CN116380756A - Method for detecting bacterial antibiotic resistance in viable and non-culturable state based on flow cytometry and application thereof - Google Patents
Method for detecting bacterial antibiotic resistance in viable and non-culturable state based on flow cytometry and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of environmental microorganisms, and discloses a method for detecting bacterial antibiotic resistance in a living non-culturable state based on flow cytometry and application thereof. The method obtains pure living bacteria in a non-viable state through a disinfection technology, adjusts the concentration of the bacteria, and incubates with antibiotics with different concentrations in equal volumes. After the incubation is completed, centrifuging, washing and re-suspending, adding SYTO 9 and PI staining agents according to the proportion, incubating in a dark place at a proper temperature, adjusting voltage parameters of FSC, SSC, FITC and PI of a flow cytometer, and analyzing the survival rate of the bacteria in a viable and non-culturable state to determine the antibiotic resistance of the bacteria. The method has the advantages of strong operability, rapidness, high flux, high accuracy, high sensitivity and wide application range, can detect the drug resistance of different kinds of living bacteria in a non-culturable state to different kinds of antibiotics, and can be applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention belongs to the application in the technical field of environmental microorganisms, and in particular relates to a method for detecting bacterial antibiotic resistance in a living non-culturable state based on flow cytometry and application thereof.
Background
Antibiotic resistance has become an increasingly global public health problem that threatens human health and public health safety. Although antibiotic resistance is a major concern worldwide, the fact that bacteria can acquire antibiotic resistance by entering a dormant state is still ignored. Among them, living, non-culturable bacteria are a typical dormant subpopulation. In fact, to evade the damage caused by disinfection techniques, bacteria are able to globally regulate and generate a series of stress responses to enter a viable, non-culturable dormant state. After entering the viable, non-culturable state, the bacteria remain viable but lose their ability to grow on standard media. It exhibits lower metabolic activity and higher efflux activity, thus obtaining temporary antibiotic resistance, and may progress even further to antibiotic resistance. This short-term antibiotic resistance allows the live, non-culturable bacteria to resist antibiotics, which cannot be completely eliminated by traditional disinfection means, which is prone to persistent and recurrent bacterial infections after resuscitation. It is therefore necessary to apply and develop new methods to further determine the antibiotic resistance of living bacteria in a non-culturable state, which is important to create new effective intervention methods to ensure water and food safety, reducing public health risks.
However, due to the non-culturability of the living non-culturable state bacteria, the antibiotic resistance thereof cannot be detected using the conventional culture-based method, which is disadvantageous in research of the antibiotic resistance of the living non-culturable state bacteria and difficult to develop an effective control method. Therefore, it is currently highly desirable to establish a detection method for accurately adjusting various parameters to efficiently detect antibiotic resistance of living bacteria in a non-culturable state; the detection method has the advantages of low cost, easily available raw materials, wide application range, high flux, strong operability and relatively environmental protection, and can be aimed at different types of bacteria.
Disclosure of Invention
In order to solve the problem that the antibiotic resistance of the living bacteria in the non-culturable state is difficult to detect, the invention provides a novel method for detecting the antibiotic resistance of the living bacteria in the non-culturable state based on flow cytometry. The method can rapidly and high-flux detect the antibiotic resistance of the bacteria in a viable but non-culture state.
The invention adopts the following specific scheme:
a method for detecting bacterial antibiotic resistance in a viable, non-culturable state based on flow cytometry, comprising the steps of: s1, obtaining pure living bacteria in a non-culturable state through a disinfection technology, adjusting the concentration of the bacteria in the living bacteria in the non-culturable state, and then uniformly mixing antibiotics with the same volume and different concentrations, and incubating;
s2, centrifuging, washing, re-suspending, adding SYTO 9 and PI staining agents, incubating in a dark place, adjusting FSC and SSC voltages of a flow cytometer to circle a target object, adjusting FITC and PI pathway voltages to proper parameters, drawing a cross gate, analyzing the survival rate of bacteria, observing the ratio of a live bacterial group (bacteria only stained with the SYTO 9 staining agents), taking the concentration of antibiotics corresponding to a turning point of the survival rate of bacteria of an experimental group relative to a control group of less than 10% as the minimum sterilization concentration of the bacteria in a living non-culturable state, and then determining the antibiotic resistance of the bacteria in the living non-culturable state.
Preferably, the bacteria in step S1 are one or more of escherichia coli, pseudomonas aeruginosa, salmonella or enterococcus faecalis.
Preferably, the concentration of the viable, non-culturable state bacteria described in step S1 is 10 5 ～10 10 CFU/mL。
Preferably, the antibiotic in step S1 is one or more of cefotaxime, polymyxin, kanamycin, and tetracycline.
Preferably, the concentration of the antibiotic in step S1 is 0.001-200000 mg/L.
