A rapid detection method of active resistant bacteria in water environment based on metabolic markers
By combining metabolic labeling methods with flow cytometry or fluorescence microscopy, we can rapidly detect viable resistant bacteria in the aquatic environment, solving the problems of long processing time and false negatives of traditional methods, and achieving efficient detection of VBNC-state bacteria.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- UNIV OF SCI & TECH OF CHINA
- Filing Date
- 2026-04-23
- Publication Date
- 2026-07-21
AI Technical Summary
Existing technologies are insufficient for the rapid and accurate detection of viable resistant bacteria in aquatic environments, especially unculturable bacteria (VBNC), and traditional methods are time-consuming or prone to producing false negative results.
This method employs a metabolic labeling approach, using non-natural amino acid probes and fluorescent probes combined with click reactions to detect viable resistant bacteria in water samples via flow cytometry or fluorescence microscopy. The process includes co-incubation, fluorescent labeling, and signal detection steps, and is applicable to various antibiotic mechanisms of action.
It enables rapid differentiation between sensitive and resistant bacteria within 20 minutes, is suitable for complex water samples, can detect VBNC-state bacteria, and can achieve quantitative analysis and visualization at the single-cell level by combining flow cytometry or fluorescence microscopy, thus improving the sensitivity and accuracy of detection.
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Figure CN122430291A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of environmental microbial detection technology, and in particular to a rapid detection method for active resistant bacteria in aquatic environments based on metabolic markers. Background Technology
[0002] The aquatic environment is a significant reservoir of antibiotics, resistance genes, and resistant bacteria. The continuous release of large amounts of residual antibiotics and their metabolites into the aquatic environment significantly promotes the proliferation and spread of resistant bacteria. Accurate detection and quantification of active resistant bacteria in the aquatic environment are crucial for understanding the occurrence, fate, and migration of resistant bacteria, as well as for assessing public health risks and developing effective prevention and control strategies.
[0003] Currently, common methods for detecting resistant bacteria in water samples mainly include growth-based phenotypic assays and molecular-based genotypic assays. Growth-based phenotypic assays, such as the disk diffusion method and broth dilution method, provide reliable results, but typically require 24-72 hours of incubation time, are cumbersome, and can only detect culturable bacteria, failing to cover live unculturable (VBNC) bacteria—bacteria commonly found in environmental water samples that, while metabolically active, cannot form colonies on conventional culture media. Molecular-based genotypic assays, such as PCR, real-time quantitative PCR, and DNA sequencing, offer rapid detection, but primarily rely on known resistance gene sequences, making it impossible to distinguish whether the resistance gene truly expresses a resistance phenotype, and prone to false negatives for novel resistance mechanisms. Furthermore, molecular detection methods struggle to directly link resistance genes to active host strains.
[0004] In recent years, detection methods based on bacterial metabolic activity have attracted attention. These methods do not rely on bacterial proliferation and are expected to significantly shorten detection time. However, existing metabolic activity detection methods are mainly for pathogens in clinical samples (such as urine and blood). Their applicability and detection effectiveness for aquatic environmental samples, which are complex in composition, highly diverse in bacterial communities, and exhibit significant differences in bacterial activity, remain unclear. Aquatic environmental samples contain a wide variety of bacteria, including a large number of low-activity or VBNC bacteria, and contain various interfering substances, placing higher demands on the sensitivity, anti-interference ability, and universality of detection methods.
[0005] Therefore, developing a rapid, culture-independent, and applicable method for detecting viable resistant bacteria in complex water samples, capable of detecting VBNC-state bacteria, is a pressing technical problem to be solved in this field. Summary of the Invention
[0006] The purpose of this invention is to provide a method for the rapid detection of active resistant bacteria in aquatic environments based on bioorthogonal non-natural amino acid labeling, which does not rely on bacterial culture.
