Method for predicting parallel transfer risk of drug-resistant genes of environmental microorganisms
By calculating the API value by detecting the MIC values of donor and recipient bacteria, and combining it with a fluorescence reporter system, the problem of the difficulty in monitoring parallel transfer of drug resistance genes by traditional methods has been solved, and efficient and accurate prediction and evaluation of drug resistance genes in environmental samples has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-23
- Publication Date
- 2026-03-13
AI Technical Summary
Traditional methods are difficult to monitor parallel gene transfer of drug resistance genes in complex environmental samples in real time and efficiently, and cannot meet the needs of large-scale environmental pollutant monitoring.
By detecting the minimum inhibitory concentration (MIC) values of donor and recipient bacteria, calculating the asymmetric pressure index (API value), and combining this with a fluorescence reporter system, high-throughput screening for parallel transfer of drug-resistant genes in environmental samples can be achieved.
It enables accurate identification of parallel gene transfer events in a short time, improves research throughput and efficiency, and can simultaneously assess a large number of environmental pollutants, meeting the needs of large-scale environmental risk assessment.
Smart Images
Figure CN121653149A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for predicting the risk of parallel transfer of antibiotic resistance genes in environmental microorganisms. Background Technology
[0002] The spread of antibiotic resistance (AMR) has become a major threat to global public health. Antimicrobial resistance genes (ARGs) in the environment, as mobile genetic material, are most dangerous because they can spread between different bacteria, and even across different species or phyla, via horizontal gene transfer (HGT). HGT is a major driver of resistance spread in the environment, and its mechanisms include conjugation, transformation, and transduction, enabling the rapid spread of resistance genes between different species and even different phyla of microorganisms. However, traditional HGT detection methods (such as PCR amplification, fluorescent labeling, or genome alignment) typically rely on specific experimental conditions or model strains, making it difficult to monitor gene transfer events in complex environmental samples in a real-time and efficient manner. Furthermore, these methods have low throughput, are time-consuming, and cannot meet the needs of large-scale environmental pollutant monitoring, limiting a comprehensive understanding of HGT dynamics in the environment. Summary of the Invention
[0003] To address the aforementioned technical problems, this invention provides a method for predicting the risk of parallel transfer of antibiotic resistance genes in environmental microorganisms. By detecting the MIC value of bacteria, parallel gene transfer can be detected, which can accurately determine the occurrence of parallel gene transfer events in a short time and simultaneously achieve high-throughput screening of large batches of environmental samples.
[0004] The first objective of this invention is to provide a method for predicting the risk of parallel transfer of antibiotic resistance genes in environmental microorganisms, comprising the following steps:
[0005] Step S1: Introduce a conjugation plasmid into the donor bacteria, wherein the conjugation plasmid expresses a fluorescent protein, and the donor bacteria are Gram-negative bacteria;
[0006] Step S2: Select recipient bacteria, wherein the recipient bacteria are pure cultured single strains or mixed microbial colonies from environmental sources;
[0007] Step S3: Co-culture the donor bacteria and the recipient bacteria, and determine the minimum inhibitory concentration (MIC) values of the donor bacteria and the recipient bacteria respectively under the condition of environmental pollutants.
[0008] Step S4: Calculate the asymmetric stress index (API) value. The API value is the ratio of the MIC value of the recipient bacterium to the MIC value of the donor bacterium. When the API value is greater than or equal to 100, it is determined that the donor bacterium and the recipient bacterium will undergo parallel transfer of drug resistance genes under the condition of the presence of the environmental pollutants.
[0009] Furthermore, the conjugation plasmid uses the RP4 plasmid as its backbone.
[0010] Furthermore, the donor bacteria's genome suppresses the expression of fluorescent protein by the RP4 plasmid.
[0011] Furthermore, the fluorescent protein is a green fluorescent protein.
[0012] In one embodiment of the present invention, the fluorescent protein is green fluorescent protein (GFP).
[0013] Furthermore, the genome of the donor bacterium encodes a gene that expresses a red fluorescent protein.
