CN111826415A - Method for detecting inhibition of antibacterial drugs on bacteria - Google Patents
Method for detecting inhibition of antibacterial drugs on bacteria Download PDFInfo
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Abstract
The invention provides a method for detecting the inhibition of an antibacterial drug on bacteria, which comprises the following steps: adding an antibacterial agent with a preset concentration into bacteria to be detected, setting the bacteria as a bacteria medicine mixture, and setting the bacteria to be detected without adding the antibacterial agent as a positive control; obtaining a current number of the bacteria of the bacterial drug mixture and a current number of the bacteria of the positive control when a first predetermined time is reached after a time interval from the addition of the antibacterial drug; determining the inhibition or partial inhibition or non-inhibition of said bacteria by said predetermined concentration of said antibacterial agent based on the ratio of the current number of said bacteria of said bacterial drug mixture to the current number of said bacteria of said positive control. The most outstanding beneficial effects of the invention are as follows: the breakthrough of the report time can obtain the result of bacteria inhibition detection of the antibacterial agent within 1-2 hours after obtaining the pure culture, and has low cost and easy automation.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a method for detecting the inhibition of bacteria by an antibacterial drug.
Background
The problem of bacterial drug resistance is more and more serious, the bacterial drug resistance is rapidly and widely spread in the global scope, governments of various countries pay more attention, China also puts a great deal of management regulations, reasonable use of antibiotics is the most core work for bacterial drug resistance, and rapid antibiotic sensitivity test is the most important.
In view of the development of mass spectrometry and nucleic acid technology, the rapidity of bacterial identification has been substantially realized (completed within 1-2 hours after the day of the outcome), and thus the development of new rapid antibiotic sensitivity tests is more urgent and practical. Switching empirical broad-spectrum antibiotic therapy to targeted therapy as soon as possible is a basic stone for antibiotic management, but the current drug sensitivity test report time restricts clinical practice, and the report time of the traditional manual method is too long, so that the current clinical fully-automatic drug sensitivity test methods are mostly adopted, wherein the ViTEK system of French Merrier company and the Phoenix system of American BD company are the fastest detection systems, the reliability and the accuracy of the two systems are proved, but the average time Phoenix is 12.1 hours, and the Vitek2 is 9.8 hours, and the report time only can be specifically selected by a doctor on the next day in consideration of the daily workflow and the work and rest time.
At present, in order to shorten the time of drug sensitivity test report, a great deal of research is carried out at home and abroad, and various methods such as a mass spectrometry method, a flow cytometry method, a vibration cantilever microorganism cell weighing method, an isothermal micro-production thermal method, a magnetic bead rotation method, a droplet detection method, a real-time PCR method, a microarray method, a conductivity method, a surface plasma resonance method, an RNA sequencing method, a phage method, a real-time microscopy method and a microscopic acoustic wave method are developed, but the methods are only in a research stage, only small sample analysis is carried out, and all the methods need professional technical personnel to operate, equipment is expensive, and the methods are non-traditional special equipment, and have the defects of complex operation, unstable performance, high cost, inconvenient use and poor judgment of practical prospects.
The rapid drug sensitivity test can be divided into two major types, namely a phenotypic method and a nonphenotypic method. Non-phenotypic methods are mainly nucleic acid-based molecular biology methods such as real-time PCR, microarray, RNA sequencing, transcriptome and whole genome sequencing, etc. with the advantages: 1. short time, such as multiple PCR of direct positive blood culture can detect multiple drug resistant genes; 2. quantitative analysis can be realized by digital PCR; 3. the corresponding drug resistance mechanism is clear. The disadvantages are: 1. the drug resistance mechanism of bacteria is complex and huge, and if the bacteria are comprehensively used in clinic, the economic efficiency and the rapidity are affected due to overlarge workload; 2. the problem of consistency of gene detection results and phenotypes of drug resistance gene detection also needs a large amount of verification work due to the problem of genetic heterogeneity; 3. the new drug resistance mechanism cannot be detected, and the acute discovery of the new drug resistance mechanism is urgently needed clinically; 4. the traditional Chinese medicine composition is not applied in clinical practice, is immature, needs further clinical observation, and can be actually applied clinically after being approved and standardized by global experts.