Preferably, the incubation time of the antibiotics described in step S1 is 1 to 48 hours.
Preferably, the mixing volume ratio of SYTO 9 stain to bacterial suspension in step S2 is 1:1-1000000.
Preferably, the mixing volume ratio of the PI staining agent to the bacterial suspension in the step S2 is 1:1-1000000.
Preferably, the incubation temperature in step S2 is 4-40 ℃.
Preferably, the stain incubation time in step S2 is 1 to 240min.
Preferably, the flow cytometer parameters in step S3: the FSC voltage is 10-600V; the SSC voltage is 10-600V; the FITC channel voltage is 10-600V; the PI channel voltage is 10-600V.
The method for detecting the bacterial antibiotic resistance in the living non-culturable state based on the flow cytometry is applied to the technical field of environmental microorganisms.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a method for detecting the antibiotic resistance of living bacteria in a non-culturable state by using a flow cytometer, which solves the problem that the antibiotic resistance of the living bacteria in the non-culturable state cannot be detected by using the traditional drug sensitive experiment based on the culturable capacity.
2. The method for detecting the antibiotic resistance of the living bacteria in the non-culturable state has wide application range and can detect the antibiotic resistance of the living bacteria in the non-culturable state of different bacterial types.
3. The method for detecting the antibiotic resistance of the living bacteria in the non-culturable state can detect the antibiotic resistance of the living bacteria in the non-culturable state to different antibiotics.
4. The method for detecting the antibiotic resistance of the living bacteria in the non-culturable state is stable, the sample consumption is small, the detection speed can be greatly improved, the high-flux detection can be realized, and the detection accuracy and the sensitivity are high.
Drawings
FIG. 1 shows the results of antibiotic resistance test of live, non-culturable bacteria in example 1.
Detailed Description
A method for detecting bacterial antibiotic resistance in a viable, non-culturable state based on flow cytometry, comprising the steps of:
s1, obtaining pure living bacteria in a non-culturable state through a disinfection technology, adjusting the concentration of the bacteria in the living bacteria in the non-culturable state, and then uniformly mixing antibiotics with the same volume and different concentrations, and incubating;
s2, centrifuging, washing and re-suspending, adding SYTO 9 and PI staining agents, incubating in a dark place at a proper temperature, adjusting FSC and SSC voltages of a flow cytometer to circle a target object, adjusting FITC and PI channel voltages to proper parameters, drawing a cross gate, analyzing the bacterial survival rate, observing the ratio of a living bacterial group (bacteria only stained with the SYTO 9 staining agents), taking the concentration of antibiotics corresponding to a turning point of the bacterial survival rate of an experimental group relative to a control group of less than 10% as the minimum sterilization concentration of the living bacteria in a non-culturable state, and then determining the antibiotic resistance of the living bacteria in the non-culturable state.
The invention provides a method for detecting antibiotic resistance of living bacteria in a non-culturable state, which obtains pure bacteria in a living non-culturable state through a disinfection technology. The antibiotic resistance of the bacteria in the viable but non-culturable state is detected by adjusting the conditions of concentration, antibiotic concentration, incubation time and the like of the bacteria in the viable but non-culturable state, and then adjusting the concentration of the coloring agent, the coloring incubation temperature and the incubation time. Based on the concentration of the bacteria in the most suitable conditions to be explored, the concentration of the bacteria in the viable, non-culturable state is 10 5 ～10 10 CFU/mL, antibiotic concentration of 0.001-200000 mg/L, antibiotic incubation time of 1-48 h, SYTO 9 stain and bacterial suspension mixing volume ratio of 1:1-1000000, PI stain and bacterial suspension mixing volume ratio of 1:1-1000000, incubation temperature of 4-40 ℃, dyeing incubation of 1-240 min, FSC voltage of 10-600V, SSC voltage of 10-600V, FITC pathway voltage of 10-600V, PI pathway voltage of 10-600V, and detection of antibiotic resistance of viable, non-culturable bacteria under appropriate conditions.
The present invention is further illustrated below in conjunction with specific examples, but should not be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present patent are those conventional in the art.
Example 1
1. Obtaining viable non-culturable bacteria, and adjusting the concentration of the viable non-culturable bacteria to 10 8 CFU/mL, then adding 0, 256, 1280, 2560 and 12800mg/L of cefotaxime antibiotics into the culture medium respectively, finally adding the same volume of viable and non-culturable bacteria in sequence, mixing uniformly, namely, the final concentration of the cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, and incubating for 24 hours.
