Method for in-situ detection of drug sensitivity of mixed flora based on combination of metabolic marker and fluorescence in-situ hybridization

Abstract

The invention discloses a method for in-situ detection of drug sensitivity of different types of bacteria in a mixed flora based on metabolic labeling combined with fluorescence in-situ hybridization, and belongs to the technical field of microbiological detection. The activity change condition of all bacteria in a flora under the action of an antibacterial drug is quantified through a D-type amino acid fluorescent probe (FDAA) metabolism labeling method, target bacteria are recognized through a fluorescence in-situ hybridization technology, and the drug sensitivity of the bacteria is detected in a manner of detecting the fluorescence intensity change of the corresponding bacteria in combination with a flow cytometry. The method can be used for rapidly and effectively performing species identification on various pathogenic bacteria in a mixed flora sample at the flora level and simultaneously determining and evaluating the antibacterial drug sensitivity of the pathogenic bacteria. The method is high in detection sensitivity, the minimum inhibitory concentration (MIC) of low-abundance target bacteria (larger than or equal to 0.5%) in a flora system can be accurately measured, and antibacterial spectrums of almost all bacteria in the complex flora system are established; the kit can be used for drug sensitivity detection of aerobic bacteria and anaerobic bacteria, can also be used for antibiotic sensitivity determination of flora level of microorganisms which cannot be cultured at present, and can reflect MIC of bacteria at flora level. The method has very high application value in rapid drug sensitivity detection of clinical mixed infection pathogenic bacteria and establishment of a bacterial antibacterial spectrum in a complex environment sample.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and particularly relates to a method for in-situ detection of drug susceptibility of mixed flora based on metabolic labeling combined with fluorescence in-situ hybridization. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and does not necessarily constitute an admission or imply in any form that this information forms the prior art already known to those of ordinary skill in the art.

[0003] Bacterial drug resistance has become one of the most serious global public health problems in the 21st century. The abuse of antibiotics in medical and aquaculture industries has enabled many pathogenic bacteria to acquire and spread drug resistance through horizontal and vertical gene transfer, rendering traditional antibiotic treatments gradually ineffective. This problem poses a huge challenge to clinical diagnosis and treatment. Especially in complex mixed infection cases, the difficulty of diagnosis and treatment increases significantly because different treatment strategies need to be adopted for different pathogenic bacteria. In addition, the complex interactions among pathogenic bacteria in the flora (such as certain pathogenic bacteria may promote or inhibit the growth of other pathogenic bacteria) further increase the complexity of research and diagnosis. Exploring these interaction mechanisms may provide new solutions for the diagnosis and treatment of mixed infections.

[0004] Bacterial antibiotics susceptibility test (AST) is a key basis for the rational use of antibiotics in current clinical practice. By detecting the minimum inhibitory concentration (MIC) of different microorganisms against different antibiotics, the clinical medication strategy can be determined. [1] Clinically existing AST technologies can mainly be divided into two methods: traditional phenotype-based detection and molecular biology analysis-based methods.

[0005] Traditional phenotype-based AST methods (such as agar dilution method, disk diffusion method, and microbroth dilution method) are only applicable to known types of bacteria and are difficult to be used for drug susceptibility determination of unculturable bacteria. These methods usually take a long time (≥16h), are complex to operate, and are difficult to meet the requirements of rapid clinical diagnosis. [2][3] In addition, the limitations of traditional phenotype detection methods have led to more widespread use of broad-spectrum antibiotics in clinical treatment, thus exacerbating the generation and spread of drug resistance. Even for culturable pathogenic bacteria, whether the MIC values of the isolated bacteria obtained by traditional methods can accurately reflect their drug susceptibility in a mixed infection environment still lacks effective research means.

[0006] With the continuous development of molecular biology techniques, a series of innovative methods for drug susceptibility detection in mixed samples have been proposed [4] , mainly by means of mass spectrometry (≤4h), sequencing (≤12h), fluorescence in situ hybridization (≤6h), etc., and combined with phenotypic research methods such as microfluidic microscopy imaging, to separate some bacteria in complex microbial samples and observe the phenotype to determine their drug susceptibility [5] . Most of the existing methods for determining the antibiotic susceptibility of bacteria in mixed samples inevitably have the problem of how to separate the target bacteria from the mixed samples and perform phenotypic observation to obtain their specific MIC. Among them, the methods for separating bacteria in mixed samples mainly include microfluidic chip capture and magnetic enrichment separation methods. On the basis of bacterial separation, mass spectrometry, sequencing or fluorescence in situ hybridization methods are combined to identify the separated bacteria, and at the same time, microscopic imaging technology is used to observe the phenotypic characteristics of the separated bacteria under the action of antibiotics, so as to judge their specific MIC. Microfluidic microscopy imaging provides an intuitive visualization means for the drug susceptibility test of bacteria in mixed samples, allowing direct observation of the effects of antibiotics on bacterial growth and morphology[6] [7][8] , but its current popularity is limited, facing the problems of high technical difficulty and high cost. In addition, it involves complex and cumbersome image analysis and processing, and because this technology relies on observing the growth and morphological changes of bacteria, it may not be able to accurately identify those bacteria that do not grow but still have metabolic activity, easily leading to misjudgment of bacterial drug resistance and being difficult to be effectively applied in clinical diagnosis and treatment

[0007] The emerging technologies for determining the drug susceptibility of bacteria in mixed samples often rely on special instruments and technologies. While the operation is complex and cumbersome, they can only judge the MIC of some bacteria with obvious phenotypic drug susceptibility information in the microbial community. The obtained antibacterial spectrum resolution is limited and the accuracy needs to be improved, making it difficult to be popularized and applied in clinical diagnosis

[0008] Therefore, it is necessary to develop a new method for rapid in-situ detection of the drug susceptibility of different types of bacteria in mixed microbial communities to solve the above problems Summary of the Invention

[0009] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a method for rapid in-situ detection of the drug susceptibility of different bacteria in mixed microbial communities based on metabolic labeling combined with fluorescence in situ hybridization (≤8 hours). Through this method, various target bacteria can be identified in the mixed microbial community, the metabolic activity changes of various bacteria (including non-culturable bacteria) in the mixed microbial community under the action of antibacterial drugs can be traced and their MIC can be obtained, as well as the rapid identification and MIC determination of target pathogenic bacteria in the mixed microbial community

[0010] To achieve the above purpose, the present invention adopts the following technical solutions

[0011] A method for in-situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in-situ hybridization, comprising the following steps: using a fluorescent D-amino acid probe (FDAA) to perform metabolic labeling on a mixed flora sample treated with different concentrations of antibiotics to reflect the change in bacterial metabolic activity under the action of antibiotics, using fluorescence in-situ hybridization probes (FISH) of different types of bacteria to perform fluorescence in-situ hybridization on the metabolically labeled mixed flora sample to identify and recognize target bacteria, detecting the metabolically labeled and fluorescence in-situ hybridized mixed flora sample by a flow cytometer, and determining the minimum inhibitory concentration (MIC) of each bacterium in the mixed flora sample according to its population separation and fluorescence intensity change;

[0012] Wherein, the fluorescent D-amino acid probe is connected to the peptidoglycan structure of bacteria, the fluorescence in-situ hybridization probe specifically binds to the 16S rDNA of target bacteria, the mixed flora sample includes a complex flora sample and a simple flora sample, the types of bacteria in the complex flora sample are unknown, the types of bacteria in the simple flora sample are known or selected from any N of X types of bacteria with known types, wherein, 2≤X<10, 2≤N≤X.