The phenotypic drug susceptibility test directly observes the response of bacteria to drugs in vitro and can directly observe the sensitivity and tolerance of bacteria to antibiotics, and the traditional phenotypic drug susceptibility test is fully developed, examined and verified and is fully proved in clinical practice, so that the phenotypic drug susceptibility test becomes a reference standard of the AST method. Before the gene-based non-phenotypic drug sensitivity test is not perfect and definite, the 'intermediate technology', namely the technology developed on the basis of the traditional culture method, can be implemented earlier, has great feasibility and is expected.
Therefore, it is very important to provide an economical and fast drug sensitivity test scheme.
Disclosure of Invention
The invention provides a method for detecting the inhibition of bacteria by an antibacterial agent, which at least solves the technical problem that the result time of the method for detecting the inhibition of bacteria by the antibacterial agent in the prior art is long, and doctors can only pertinently select the medicine in the next day.
The invention provides a method for detecting the inhibition of an antibacterial drug on bacteria, which comprises the following steps: adding an antibacterial agent with a preset concentration into bacteria to be detected, setting the bacteria as a bacteria medicine mixture, and setting the bacteria to be detected without adding the antibacterial agent as a positive control;
obtaining a current number of the bacteria of the bacterial drug mixture and a current number of the bacteria of the positive control when a first predetermined time is reached after a time interval from the addition of the antibacterial drug;
determining the inhibition or partial inhibition or non-inhibition of said bacteria by said predetermined concentration of said antibacterial agent based on the ratio of the current number of said bacteria of said bacterial drug mixture to the current number of said bacteria of said positive control.
Optionally, determining that the predetermined concentration of the antibacterial drug inhibits the bacteria if a ratio of a current number of the bacteria of the bacterial drug mixture to a current number of the bacteria of the positive control is equal to a first predetermined threshold.
Optionally, the first predetermined threshold is any one of values from 0 to 0.6.
Optionally, the first predetermined threshold is any one of values from 0 to 0.4.
Optionally, in a case where a ratio of a current number of the bacteria of the bacterial drug mixture to a current number of the bacteria of the positive control is equal to a second predetermined threshold value, determining that the predetermined concentration of the antibacterial drug has partial inhibition but not inhibition on the bacteria;
obtaining a second current quantity of said bacteria of said bacterial drug mixture and a second current quantity of said bacteria of said positive control when a second predetermined time is reached spaced from the time of addition of said antibacterial drug, wherein said second predetermined time is greater than said first predetermined time;
determining that the predetermined concentration of the antimicrobial drug is inhibitory to the bacteria if a ratio of a second present number of the bacteria of the bacterial drug mixture to a second present number of the bacteria of the positive control is equal to the first predetermined threshold.
Optionally, in a case where a ratio of a current number of the bacteria of the bacterial drug mixture to a current number of the bacteria of the positive control is greater than a second predetermined threshold, it is determined that the predetermined concentration of the antibacterial drug does not inhibit the bacteria.
Optionally, the first predetermined time period is any value from 0 to 1.5 hours, and the first predetermined time period is not equal to 0 hour.
Optionally, the second predetermined threshold is any one of values from 0.4 to 0.8.
Optionally, the current number of the bacteria of the bacterial drug mixture and the current number of the bacteria of the positive control are obtained by a resistance counting method.
Optionally, the method for detecting the inhibition of the antibacterial agent on bacteria comprises the following detection steps:
a. preparing the bacterial strains: inoculating the bacterial strain on a culture medium, and incubating for 15 hours to 24 hours at a temperature of 20 ℃ to 40 ℃ for later use;
b. preparing the bacterial drug mixture and the positive control, and incubating at 20-40 ℃;
c. obtaining a current or second current number of said bacteria of said bacterial drug mixture and a current or second current number of said bacteria of said positive control by said resistance counting method after said first or second predetermined period of time;
d. determining that the predetermined concentration of the antibacterial agent inhibits the bacteria when a ratio of the current or second current number of the bacteria of the bacterial drug mixture to the current or second current number of the bacteria of the positive control is equal to any one of values from 0 to 0.4.
