CN110157773B - Drug resistance judgment standard test method for mycoplasma gallisepticum to tylosin - Google Patents
Drug resistance judgment standard test method for mycoplasma gallisepticum to tylosin Download PDFInfo
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Abstract
The invention discloses a drug resistance judgment standard test method of mycoplasma gallisepticum to tylosin, which comprises the steps of establishing a drug resistance detection method of tylosin to mycoplasma gallisepticum, establishing a wild critical value of tylosin to mycoplasma gallisepticum, establishing a pharmacodynamic critical value of tylosin to mycoplasma gallisepticum, establishing a clinical critical value of tylosin to mycoplasma gallisepticum and establishing a flow chart by referring to a break point published by CLSI, and the method accords with COWT＞COPD＞COCLSelecting a wild critical value for the corresponding breakpoint value to obtain a drug resistance judgment standard of the mycoplasma gallisepticum to tylosin; the method can obtain the drug resistance judgment standard of the mycoplasma gallisepticum to tylosin, can provide stable drug data support for scientific culture, can scientifically guide clinical drug, has high safety, can effectively slow down the generation of drug resistance of the mycoplasma gallisepticum to tylosin, and protects and maintains the effectiveness of tylosin.
Description
Technical Field
The invention relates to the technical field of drug resistance judgment, in particular to a drug resistance judgment standard test method of mycoplasma gallisepticum to tylosin.
Background
The mycoplasma of infected chicken is mainly divided into two types, namely, mycoplasma gallisepticum and mycoplasma synoviae, wherein the mycoplasma gallisepticum is also called mycoplasma gallisepticum and mainly infects chicken and turkey to cause chronic respiratory system infectious diseases of chicken, while the mycoplasma synoviae infects the chicken to cause the chicken to have symptoms such as arthritis, and tylosin is used as a special drug for animals, has a good treatment effect on respiratory diseases of livestock and poultry, is not easy to generate drug residues, has a weak toxic effect and extremely high safety, and further promotes the clinical application of the livestock and poultry in veterinary medicine.
Unreasonable medicine use of the current breeding industry in China already causes the generation of a medicine resistance phenomenon, medicine resistance detection has necessity and urgency, in order to slow down the generation of the medicine resistance of mycoplasma gallisepticum to tylosin, protect and maintain the effectiveness of tylosin, a medicine resistance monitoring measure needs to be carried out on the mycoplasma gallisepticum, a medicine resistance judgment standard is a scientific means for carrying out effective medicine resistance monitoring on bacteria, is a scientific criterion for judging the medicine resistance of the bacteria, analyzes the current situation and the development rule of the medicine resistance in order to monitor the generation of the medicine resistance, and guides clinical medicine more scientifically.
Disclosure of Invention
Aiming at the problems, the invention provides a drug resistance judgment standard test method of mycoplasma gallisepticum to tylosin, the drug resistance judgment standard of the mycoplasma gallisepticum to tylosin can be obtained by the method, stable drug data support can be provided for scientific culture, clinical medication can be more scientifically guided, the safety is high, the generation of drug resistance of the mycoplasma gallisepticum to tylosin can be effectively slowed down, and the effectiveness of the tylosin is protected and maintained.