2. Centrifuging, washing, re-suspending, uniformly mixing SYTO 9 stain and PI stain with a bacterial suspension, wherein the mixing volume ratio of SYTO 9 stain to bacterial suspension is 1:10000, the uniform mixing volume ratio of PI stain is 1:10000, incubating for 15min at 4 ℃ in a dark place, adjusting parameters on a flow cytometer, adjusting FSC parameters to 500V, SSC parameters to 400V, selecting a target object in a circle, adjusting FITC channel voltage to 400V, adjusting PI channel voltage to 500V, drawing a cross gate, analyzing bacterial survival rate, observing the ratio of a living bacterial group (bacteria only infected with SYTO 9 stain), taking the antibiotic concentration corresponding to a turning point of which the bacterial survival rate of different concentration experiment groups is less than 10% relative to that of a control group as the minimum sterilization concentration of the bacteria, and determining the antibiotic resistance of the bacteria in a living non-culturable state.
FIG. 1 shows the results of detection of antibiotic resistance of a living, non-culturable state bacterium based on activity in example 1. As can be seen from FIG. 1, the concentration of bacteria in the viable, non-culturable state is 10 8 CFU/mL, final concentration of cefotaxime antibiotic in incubation process is 0, 128, 640, 1280, 6400mg/L, antibiotic incubation time is 24h, mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, PI coloring agent uniform mixing volume ratio is 1:10000, coloring agent incubation temperature is 4 ℃, coloring agent light-shielding incubation time is 15min, FSC parameter is 500V, SSC parameter is 400V, FITC channel voltage is 400V, PI is communicatedThe antibiotic resistance of the bacteria in the viable, non-culturable state was determined to be 1280mg/L at a voltage of 500V.
Example 2
The difference from example 1 is that: the bacterial species in step 1 of this example is Pseudomonas aeruginosa.
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 4 ℃, the light-shielding incubation time of the coloring agent is 15min, FSC parameters are 500V, SSC parameters are 400V, FITC channel voltage is 400V, and the voltage of PI channel is 500V, so that the antibiotic resistance of the bacteria in the living non-culturable state is 1280mg/L, and the detection of the antibiotic resistance of the bacteria in the living non-culturable state under the optimal bacterial strain is feasible.
Example 3
The difference from example 1 is that: the antibiotic in step 1 in this example is polymyxin.
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of polymyxin antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 4 ℃, the light-shielding incubation time of the coloring agent is 15min, FSC parameters are 500V, SSC parameters are 400V, the FITC channel voltage is 400V, and the voltage of the PI channel is 500V, so that the antibiotic resistance of the bacteria in the living non-culturable state is 128mg/L, and the detection of the antibiotic resistance of the bacteria in the living non-culturable state under the most suitable antibiotics is feasible.
Example 4
The difference from example 1 is that in step 2 of this example, the mixing volume ratio of SYTO 9 to bacterial suspension is different (the mixing volume ratio of SYTO 9 to bacterial suspension is 1:1000).
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:1000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 4 ℃, the light-shielding incubation time of the coloring agent is 15min, FSC parameters are 500V, SSC parameters are 400V, the FITC channel voltage is 400V, and the voltage of the PI channel is 500V, so that the antibiotic resistance of the bacteria in a living non-culturable state is 1280mg/L, and the detection of the antibiotic resistance of the bacteria in the living non-culturable state under the optimal SYTO 9 dye concentration is feasible.
Example 5
The difference from example 1 is that: in this example, the ratio of the PI dye to the bacterial suspension in step 2 was different (the ratio of PI to bacterial suspension was 1:100000)
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI coloring agent is 1:100000, the incubation temperature of the coloring agent is 4 ℃, the light-shielding incubation time of the coloring agent is 15min, FSC parameters are 500V, SSC parameters are 400V, the FITC channel voltage is 400V, and the voltage of a PI channel is 500V, so that the antibiotic resistance of the bacteria in a living non-culturable state is 1280mg/L, and the detection of the antibiotic resistance of the bacteria in the living non-culturable state under the optimal PI dye concentration is feasible.
Example 6
The difference from example 1 is that the incubation temperature of the stain in step 2 in this example is different (temperature 25 ℃).
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 CFU/mL, final concentration of cefotaxime antibiotic during incubation is 0, 128, 640, 1280, 6400mg/L, antibiotic incubation timeFor 24h, the mixing volume ratio of SYTO 9 stain and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI stain is 1:10000, the incubation temperature of the stain is 25 ℃, the incubation time of the stain is 15min, FSC parameters are 500V, SSC parameters are 400V, FITC channel voltage is 400V, and the voltage of PI channel is 500V, the antibiotic resistance of the bacteria in a viable and non-culturable state is 1280mg/L, which indicates that the detection of the antibiotic resistance of the bacteria in the viable and non-culturable state is feasible at the optimal incubation temperature of the stain.
Example 7
The difference from example 1 is that the stain incubation time in step 2 in this example is different (30 min).