[0007] The present invention provides a rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers, which is achieved through the following technical solution: A rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers includes the following steps: (a) Sample pretreatment: Collect water samples to be tested and filter to remove large particulate impurities; (b) Co-incubation labeling: Non-natural amino acid probes and target antibiotics were added simultaneously to the pretreated water sample for co-incubation; The non-natural amino acid probe is a methionine analog containing an alkyne or azide group; (c) Fluorescent labeling: After incubation, a click reaction system containing a copper catalyst, ligand, reducing agent and fluorescent probe is added to carry out a bioorthogonal reaction; The fluorescent probe contains an azide or alkynyl group that is complementary to the non-natural amino acid probe. (d) Signal detection and determination: The fluorescence signal of the bacterial cells after the reaction was detected, and the antibiotic sensitivity of the strain was determined by comparing it with the control group without antibiotics.
[0008] Preferably, in the detection step (d), the fluorescence signal of the bacterial community is obtained at the single-cell level by flow cytometry, and the proportion of active resistant bacteria in the sample is quantified based on a preset fluorescence intensity threshold.
[0009] Preferably, in the detection step (d), the fluorescence signal of the bacteria is observed by a fluorescence microscope to achieve a visual qualitative analysis of the active resistant bacteria.
[0010] Preferably, in the co-incubation step (b), the concentration of the target antibiotic added is 8-12 times, more preferably 10 times, the minimum inhibitory concentration (MIC) specified by the Clinical and Laboratory Standards Institute (CLSI). The inventors have found that this concentration can effectively inhibit the metabolic activity of sensitive bacteria in the shortest time without affecting resistant bacteria, thereby achieving the fastest differentiation.
[0011] Preferably, in the co-incubation step (b), the incubation time is 20 minutes to 4 hours. For rapidly growing bacteria (such as Escherichia coli), labeling can be completed in 20 minutes; for slow-growing microorganisms in environmental water samples, the incubation time can be appropriately extended according to the characteristics of the sample.
[0012] Preferably, the non-natural amino acid probe is L-homopropylglycine, and its final concentration in the co-incubation system is 0.05 mM to 0.5 mM.
[0013] Preferably, the fluorescent probe is 3-azido-7-hydroxycoumarin.
[0014] The method of this invention is applicable to antibiotics with a variety of mechanisms of action, including but not limited to antibiotics that inhibit bacterial protein synthesis (such as kanamycin, chloramphenicol, and gentamicin) and antibiotics that inhibit bacterial DNA synthesis (such as ciprofloxacin).
[0015] The present invention provides a rapid detection method for active resistant bacteria in aquatic environments based on metabolic markers, which further includes a step of restaining the sample with a DNA fluorescent dye before signal detection, in order to distinguish bacterial cells from non-biological particles in flow cytometry analysis.
[0016] In summary, this invention offers the following advantages: it can rapidly distinguish between sensitive and resistant bacteria within 20 minutes; it is applicable to antibiotics with various mechanisms of action; combined with flow cytometry or fluorescence microscopy, it enables quantitative analysis and visualization at the single-cell level; and it can effectively detect live, non-culturable resistant bacteria in water samples. This invention is simple, rapid, and efficient, providing a powerful tool for monitoring active resistant bacteria in aquatic environments. Attached Figure Description
[0017] Figure 1 The results of detecting antibiotic susceptibility of different strains using the method of this invention are shown. (a) Relative fluorescence intensity of *E. coli* MG1655, *E. coli* KanR, *E. coli* ChlR, and *E. coli* GenR cells labeled by the method of this invention under different antibiotic treatments. (b) Heatmap of MIC results detected by the broth microdilution method. The table on the right shows the CLSI classification of each strain to different antibiotics. The purple-red dashed line (60-80%) is the standard for judging antibiotic susceptibility. Error bars represent the standard deviation of three replicate experiments. Con: Control (no antibiotic); Kan: Kanamycin; Chl: Chloramphenicol; Gen: Gentamicin; Cip: Ciprofloxacin.