Optionally, in the step a, the bacterial strain is inoculated on a blood agar culture medium and incubated at 37 ℃ (DEG C) for 18 hours; and/or
In the step b, preparing the bacterial drug mixture and the positive control, and incubating at 37 ℃; and/or
In the step c, the first predetermined time period is 0.5 hour, 1 hour or 1.5 hours; the second predetermined period of time is 2 hours or 2.5 hours or 3 hours.
Optionally, the current number of the bacteria of the bacterial drug mixture and the current number of the bacteria of the positive control are obtained by a flow-type bacteria counting method, a microscopic bacteria counting method, a counter measuring method, an electron counter counting method, a live cell counting method, or a cell gravimetric method.
The essence of the drug sensitivity test of the invention is to observe the influence of antibiotics on the growth, metabolism and reproduction of bacteria, and to infer the effectiveness of future medication according to the condition of the influence of drugs on the growth, metabolism and reproduction of bacteria (i.e. the inhibition condition of bacteria) observed in vitro tests and the clinical and pharmacokinetic conditions. The traditional method monitors the killing effect of antibiotics on bacteria through the change of the number of bacteria in a liquid or solid culture medium, and observes that bacteria groups can quickly detect the influence of drugs on the bacteria at an early stage if each bacteria individual can be accurately monitored in number, but not the change trend detection of the total number of the bacteria groups, so that the time of drug sensitivity test has a great breakthrough.
Specifically, one of the technical schemes of the invention is to creatively apply a resistance counting method (Coulter principle) which is the most mature, reliable, fast and economical in human blood cell counting to an antibacterial drug inhibition detection method, so as to realize the detection of a fast antibiotic sensitivity test, and when antibacterial drugs with different concentrations are added during the growth of bacteria, the antibacterial drugs with more than a certain concentration can be found to inhibit the growth and the reproduction of the bacteria, so that the minimum inhibitory concentration is determined. The traditional method is to determine the minimum inhibitory concentration through the turbidity change of broth after the bacteria growth, which needs a long time, usually 18 hours, in recent years some commercial companies optimize, use a more sensitive turbidimeter or add redox indicators to try to find the bacteria growth or inhibition early, these methods also need 10 hours to report, because the antibacterial drugs and the bacteria contact a short time to generate the effect, so it is very practical to find the method or scheme for quickly determining the effect and the influence of the drugs on the bacteria, and can determine the sensitivity of the bacteria to the drugs in a short time, the invention can count the bacterial cells quantitatively in a short time by using a resistance counting method, can quickly determine the sensitivity of the antibacterial drugs, and determine the inhibition of the antibiotics on the bacteria by analysis and comparison of the change of the bacterial number, and the sensitivity of the antibacterial agent is rapidly determined. The method is very suitable for quick drug sensitivity test, and the result is stable and reliable.
The most outstanding beneficial effects of the technical scheme of the invention are as follows:
(1) the breakthrough in time is reported. Reports can be made within 1-2 hours after obtaining a pure culture.
(2) And the practicability is broken through. But the real advantage is that the rapid identification of bacteria (completed within 1-2 hours) is matched, and the targeted antibiotic treatment can be realized on the day of obtaining pure culture of bacteria in consideration of daily work flow and work and rest time, so that the mortality and medical cost of patients can be reduced, and the increase of the drug resistance of bacteria can be slowed down.
(3) The technology is mature, stable and reliable.
(4) The principle is close to the international standardized scheme (the broth dilution method drug sensitivity test), and the conversion is strong in clinical practicability.
(5) The cost is low.
(6) Easy to be automated.