[0013] Further, the flow detection includes: counting the bacteria with fluorescence signals of both the fluorescent D-amino acid probe and the fluorescence in-situ hybridization probe by a flow cytometer, and determining the minimum inhibitory concentration of each bacterium in the mixed flora sample against different antibiotics according to the change in the median value of the fluorescence intensity of the fluorescent D-amino acid probe of the counted bacteria.

[0014] Further, the method also includes bacterial species identification and probe determination. According to the identification result of the bacterial species in the mixed flora sample, the corresponding fluorescence in-situ hybridization probe is selected or designed. The methods for bacterial species identification include 16S rDNA sequencing and fluorescence in-situ hybridization. 16S rDNA sequencing is used to preliminarily determine the bacterial species in the complex flora sample to facilitate the design of fluorescence in-situ hybridization probes for various bacteria in the complex flora system. The general situation of the bacterial species in the simple sample is known and 16S rDNA sequencing is not required. Finally, the bacterial species in the flora sample are identified based on fluorescence in-situ hybridization.

[0015] Further, the method also includes: after performing metabolic labeling on the mixed flora sample with the fluorescent D-amino acid probe, performing fixation treatment and permeabilization treatment on the mixed flora sample. The permeabilization treatment includes methods such as ethanol permeabilization, lysozyme permeabilization, Tween-20 permeabilization, Triton X-100 permeabilization, etc.

[0016] In some embodiments, paraformaldehyde is used to fix bacteria to protect their morphology and structure; ethanol is used for permeabilization treatment to increase the permeability of the bacterial cell membrane and allow the FISH probe to enter the bacteria and hybridize with the target sequence.

[0017] In some embodiments, lysozyme is used for permeabilization treatment; adding an appropriate concentration of lysozyme can help permeabilize the bacterial cell wall while maintaining the integrity of the bacterial morphology, shorten the time required for fixation and permeabilization treatment, facilitate subsequent fluorescence in situ hybridization, and improve the detection efficiency.

[0018] Further, the simple flora sample is selected from any one of samples such as clinical urine samples, blood samples, bronchoalveolar lavage fluid samples, drainage fluid samples, cerebrospinal fluid samples, etc.

[0019] Further, the complex flora sample is selected from any one of samples such as intestinal flora samples, saliva samples, sputum samples, vaginal secretion samples, biopsy tissue samples, air samples, soil samples, water resource samples, etc.

[0020] Further, the fluorescent D-amino acid probe includes probes such as Cy5-D-amino acid probe (EDA-Cy5, Cy5-ethylenediamine-D-alanine), FAM-D-amino acid probe (FDAA-FAM, FAM-D-alanine or FAM-D-lysine), TAMRA-D-amino acid probe (TADA, TAMRA-D-alanine), Rhodamine-D-amino acid probe (RADA, Rhodamine-D-alanine), etc. The fluorescent D-amino acid probe has stable metabolic labeling and strong signals, and the fluorescence in situ hybridization technology that strictly follows the base complementary pairing principle ensures a high degree of reliability of the results, with basically no background signal interference, and with the help of a flow cytometer, the drug sensitivity of target bacteria accounting for 0.5% in the mixed sample can be analyzed at the lowest, with high sensitivity.

[0021] The present invention also provides an application of the method for in-situ detection of antimicrobial drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization as described above, and uses the method to perform in-situ detection of antimicrobial drug sensitivity on the mixed flora sample.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention conducts bacterial drug sensitivity detection by means of the FDAA metabolic labeling method, fluorescence in situ hybridization technology and detecting the corresponding fluorescence intensity in combination with a flow cytometer, eliminating the complex and cumbersome steps of isolating and identifying target bacteria, and being able to quickly and effectively identify various pathogenic bacteria in a mixed flora sample and simultaneously determine their antimicrobial susceptibilities in situ. The present invention can accurately and intuitively present the sensitive and resistant degrees of bacteria in different mixed floras to various antimicrobial drugs at the flora level. Through FDAA metabolic labeling, bacteria in a metabolically active but non-growing state can be effectively identified and their MICs can be determined, with relatively high detection sensitivity. For target bacteria with low abundance (≥0.5%) in the flora system, their MICs can also be accurately determined, establishing a wide antimicrobial spectrum; drug sensitivity detection can be performed on both aerobic and anaerobic bacteria, and antimicrobial susceptibility determination at the flora level can also be performed on currently unculturable microorganisms, which can reflect the MICs of bacteria in the population and has high application value. In addition, the present invention has good compatibility with existing instruments and equipment, uses fewer instruments and has lower costs, making it more popularizable and capable of being effectively applied to clinical diagnosis and treatment.

[0024] The present invention can determine the MICs of non-separable and unculturable bacteria that cannot be determined by traditional AST strategies, can achieve rapid identification of pathogenic bacteria in complex samples and simultaneously evaluate their MICs in the flora environment, so as to effectively and pertinently provide personalized treatment plans for different clinical patients and give accurate and reasonable antibiotic treatment methods. In addition, by analyzing and comparing the MIC differences of the same pathogenic bacteria in different mixed infection samples and when cultured alone in vitro, the influence of the interaction between bacteria on their drug resistance can be further studied, the drug resistance mechanism and transmission mode of bacteria can be studied, and more reasonable and effective strategies can be formulated for clinical treatment to help alleviate the losses caused by antibiotic resistance to global health and the economy.

[0025] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and describes them in detail in conjunction with the drawings as follows. Brief Description of the Drawings

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, some of the following drawings are embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is a schematic diagram of the relationship between the fluorescence signal intensity of bacteria and the growth curve and growth rate (metabolic activity) and the structure of the D-amino acid probe;

[0028] Figure 2 To simulate the antibiotic susceptibility test of target microorganisms in vitro in the microbiota by combining the FDAA metabolic labeling and FISH method (FISH-FaAST);

[0029] Figure 3 To show the MIC expression of the same bacteria to levofloxacin in pure bacterial samples and mixed bacterial samples;

[0030] Figure 4 To show the flow chart of FISH-FaAST for bacteria in the gut microbiota;

[0031] Figure 5 To show the results of flow cytometry drug sensitivity test of bacteria C. perfringens against antibiotics Moxifloxacin, Cefoxitin, Linezolid, Metronidazole in human gut microbiota sample Sample3 by FISH-FaAST method;

[0032] Figure 6 To show the results of flow cytometry drug sensitivity test of bacteria E. coli against antibiotics Moxifloxacin, Cefoxitin, Vancomycin, Linezolid, Metronidazole in human gut microbiota sample Sample3 by FISH-FaAST method;

[0033] Figure 7 To show the results of flow cytometry drug sensitivity test of four uncultured bacterium against antibiotics Moxifloxacin, Meropenem, Vancomycin in human gut microbiota samples Sample1 and Sample2 by FISH-FaAST method;

[0034] Figure 8 To show the flow chart of the mixed infection experiment;

[0035] Figure 9 To show the identification of pathogenic bacteria and the results of antibiotic sensitivity determination in clinical mixed infection blood samples. Detailed implementation manners

[0036] To better describe the present invention, the following further explanations are given through specific examples. The methods in the following examples are all conventional methods unless otherwise specified.

