Ultrahigh-flux screening method for heterogeneous vancomycin-mediated drug-resistant staphylococcus aureus based on fluorescence activated microdroplet sorting

Abstract

The invention belongs to the technical field of detection, and particularly relates to a heterogeneity vancomycin intermediate drug-resistant staphylococcus aureus ultrahigh-flux screening method based on fluorescence activation microdroplet sorting. Comprising the following steps: S1, culturing a cryopreserved heterogeneous vancomycin-mediated drug-resistant staphylococcus aureus (hVISA) strain, and then collecting; s2, mixing the bacteria collected in the step S1 with a fluorescent probe and vancomycin, and generating microdroplets in a microfluidic device; and S3, sorting the microdroplets generated in the S2 through a microfluidic device, and collecting to obtain the heterogeneous vancomycin intermediate drug-resistant staphylococcus aureus. In the invention, a fluorescence resonance energy transfer (FRET)-based RNA probe is selected to monitor the growth of bacteria. These probes are encapsulated in nanoliter-scale droplets together with bacteria and antibiotics. Therefore, the fluorescent microdroplets containing the growing bacteria can be distinguished from the microdroplets not containing the bacteria, so that more drug-resistant florae can be enriched.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for ultra-high-throughput screening of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting. Background Art

[0002] Staphylococcus aureus is a pathogen with great harm, capable of causing various infections ranging from abscesses, osteomyelitis to bacteremia. However, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has made treatment more complicated because it is resistant to almost all β-lactam antibiotics, posing a great challenge to clinicians. For the treatment of MRSA, vancomycin has become the last line of defense. The abuse of antibiotics has led to the emergence of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus (hVISA), which is associated with persistent infections, treatment failures and prolonged hospital stays. However, it is still difficult to accurately identify these strains.

[0003] Compared with other vancomycin-insensitive Staphylococcus aureus such as vancomycin-intermediate resistant Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA), hVISA is not rare. The prevalence of hVISA in MRSA strains varies widely, ranging from 0% to 93.0%, depending on the experimental method and geographical factors. Unfortunately, conventional drug resistance detection methods, such as agar dilution method or broth microdilution method, cannot reliably distinguish hVISA and vancomycin-susceptible Staphylococcus aureus (VSSA). Part of the reason is that in the hVISA population, only a small fraction of VISA strains grow slowly and are often outcompeted by VSSA strains in a short time. Other screening methods, such as brain heart infusion agar supplemented with 4 μg / mL vancomycin (BHI-V4), or brain heart infusion agar supplemented with 5 μg / mL teicoplanin (BHIT5), and macro E-test (MET), although more convenient to operate, have limitations in sensitivity and specificity compared with the gold standard "population analysis - area under the curve (PAP-AUC)". However, the PAP-AUC method requires culturing bacteria on plates containing different concentrations of vancomycin for 48 hours and then counting the bacteria at different concentrations of vancomycin. Its cumbersome operation and complex process make it unsuitable for routine clinical use. Therefore, there is an urgent need for a rapid and effective detection method to identify the "culprit" and avoid drug abuse.

[0004] Microfluidic technology can enrich target bacterial strains through advanced screening techniques. Currently, high-throughput screening techniques have become increasingly sophisticated and are widely used for efficient screening of microorganisms. Among them, the most commonly used method is fluorescence-activated droplet sorting (FADS). In FADS, droplets are detected in real time through a flow system. Each droplet is rapidly scanned by a laser, and the fluorescence signal is measured by a photomultiplier tube (PMT), greatly increasing the throughput and allowing thousands of droplets to be screened per second. Fluorescent markers are crucial for detecting bacterial growth, and the most widely used ones are resazurin and nucleic acid probes. Bacterial metabolites can convert non-fluorescent resazurin into the fluorescent compound resorufin, making resorufin an effective marker for identifying droplets containing actively growing bacteria. However, resorufin is known to leak from droplets over time, limiting the incubation time to only a few hours.

[0005] Although the use of microfluidic devices for screening drug-resistant bacteria is still relatively rare, there have been some notable studies. Liu et al. published an article, "High-throughput screening of antibiotic-resistant bacteria in ", in which a microfluidic-based droplet platform was developed for the high-throughput evaluation and isolation of Escherichia coli resistant to fusidic acid.