The invention provides a drug resistance judgment standard test method of mycoplasma gallisepticum to tylosin, which comprises the following steps:
the method comprises the following steps: establishing a drug resistance detection method of tylosin to mycoplasma gallisepticum: the method comprises the following steps of (1) determining the MIC of tylosin to clinically isolated mycoplasma gallisepticum by taking staphylococcus aureus ATCC29213 as a quality control strain and taking tylosin as a quality control drug;
step two: establishing a wild-type cutoff value of tylosin for Mycoplasma gallisepticum, wherein the wild-type cutoff value is COWTRepresents: firstly, 107 strains of mycoplasma gallisepticum are separated and obtained from clinically infected chickens, the 107 strains of mycoplasma gallisepticum and 4 strains of standard strains stored in a laboratory form 111 strains of mycoplasma gallisepticum, then the MIC value of tylosin to the 111 strains of mycoplasma gallisepticum is determined by adopting a broth dilution method, the obtained MIC data is introduced into Ecoffinder software, nonlinear regression simulation is carried out, and wild type critical values under different confidence intervals are obtained;
step three: establishing pharmacodynamic critical value of tylosin to mycoplasma gallisepticum, wherein the pharmacodynamic critical value is usedCOPDRepresents: selects MIC90The strain is subjected to chick embryo virulence test, SPF-level chick embryos are selected to be subjected to chick embryo infection test, the chick embryos are incubated to 7 days old, the chick embryos are found under strong light and the positions of the chick embryos and the air chamber are marked, a yolk sac inoculation method is selected, and the inoculation dose is 106The CCU observes the chick embryo condition every day, records the death number of the chick embryos every day, finally selects the M17 strain to perform a PK-PD test, establishes a pharmacodynamic critical value, performs Monte Carlo simulation on the obtained PK-PD data by using Crystalball7 software, and selects the maximum MIC value corresponding to the standard reaching rate of more than 90 percent as the pharmacodynamic critical value;
step four: establishing a clinical critical value of tylosin to mycoplasma gallisepticum, wherein the clinical critical value is COCLRepresents: selecting 5 different MIC strains to perform chick embryo virulence test, selecting SPF (specific pathogen free) chick embryos to perform virulence identification on mycoplasma gallisepticum, incubating the chick embryos to 7 days old, finding out the chick embryo under strong light and marking the position of the chick embryo and an air chamber, and selecting a yolk sac inoculation method in the test, wherein the inoculation dose is 106CCU, observing the chick embryo condition every day, recording the death number of the chick embryos every day, finally selecting M1, M11, M17, M23 and M24 strains to carry out clinical treatment tests, and making a clinical critical value of tylosin to mycoplasma gallisepticum, wherein the clinical critical value made by the clinical tests can be verified by a 'Window W' calculation mode, a nonlinear regression mode and a binary tree (CART) analysis mode;
step five: and (3) establishing a final break point by utilizing a break point establishing flow chart according to a wild critical value of the tylosin to the mycoplasma gallisepticum, a pharmacodynamic critical value of the tylosin to the mycoplasma gallisepticum and a clinical critical value of the tylosin to the mycoplasma gallisepticum, so as to obtain a drug resistance judgment standard of the mycoplasma gallisepticum to the tylosin.
The further improvement lies in that: when the method for detecting the drug resistance of the tylosin to the mycoplasma gallisepticum is established in the first step, the mycoplasma gallisepticum can metabolize glucose to produce acid, the characteristic can cause the pH of a liquid culture medium to change, a color change unit titer test is required to be adopted for detection, and then the method is applied to indirect determination of the content of the culture.
The further improvement lies in that: and the separation and acquisition process of the mycoplasma gallisepticum in the second step comprises strain separation, strain purification, strain identification and strain preservation.
The further improvement lies in that: in the second step, firstly, the normal distribution of the wild strains is tested by logarithmic transformation, the MIC range of the wild strains belonging to the normal distribution is selected, the MIC distribution is determined to belong to the normal distribution, then, the nonlinear regression analysis is carried out, the MIC data converted into the logarithmic distribution is subjected to multiple fitting by using the nonlinear regression method, firstly, fitting is carried out from the minimum MIC to the MIC with the highest distribution frequency, then, an MIC gradient is increased progressively for each fitting until the difference between the number of the fitted strains and the number of the actual strains is minimum, then, the optimal fitting range is determined to be the minimum MIC to the MIC with the lowest difference between the number of the strains, then, the NORMINV function in an Excel formula is used for verifying the maximum upper limit value of the wild strains, then, the probability of verifying the maximum upper limit is determined by the NORMDST function, the finally determined critical value at least comprises 95 percent of the wild strains, and the statistical analysis steps are integrated, directly analyzing the MIC distribution data of the wild strain by using Excel software ECOFFinder analysis software to obtain the wild critical value of the tylosin to the mycoplasma gallisepticum.
The further improvement lies in that: the chicken embryo virulence test in the third step comprises the following specific processes: selecting SPF-grade chick embryo to perform chick embryo infection test, incubating the chick embryo to 7 days old, finding out the chick embryo under strong light and marking the position of the chick embryo and an air chamber, performing test on an aseptic operating platform, sterilizing the surface of the egg with about 75% of medical alcohol, wherein the marks of the chick embryo and the air chamber cannot be wiped off, and selecting yolk sac for inoculation in the test, wherein the inoculation dose is 106CCU, the inoculation method is as follows: and (3) after an eggshell at the end of the air chamber is disinfected, punching a small hole in the center of the air chamber, vertically puncturing the eggshell by about 3cm by using a No. 7 syringe needle, injecting about 0.2mL of inoculated bacteria liquid, sealing the port by paraffin, continuously hatching at 37 ℃ and turning the eggshell 2 times every day.