[0037] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified.

[0038] The following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0039] The present invention provides a method for in-situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in-situ hybridization. Through this method, various target bacteria can be identified in complex flora, the metabolic activity changes of various bacteria (including non-culturable bacteria) in the mixed flora under the action of antibiotics can be traced and their MICs can be obtained, as well as the rapid identification and MIC determination of target pathogenic bacteria in the mixed flora. This method is mainly based on the following technologies:

[0040] 1. Flow cytometry:

[0041] Flow cytometry (FCM) is a mature technology that uses a flow cytometer to perform rapid, sensitive, and accurate multi-parameter quantitative analysis and sorting of cells or biological particles in a rapidly flowing state. Fluorescent dyes on cells or in cells are excited by a laser and emit fluorescence, which is detected by a detector and converted into a digital signal that can be recognized by a computer through photoelectric conversion. The intensity of the fluorescence signal represents the amount of fluorescent dye labeled on the cells.

[0042] 2. FISH-FaAST (fluorescence in-situ hybridization combined with FDAA metabolic labeling):

[0043] The present invention is based on fluorescent D - amino acids (FDAAs) to evaluate the bacterial metabolic activity of a mixed flora sample treated with an antibiotic gradient. The metabolic activity of bacteria in the mixed - infection sample is presented by the FDAA fluorescence signal. Combining with the fluorescent in situ hybridization technique (FISH), a second - type fluorescence labeling is performed on the target pathogenic bacteria in the mixed - infection sample after FDAA metabolic labeling. Finally, the double - fluorescence - labeled part in the mixed sample is detected by a flow cytometer (CytoFLEX) (i.e., the target pathogenic bacteria with metabolic activity in the mixed sample after antibiotic treatment, having both FDAA and FISH fluorescence signals). The median value of the FDAA fluorescence intensity of the double - fluorescence - labeled part under different antibiotic concentration treatments is statistically analyzed to analyze the drug sensitivity of the target bacteria in the mixed - infection sample. As the antibiotic concentration increases, when the median value of the fluorescence intensity significantly decreases for the first time, the corresponding antibiotic concentration is the MIC of the target pathogenic bacteria.

[0044] Among them, D - amino acid (DAA), especially D - alanine, is a special amino acid at the end of the pentapeptide structure in bacterial peptidoglycan. During the synthesis and metabolism of peptidoglycan, bacteria will perform cross - linking, hydrolysis (removing the terminal D - alanine), substitution (with other D - amino acids (DAA) in the surrounding environment), etc. at this position through the transpeptidase domain in enzymes such as penicillin - binding proteins (PBPs). In the present invention, a fluorescent group is modified on the side chain of D - amino acid and connected to the bacterial peptidoglycan structure through a metabolic labeling method. The higher the fluorescence intensity of FDAA on the flow cytometer, the more FDAA bound to the bacterial peptidoglycan, which means the higher the bacterial metabolic activity. [9] . At the same time, with the help of the fluorescent in situ hybridization technique, a specific sequence with a fluorescent group modified at the 5' end strictly follows the base - complementary pairing principle and specifically binds to the 16S rDNA of the target bacteria in the mixed - infection sample treated with an antibiotic gradient and FDAA - labeled. Thus, the target pathogenic bacteria can be identified and detected through the corresponding channels of the flow cytometer. Finally, the median value of the FDAA fluorescence intensity of the double - positive signal part with both FDAA metabolic labeling and FISH labeling is analyzed to quickly obtain the drug resistance of various pathogenic bacteria to different antibiotics and the corresponding MIC in a complex mixed - flora sample.

[0045] By fluorescently labeling D - type amino acids (such as Cy5 - FDAA) to label bacterial peptidoglycan, detecting the metabolic activity of bacteria has the characteristics of stable labeling and strong signals. In addition, specific FISH probes with FAM fluorophores are used to help identify specific bacterial species, and flow cytometry is used to detect double - fluorescence signals (FDAA and FISH) to determine the MIC of target pathogenic bacteria. The drug sensitivity of target bacteria with a proportion of 0.5% in mixed samples can be analyzed at the lowest. Compared with traditional drug susceptibility testing methods, the FISH - FaAST method does not require bacterial isolation and culture, can directly analyze clinical mixed - infection samples, evaluate the drug resistance of target bacteria to different antibiotics at the complex flora level, help analyze the impact of the interaction of different pathogenic bacteria on drug resistance, rather than just studying the drug resistance of target bacteria growing alone against antibiotics, which is closer to the actual clinical environment; it can achieve rapid detection of bacterial drug sensitivity, can complete antibiotic sensitivity testing within 8 hours, is also applicable to unculturable bacteria, and solves the limitations of traditional AST methods; in addition, this technology has high sensitivity and can detect target bacteria with low abundance (0.5%).

[0046] Bacterial antibiotic susceptibility test (AST) is a prerequisite for the precise use of antibiotics. Based on FDAA metabolic labeling and combined with fluorescence in situ hybridization method, the present invention provides a strategy for effectively determining the antibiotic susceptibility of various pathogenic bacteria in various clinical mixed - infection samples or other complex flora samples at the flora level. It can measure the MIC of unculturable bacteria that cannot be measured by traditional AST strategies, can achieve rapid identification of pathogenic bacteria in complex samples and evaluate their MIC in the flora environment, so as to effectively and specifically provide personalized treatment plans for different clinical patients and give precise and reasonable antibiotic treatment methods. In addition, by analyzing and comparing the MIC differences of the same pathogenic bacteria in different mixed - infection samples and when isolated and cultured, the impact of the interaction between bacteria on their drug resistance can be further studied, the mechanism and transmission mode of bacterial drug resistance can be studied, and more reasonable and effective strategies can be formulated for clinical treatment to help alleviate the losses caused by antibiotic resistance to global health and economy.

[0047] Taking intestinal flora samples and clinical mixed - infection samples as examples, the process and principle of in - situ detection of antibiotic sensitivity of complex flora samples and simple flora samples using this method are respectively elaborated:

[0048] (I) Determination of drug sensitivity of target bacteria in samples with high diversity such as intestinal flora

[0049] 1) Sample pre - culture and antibiotic preparation: Take the fecal sample provided by healthy volunteers, resuspend it in modified GAM liquid medium, and place it in an anaerobic workstation for anaerobic culture at 37°C for about 48 h. Adjust the bacterial density of the culture to OD 600 = 0.1, and store the remaining fecal sample at - 80°C. Prepare various antibiotic solutions with two - fold serial dilutions (0.125 - 128 μg / ml) in advance.

