PBP drug is at least one of berberine hydrochloride, meropenem, mecillin, sulbactam, ceftazidime, and aztreonam.

14. The method for determining the administration time of multidrug-resistant Acinetobacter baumannii according to claim 13, characterized in that, The targeted PBP drug is berberine hydrochloride / meropenem, berberine hydrochloride / meropenem / mexilin, berberine hydrochloride / meropenem / sulbactam, or berberine hydrochloride / meropenem / mexilin / sulbactam.

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