Drawings
The above and other objects, features and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description read in conjunction with the accompanying drawings. Several embodiments of the invention are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which:
FIG. 1 is a schematic diagram showing the variation of the bacterial load at different times in an alternative E.coli broth culture according to an embodiment of the present invention;
FIG. 2 is a schematic representation of an alternative Escherichia coli broth culture showing variations in turbidity over time in accordance with embodiments of the present invention;
FIG. 3 is a graphical representation of the results of turbidity vs. bacterial count observations of an alternative broth bacterial culture in accordance with an embodiment of the present invention;
FIG. 4 is a graph showing the results of the growth experiment in example 3 of the present invention;
FIG. 5 is a graph showing the results of comparison between 2h and 24h in example 3 of the present invention;
FIG. 6 is a graph showing the results of the sensitivity matching rates in example 3 of the present invention.
The following detailed description is intended to further illustrate but not limit the invention, the following example being only one preferred embodiment of the invention.
Detailed Description
The principles and spirit of the present invention will be described with reference to a number of exemplary embodiments. It is understood that these embodiments are given solely for the purpose of enabling those skilled in the art to better understand and to practice the invention, and are not intended to limit the scope of the invention in any way. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Example 1
The purpose is as follows: and (3) searching a bacterial change sensitivity index during broth bacterial culture, determining a theoretical basis for a rapid drug sensitivity test, and establishing a bacterial drug sensitivity detection method.
Materials and methods:
1. preparing strains:
three standard strains (ATCC 25922 Escherichia coli, ATCC 25923 Staphylococcus aureus, ATCC27853 Pseudomonas aeruginosa) were transferred and incubated at 37 ℃ for 18 hours.
2. Broth preparation:
the prepared strain is ground on the bottle wall of AST (antibiotic susceptibility test) broth, mixed evenly, covered with a bottle cap, and measured for turbidity of 0.5 McLem unit by a turbidimeter (Phoenix Spec Nephelomter, BD Co.) for later use. The bacteria to be tested were inoculated at the inoculation concentration for the drug sensitivity test by CLSI (american clinical laboratory standards organization) broth dilution and incubated in an incubator at 37 ℃ (celsius).
3. Bacterial count
The cells were counted by resistance counting (resistance-3000 type RC-type (Coulter) particle counter, Kyohima) at 0 min, 10 min, 30 min, 60 min, 90 min and 120 min in an incubator at 37 ℃. And measuring 2 times, taking an average value, and recording data.
4. Turbidity measurement
Incubate at 37 ℃ for 2 turbidity measurements using a turbidimeter (Phoenix Spec nephelometer, BD Co.) at 0 min, 10 min, 30 min, 60 min, 90 min, 120 min, average and record data.
Results
Table 1 attached observation of turbidity and number of bacteria in broth culture:
TABLE 1
The change of the bacterial quantity of the Escherichia coli broth in different times is shown in figure 1;
the change in turbidity of the Escherichia coli broth over time is shown in FIG. 2.
And (4) conclusion:
1. when the bacteria are cultured in broth, the difference of different methods for observing bacteria is large, the conventional colony bacteria has poor sensitivity in growth turbidimetry, and the difference cannot be measured within 120 minutes.
2. The bacterial count method can measure significant differences within 30 minutes of culture in broth, demonstrating the feasibility of the bacterial drug susceptibility assay.
Example 2
Ampicillin inhibition assay for ATCC25922 escherichia coli (by resistance counting):
materials and methods:
1. preparing strains:
standard strain ATCC25922 Escherichia coli was transferred and incubated at 37 ℃ for 18 hours.
2. Broth preparation:
10 of the drug sensitive test tubes (or cups) contained the required antibiotic at double dilution (different drug concentrations refer to us CLSI standard); the eleventh tube contained no antibiotic as a Positive Control (PC); the twelfth tube without bacterial suspension served as Negative Control (NC).
The prepared strain is ground on the bottle wall of MH (antibiotic susceptibility) broth, mixed evenly, covered with a bottle cap, and measured for turbidity with a turbidimeter (Phoenix Spec Nephelomter, BD Co.) to 0.5 McLet unit for later use.