[0050] 2) Antibiotic treatment and FDAA metabolic labeling: In the anaerobic workstation, mix the whole fecal culture with the gradient - diluted antibiotic solution in equal volume (500 μl), and add 15 μl of 10 mM FDAA probe. Incubate anaerobically at 37°C and 200 rpm for 2 - 4 h for metabolic labeling. At the same time, take a part of the whole fecal culture for 16S rDNA sequencing to identify the types of bacteria in the flora after the whole culture, confirm the microbial diversity in the culture, and design and select FISH probes according to the sequencing results. Then centrifuge the sample to remove the supernatant, and resuspend it in 1 ml of PBS for the first wash to remove the residual and non - specifically - bound FDAA probes in the sample during the FDAA labeling process. Repeat the washing step twice.

[0051] 3) Fixation and permeabilization: After the second wash, resuspend the sample in 200 μl of 4% PFA, and incubate with gentle rotation in the dark for 2 h for bacterial fixation to protect the morphology and structure of bacteria. After fixation, centrifuge the sample, remove the supernatant, resuspend it in PBS buffer, and add an equal volume of absolute ethanol. Freeze - permeabilize at - 30°C for more than 24 h to increase the permeability of the bacterial cell membrane and allow the FISH probe to enter the bacteria and hybridize with the target sequence.

[0052] 4) Probe search and design: By combining the results of the full - length microbial diversity analysis of the culture, search the literature for suitable probes or use bioinformatics and ARB software for probe design.

[0053] 5) Fluorescence in situ hybridization: Take about 50 μl of the sample frozen and permeabilized in the - 30°C refrigerator into a new 1.5 ml centrifuge tube, centrifuge it, remove the supernatant, and resuspend it in the hybridization solution with the corresponding formamide concentration. Incubate at the specified temperature at 800 rpm in the dark for 6 h. After hybridization, centrifuge the sample and resuspend it in 400 μl of the hybridization wash solution with 0% formamide. Put it into a thermostatic mixer, adjust the temperature to two degrees higher than the hybridization temperature, and incubate and wash at 800 rpm for 15 min. Repeat the washing twice, then centrifuge to remove the supernatant, and resuspend it in an appropriate volume of filtered PBS.

[0054] 6) Flow cytometry analysis: Finally, flow cytometry was used to detect the fluorescence intensity of metabolic markers of various bacteria under the action of drugs at different concentrations. The part with both FDAA and FISH fluorescence signals was counted. According to the change in the median value of the FDAA fluorescence intensity in this part, the MIC of each bacterium against different antibiotics was determined. The part with both FDAA and FISH signals was detected by flow cytometry (the gating in flow cytometry can be defined according to the negative group corresponding to each antibiotic concentration, that is, the negative group that has only been treated with gradient antibiotics without FDAA metabolic labeling and FISH labeling). The median value of the FDAA fluorescence signal in the double-fluorescence positive part in different antibiotic concentration groups was counted. Combining with the definition of MIC (minimum inhibitory concentration), as the antibiotic concentration increases, when the median value of the fluorescence intensity begins to decrease steadily, it indicates that the growth of the target bacteria in the bacterial population starts to be inhibited at the antibiotic concentration corresponding to this point, that is, the MIC value of the target bacteria in the bacterial population against this antibiotic.

[0055] (2) Rapid determination of the drug sensitivity of target pathogens in samples with low diversity such as clinical mixed infection samples:

[0056] 1) Preliminary judgment of the types of bacteria in the sample: The clinical mixed infection sample was mixed evenly in modified GAM medium for bacterial proliferation. After culturing and proliferating to an appropriate bacterial density, 4% paraformaldehyde and 75% ethanol were used for bacterial fixation and permeabilization respectively. Several FISH probes targeting different common pathogens were added for fluorescence in situ hybridization to identify the pathogens in the mixed infection sample;

[0057] 2) Antibiotic gradient treatment and FDAA metabolic labeling: The remaining culture sample was mixed with an equal volume (500 μl) of various gradient-diluted antibiotic solutions, and 15 μl of 10 mM FDAA probe was added. Incubate at 37 °C

[0058] at 200 rpm for 2 - 4 h for metabolic labeling;

[0059] 3) Fixation, permeabilization and lysozyme treatment: Subsequently, the sample was centrifuged to remove the supernatant, resuspended in 4% PFA and incubated with gentle rotation in the dark for 4 min. Centrifuge again and resuspend in 1xPBS, incubate with shaking at 37 °C and 800 rpm for 3 min. Immediately centrifuge the sample and resuspend in 70% ethanol for permeabilization for 4 min. Centrifuge again and resuspend in 1xPBS, incubate with shaking at 37 °C and 800 rpm for 3 min. Centrifuge the sample and resuspend in 2 mg / ml lysozyme to treat the bacteria, and then wash twice with filtered PBS;

[0060] 4) Fluorescence in situ hybridization: Finally, resuspend in 400 μl of hybridization solution containing 20% formamide, and add several 4 μl of 100 μM FISH probes respectively. Incubate at 46 °C with 800 rpm for 30 min for tracking the target bacteria in clinical mixed infection samples. Subsequently, wash the samples with hybridization solution containing 0% formamide to remove residual FISH probes;

[0061] 5) Flow cytometry analysis: Wash the samples twice with filtered PBS solution, and then perform fluorescence analysis with a flow cytometer. Count the part with both FDAA and FISH fluorescence signals, and determine the MIC values of the target pathogenic bacteria in clinical mixed infection samples against different antibiotics according to the median value of FDAA fluorescence intensity to assist clinical diagnosis and treatment.

[0062] The above is the process and principle of in-situ detection of antibiotic sensitivity for complex microbial community samples (intestinal microbial community samples) and simple microbial community samples (clinical mixed infection samples) using this method. By making simple improvements to the above experimental steps in combination with existing technologies, in-situ detection of antibiotic sensitivity and in-situ detection of sensitivity to other antibacterial drugs (chemically synthesized drugs such as sulfonamides, imidazoles, nitroimidazoles, quinolones, etc.) can be achieved for any mixed microbial community samples (clinical urine samples, blood samples, bronchoalveolar lavage fluid samples, drainage fluid samples, cerebrospinal fluid samples, intestinal microbial community samples, saliva samples, sputum samples, vaginal secretion samples, biopsy tissue samples, air samples, soil samples, water resource samples, etc.).