[0014] In one embodiment of the present invention, the red fluorescent protein is mCherry.
[0015] Furthermore, it also includes a step of verification by fluorescence signal, which verifies whether parallel transfer of the drug resistance gene has occurred by detecting whether the recipient bacteria express the fluorescent reporter gene.
[0016] Furthermore, the environmental pollutants are selected from one or more of antibiotics, emerging pollutants, benzene compounds, plastic additives, heavy metal ions, pesticides, and nanoparticles.
[0017] Furthermore, the environmental pollutant is selected from one or more of ceftriaxone, cefazolin, cefotaxime sodium, and cefepime.
[0018] In one embodiment of the present invention, the antibiotics ceftriaxone, cefazolin, cefotaxime sodium, and cefepime exhibited a phenomenon that promoted parallel gene transfer at environmentally relevant concentrations.
[0019] A second object of the present invention is to provide a kit for implementing the above-described prediction method, the kit comprising the following components:
[0020] (1) A donor bacterium carrying a conjugation plasmid, wherein the donor bacterium is a Gram-negative bacterium and the conjugation plasmid expresses a fluorescent reporter protein;
[0021] (2) One or more environmental pollutants;
[0022] (3) Reagents used to detect the minimum inhibitory concentration of donor and recipient bacteria.
[0023] Furthermore, the donor bacterial genome inhibits the expression of fluorescent reporter proteins by the conjugative plasmid.
[0024] A third objective of this invention is to provide the application of the above-described kit in predicting whether parallel transfer of drug resistance genes occurs in environmental microorganisms.
[0025] The beneficial effects of this invention are:
[0026] This invention is the first to propose and experimentally verify that the asymmetric pressure index (API) can be used to accurately predict the occurrence of parallel gene transfer. This index has a clear physical meaning and statistical basis, and the prediction results have been intuitively and reliably verified using a fluorescence reporter system, ensuring the scientific rigor and accuracy of the method. Compared to traditional conjugation experiments or molecular biology methods, this invention uses a broth dilution method to detect MICs, which can be operated on miniaturized platforms such as 384-well plates. The results are intuitive and reliable, avoiding false positives that may occur with molecular methods. It can simultaneously and rapidly screen hundreds of pollutant-microbe combinations, and can simultaneously assess a large number of environmental pollutants, covering various types such as antibiotics, nanomaterials, and heavy metals, greatly improving research throughput and efficiency, and meeting the needs of large-scale environmental risk assessment. The recipient bacteria to be detected are not limited to laboratory model strains; mixed microbial communities in environmental samples can be directly used as recipients. This provides a novel and efficient quantitative prediction tool and product for monitoring the risk of antibiotic resistance gene transmission in environmental microorganisms, which has important theoretical and practical significance for curbing the spread of antibiotic resistance. Attached Figure Description
[0027] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:
[0028] Figure 1 This is a statistical result of the MIC values of donor and recipient bacteria after treatment with different environmental pollutants in Example 1 of the present invention;
[0029] Figure 2 This is the result of the transfer rate under different environmental pollutant treatments verified by fluorescence in Example 2 of the present invention;
[0030] Figure 3 The experimental results of HGT detection by the fluorescence reporter system in Example 2 of the present invention are shown, where A is the donor-receptor mixed uninduced group and B is the donor-receptor mixed induced group;
[0031] Figure 4 The results of environmental pollutant treatment of the mixture of donor bacteria and soil mixed bacteria in Example 3 of the present invention are shown, where A and C are control groups, B is the soil mixed bacteria group, and D is the clinical PAO1 group. Detailed Implementation
[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0033] The minimum inhibitory concentration (MIC) determination involved in the examples was performed using the broth dilution method.