[0006] However, the following problems still exist in this article:

[0007] 1. This article targets Escherichia coli resistant to fusidic acid. Clinically, the main treatment for E. coli is cephalosporin antibiotics, so this article is only a proof of concept and has no practical clinical significance.

[0008] 2. Different sizes of microfluidic droplets are generated: In this article, in order to clearly observe the bacterial colonies generated in the droplets during screening, the droplet size of 330 pL was used.

[0009] 3. Fluorescent probes: This article did not use fluorescent probes. During the screening process, the difference in transmitted light was detected, which required long-term bacterial growth (overnight, about 8 - 12 hours). Summary of the Invention

[0010] To overcome the limitations of the above screening methods, the present invention innovatively introduces microfluidic technology. This method can achieve single-cell culture and analysis, providing new possibilities for microbiome research. The system can efficiently generate water-in-oil (w / o) microdroplets with volumes ranging from thousands to millions of picoliters to nanoliters. Bacteria in the bacterial population are randomly encapsulated into these microdroplets according to Poisson distribution. Each microdroplet serves as an independent microculture chamber, providing an independent environment for bacterial growth and product accumulation, and eliminating the interference caused by resource competition and inhibition among bacteria in the conventional method during the entire screening process. In the present invention, a microfluidic device is used to separate bacteria with different drug resistances in the hVISA population and culture them separately in different microdroplets, avoiding cross-interference. The microbial population from each microdroplet is single, facilitating the isolation of bacteria with heterogeneous characteristics.

[0011] The present invention relates to a method for ultra-high-throughput screening of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated microdroplet sorting, including:

[0012] S1: Cultivate the cryopreserved hVISA strain and then collect it;

[0013] S2: Mix the bacteria collected in S1 with a fluorescent probe and vancomycin, and generate microdroplets in a microfluidic device;

[0014] S3: Sort the microdroplets generated in step 2 through a microfluidic device.

[0015] Preferably, S2 includes:

[0016] S2.1: Mix the bacteria collected in S1 with fresh culture medium, continue to culture, centrifuge to collect bacterial cells, and then resuspend them in fresh culture medium to obtain a bacterial suspension; Mix the bacterial suspension, fluorescent probe, and BHI containing vancomycin to obtain a bacterial mixture;

[0017] S2.2: Use the microdroplet generation oil as the continuous oil phase, and the microfluidic device encapsulates the bacterial mixture in S2.1 with the continuous oil phase.

[0018] Preferably, in S2.1, the bacteria collected in S1 are mixed with fresh culture medium at a volume ratio of 1:50.

[0019] Preferably, in S2.1, the mixed bacteria are continuously cultured in a constant temperature shaker at 37 °C at 200 rpm for 2 hours.

[0020] Preferably, in S2.1, the volume ratio of the bacterial suspension, fluorescent probe, and BHI containing vancomycin is 1:1:48.

[0021] Preferably, in S2.2, the flow rate ratio of the bacterial mixture to the continuous oil phase is 1:2.

[0022] Preferably, S3 includes:

[0023] S3.1: Pump the droplets into the sorting chip of the microfluidic device;

[0024] S3.2: When the droplets pass through the sorting chip, collect the fluorescence signals and analyze them using data acquisition software, and set the sorting threshold according to the fluorescence signals;

[0025] S3.3: Judge whether to sort the droplets according to the fluorescence signal intensity. The positive droplets are collected into the sorting channel by applying a high-voltage pulse across the electrodes, while the negative droplets flow into the waste channel passively; the droplets in the sorting channel and the waste channel are respectively collected in different Eppendorf tubes. Among them, the heterogeneous vancomycin-intermediate resistant Staphylococcus aureus is collected from the sorting channel.

[0026] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages:

[0027] 1. In the present invention, FRET-based RNA probes are selected to monitor the growth of bacteria. These probes are encapsulated together with bacteria and antibiotics in nanoliter-scale droplets and then cultured at a specific antibiotic concentration. When the intermediate-resistant bacteria in the bacterial population proliferate, the RNase (RNA enzyme that can cleave RNA) released by the bacteria will cleave the FRET probe, resulting in an increase in fluorescence intensity. Therefore, the fluorescent droplets containing growing bacteria can be distinguished from the quenched droplets without bacteria, and more drug-resistant bacterial populations can be enriched thereby.