[0063] The following further elaborates the present invention with specific embodiments:

[0064] Example 1 Verification of method feasibility (simulated microbial community):

[0065] Take 3 clinically isolated strains, culture and treat them with antibiotics respectively. Obtain the growth inhibition effect of the corresponding antibiotics on bacteria by using the FDAA metabolic labeling method. After mixing the samples, use the FISH labeling method and obtain the MIC of the target bacteria treated with antibiotics from the simulated microbial community with a flow cytometer. The specific steps are as follows:

[0066] 1.1 Take clinically isolated Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and culture them respectively;

[0067] 1.2 Scrape an appropriate amount of colonies from each isolated culture into a 15 ml centrifuge tube containing 5 ml of LB medium, and control the bacterial density to 0.1 (OD 600 = 0.1);

[0068] 1.3 Prepare an antibiotic levofloxacin solution diluted in a two-fold dilution series (0 - 128 μg / ml), and dispense 500 μl of each concentration into 1.5 ml centrifuge tubes;

[0069] 1.4 Take 500 μl of each bacterial solution with OD 600 = 0.1 and mix it with 500 μl of the antibiotic levofloxacin solution at 0 - 128 μg / ml (total volume 1 ml);

[0070] 1.5 Add 15 μl of 10 mM Cy5 - DAA probe (final concentration 0.15 mM, see the structural diagram in Figure 1 b);

[0071] 1.6 Incubate in the dark at 37 °C with 200 rpm for 2 h;

[0072] 1.7 After incubation, mix the bacterial solutions in the 3 centrifuge tubes, place them in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0073] 1.8 Add 1 ml of PBS, pipette to mix well, and place it in the centrifuge again, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0074] 1.9 Add 1 ml of PBS, pipette to mix well, and place it in the centrifuge again, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0075] 1.10 Add 200 μl of 4% PFA, pipette to mix well, and incubate with rotation in the dark for 2 h for bacterial fixation;

[0076] 1.11 Place the fixed sample in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0077] 1.12 Add an equal volume of absolute ethanol and PBS (total volume 800 μl), place it at - 30 °C for fixation and permeabilization for more than 24 h;

[0078] 1.13 Take 50 μl of the mixed bacterial sample for each antibiotic gradient and dispense it into different 1.5 ml centrifuge tubes,

[0079] Centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0080] 1.14 Add 400 μl of the hybridization solution with the corresponding formamide concentration, pipette to mix well, then add 4 μl of the FISH probe for the target bacteria at 100 μM, and incubate and hybridize at 800 rpm at the corresponding temperature for 6 h;

[0081] 1.15 Place it in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, remove the supernatant, add 1 ml of filtered PBS, and pipette to mix well;

[0082] 1.16 Centrifuge at 13,000 rpm for 3 min at room temperature, remove the supernatant, add 1 ml of filtered PBS, and pipette to mix well.

[0083] 1.17 Centrifuge at 13,000 rpm for 3 min at room temperature, remove the supernatant, add 1 ml of filtered PBS, and pipette to mix well.

[0084] 1.18 Centrifuge at 13,000 rpm for 3 min at room temperature, remove the supernatant, add 200 μl of filtered PBS, and pipette to mix well to prepare a bacterial suspension.

[0085] 1.19 Use a flow cytometer to detect the fluorescence intensity of each sample, and statistically analyze the median value of the APC fluorescence intensity of the part with both FDAA and FISH positive signals.

[0086] The process of the above-mentioned method combining FDAA metabolic labeling and FISH method (FISH-FaAST) for testing the antibiotic sensitivity of target microorganisms in in vitro simulated microbial communities is as Figure 2 shown. The MIC expressions of three bacteria, Ec11, Kp9, and Ab16, for levofloxacin in pure bacterial samples and mixed bacterial samples are as Figure 3 shown. The concentration settings of levofloxacin and the MIC determination results of three bacteria, Ec11, Kp9, and Ab16, for levofloxacin in pure bacterial samples and mixed bacterial samples are shown in Table 1.

[0087] Table 1 Antibiotic concentration settings and MIC determination results

[0088]

[0089] Please refer to Figure 3 , the three pictures in the first row refer to the MIC of levofloxacin measured in pure bacterial samples of Ec11, Kp9, and Ab16. The points indicated by the red arrows represent the MIC values. Among them, Ec11 is sensitive to levofloxacin (MIC: 0.5), and Kp9 and Ab16 are resistant to levofloxacin (MIC ≥ 64). As Figure 3 can be seen, the MIC of the three bacteria for levofloxacin in the simulated microbial community is consistent with the results measured in pure bacteria. As can be seen from Table 1, the three bacteria have consistent MIC expressions for the same antibiotic in pure and mixed samples, indicating that the method for in-situ detection of drug sensitivity of mixed microbial communities based on metabolic labeling combined with fluorescence in-situ hybridization provided by the present invention is feasible and the results are reliable.

[0090] Example 2 Determination of MIC of target bacteria in intestinal microbiota samples

[0091] Please refer to Figure 4, the response of different bacteria to the inhibitory effects of different antibiotics at the microbial community level was determined by the FISH-FaAST method in intestinal microbiota samples, and the specific process is as follows:

[0092] 2.1 Samples were taken from 3 healthy young volunteers (3 g / person). A part of the 3 collected fresh fecal samples was directly resuspended in modified GAM medium and incubated at 37 °C for more than 48 hours in a Concept-400 anaerobic workstation. The fecal re-cultures and the remaining original fecal samples were subjected to full-length microbial diversity analysis, and corresponding FISH probes were selected and designed according to the sequencing results;

[0093] 2.2 The re-cultured fecal samples were diluted with modified GAM medium in an anaerobic workstation, and the bacterial density was adjusted to OD 600 = 0.1;

[0094] 2.3 The bacterial suspension was mixed with 6 antibiotics (Moxifloxacin, Meropenem, Cefoxitin, Vancomycin, Linezolid,

[0095] Metronidazole) pre-prepared by two-fold serial dilution in equal volume (the total volume of each antibiotic-bacterial suspension mixture was 1 ml);

[0096] 2.4 15 μl of 10 mM FDAA probe was added respectively, and incubated in the dark at 37 °C

[0097] at 120 rpm for 4 hours;

[0098] 2.5 After incubation, the samples were placed in a centrifuge and centrifuged at 13,000 rpm at room temperature for 3 min, then returned to the anaerobic workstation to remove the supernatant;

[0099] 2.6 1 ml of PBS was added, pipetted and mixed evenly, and then placed in a centrifuge again, centrifuged at 13,000 rpm at room temperature for 3 min, returned to the anaerobic workstation to remove the supernatant;

[0100] 2.7 1 ml of PBS was added, pipetted and mixed evenly, and then placed in a centrifuge again, centrifuged at 13,000 rpm at room temperature for 3 min, returned to the anaerobic workstation to remove the supernatant;

[0101] 2.8 200 μl of 4% PFA was added, pipetted and mixed evenly, and incubated with gentle rotation in the dark for 2 h for bacterial fixation;

[0102] 2.9 Place the fixed sample in a centrifuge and centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant; 2.10 Add an equal volume of absolute ethanol and PBS (total volume 800 μl) to each sample, and place it at -30 °C for fixation and permeabilization for more than 24 h;