The test bacteria were inoculated at the inoculation concentration for the drug sensitivity test by CLSI (standard tissue in the clinical laboratory of the usa) broth dilution method and incubated in an incubator at 37 ℃.
Preparing bacterial liquid: and selecting the bacterial colonies for later use to prepare a bacterial suspension, wherein the concentration of the bacterial suspension is 0.5 McLee unit. The colony suspension is added to broths containing various antibiotics at different concentrations (MH) and contains, after each inoculation, 1X10^4cfu/ml to 5X10^7cfu/ml, optimally 5X10^6cfu/ml (colony forming units/ml), i.e., optimally 5X10^6 colony forming units per ml.
3. Resistance counting method (Kuert principle)
The cells were counted by resistance counting (RC-3000 type resistance method (Coulter) particle counter, Kyoto-Marek) at 0 min, 10 min, 30 min, 60 min, 90 min and 120 min in an incubator at 37 ℃. And measuring 2 times, taking an average value, and recording data.
Appendix 2ATCC 25922 bacterial counts of Escherichia coli for the ampicillin rapid susceptibility test vary as shown in the following table:
TABLE 2
The results of the observations of the turbidity of the broth bacterial cultures as a function of the number of bacteria are shown in FIG. 3, in which the abscissa represents the concentration values of the antibiotic drugs, corresponding to the first row of values in the attached Table 2, in μ g/ml (micrograms per ml) and the ordinate represents the value of the number of bacteria, in units per μ l (each per microliter).
A total of 12 tubes were tested, the eleventh tube was used as Positive Control (PC), the twelfth tube was used as Negative Control (NC), and the other 10 tubes were test tubes, and 12 tubes were tested at each test time point.
The results of the resistance method susceptibility test are shown in FIG. 3, which shows that the bacterium ATCC25922 Escherichia coli in this example is inhibited by ampicillin, and the minimum inhibitory concentration is 4 μ g/ml.
In addition, the bacteria in this example were simultaneously subjected to drug susceptibility tests by three other methods, namely, ampicillin inhibition of the bacteria ATCC25922 Escherichia coli in this example was detected according to the VITEK microorganism identification drug susceptibility system operating manual of Merrill, France, and the detection result was a minimum inhibitory concentration of 4. mu.g/ml (micrograms per milliliter); according to the test method, the method is detailed in the operation manual of an ester drug sensitive kit of American Saimer Feilk company, the inhibition of ampicillin on the bacterium ATCC25922 Escherichia coli in the embodiment is detected, and the detection result is the minimum inhibitory concentration of 2 mu g/ml; ampicillin was tested for inhibition of the bacterium ATCC25922 Escherichia coli of this example according to the broth dilution susceptibility test, which is specified in the American society for clinical laboratory standards for testing for broth dilution susceptibility test, at a minimum inhibitory concentration of 4. mu.g/ml, which was found to be consistent and sensitive.
And (4) conclusion:
the MIC (minimum inhibitory concentration) was determined within 1.60 minutes.
2. The MIC (minimum inhibitory concentration) measured within 60 minutes in the embodiment of the invention is consistent with the drug sensitivity test results of a VITEK (drug sensitivity test method of Merrier company), an Etest method and a broth dilution method, namely, the feasibility of the bacterial drug sensitivity test method is re-demonstrated, and the beneficial effect of rapidly obtaining the bacterial drug sensitivity result is obvious.
As is clear from examples 1 and 2, the results of the method for detecting the inhibition of bacteria by the antibacterial agent of the present invention were analyzed in comparison with the results of the conventional methods by comparing the results with the results of VITEK (MerrieA susceptibility test method), Etest and BMD (broth dilution method), and the comparative standards were determined according to the FDA (American food and drug administration).
Compared with a positive control, the observation of 20%, 40%, 60% or 80% reduction of cell count is used as a judgment point of drug sensitive result, and compared with the consistency of the traditional method, the conclusion is that the accuracy reaches 100% when the cell count is reduced by more than 40% to 60%, wherein the accuracy is optimal when the cell count is reduced by more than 60%, the time for different bacteria to reach the cell count is different by 40% to 60%, the clinical common bacteria can be completed within 90 to 120 minutes, and most clinical common bacteria can be completed within 90 minutes.