[0018] Figure 2 This image shows the results of the method of the present invention combined with fluorescence microscopy for visual detection of viable resistant bacteria in mixed bacterial samples. Specifically, (a) shows the results of the method of the present invention for detecting chloramphenicol-sensitive *Escherichia coli* (E. coli) in the presence of antibiotics. E. coli ChlS-GFP and chloramphenicol-resistant Escherichia coli (ChlS-GFP) E. coli (a) Schematic diagram of selective labeling of mixed bacterial samples (ChlR); (b), (c), and (d) are fluorescence microscopy images of the above mixed bacterial samples after incubation for 20, 40, and 60 minutes in M9 medium containing 0.5 mM L-high-propargylglycine and 10 times the CLSI minimum inhibitory concentration of chloramphenicol. The right side shows the combined bright-field and fluorescence channel images at each time point. In the figures, blue fluorescence represents chloramphenicol-resistant bacteria with metabolic activity successfully labeled by the method of this invention, and green fluorescence (GFP labeling) represents chloramphenicol-sensitive bacteria. Scale bar: 10 μm.
[0019] Figure 3 This is a graph showing the quantitative analysis results of the proportion of resistant bacteria in mixed bacterial samples detected by the method of this invention combined with flow cytometry. (a)-(f) represent different proportions of chloramphenicol-resistant Escherichia coli (E. coli). E. coli ChlR) and chloramphenicol-sensitive Escherichia coli ( E. coli Flow cytometry plot of MG1655 (ChlS) mixture incubated for 3 hours in actual water samples filtered through 0.22 μm. The incubation system contained 0.05 mM L-homopropylglycine and 10 times the CLSI minimum inhibitory concentration of chloramphenicol. The theoretical ratios of ChlR to ChlS were: (a) 0%:100%, (b) 2%:98%, (c) 5%:95%, (d) 15%:85%, (e) 25%:75%, and (f) 30%:70%. Figure 3 In the diagram, BONCAT+ phylum (blue area) represents active bacteria successfully labeled by the method of this invention, whose fluorescence intensity exceeds the negative control threshold. (g) is the background fluorescence level determined by culturing Escherichia coli MG1655 in filtered water samples without HPG and chloramphenicol (negative control), used to set the fluorescence intensity threshold of BONCAT+ phylum; (h) is the proportion of BONCAT+ cells detected after incubating Escherichia coli MG1655 in filtered water samples containing 0.05 mM L-high-propargylglycine and without chloramphenicol (positive control), representing the normal labeling efficiency of the bacterial community under antibiotic pressure. (i) is the linear correspondence between the relative proportion of labeled cells detected by flow cytometry and the theoretical proportion of chloramphenicol-resistant Escherichia coli (ChlR). The relative proportion of labeled cells represents the ratio of the proportion of labeled cells under chloramphenicol conditions to the proportion of labeled cells under chloramphenicol-free conditions (positive control). Error bars represent the standard deviation of three replicate experiments.
[0020] Figure 4 This is a comparison chart showing the results of detecting the proportion of chloramphenicol-resistant bacteria in actual water samples using the method of this invention and the traditional plate count method. The values above the histogram represent the proportion of chloramphenicol-resistant bacteria detected by the respective methods. Parallel detection was performed using the method of this invention and the traditional plate count method for water samples from Chaohu Lake and hospital wastewater, respectively. The proportion of chloramphenicol-resistant bacteria in the actual water samples is based on... Figure 3 The standard curve shown in (i) is calculated. Detailed Implementation
[0021] To further understand the inventiveness and technical advancements of this invention, the preferred embodiments of this invention will be discussed in detail below with reference to examples and comparative examples.