[0028] 2. The present invention is the first to apply the microfluidic platform to study hVISA. Initially, high-throughput droplet generation achieved single-droplet encapsulation, and then by utilizing the drug resistance differences of bacteria to specific drug concentrations, bacterial populations were screened and enriched from sensitive populations. This process promoted the formation of new bacterial populations.

[0029] 3. The present invention studies hVISA, which is a transitional state between methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate resistant Staphylococcus aureus (VISA). Since vancomycin is the last line of defense for MRSA treatment and there are a small number of VISA in hVISA, it is not effective for vancomycin treatment, resulting in protracted Staphylococcus aureus bloodstream infections and prolonged hospital stays for patients. Most problematically, hVISA is detected as vancomycin-sensitive by conventional drug susceptibility testing methods, so it may lead to the abuse of vancomycin and further aggravate the condition.

[0030] 4. The microdroplets generated by the chip adopted in the present invention can reach a minimum of 35.56 pL. At the same volume, more microdroplets can be generated by us; at the same flow rate, the flux is better. This will enable each single bacterium in hVISA to be encapsulated within a short time, providing a prerequisite for screening drug-resistant bacteria.

[0031] 5. The present invention adopts a fluorescent probe, which is more sensitive and can show fluorescence as fast as within 4 hours. The fluorescence value is sufficient for screening positive microdroplets at 6 hours, and the fluorescence is relatively stable, lasting for at least 7 days.

[0032] 6. Stability of the microdroplets of the present invention: The larger the microdroplets prepared in the present invention, the easier they are to break and fuse. Therefore, the microdroplets generated by us are more stable.

[0033] 7. Verification with standard strains and clinical strains: The present invention uses the hVISA standard strain Mu3 for relevant experiments, and this strain of this research is not a conventional standard strain. In addition, 15 hVISA strains isolated previously are also used in our research for verification. Description of the Drawings

[0034] The drawings are used to provide further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention.

[0035] In the drawings:

[0036] Figure 1 The occurrence, definition and screening process of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus (hVISA) of the present invention. MRSA: Methicillin-resistant Staphylococcus aureus; VISA: Vancomycin-intermediate resistant Staphylococcus aureus.

[0037] Figure 2 Selection of microdroplet size and bacterial concentration. (A-L) Microdroplets generated at different times at different channel depths (A-D, 0 hours; E-H, 12 hours; I-L, 7 days, scale bar = 100 μm). (A-D, M) Comparison of microdroplet sizes generated at different channel depths. (N) Comparison of microdroplet fluxes at different channel depths based on microdroplet sizes at 0 hour. (I-L, O) Comparison of different microdroplet stabilities (proportion of broken and fused microdroplets). Comparison of the coefficient of variation (CV) of microdroplets generated at different channel depths at 0 hour, 12 hours and 7 days. (Q) Microdroplets at different bacterial concentrations, scale bar = 100 μm. (R) Proportion of positive microdroplets at different bacterial concentrations. (S) Distribution of the number of bacteria in microdroplets based on the Poisson statistical model.

[0038] Figure 3(A) Schematic diagram of the fluorescence probe principle. (B) ELISA test results show that fluorescence increases with bacterial growth. (C) Fluorescence changes of microdroplets from 0 hour to 7 days (merged figure, scale bar = 100 μm). (D) Fluorescence of positive microdroplets starts to increase at 4 hours, stabilizes at 6 hours, reaches a plateau at 12 hours, and remains stable until day 7. (E) The fluorescence ratio of positive microdroplets to negative microdroplets changes over time and reaches a peak at around 24 hours. (F) The fluorescence intensity of positive microdroplets is significantly higher than that of negative microdroplets. Data were analyzed by unpaired t-test, ****p < 0.0001.

[0039] Figure 4 Determination of drug screening concentration. Images (merged figure, scale bar = 100 μm) and bar graph of fluorescence microdroplets at different concentrations of vancomycin (VAN0 - VAN8, 0 - 8 μg / mL). NC: negative control without bacteria. Data were analyzed by unpaired t-test, *p < 0.05; **0.001 < p < 0.05; ***p < 0.001; ns: not significant.

[0040] Figure 5 Specific verification of VAN4 (merged figure, scale bar = 100 μm). +VAN4: culture medium containing 4 μg / mL vancomycin; -VAN4: culture medium without 4 μg / mL vancomycin.