[0103] 2.11 Take 50 μl of the mixed bacterial sample for each antibiotic gradient, aliquot it into different 1.5 ml centrifuge tubes, centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant;

[0104] 2.12 Add 400 μl of the hybridization solution corresponding to the formamide concentration, pipette and mix well, then add 4 μl of the FISH probe for the target bacterium at 100 μM, and incubate and hybridize at 800 rpm at the corresponding temperature for 6 h (the FISH probes used are shown in Table 2);

[0105] 2.13 Place it in a centrifuge, centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant, add 1 ml of filtered PBS, and pipette and mix well;

[0106] 2.14 Centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant, add 1 ml of filtered PBS, and pipette and mix well;

[0107] 2.15 Centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant, add 1 ml of filtered PBS, and pipette and mix well;

[0108] 2.16 Centrifuge at 13,000 rpm at room temperature for 3 min to remove the supernatant, add 200 μl of filtered PBS, and pipette and mix well to prepare a bacterial suspension;

[0109] 2.17 Use a flow cytometer to detect the fluorescence intensity of each sample, and statistically analyze the median value of the APC (Cy5) fluorescence intensity of the part with both FDAA and FISH positive signals (the antibiotic concentration gradient and the detection results are shown in Table 3).

[0110] Table 2 Design and selection of FISH probes

[0111]

[0112]

[0113] As shown in the above table, the intestinal flora obtained by overall culturing of fecal samples provided by healthy volunteers was subjected to microbial full-length diversity testing (16S rDNA sequencing). According to the returned sequencing results, FISH probes were designed at the species level with the help of ARB software. The probes were all compared by blast, and the results were good. They were specifically verified by flow cytometry and confocal microscopy, and all had good specificity and labeling effects.

[0114] Table 3 shows the consistency and differences between the MIC results of the target bacteria in gut microbiota sample 3 measured by the method shown in the present invention and the results obtained by two other methods (the method in the reference * and the agar method).

[0115] Table 3 Antibiotic concentration gradient settings and MIC results (taking sample 3 as an example)

[0116]

[0117] Reference *: Maier, L., Goemans, C. V., Wirbel, J. et al. Unravelling the collateral damage of antibiotics on gut bacteria. Nature 599, 120–124 (2021).

[0118] Agar method *: Standard agar dilution method. Single bacteria isolated after reculturing using human fecal samples were used to verify the consistency and differences between the results obtained by the FISH-FaAST method of the present invention and the standard agar dilution method.

[0119] Note: There is some overlap between the results in the reference and those of the present invention, so there are some results without references; the agar dilution method belongs to the traditional phenotype-based drug sensitivity test. Some bacteria cannot be isolated and cultured, and only some of the results of the agar method can be used for reference and comparison. Compared with the traditional drug sensitivity detection method, the FISH-FaAST method does not require bacterial isolation and culture, can directly analyze clinical mixed infection samples, evaluate the drug resistance of target bacteria to different antibiotics at the complex microbiota level, help analyze the influence of the interaction of different pathogenic bacteria on drug resistance, rather than only studying the drug resistance of target bacteria under single growth, and is closer to the actual clinical environment; it can achieve rapid detection of bacterial drug sensitivity, can complete the antibiotic sensitivity test within 8 hours, is also applicable to unculturable bacteria, and solves the limitations of the traditional AST method; in addition, this technology has high sensitivity and can detect target bacteria with low abundance (0.5%).

[0120] Flow cytometry drug sensitivity test results of bacteria C. perfringens against antibiotics Moxifloxacin, Cefoxitin, Linezolid, Metronidazole in human gut microbiota sample Sample3 measured by the FISH-FaAST method are as Figure 5As shown. By the FISH-FaAST method of the present invention, the antibiotic concentration corresponding to the first point when the FDAA signal starts to decline is determined as the MIC of this bacterium against this antibiotic. Therefore, the MIC of C. perfringens in Sample3 against MXF (moxifloxacin) is 1, the MIC against LZD (linezolid) is 4, the MIC against MTZ (metronidazole) is 8, and the MIC against FOX (cefoxitin) is 2. Most of the MIC results measured in the flora are consistent with the standard MIC results of EUCAST.

[0121] The results of flow cytometry drug sensitivity testing of the bacterium E. coli in the human intestinal flora sample Sample3 against the antibiotics Moxifloxacin, Cefoxitin, Vancomycin, Linezolid, and Metronidazole by the FISH-FaAST method are as Figure 6 shown. The MIC of E. coli in Sample3 against VAN (vancomycin) is ≥128, the MIC against MXF (moxifloxacin) is 8, the MIC against FOX (cefoxitin) is 16, the MIC against MTZ (metronidazole) is ≥128, and the MIC against LZD (linezolid) is 64. The MIC of E. coli measured in the flora is overall consistent with the EUCAST results, with only differences in the part for MXF.

[0122] The results of flow cytometry drug sensitivity testing of four uncultured bacteria in the human intestinal flora samples Sample1 and Sample2 against the antibiotics Moxifloxacin, Meropenem, and Vancomycin by the FISH-FaAST method are as Figure 7 shown. Several current uncultured bacteria in the human intestinal flora samples Sample1 and Sample2 are determined by full-length microbial diversity analysis, and specific FISH probes OTU11-R5, OTU21-R1, and OTU15-R3 are designed and then FISH-FaAST determination is carried out. This part is the MIC determination of bacteria that have not been isolated and cultured yet, so there is no specific MIC result reference. By the FISH-FaAST method, the MIC of Uncultured bacterium OTU11 in Sample 1 against Moxifloxacin is 1, the MIC against Meropenem is 0.5, and the MIC against Vancomycin is 0.5 (attached Figure 7(The first three figures in the first row); The MIC of Uncultured bacterium OTU 21 in Sample 1 for Meropenem is 0.5 (attached Figure 7 (in the middle of the second row); The MIC of Uncultured bacterium OTU15 in Sample 2 for Moxifloxacin is 32, for Meropenem is 0.125, and for Vancomycin is ≥128; The MIC of Uncultured bacterium OTU21 in Sample 2 for Meropenem is 1, and for Vancomycin is 0.5.