[0034] Example 1: Characterizing parallel gene transfer using MIC ratios among bacteria
[0035] Donor and recipient bacteria were inoculated into LB medium, with *E. coli* K12MG1655 as the donor and *E. coli* ATCC25922 as the recipient, and cultured at 37°C with shaking until the logarithmic growth phase. Seven 50 / 50 dilutions of corresponding environmental contaminants were then transferred to 384-well plates using a power pipette. The inoculation apparatus was sterilized with alcohol and then irradiated with UV for 30 min. Inoculation was then performed at the corresponding well positions using the inoculation apparatus. After inoculation, the plates were incubated at 37°C for 12 h, and the absorbance (OD) was measured. 600 The method involves measuring the MIC values of donor and recipient bacteria under different materials to obtain the corresponding API (where API = donor bacteria MIC value / recipient bacteria MIC value). The API value is then used to determine whether parallel genes have been transferred.
[0036] Experimental results are as follows Figure 1 As shown, the unit of MIC is μg / mL. The results show that there are differences in the API values of donor and recipient bacteria after treatment with different environmental pollutants.
[0037] Example 2: Fluorescent labeling to verify parallel gene transfer between bacteria
[0038] The donor bacteria *Escherichia coli* K12MG1655 and the recipient bacteria *Escherichia coli* ATCC25922 used in Example 1 were mixed in a 1:1 ratio (10... 7 Samples (CFU / mL) were mixed in LB medium and treated with different environmental contaminants at concentrations ranging from 0-40 mg / L. After incubation for 24 hours, the samples were washed twice with PBS, fixed and stained with Hoechst 33342, and then diluted 1000-fold with PBS for analysis using a confocal high-content screening microscope. Two replicates were set up for each concentration. The transfer rates of different environmental contaminants are shown in the figure. Figure 2 As shown.
[0039] Among the various environmental pollutants tested, four cephalosporin antibiotics significantly enhanced parallel gene transfer: ceftriaxone, cefazoloxime, cefotaxime sodium, and cefepime. These antibiotics promoted parallel gene transfer at environmentally relevant concentrations, while other tested pollutants did not show a significant effect. This is consistent with the API results obtained in Example 1, validating the reliability of the method for detecting parallel genes using bacterial MIC values. Therefore, parallel gene transfer is considered to have occurred between donor and recipient bacteria when the API values of both the donor and recipient bacteria are not lower than 100.
[0040] Further, using *E. coli* K12MG1655 carrying the conjugative plasmid RP4 (which contains three main parts: the IncP1 conjugative plasmid backbone structure; kanamycin, tetracycline, and ampicillin resistance tags; and a LacI-repressible PA1 / O4 / O3 promoter that regulates GFP gene expression, see the literature *Antibiotic Resistance Gene-Carrying Plasmid Spreads into the Plant Endophytic Bacteria using Soil Bacteria as Carriers*, *Environmental Science & Technology*), mCherry and lacI were modified on its chromosome. q Two gene edits were performed (expression of red fluorescent protein, LacI protein, and kanamycin resistance; LacI protein inhibited the expression of the GFP promoter on the RP4 plasmid). The recipient bacterium was ceftriaxone-resistant Escherichia coli E. coli K12MG1655 (res Cef), without fluorescent labeling. The donor and recipient bacteria were mixed at a 1:1 ratio (10T...). 7 The bacterial culture (CFU / mL) was mixed in LB medium, and 40 mg / L ceftriaxone (Cef) was added to induce conjugation. After incubation for 24 hours, the cells were washed twice with PBS, and the DNA of all bacteria was stained with Hoechst 33342 fluorescent dye (blue fluorescence). 10 μL of the bacterial culture was added to a glass slide and observed using an Olympus FV1200 confocal microscope. The experimental results are as follows: Figure 3 As shown, the successfully conjugated recipient bacteria exhibit green fluorescence ( Figure 3 B), unconjugated recipient bacteria show only blue fluorescence, while donor bacteria show both red and blue fluorescence. Figure 3 A). Ceftriaxone showed a promoting effect on parallel gene transfer at environmentally relevant concentrations, consistent with the above experimental results.
[0041] Example 3: Using soil mixed bacteria and clinically resistant bacteria as recipient bacteria to detect parallel gene transfer.