[0041] Figure 6 (A) Time-lapse fluorescence sequence (1000 ms) of the standard hVISA strain Mu3 screened; (B) histogram of fluorescence signals after Mu3 screening; (C) after screening, the minimum inhibitory concentration (MIC) of the microbial population against vancomycin increased from 2 μg / mL to 3 μg / mL (Etest assay).

[0042] Figure 7 Clinical strain verification: (A) Comparison of MIC between the original strain and the screened strain (E-test drug susceptibility test); (B) bar graph of the comparison of MIC between the original strain and the screened strain.

[0043] Figure 8 Comparison of fluorescence probe performance: A, microdroplet images of different probes, scale bar = 100 μm, B, peak graphs of different probes; C, fluorescence intensities of different probes in positive and negative microdroplets. Data were analyzed by unpaired t-test, ***p < 0.001.

[0044] Figure 9 Microdroplet generation chip.

[0045] Figure 10 Sorting chip structure. Specific implementation manner

[0046] The following is combined with the appendixFigures 1 - 10 The preferred embodiments of the present invention will be described. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0047] In the following embodiments, the test methods and detection methods are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0048] Embodiment

[0049] Experimental preparation

[0050] 1. Bacterial culture and sample preparation

[0051] The bacterial strains used in the present invention were prepared according to the standard protocol of the American Type Culture Collection (ATCC). The standard strains Mu3 (hVISA) and Mu50 (VISA) were provided by the First Affiliated Hospital of the University of Science and Technology of China. Other hVISA strains were from previous studies in our laboratory (Cheng X, Ma L, Wang Y, Sun W, Su J. Prevalence and molecular characteristics of heterogeneous vancomycin intermediate Staphylococcus aureus in a tertiary care center of northern China. Diagn Microbiol Infect Dis. 2024 Mar;108(3):116180. doi:10.1016 / j.diagmicrobio.2024.116180. Epub 2024 Jan 4. PMID: 38183897.). The strains ATCC29213, ATCC25923, ATCC43300, and RN4220 were stored in our laboratory. Vancomycin was purchased from the National Institutes for Food and Drug Control, China.

[0052] 2. Microfluidic device

[0053] The microfluidic device is a commercially available device, and the fluid flow in the microfluidic device is regulated by an injection pump (Pump11 PicoPlus Elite, Harvard Apparatus, Holliston, MA, USA). Sealed glass syringes (Gaoge, China) with a volume of 250 μL or 500 μL were used to dispense solutions.

[0054] Based on the above preparations, the technical solution of this application is specifically as follows:

[0055] A method for ultra-high throughput screening of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting, comprising:

[0056] S1: Respectively culture the cryopreserved standard strains Mu3 (hVISA, heterogeneous vancomycin-intermediate resistant Staphylococcus aureus), Mu50 (VISA), ATCC29213, ATCC25923, ATCC43300, and RN4220 strains in a constant temperature shaker (about 200 rpm) at 37 °C for 12 hours, and then collect them; among them, the culture medium is a conventional BHI medium;

[0057] S2: Mix the bacteria collected in S1 with a fluorescent probe and vancomycin to generate droplets in a microfluidic device;

[0058] S2.1: Mix the bacteria collected in S1 with fresh culture medium (BHI medium) at a volume ratio of 1:50, continue to culture in a constant temperature shaker at 200 rpm at 37 °C for 2 hours, centrifuge to collect the bacterial cells, and then resuspend them in fresh culture medium to obtain a bacterial suspension of 5*10^7 CFU / mL; mix the bacterial suspension, the fluorescent probe, and BHI containing vancomycin to obtain a bacterial mixture.

[0059] Among them, the volume ratio of the bacterial suspension, the fluorescent probe, and BHI containing vancomycin is 1:1:48.

[0060] In the finally prepared bacterial mixture, the concentration of the bacterial suspension is 10^6 CFU / mL, the fluorescent probe is 2 μM, and vancomycin is 4 μg / mL.

[0061] Among them, the fluorescent probe includes the following three types:

[0062] Probe 1: RNase Activity Fluorescent Detection Kit P0347S, Beyotime Biotechnology, China;

[0063] Probe 2: RNaseAlert Laboratory Detection Kit 4479768, Invitrogen&trade, USA;

[0064] Probe 3: 5'-Alexa488-UCUCGGUGCGUUG-BHQ1-3', Japan; the probe used in this invention patent.