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 25 ℃, the light-shielding incubation time of the coloring agent is 30min, FSC parameters are 500V, SSC parameters are 400V, the FITC channel voltage is 400V, and the voltage of the PI channel is 500V, so that the antibiotic resistance of the bacteria in a living non-culturable state is 1280mg/L, and the detection of the antibiotic resistance of the bacteria in the living non-culturable state under the optimal coloring agent incubation time is feasible.
Example 8
The difference from example 1 is that the FITC pathway voltage in step 2 in this example is different (FITC pathway voltage 500V).
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotic in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of the antibiotic is 24 hours, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:10000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 25 ℃, the light-proof incubation time of the coloring agent is 30min, FSC parameters are 500V, SSC parameters are 400V, the voltage of a FITC channel is 500V, and the voltage of a PI channel is 500V,determining that the antibiotic resistance of the viable, non-culturable bacteria is 1280mg/L indicates that detection of antibiotic resistance of the viable, non-culturable bacteria is feasible at the optimal stain incubation time.
Comparative example
The difference from example 1 is that: in step 2 of this example, the ratio of SYTO 9 to bacterial suspension was different (the ratio of SYTO 9 to bacterial suspension was 1:100000000).
The results of the examination showed that the concentration of the bacteria in the viable, non-culturable state was 10 8 The final concentration of cefotaxime antibiotics in the incubation process is 0, 128, 640, 1280 and 6400mg/L, the incubation time of antibiotics is 24h, the mixing volume ratio of SYTO 9 coloring agent and bacterial suspension is 1:100000000, the uniform mixing volume ratio of PI coloring agent is 1:10000, the incubation temperature of the coloring agent is 25 ℃, the light-shielding incubation time of the coloring agent is 30min, FSC parameters are 500V, SSC parameters are 400V, the voltage of a FITC channel is 500V, the staining effect is poor under the condition that the voltage of a PI channel is 500V, the real proportion of living bacteria is difficult to observe on a flow cytometer, and the antibiotic resistance of living bacteria in a non-culturable state cannot be judged.
The detection method is optimized, and practices show that the ratio of the living bacteria to the living bacteria is difficult to observe, and the antibiotic resistance of the bacteria in a living non-culturable state cannot be effectively judged by changing parameter conditions, such as too high or too low mixing proportion of the coloring agent, excessive or insufficient incubation reaction, unreasonable parameter setting of a flow cytometer and the like.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (10)
1. A method for detecting bacterial antibiotic resistance in a viable, non-culturable state based on flow cytometry, characterized by: the method comprises the following steps:
s1, obtaining pure living bacteria in a non-culturable state through a disinfection technology, adjusting the concentration of the bacteria in the living bacteria in the non-culturable state, and then uniformly mixing antibiotics with the same volume and different concentrations, and incubating;
s2, centrifuging, washing, re-suspending, adding SYTO 9 and PI staining agents, incubating in a dark place, adjusting FSC and SSC voltages of a flow cytometer to circle and select a target object, adjusting FITC and PI channel voltages to proper parameters, drawing a cross gate, analyzing bacterial survival rate, and observing the ratio of a live bacterial group, wherein the live bacterial group is bacteria only stained with the SYTO 9 staining agents; the antibiotic concentration corresponding to the turning point at which the bacterial survival rate of the experimental group relative to the control group is less than 10% is taken as the minimum sterilization concentration of the living non-culturable state bacteria, and then the antibiotic resistance of the living non-culturable state bacteria is determined.
2. The method according to claim 1, characterized in that: the bacteria in the step S1 are one or more of escherichia coli, pseudomonas aeruginosa, salmonella or enterococcus faecalis.
3. The method according to claim 1, characterized in that: the concentration of the viable, non-culturable state bacteria described in step S1 is 10 5 ～10 10 CFU/mL。
4. The method according to claim 1, characterized in that: in the step S1, the antibiotic is one or more of cefotaxime, polymyxin, kanamycin or tetracycline.
5. The method according to claim 4, wherein: the concentration of the antibiotics is 0.001-200000 mg/L; the incubation time of the antibiotics is 1-48 h.
6. The method according to claim 1, characterized in that: in the step S2, the mixing volume ratio of the SYTO 9 coloring agent to the bacterial suspension is 1:1-1000000.
7. The method according to claim 1, characterized in that: the mixing volume ratio of the PI coloring agent to the bacterial suspension in the step S2 is 1:1-1000000.
8. The method according to claim 1, characterized in that: in the step S2, the incubation temperature is 4-40 ℃ and the incubation time is 1-240 min.
9. The method according to claim 1, characterized in that: in step S3, the flow cytometer parameters: the FSC voltage is 10-600V; the SSC voltage is 10-600V; the FITC channel voltage is 10-600V;
the PI channel voltage is 10-600V.
10. Use of a method for detecting bacterial antibiotic resistance in a viable, non-culturable state based on flow cytometry according to any one of claims 1-9 in the field of environmental microbiology.
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