[0123] Example 3 Determination of Antibiotic Sensitivity of Multiple Target Pathogens in Clinical Mixed Infection Samples

[0124] 3.1 Please refer to Figure 8 , take clinical mixed infection samples, such as drainage fluid, bronchoalveolar lavage fluid, blood, and perform pretreatment on the samples:

[0125] 1) Filter the mixed infection sample using a 40μm cell strainer to reduce agglomeration and large tissue in the sample;

[0126] 2) Take the filtered filtrate, centrifuge at 600×g for 8 min, and take the supernatant;

[0127] 3) Add 3 volumes of red cell lysis buffer, and let it stand until the solution becomes clear;

[0128] 4) Place the sample in a centrifuge, centrifuge at 4000 rpm at room temperature for 20 min, and remove the supernatant;

[0129] 5) Resuspend in CAMHB medium;

[0130] 3.2 Add the clinical mixed infection sample to CAMHB medium for overall culture and proliferation;

[0131] 3.3 Bacterial identification: Take an appropriate amount of the proliferated culture into a 1.5 ml centrifuge tube;

[0132] 1) Place it in a centrifuge and centrifuge at 13000 rpm at room temperature for 3 min;

[0133] 2) Resuspend in 200 μl of 4% PFA, and incubate with rotation in the dark for 4 min for bacterial fixation;

[0134] 3) Place it in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0135] 4) Resuspend in 1 ml of filtered PBS, pipette to mix evenly, centrifuge at 13,000 rpm for 3 min at room temperature, and discard the supernatant;

[0136] 5) Resuspend in 1 ml of filtered PBS, pipette to mix well, centrifuge at 13,000 rpm for 3 min at room temperature, and discard the supernatant;

[0137] 6) Resuspend in 800 μl of 75% ethanol for permeabilization for 4 min;

[0138] 7) Place in a centrifuge, centrifuge at 13,000 rpm for 3 min at room temperature, and discard the supernatant;

[0139] 8) Resuspend in 200 μl of 2 mg / ml lysozyme solution and react for 3 min;

[0140] 9) Take 50 μl of the sample treated with lysozyme, centrifuge at 13,000 rpm for 3 min at room temperature, and discard the supernatant;

[0141] 10) Resuspend in 400 μl of hybridization solution with 20% FA, add 4 μl of several groups of FISH probes, and hybridize at 46 °C

[0142] at 800 rpm for 6 h;

[0143] 11) Place in a centrifuge and centrifuge at 13,000 rpm for 3 min at room temperature;

[0144] 12) Resuspend in 400 μl of 0% FA preheated to 48 °C, incubate and wash at 48 °C at 800 rpm for 15 min;

[0145] 13) Place in a centrifuge and centrifuge at 13,000 rpm for 3 min at room temperature;

[0146] 14) Resuspend in 400 μl of 0% FA preheated to 48 °C, incubate and wash at 48 °C at 800 rpm for 15 min;

[0147] 15) Place in a centrifuge and centrifuge at 13,000 rpm for 3 min at room temperature;

[0148] 16) Resuspend in 50 μl of filtered PBS, and analyze using a flow cytometer to identify the types of pathogenic bacteria in the mixed infection sample;

[0149] 3.4 Freeze 1 / 2 of the remaining proliferated culture in an -80 °C refrigerator, and use the remaining part for antibiotic susceptibility testing;

[0150] 3.5 Antibiotic susceptibility testing: Prepare two-fold serial dilution solutions of several antibiotics (0.125 - 128 μg / ml, 0 μg / ml), aliquot 500 μl of each gradient of each antibiotic into 1.5 ml centrifuge tubes, and label them;

[0151] 3.6 Add 500 μl of the bacterial suspension of equal volume to the antibiotic solutions of each gradient, and simultaneously add 15 μl

[0152] of 10 mM Cy5-DAA probe (final concentration 0.15 mM), incubate at 37 °C with 200 rpm for 2 h; after incubation, (the antibiotic susceptibility test of the pathogenic bacteria in the mixed infection sample can be carried out simultaneously with the bacterial identification. When the identification result in step 2 is returned, the co-incubation time of the sample + antibiotic + FDAA in the susceptibility test just ends)

[0153] 1) After the co-incubation of the bacterial sample and the antibiotic + FDAA ends, wash the sample twice with filtered PBS to remove the free DAA probe;

[0154] 2) Place the sample in a centrifuge and centrifuge at 13000 rpm at room temperature for 3 min;

[0155] 3) Resuspend in 200 μl of 4% PFA, and incubate with rotation in the dark for 4 min for bacterial fixation;

[0156] 4) Place the sample in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0157] 5) Resuspend in 1 ml of filtered PBS, pipette to mix well, then centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0158] 6) Resuspend in 1 ml of filtered PBS, pipette to mix well, then centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0159] 7) Resuspend in 800 μl of 75% ethanol for permeabilization for 4 min;

[0160] 8) Place the sample in a centrifuge, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0161] 9) Resuspend in 200 μl of 2 mg / ml lysozyme solution and react for 3 min;

[0162] 10) Take 50 μl of the sample after lysozyme treatment, centrifuge at 13000 rpm at room temperature for 3 min, and remove the supernatant;

[0163] 11) Resuspend in 400 μl of 20% FA hybridization solution, add several sets of FISH probes (each with a different type of fluorophore) that have been confirmed to contain the target pathogenic bacteria in the sample through the previous steps, 4 μl each, and hybridize at the designated temperature with 800 rpm for 6 h;

[0164] 12) Place in a centrifuge and centrifuge at 13000 rpm at room temperature for 3 min;

[0165] 13) Resuspend in 400 μl of 20% FA pre-warmed to 48°C, incubate and wash at 800 rpm for 15 min at 48°C;

[0166] 14) Place in a centrifuge and centrifuge at 13,000 rpm for 3 min at room temperature;

[0167] 15) Resuspend in 400 μl of 0% FA pre-warmed to 48°C, incubate and wash at 800 rpm for 15 min at 48°C;

[0168] 3.7 Place in a centrifuge and centrifuge at 13,000 rpm for 3 min at room temperature;

[0169] 3.8 Resuspend in 50 μl of filtered PBS, analyze the parts where different fluorescence signals are all positive with the APC signal by flow cytometry, count the median value of the APC fluorescence intensity of these parts, and determine the MIC of each target pathogen (the antibiotic concentrations and MIC determination results are shown in Table 4).

[0170] Table 4 Antibiotic concentration settings and MIC references and determination results

[0171]

[0172] With the optimized FISH-FaAST method, identify the pathogens in clinical mixed-infection blood samples and evaluate the resistance to levofloxacin. The identification and evaluation results are as Figure 9 shown. Among them, Escherichia coli and Enterococcus faecalis were identified in the mixed-infection blood sample Co-infection1, and the MICs of the two bacteria against Levofloxacin were determined; Enterococcus faecium was identified in both the mixed-infection blood samples Co-infection2 and Co-infection3, and the MICs of it against Levofloxacin were determined respectively. The resistance determination results of Enterococcus faecium to Levofloxacin in the two mixed-infection samples were consistent, and both were resistant bacteria.