[0065] This invention preferably uses Probe 3.

[0066] At the same time, the culture medium without bacteria is used as a negative control. (Randomly count the positive droplets in three fields of view to observe the inhibitory effect of the drug on the bacteria.

[0067] In addition, the selected drug screening concentrations were also specifically verified against the standard strains Mu50, Mu3, ATCC29213, ATCC25923, ATCC43300, and RN4220.

[0068] Step 2.2: Use of the microfluidic device (Brand name) droplet generation oil was used as the continuous oil phase, and the microfluidic device encapsulated the bacterial mixture of S2.1 and the continuous oil phase at a flow rate ratio of 1:2. After 24 hours of cultivation, the percentage of positive droplets was counted.

[0069] According to the Poisson distribution formula (probability X = k = f(λ; k) = e^(-λ)λ^k / k!), a suitable bacterial suspension was selected for droplet generation. The growth of bacteria was confirmed by an inverted fluorescence microscope. According to Poisson distribution analysis, 10^6 CFU / mL (λ = 0.51) was selected as the optimal concentration. At this concentration, 60% of the droplets were empty, and 30.65% of the droplets encapsulated a single bacterium, meeting the requirements for single-cell analysis.

[0070] A droplet generation chip was installed in the microfluidic device, and the depth of the droplet generation chip affected the size of the generated droplets.

[0071] The chip used in the present invention is as Figure 9 , see specifically the reference: Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, Mazutis L. 2017. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc 12:44 - 73.

[0072] The generated droplets can reach a minimum of 35.56 pL. At the same volume, we generated more droplets; at the same flow rate, the throughput was better. This will enable each bacterium in hVISA to be encapsulated within a short time, providing a prerequisite for screening drug-resistant bacteria.

[0073] The droplets were divided into four groups according to different depths of the droplet generation chip (20μm, 25μm, 30μm, 35μm) to evaluate the effects of these depths on droplet size, flow rate, stability, and coefficient of variation (CV). The size of the droplets was evaluated by randomly selecting 11 droplets from each group and measuring their diameters. The flow rate was calculated based on the average droplet diameter.

[0074] To evaluate stability, we observed the fusion and rupture percentages of the droplets generated at different channel depths after 7 days of cultivation. In addition, the CVs of the droplets generated at different channel depths were also compared at 0 hours, 12 hours, and 7 days.

[0075] Step 3: Sort the droplets generated in Step 2 through a microfluidic device, and collect heterogeneous vancomycin-intermediate resistant Staphylococcus aureus.

[0076] The flowchart of the ultra-high-throughput screening of hVISA based on fluorescence-activated droplet sorting is shown in Figure 1 , and the microfluidic chip device for droplet sorting has a pair of electrodes and bifurcated outlet channels.

[0077] As Figure 10 , the microfluidic chip is respectively connected with 250 μL syringes loaded with droplets and two fluorinated oils by Teflon tubes, and the syringes are mounted on an injection pump to control the flow rate; at the same time, physiological saline is connected to the positive and negative electrodes of the chip respectively; the microfluidic chip is also connected with a high-speed camera and a photoelectric converter, and the high-speed camera and the photoelectric converter are connected to a display.

[0078] Step 3.1: Pump the droplets into the sorting chip through the injection pump of the microfluidic device.

[0079] Step 3.2: When the droplets flow through the laser detection point in the sorting chip (the incident 490 nm excitation light irradiates the droplets to form an outgoing light), signal acquisition is completed. The digital acquisition card converts the collected fluorescence signal into a digital signal, and analyzes it using the data acquisition software programmed with LabVIEW. Set a reasonable sorting threshold according to the fluorescence signal to complete the sorting of the droplets. The entire process of the droplets entering the sorting chip is completed by a high-speed camera for image acquisition.

[0080] Step 3.3: Determine whether the droplets are sorted by the fluorescence signal intensity. The positive droplets are collected into the sorting channel by applying a high-voltage pulse across the electrodes, while the negative droplets flow passively into the waste channel; the droplets in the sorting channel and the waste channel are respectively collected in different Eppendorf tubes, where the heterogeneous vancomycin-intermediate resistant Staphylococcus aureus is collected from the sorting channel.