[0022] Example: A rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers, comprising the following steps: (a) Sample pretreatment: Collect water samples to be tested and filter to remove large particulate impurities; (b) Co-incubation labeling: Non-natural amino acid probes and target antibiotics were added simultaneously to the pretreated water sample. The concentration of the target antibiotic was 8-12 times the minimum inhibitory concentration specified by the Clinical and Laboratory Standards Association. Co-incubation was performed for 20 min to 4 h. The non-natural amino acid probe is a methionine analog containing an alkyne or azide group. Preferably, the non-natural amino acid probe is L-homopropylglycine, and the final concentration in the co-incubation system is 0.05 to 0.5 mM. (c) Fluorescent labeling: Add a click reaction system containing a copper catalyst, ligand, reducing agent and fluorescent probe to fluorescently label the newly synthesized proteins in the bacterial cells after incubation in step (b); The fluorescent probe contains an azide or alkynyl group that is complementary to the non-natural amino acid probe. Preferably, the fluorescent probe is 3-azido-7-hydroxycoumarin. (d) Signal detection and determination: The fluorescence signal of the bacterial cells after the reaction is detected, and the antibiotic sensitivity of the strain is determined by comparing it with the control group without antibiotics. Preferably, the fluorescence signal of the bacterial community is obtained at the single-cell level by flow cytometry, and the proportion of active resistant bacteria in the sample is quantified based on a preset fluorescence intensity threshold. Alternatively, the fluorescence signal of the bacterial cells is observed by fluorescence microscopy to achieve a visual qualitative analysis of the active resistant bacteria.
[0023] Preferably, the method further includes step (c'), which involves restaining the sample with a DNA fluorescent dye prior to signal detection in step (d) to distinguish bacterial cells from non-biological particles in flow cytometry analysis.
[0024] The rapid detection method for active resistant bacteria in aquatic environments based on metabolic markers in this invention is applicable to antibiotics with various mechanisms of action, including antibiotics that inhibit bacterial protein synthesis and antibiotics that inhibit bacterial DNA synthesis.
[0025] Example 1: This example verifies the feasibility of the method of the present invention in distinguishing between known sensitive and resistant bacteria, and establishes the discrimination criteria.
[0026] Selected susceptible Escherichia coli ( E. coli MG1655) and resistant Escherichia coli ( E. coli ChlR) was used as a model bacterium. Each strain (approximately 10) was used as a 8(CFU / mL) was suspended in M9 medium containing 0.5 mM L-holypropylglycine and 10× minimum inhibitory concentration (MIC) chloramphenicol, and incubated at 37°C for 20 minutes. A control group without antibiotics was included. After incubation, the cells were centrifuged and washed, and then PBS solution containing 200 μM copper sulfate, 500 μM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 5 mM sodium ascorbate, and 10 μM 3-azido-7-hydroxycoumarin was added. The reaction was carried out at room temperature in the dark for 30 minutes. Fluorescence intensity was detected using a microplate reader (excitation wavelength 405 nm, emission wavelength 480 nm). The relative fluorescence intensity (the ratio of fluorescence intensity of the antibiotic-treated group to that of the control group) was calculated.
[0027] See Figure 1 The results showed that the relative fluorescence intensity of sensitive strains was below 60%, while that of resistant strains was above 80%. Therefore, this invention defines a relative fluorescence intensity ≤60% as "sensitive" and ≥80% as "resistant". This criterion is consistent with the results of the traditional broth dilution method (MIC method), but the detection time is shortened from overnight culture in the traditional method to within 1 hour.
[0028] Example 2: Applicability verification of multiple antibiotics.
[0029] This embodiment verifies the applicability of the method of the present invention to antibiotics with multiple mechanisms of action.
[0030] Selected susceptible Escherichia coli ( E. coli MG1655) and multiple resistant Escherichia coli ( E. coli KanR, E. coli ChlR、 E. coli GenR was used as the model bacteria. Four antibiotics with different mechanisms of action—kanamycin (inhibits protein synthesis), chloramphenicol (inhibits protein synthesis), gentamicin (inhibits protein synthesis), and ciprofloxacin (inhibits DNA synthesis)—were used for testing. The procedures were the same as in Example 1.