[0042] The MIC values of the donor bacterium *Escherichia coli* K12MG1655 carrying the aforementioned conjugative plasmid RP4 were measured separately in soil mixtures with soil samples from the Dushu Lake area and with clinically resistant *Pseudomonas aeruginosa* POA1, respectively, under the induction of cefotaxime sodium and dioctyl phthalate (DEHP). The corresponding API values were obtained (where API = donor bacterium MIC value / recipient bacterium MIC value). The API values were used to determine whether parallel gene transfer had occurred.
[0043] The experimental results are shown in Table 1. The API value of the two recipient bacteria (soil mixed bacteria and clinical PAO1) cefotaxime sodium group was 100, and parallel gene transfer occurred between the donor and recipient bacteria induced by cefotaxime sodium.
[0044] Table 1. MIC detection results of soil mixed bacteria and PAO1
[0045]
[0046] Mixtures of donor bacteria and soil-borne bacteria, as well as mixtures of donor bacteria and clinically resistant PAO1, were treated with the environmental pollutants cefotaxime sodium and ceftriaxone sodium for 24 h. After fixation and staining with Hoechst 33342, the samples were diluted 1000-fold with PBS and analyzed using a confocal high-content screening microscope. The experimental results are as follows: Figure 4 As shown, soil mixed bacteria and clinical PAO1 recipient bacteria that successfully conjugated after induction with cefotaxime sodium and ceftriaxone sodium both showed green fluorescence, proving that parallel gene transfer had occurred.
[0047] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for predicting the risk of parallel transfer of antibiotic resistance genes in environmental microorganisms, characterized in that, Includes the following steps: Step S1: Introduce a conjugation plasmid into the donor bacteria, wherein the conjugation plasmid expresses a fluorescent protein, and the donor bacteria are Gram-negative bacteria; Step S2: Select recipient bacteria, wherein the recipient bacteria are pure cultured single strains or mixed microbial colonies from environmental sources; Step S3: Co-culture the donor bacteria and the recipient bacteria, and determine the minimum inhibitory concentration (MIC) values of the donor bacteria and the recipient bacteria respectively under the condition of environmental pollutants. Step S4: Calculate the asymmetric stress index (API) value. The API value is the ratio of the MIC value of the recipient bacterium to the MIC value of the donor bacterium. When the API value is greater than or equal to 100, it is determined that the donor bacterium and the recipient bacterium will undergo parallel transfer of drug resistance genes under the condition of the presence of the environmental pollutants.
2. The prediction method according to claim 1, characterized in that: The conjugative plasmid uses the RP4 plasmid as its backbone.
3. The prediction method according to claim 2, characterized in that: The donor bacteria's genome inhibits the expression of fluorescent protein by the RP4 plasmid.
4. The prediction method according to claim 1, characterized in that: The fluorescent protein is green fluorescent protein.
5. The prediction method according to claim 4, characterized in that: The genome of the donor bacteria encodes a gene that expresses red fluorescent protein.
6. The prediction method according to claim 5, characterized in that: It also includes a step of verification by fluorescence signal, which verifies whether parallel transfer of the drug resistance gene has occurred by detecting whether the recipient bacteria express the fluorescent reporter gene.
7. The prediction method according to claim 1, characterized in that: The environmental pollutants are selected from one or more of the following: antibiotics, emerging pollutants, benzene compounds, plastic additives, heavy metal ions, pesticides, and nanoparticles.
8. A kit for implementing the prediction method according to any one of claims 1-7, characterized in that, The kit includes the following components: (1) A donor bacterium carrying a conjugation plasmid, wherein the donor bacterium is a Gram-negative bacterium and the conjugation plasmid expresses a fluorescent reporter protein; (2) One or more environmental pollutants; (3) Reagents used to detect the minimum inhibitory concentration of donor and recipient bacteria.
9. The reagent kit according to claim 8, characterized in that: The donor bacterial genome inhibits the expression of fluorescent reporter protein by the conjugative plasmid.
10. The use of the kit of claim 8 or 9 in predicting whether parallel transfer of drug resistance genes occurs in environmental microorganisms.