[0081] Use 1H,1H,2H,2H-perfluorooctanol (PFO, Shanghai Aladdin Biochemical Technology Co., Ltd.) for demulsification, recover the microbial cells and culture them on BHI-V4. Then, use the same bacterial suspension with a turbidity of 0.5 McFarland to perform the vancomycin E test strip experiment on a conventional Mueller-Hinton agar plate (Oxoid, UK). After culturing at 37 °C for 24 hours, measure the MIC and compare the MIC of the original strain with that of the screened strain.

[0082] Results:

[0083] 1. Chips with different channel depths were used to generate microdroplets, and all chip designs successfully and stably generated microdroplets ( Figure 2 as shown in A-P of Figure 2 ). However, a shallower channel depth led to smaller microdroplet diameters. At channel depths of 20μm, 25μm, 30μm, and 35μm, the diameters of the microdroplets were 35.56±2.02μm, 38.16±1.10μm, 43.64±0.73μm, and 51.73±1.09μm respectively ( Figure 2 as shown in M of Figure 2 ). In terms of throughput, the volumes of the microdroplets generated in groups i and ii were similar, with throughputs of 4629.6 / s and 3472.2 / s respectively, while the throughput of group iii was 1984.1 / s and that of group iv was 984.1 / s (

[0084] as shown in N of

[0085] ). After 7 days of culture, the stability of the microdroplets was related to their volume, and larger microdroplets were more prone to rupture and fusion (

[0086] as shown in O of Figure 8 ).

[0087] In addition, by comparing the changes in the coefficient of variation (CV) of the microdroplet diameters at 0 hours, 12 hours, and 7 days, we observed that the CV generally increased over time, especially in groups iii and iv. Interestingly, in groups i and ii, the CV at 7 days was lower than that at 12 hours, indicating that smaller microdroplets tended to shrink with increasing culture time. These findings suggest that channel depths of 20μm and 25μm (groups i and ii) have higher microdroplet throughputs and better stability, making them suitable for further research on this system. Figure 3 as shown in A of

[0088] : Based on Förster resonance energy transfer (FRET), RNA probes are short oligoribonucleic acids labeled with a fluorophore at the 5' end and a quencher at the 3' end. When the fluorophore and quencher are in close proximity, fluorescence is usually quenched. However, when the RNA probe is cleaved by RNase derived from growing bacteria, the fluorophore and quencher are spatially separated, resulting in an increase in fluorescence. Figure 3 as shown in B of Figure 3In addition, the fluorescence intensities of positive and negative droplets also changed over time, and the fluorescence ratio at 24 hours was up to 6.27. To shorten the culture time, we selected 12 hours as the standard culture time, at which the fluorescence intensity of positive droplets was significantly higher than that of negative droplets.

[0089] 3. Determination of drug screening concentration and specificity evaluation

[0090] To evaluate the inhibitory effect of drugs on sensitive bacteria in the hVISA population and improve the screening efficiency of drug-resistant strains, we cultured the standard hVISA strain in media containing different drug concentrations for up to 12 hours. As Figure 4 shown, the increase in drug concentration led to a decrease in the number of positive droplets. Specifically, at 4 μg / mL (VAN4), the number of positive droplets was significantly lower than that at VAN3 (P = 0.0128), but not significantly different from that at VAN6 (P = 0.0668). Based on these results, we selected VAN4 as the optimal concentration for drug screening.

[0091] In addition, we tested the specificity of VAN4 against VISA (Mu50), hVISA (Mu3), and VSSA (ATCC29213, ATCC25923, ATCC43300, and RN4220) strains ( Figure 5 ). We observed that VAN4 partially inhibited the hVISA strain, but had no effect on the VISA strain. In contrast, VAN4 completely inhibited the VSSA strain. These results indicate that VAN4 can effectively distinguish sensitive strains from drug-resistant strains and is suitable for screening drug-resistant strains in the hVISA population.

[0092] 4. Standard strain screening and detection

[0093] We used the standard hVISA strain Mu3 to test the performance of the system with optimized droplet encapsulation, culture, and sorting parameters. Droplets were generated on a chip with a channel depth of 20 μm, containing vancomycin (final concentration 4 μg / mL), a fluorescent probe, bacteria (final concentration 10^6 CFU / mL), and BHI medium. After 12 hours of culture, the droplets were introduced into a fluorescence detection and sorting biochip for sorting.