[0031] See Figure 1 The results showed that for four different mechanisms of action of antibiotics, the relative fluorescence intensity of sensitive strains was below 60%, while the relative fluorescence intensity of resistant strains was above 80%. This indicates that the method of the present invention is applicable to antibiotics with multiple mechanisms of action and is not limited by the target of antibiotic action.
[0032] Example 3: Visual detection combined with fluorescence microscopy.
[0033] This embodiment demonstrates that the method of the present invention, combined with fluorescence microscopy, can achieve single-cell level visual qualitative analysis of active resistant bacteria.
[0034] Chloramphenicol-sensitive Escherichia coli (GFP-labeled) and chloramphenicol-resistant Escherichia coli were mixed at a 1:1 ratio and suspended in a culture medium containing 0.5 mM L-high-propargylglycine and 10× minimum inhibitory concentration of chloramphenicol, and incubated at 37°C for 20 minutes. After fluorescent labeling according to the steps in Example 1, the bacteria were observed using a fluorescence microscope (BX51, Olympus). The blue fluorescence channel (excitation wavelength 330-385 nm, emission wavelength 420 nm) was used to observe BONCAT-labeled resistant bacteria, and the green fluorescence channel (excitation wavelength 460-490 nm, emission wavelength 520 nm) was used to observe GFP-labeled sensitive bacteria.
[0035] See Figure 2 The results showed that, in the presence of antibiotics, only chloramphenicol-resistant strains exhibited blue fluorescence, while chloramphenicol-sensitive strains exhibited green fluorescence, and the two could be clearly distinguished through different fluorescence channels. This indicates that the method of the present invention can intuitively and rapidly identify resistant bacteria in mixed bacterial communities at the single-cell level.
[0036] Example 4: Quantitative analysis of mixed bacterial samples and detection of bacterial community heterogeneity using flow cytometry.
[0037] This embodiment demonstrates that the method of the present invention, combined with flow cytometry, can quantitatively analyze the proportion of resistant bacteria in mixed bacterial samples and reveal the resistance heterogeneity of the bacterial community.
[0038] Chloramphenicol-resistant Escherichia coli and chloramphenicol-sensitive Escherichia coli were mixed in different proportions (0%, 10%, 50%, 90%, 100%), with a total bacterial count of 10. 5 CFU / mL was resuspended in the filtered water sample. A final concentration of 0.05 mM L-homopropylglycine and 10 × minimum inhibitory concentration (MIC) of chloramphenicol were added to the sample, and the mixture was incubated at room temperature for 3 h. Fluorescent labeling was performed according to the procedure in Example 1. The samples were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter) with an excitation wavelength of 405 nm, and fluorescence was detected at 450 / 40 nm. A negative gate was established using the sample without L-homopropylglycine; cells with fluorescence intensity exceeding the negative gate threshold were considered labeled viable bacteria.
[0039] See Figure 3 The results showed that, in the presence of antibiotics, the proportion of labeled positive cells had a good linear relationship with the actual proportion of resistant bacteria added to the sample (R0). 2 =0.986). This indicates that this method can quantitatively detect the proportion of resistant bacteria in mixed bacterial samples. The single-cell analysis capability of flow cytometry can also simultaneously obtain fluorescence intensity distribution maps of the bacterial community, reflecting the differences in metabolic activity among different cells in the community, thereby enabling rapid assessment of the heterogeneity of resistant bacterial communities.
[0040] Example 5: Actual water sample testing and comparison with plate counting method This embodiment verifies the feasibility of the method of the present invention in detecting resistant bacteria in real environmental samples (lake water, hospital wastewater) and demonstrates its advantages in detecting VBNC-state bacteria.