Example 3
1. Growth experiment
1.1 purpose of the experiment
Six common strains (ATCC29212, ATCC29213, ATCC27853, ATCC25922, Klebsiella pneumoniae ATCC700603 and Acinetobacter baumannii) which are commonly used in clinic are inoculated according to the CLSI standard requirements, and the growth trend is recorded for determining whether the growth is possible or not for 2 h.
1.2 Experimental methods
1.2.1 negative controls: detecting the culture medium without inoculation, detecting by using a resistance bacteria counter, and recording the number of particles;
1.2.2 Strain preparation: according to the CLSI standard requirement, selecting fresh strains within 24h of 0.5 McLee, and adding 100ul of the fresh strains into 10ml of culture medium;
1.2.3 recording the result at 0h/0.5h/1h/1.5h respectively, simultaneously removing the negative background, and analyzing the result.
2.3 the experimental result is shown in fig. 4, and the growth experimental result shows that the bacteria have changed obviously in 2h, and the change can be clearly captured by the resistance counting method, so that the feasibility of resistance counting is shown in the observation and detection angle.
2.2h and 24h drug sensitivity comparison results (from the results of drug sensitivity in two time periods, the feasibility of performing bacterial drug sensitivity by the resistance method is determined).
2.1 purpose of the experiment
And respectively selecting a group of A drugs aiming at the clinical common standard strains to compare results, and inspecting the consistency results of 2h and 24 h. The strains and corresponding antibiotics are shown in Table 3.
TABLE 3
| Strain name | Antibiotic |
| Pseudomonas aeruginosa (ATCC27853) | Ceftazidime |
| Staphylococcus aureus (ATCC29213) | Erythromycin |
| Enterococcus faecalis (ATCC29212) | Penicillin |
| Klebsiella pneumoniae | Gentamicin |
| Acinetobacter baumannii | Meropenem |
2.2 Experimental methods
2.2.1 Strain preparation: respectively picking fresh strains within 24h of 0.5 McLee according to CLSI standard requirements and a strain name list 3;
2.2.2 inoculation: respectively adding 100ul of 0.5M bacterium suspension into a prepared gradient antibiotic 48-pore plate, and adding 500ul of the suspension into each pore;
2.2.3 incubation and detection: incubating at 37 ℃ for 2h, detecting by using a resistance bacteria counter, adjusting the counter to the optimal sensitive state, counting bacteria, recording the conventional drug sensitive result for 24h, and carrying out comparative analysis.
2.3 analysis of the results
TABLE 4
As can be seen from the results of fig. 5 and table 4, the first choice of drugs required by CLSI was selected for five clinically common strains to perform antibiotic susceptibility tests, MIC obtained by resistance counting (MIC was determined by using 60% of 2h positive count value as a critical point) was compared with the conventional susceptibility test method by visually observing the 24h susceptibility results, and the difference was within an acceptable range, thereby proving the feasibility of the resistance counting method in the bacterial susceptibility tests.
3. Sensitivity coincidence rate
Selecting and using a 96-hole enterobacteriaceae drug-sensitive reagent plate produced by Shandong Xinke of 11 clinical common enterobacteriaceae (2 strains of Escherichia coli, Morganella morganii, Shigella, Enterobacter aerogenes, Froude Citrobacter, 2 strains of Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae and Salmonella), inoculating a reagent plate according to the CLSI requirement, incubating for 2h at 37 ℃, inoculating two reagent plates, judging the bacterial counting result of one reagent plate in 2h, comparing the result with a positive control, preliminarily selecting a positive result value with the result value not less than 60% as the positive value, and recording the result. One reagent plate is used for recording the 24h experiment result and judging the sensitivity coincidence rate of 2h and 24h, and the result is shown in fig. 6, which shows that the drug sensitivity test of clinically common antibiotics by using a resistance counting method aiming at 11 clinically common enterobacteriaceae has higher coincidence rate compared with the traditional CLSI result.