[0094] The fluorescence time series of Mu3 screening and sorting is shown in Figure 6 A. Three bacterial states were observed in the droplets: blank, inhibited, and growing. Blank and inhibited droplets showed low fluorescence intensity and flowed into the waste channel. In contrast, growing droplets showed higher fluorescence intensity, exceeding the set sorting threshold. At this time, the voltage amplifier was activated to generate a high electric potential to introduce the target droplets into the sorting channel. The fluorescence signal distribution of the droplets was processed and a histogram was generated ( Figure 6B), the histogram clearly divides the droplets into three groups: noise (black), negative droplets (blue, including blanks and bacteria inhibitors), and positive droplets (red, representing growing bacteria).

[0095] After sorting, the MIC of the reference strain increased from 2 μg / mL to 3 μg / mL ( Figure 6 in C), demonstrating that the system can successfully identify and isolate the drug-resistant bacterial population in the hVISA population. This method is completed in approximately 12 hours, significantly faster than the gold standard PAP-AUC (which takes 48 hours). In addition, our system can not only identify drug-resistant strains but also perform sorting, while PAP-AUC is limited to identification.

[0096] 5. Validation with clinical isolates

[0097] To further test the reliability of the system, we performed further screening on previously isolated clinical strains ( Figure 7 in A). These isolates had been confirmed as hVISA by the gold standard PAP-AUC method. These isolates belonged to five different subtypes: ST239-t030, ST59-t437, ST1097-t2270, ST5-t570, and ST5-t2460. After sorting, we selected single colonies that were morphologically similar to the original strains and grew in BHI-V4 for antimicrobial susceptibility testing (AST). The results showed that the MIC of the drug-resistant population was higher than that of the initial culture ( Figure 7 in B), confirming that the system can effectively identify and isolate the drug-resistant bacterial population in clinical isolates.

[0098] As described above, the basic principles, main features, and advantages of the present invention have been described. The above embodiments and descriptions are only for describing the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A method for ultra-high throughput screening of heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting, comprising: S1: Culturing the cryopreserved hVISA strains and then collecting them; S2: Mixing the bacteria collected in S1 with a fluorescent probe and vancomycin, and generating droplets in a microfluidic device; S3: Sorting the droplets generated in S2 through the microfluidic device, and collecting heterogeneous vancomycin-intermediate resistant Staphylococcus aureus.

2. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 1, wherein S2 Including: S2.1: Mixing the bacteria collected in S1 with fresh culture medium, continuing the culture, centrifuging to collect the bacterial cells, and then resuspending them in fresh culture medium to obtain a bacterial suspension; Mixing the bacterial suspension, the fluorescent probe and the culture medium containing vancomycin to obtain a bacterial mixture; S2.2: Using a droplet generation oil as the continuous oil phase, and the microfluidic device encapsulates the bacterial mixture in S2.1 with the continuous oil phase.

3. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 2, wherein, In S2.1, the bacteria collected in S1 are mixed with the fresh culture medium at a volume ratio of 1:

50.

4. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 3, characterized in that, In S2.1, the mixed bacteria are further cultured in a constant temperature shaker at 37°C at 200 rpm for 2 hours.

5. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 4, characterized in that, In S2.1, the volume ratio of the bacterial suspension, the fluorescent probe and the culture medium containing vancomycin is 1:1:

48.

6. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 5, wherein In S2.2, the flow rate ratio of the bacterial mixture to the continuous oil phase is 1:

2.

7. The ultra-high throughput screening method for heterogeneous vancomycin-intermediate resistant Staphylococcus aureus based on fluorescence-activated droplet sorting according to claim 6, wherein S3 Including: S3.1: Pumping the droplets into the sorting chip of the microfluidic device; S3.2: When the droplets pass through the sorting chip, collecting the fluorescence signals and analyzing them using data acquisition software, and setting the sorting threshold according to the fluorescence signals; S3.3: Judging whether to sort the droplets according to the fluorescence signal intensity. The positive droplets are collected into the sorting channel by applying a high voltage pulse across the electrodes, while the negative droplets flow passively into the waste channel; The droplets in the sorting channel and the waste channel are respectively collected in different Eppendorf tubes, wherein the heterogeneous vancomycin-intermediate resistant Staphylococcus aureus is collected in the sorting channel.

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