[0173] In summary, the present invention conducts bacterial drug sensitivity detection through the FDAA metabolic labeling method, FISH fluorescence in situ hybridization technology, and by combining flow cytometry to detect the corresponding fluorescence intensity. It skips the complex and cumbersome steps of isolating and identifying the target bacteria, has good clinical application value, can identify various pathogenic bacteria in complex clinical mixed infection samples within 24 hours while measuring and evaluating their antibiotic sensitivity, and has the advantages of high efficiency, accuracy, and high sensitivity. Moreover, the present invention can accurately and intuitively present the sensitive and resistant degrees of different intestinal bacteria to various antibiotics at the microbial community level. Different from the existing technology that only observes the phenotypic characteristics of bacterial drug sensitivity, the present invention can effectively identify the MIC of bacteria with metabolic activity but no growth through FDAA metabolic labeling, with high detection sensitivity (stable fluorescence labeling, strong signal, high signal-to-noise ratio), without targeting certain specific resistance genes or proteins, and can accurately determine the MIC of low-abundance target intestinal bacteria (0.5%), with higher antibacterial spectrum resolution, and can also measure the antibiotic sensitivity of currently unculturable microorganisms at the microbial community level. In addition, the present invention has good compatibility with existing instruments and equipment, uses fewer instruments and lower costs, is more popularizable, and can be effectively applied to clinical diagnosis and treatment.

[0174] The present invention can measure the MIC of non-separable and unculturable bacteria that cannot be measured by traditional AST strategies, can achieve rapid identification of pathogenic bacteria in complex samples while evaluating their MIC in the microbial community environment, and thus can effectively and specifically provide personalized treatment plans for different clinical patients and give accurate and reasonable antibiotic treatment methods. In addition, by analyzing and comparing the MIC differences of the same pathogenic bacteria in different mixed infection samples and when cultured alone in vitro, the interaction between bacteria and its impact on drug resistance can be further studied, and the drug resistance mechanism and transmission mode of bacteria can be studied to formulate more reasonable and effective strategies for clinical treatment and help alleviate the losses caused by antibiotic resistance to global health and economy.

[0175] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0176] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

[0177] References

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[0179] concentration of antibiotics: Methods, interpretation, clinical relevance.

[0180] Pathogens, 2021, 10, 165.

[0181] [2] James H, Mary J. Antimicrobial Susceptibility Testing: A Review of General

[0182] Principles and Contemporary Practices[J]. CLIN INFECT DIS, 2009, 49(11):

[0183] 1749–1755.

[0184] [3] Galani I, Adamou P, Karaiskos I, et al. Evaluation of ComASPTM Colistin

[0185] (formerly SensiTestTM Colistin), a commercial broth microdilution-based

[0186] method to evaluate the colistin minimum inhibitory concentration for

[0187] carbapenem-resistant Klebsiella pneumoniae isolates[J]. J. Glob. Antimicrob.

[0188] Resist, 2018, 15, 123–126.

[0189] [4] Gajic I, Kabic J, Kekic D, Jovicevic M, et al. Antimicrobial Susceptibility

[0190] Testing: A Comprehensive Review of Currently Used Methods[J]. Antibiotics,

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[0192] [5] Kandavalli V, Karempudi P, Larsson J, et al. Rapid antibiotics susceptibility

[0193] testing and species identification for mixed samples. Nat Commun, 2022, 13(1): 6215.

[0195] [6] Choi J, Yoo J, Lee M, et al. A rapid antimicrobial susceptibility test based on single - cell morphological analysis[J]. SCI TRANSL MED, 2014, 6(267): 267ra174 - 267ra174.

[0198] [7] Hou Z, An Y, Hjort K, et al. Time lapse investigation of antibiotics susceptibility using a microfluidic linear gradient 3D cμlture device[J]. Lab on a Chip, 2014, 14(17): 3409 - 3418.

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Claims

1. A method for in situ detection of drug sensitivity of mixed bacterial flora based on metabolic labeling combined with fluorescence in situ hybridization, characterized in that: The method comprises the following steps: using a fluorescent D-amino acid probe (FDAA) to metabolically label mixed bacterial samples treated with different concentrations of antibiotics to reflect changes in metabolic activity of various bacteria in the samples under the action of antibiotics, using a fluorescent in situ hybridization probe to perform fluorescent in situ hybridization on various bacteria in the metabolically labeled mixed bacterial samples to identify target bacteria, and using a flow cytometer to detect changes in fluorescence intensity of various bacteria in the mixed bacterial samples that have undergone metabolic labeling and fluorescent in situ hybridization to determine the drug sensitivity of various bacteria in the mixed bacterial samples; The mixed flora sample includes a complex flora sample and a simple flora sample, the bacterial species in the complex flora sample are unknown, and the bacterial species in the simple flora sample are known or selected from any N species of X bacteria of known species, wherein 2≤X<10, 2≤N≤X.

2. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The process of detection by flow cytometry includes: counting bacteria that carry the fluorescent signals of the fluorescent D-type amino acid probe and the fluorescent in situ hybridization probe by flow cytometry, and determining the minimum inhibitory concentration (MIC) of various types of bacteria in the mixed bacterial sample to different antimicrobial drugs based on the changes in the median value of the fluorescence intensity of the fluorescent D-type amino acid probe of each type of target bacteria counted.

3. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The method also includes bacterial species identification and probe determination, and selecting or designing corresponding fluorescent in situ hybridization probes according to the identification results of bacterial species in the mixed bacterial flora sample.

4. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 3, characterized in that: The method for identifying bacterial species includes 16SrDNA sequencing and fluorescence in situ hybridization. 16SrDNA sequencing is used to preliminarily determine the bacterial species in the complex flora sample so as to design fluorescence in situ hybridization probes for various types of bacteria in the complex flora sample, and to identify the bacterial species in the mixed flora sample based on fluorescence in situ hybridization.

5. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The method further comprises: after metabolically labeling the mixed bacterial community sample using a fluorescent D-type amino acid probe, performing a fixation treatment and a permeabilization treatment on the mixed bacterial community sample.

6. The method for in situ detection of drug sensitivity of mixed bacterial flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 5, characterized in that: The permeabilization treatment includes ethanol permeabilization, lysozyme permeabilization, Tween-20 permeabilization, and TritonX-100 permeabilization.

7. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The simple flora samples include clinical mixed infection samples, clinical urine samples, blood samples, lung lavage fluid samples, drainage fluid samples, and cerebrospinal fluid samples.

8. The method for in situ detection of drug sensitivity of mixed bacterial flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The complex flora samples include intestinal flora samples, saliva samples, sputum samples, vaginal secretion samples, biopsy tissue samples, air samples, soil samples, and water resource samples.

9. The method for in situ detection of drug sensitivity of mixed flora based on metabolic labeling combined with fluorescence in situ hybridization according to claim 1, characterized in that: The fluorescent D-type amino acid probes include Cy5-D-type amino acid probes (EDA-Cy5, Cy5-ethylenediamine-D-alanine), FAM-D-type amino acid probes (FDAA-FAM, FAM-D-alanine or FAM-D-lysine), TAMRA-D-type amino acid probes (TADA, TAMRA-D-alanine), and Rhodamine-D-type amino acid probes (RADA, Rhodamine-D-alanine).

10. An application of the method for in situ detection of drug sensitivity of mixed bacterial flora based on metabolic labeling combined with fluorescence in situ hybridization as claimed in any one of claims 1 to 9, characterized in that: The method was applied to perform in situ detection of antimicrobial susceptibility in mixed flora samples.

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