Example 4
The result of the drug sensitivity test of the live bacteria and the consistency with the traditional method
1.1 purpose of the experiment
A group of A drugs are respectively selected for clinical common standard strains to compare results, consistency results of 2h and 24h are inspected, growth tendency of the strains is recorded to determine whether the 2h has the possibility of determining the growth of the strains, and the strains and corresponding antibiotics are shown in a table 5.
TABLE 5
| Strain name | Antibiotic |
| Pseudomonas aeruginosa (ATCC27853) | Ceftazidime |
| Staphylococcus aureus (ATCC29213) | Erythromycin |
| Enterococcus faecalis (ATCC29212) | Penicillin |
| Klebsiella pneumoniae | Gentamicin |
| Acinetobacter baumannii | Meropenem |
1.2 Experimental methods
1.2.1 Strain preparation: according to the CLSI standard requirements, fresh strains within 24h of 0.5 McLee are respectively picked according to the strain name list of Table 6.
1.2.2 inoculation: 100ul of 0.5M M.suspension was added to a prepared gradient antibiotic 48 well plate, 500ul was added per well.
1.2.3 incubation and detection: incubate at 37 ℃ for 2h for viable bacteria count and record 24h conventional drug sensitivity results for comparative analysis.
1.3, analyzing the result of the experiment,
1.3.1 results of the experiment
Drug sensitivity results of pseudomonas aeruginosa to ceftazidime:
quick drug sensitive result of living cells: MIC is 2; traditional drug sensitive results: MIC is 2.
The specific data of the rapid drug sensitivity of the living cells are shown in Table 6.
TABLE 6 (where the first bar is the antibiotic drug concentration value in μ g/ml and the second bar is the bacterial count value in μ l/μ l (each per microliter))
The result of drug sensitivity of staphylococcus aureus to erythromycin is as follows:
quick drug sensitive result of living cells: MIC is 0.25; traditional drug sensitive results: MIC is 0.25.
The specific data of the rapid drug sensitivity of the living cells are shown in Table 7.
Table 7 (where the first horizontal row values are antibiotic drug concentration values in μ g/ml and the second horizontal row values are bacteria number values in μ l/μ l)
The result of the drug sensitivity of enterococcus faecalis to penicillin:
quick drug sensitive result of living cells: MIC is 1; traditional drug sensitive results: MIC is 2.
The specific data of the rapid drug sensitivity of the living cells are shown in Table 8.
Table 8 (where the first horizontal row values are antibiotic drug concentration values in μ g/ml (micrograms per milliliter) and the second horizontal row values are bacteria number values in units per μ l (each per microliter))
Drug sensitivity results of klebsiella pneumoniae to gentamicin:
quick drug sensitive result of living cells: MIC is 8; traditional drug sensitive results: MIC is 8.
The specific data of the rapid drug sensitivity of the living cells are shown in Table 9.
TABLE 9 (where the first bar is the antibiotic drug concentration value in μ g/ml and the second bar is the bacteria number value in μ l/μ l (each per microliter))
The result of drug sensitivity of acinetobacter baumannii to meropenem is as follows:
quick drug sensitive result of living cells: MIC is 0.12; traditional drug sensitive results: MIC is 0.12.
Data table 10 for rapid drug susceptibility of living cells.
TABLE 10 (where the first bar is the antibiotic drug concentration value in μ g/ml and the second bar is the bacteria number value in μ l/μ l)
As can be seen from the results in tables 6 to 10, the first choice of the drugs required by CLSI was selected for five clinically common strains to perform antibiotic susceptibility tests, and the MIC obtained by the viable cell counting method (determining the MIC according to 60% of the 2h positive count value as the critical point) was compared with the traditional susceptibility test method by visually observing the 24h susceptibility results, and the difference was within the acceptable range, thereby proving the feasibility of the viable cell counting method in the bacterial susceptibility tests, which is consistent with the traditional method.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A method for detecting bacterial inhibition by an antibacterial agent, comprising:
adding an antibacterial agent with a preset concentration into bacteria to be detected, setting the bacteria as a bacteria medicine mixture, and setting the bacteria to be detected without the antibacterial agent as a positive control;
obtaining a current number of the bacteria of the bacterial drug mixture and a current number of the bacteria of the positive control when a first predetermined time is reached apart from a time of addition of the antibacterial drug;
determining the inhibition or partial inhibition or non-inhibition of the bacteria by the predetermined concentration of the antibacterial drug according to a ratio of the current number of the bacteria of the bacterial drug mixture to the current number of the bacteria of the positive control.