[0041] Water samples from a lake and wastewater from a hospital were collected and pre-filtered through a 5 μm membrane. The filtrate was then directly added to L-homophytic glycine (final concentration 0.05 mM) and chloramphenicol (10 × minimum inhibitory concentration), and incubated at room temperature for 3 hours. Labeling and flow cytometry analysis were performed according to the steps in Example 3. A conventional plate counting method (cultured on plates containing 32 μg / mL chloramphenicol) was also used for comparison.
[0042] See Figure 4 The results showed that for lake water samples, the proportion of resistant bacteria detected by the method of this invention was not significantly different from that detected by plate counting. However, for hospital wastewater samples, the proportion of resistant bacteria detected by the method of this invention (approximately 18.9%) was significantly higher than that detected by plate counting (approximately 11.2%). Analysis suggests that the difference is mainly due to the presence of a large number of live, non-culturable resistant bacteria in hospital wastewater. These bacteria possess metabolic activity but cannot form colonies on plates, making them undetectable by plate counting. The method of this invention, however, can effectively capture these bacteria by detecting protein synthesis activity. This indicates that the method of this invention has a more comprehensive detection capability in complex environmental samples.
[0043] In summary, the method of this invention can rapidly distinguish between sensitive and resistant bacteria within 20 minutes, is applicable to antibiotics with various mechanisms of action, and, when combined with flow cytometry or fluorescence microscopy, enables quantitative analysis and visualization at the single-cell level. It can also effectively detect live, non-culturable resistant bacteria in water samples. This method is simple, rapid, and efficient, providing a powerful tool for monitoring active resistant bacteria in aquatic environments.
[0044] It should be noted that this specific embodiment is merely an explanation of the technical solution of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.
Claims
1. A rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers, characterized in that: Includes the following steps: (a) Sample pretreatment: Collect water samples to be tested and filter to remove large particulate impurities; (b) Co-incubation labeling: Non-natural amino acid probes and target antibiotics were added simultaneously to the pretreated water sample for co-incubation; (c) Fluorescent labeling: Add a click reaction system containing a copper catalyst, ligand, reducing agent and fluorescent probe to fluorescently label the newly synthesized proteins in the bacterial cells after incubation in step (b); (d) Signal detection and determination: Detect the fluorescence signal of the bacterial cells after labeling in step (c), and determine the antibiotic sensitivity of the strain by comparing it with the control group without antibiotics.
2. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: In step (b), the non-natural amino acid probe is a methionine analog containing an alkyne or azide group.
3. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 2, characterized in that: In step (b), the non-natural amino acid probe is L-homopropylglycine, and the final concentration of the non-natural amino acid probe in the co-incubation system is 0.05 mM to 0.5 mM.
4. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: In step (c), the fluorescent probe contains an azide or alkynyl group that is complementary to the non-natural amino acid probe.
5. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 4, characterized in that: In step (c), the fluorescent probe is 3-azido-7-hydroxycoumarin.
6. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: In step (d), the fluorescence signal of the bacterial community is obtained at the single-cell level by flow cytometry, and the proportion of active resistant bacteria in the sample is quantified based on a preset fluorescence intensity threshold.
7. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: In step (d), the fluorescence signal of the bacteria is observed using a fluorescence microscope to achieve a visual qualitative analysis of the active resistant bacteria.
8. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: In step (b), the concentration of the target antibiotic added is 8-12 times the minimum inhibitory concentration specified by the Clinical and Laboratory Standards Association; the incubation time is 20 min to 4 h.
9. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: It also includes a step of restaining the sample with a DNA fluorescent dye before signal detection in step (d) to distinguish bacterial cells from non-biological particles in flow cytometry analysis.
10. The rapid detection method for viable resistant bacteria in an aquatic environment based on metabolic markers according to claim 1, characterized in that: The rapid detection method for viable resistant bacteria in the aquatic environment based on metabolic markers is applicable to antibiotics with multiple mechanisms of action, including antibiotics that inhibit bacterial protein synthesis and antibiotics that inhibit bacterial DNA synthesis.