2. The method of claim 1, wherein the presence of inhibition of the bacteria by the predetermined concentration of the antibacterial drug is determined if a ratio of a current number of the bacteria of the bacterial drug mixture to a current number of the bacteria of the positive control is equal to a first predetermined threshold.
3. The method of claim 2, wherein the first predetermined threshold is any one of values from 0 to 0.6.
4. The method of claim 3, wherein the first predetermined threshold is any value from 0 to 0.4.
5. The method of claim 2, wherein in the case that the ratio of the current number of bacteria of the bacterial drug mixture to the current number of bacteria of the positive control is equal to a second predetermined threshold value, determining that the predetermined concentration of the antibacterial drug has partial inhibition of the bacteria but not inhibition;
obtaining a second current amount of the bacteria of the bacterial drug mixture and a second current amount of the bacteria of the positive control when a second predetermined time is reached spaced apart from the time of addition of the antibacterial drug, wherein the second predetermined time is greater than the first predetermined time;
determining that there is inhibition of the bacteria by the predetermined concentration of the antibacterial drug where a ratio of a second current number of the bacteria of the bacterial drug mixture to a second current number of the bacteria of the positive control is equal to the first predetermined threshold.
6. The method of claim 5, wherein in the event that the ratio of the current number of bacteria of the bacterial drug mixture to the current number of bacteria of the positive control is greater than a second predetermined threshold, determining that the predetermined concentration of the antibacterial drug does not inhibit the bacteria.
7. The method of claim 1, wherein the first predetermined period is any one of 0 to 1.5 hours, and the first predetermined period is not equal to 0 hour.
8. The method of claim 5, wherein the second predetermined threshold is any one of values from 0.6 to 0.8.
9. The method of any one of claims 1 to 8, wherein the current number of bacteria of said bacterial drug mixture and the current number of bacteria of said positive control are obtained by resistance counting.
10. The method of claim 9, wherein the method for detecting the inhibition of bacteria by the antibacterial agent comprises the following steps:
a. preparing the bacterial strain: inoculating the bacterial strain on a culture medium, and incubating at a temperature of 20 to 40 degrees Celsius (C.) for 15 to 24 hours for use;
b. preparing the bacterial drug mixture and the positive control, incubating at a temperature of 20 ℃ to 40 ℃;
c. obtaining the current number or the second current number of the bacteria of the bacterial drug mixture and the current number or the second current number of the bacteria of the positive control by the resistance counting method after the first predetermined period of time or the second predetermined period of time;
d. determining that the predetermined concentration of the antibacterial drug has inhibition on the bacteria if a ratio of the current or second current number of the bacteria of the bacterial drug mixture to the current or second current number of the bacteria of the positive control is equal to any one of values from 0 to 0.4.
11. The method of claim 10,
in the step a, the bacterial strain is inoculated on a blood agar culture medium and incubated at the temperature of 37 ℃ for 18 hours; and/or
In the step b, preparing the bacterial drug mixture and the positive control, and incubating at the temperature of 37 ℃; and/or
In the step c, the first preset time period is 0.5 hour or 1 hour or 1.5 hours; the second predetermined period of time is 2 hours or 2.5 hours or 3 hours.
12. The method according to any one of claims 1 to 8, wherein the current number of said bacteria of said bacterial drug mixture and the current number of said bacteria of said positive control are obtained by flow or microscopic bacterial counting or counter assay or electron counter counting or live cell counting or cell gravimetric assay.
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