The further improvement lies in that: in the third step, the pharmacokinetic data is subjected to Monte Carlo simulation by using Crystalball7 software to obtain the standard-reaching rate of the pharmacokinetic target value under different MICs, and the maximum MIC value corresponding to the standard-reaching rate of more than 90% is selected as the pharmacodynamic critical value.
The further improvement lies in that: and when the clinical critical value of the tylosin to the mycoplasma gallisepticum is formulated in the fourth step, selecting the pathogenic mycoplasma gallisepticum according to the MIC distribution range, wherein the selection basis is as follows: the highest peak MIC, around the highest peak MIC, wild type cutoff, MIC50, MIC of MIC90, 5 MIC strains were selected for comprehensive consideration according to the specific MIC distribution.
The further improvement lies in that: the pathogenicity test of the strain in the fourth step is verified by a chicken embryo infection model test, the corresponding bacterial liquid in the logarithmic phase is inoculated to the chicken embryos through yolk sac injection, each chicken embryo is inoculated with 0.3mL of bacterial liquid with the same concentration, meanwhile, the aseptic blank broth abdominal cavity injection chicken embryos are used as a blank control group, the death condition of the chicken embryos is observed, and the strain with high mortality rate is selected.
The further improvement lies in that: the clinical critical value obtained by the clinical test in the fourth step can be substituted into a 'Window' algorithm, nonlinear regression in SPSS software and binary tree (CART) analysis of SalfordpredictiveModler software for verification, firstly, the result of clinical curative effect is analyzed by a 'Window' method, the parameter values Maxdiff and CAR are measured, the clinical critical value range is determined according to the parameter values, then, the data obtained by the nonlinear regression in SPSS software is fitted according to a formula between POC and MIC proposed by EUCAST, and Log is used2MIC is independent variable, POC is dependent variable, a model expression with the highest correlation coefficient is obtained to determine the clinical critical value range, and finally clinical test data are introduced into SalfordpredictiveModler software, and binary tree analysis (CART) is carried out by taking MIC as a predictive variable and POC as a target variable to determine the clinical critical value range.
The further improvement lies in that: comparing the wild type critical value, the pharmacodynamic critical value and the clinical critical value, wherein when the wild type critical value is equal to the clinical critical value, the value is a breakpoint value; and when the conditions are not equal, comparing the sizes of the three critical values, and determining a final breakpoint value as a drug resistance judgment standard of the mycoplasma gallisepticum to tylosin.
The invention has the beneficial effects that: the method can obtain the drug resistance judgment standard of the mycoplasma gallisepticum to tylosin, can provide stable drug data support for scientific culture, can scientifically guide clinical drug, has high safety, can effectively slow down the generation of drug resistance of the mycoplasma gallisepticum to tylosin, and protects and maintains the effectiveness of tylosin.
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FIG. 1 is a schematic flow chart of the method of the present invention for defining a break point according to CLSI.
FIG. 2 is a graph showing the results of non-linear regression using Ecoffinder software to establish a wild-type cut-off value in the method of the present invention.
FIG. 3 is a schematic diagram showing the result of the Monte Carlo simulation standard-reaching rate determination of pharmacodynamic critical value by using Crystalball7 software in the method of the present invention.
FIG. 4 is a graph showing the results of binary tree (CART) analysis using SalfordpredictiveModler software to establish clinical cut-off values in the method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to the results shown in fig. 1, 2, 3 and 4, the present example provides a standard test method for determining drug resistance of mycoplasma gallisepticum to tylosin, comprising the following steps:
the method comprises the following steps: establishing a method for detecting the drug resistance of tylosin to mycoplasma gallisepticum: the method comprises the steps of taking staphylococcus aureus ATCC29213 as a quality control strain and taking tylosin as a quality control drug, determining the MIC of the tylosin to clinically isolated mycoplasma gallisepticum, and when a method for detecting the drug resistance of the tylosin to the mycoplasma gallisepticum is established, as the mycoplasma gallisepticum can metabolize glucose to produce acid, the characteristic can cause the pH of a liquid culture medium to change, a color change unit titer test is required to be adopted for detection, and then the method is applied to indirect determination of the content of a culture;
step two: establishing a wild type critical value of tylosin to mycoplasma gallisepticum, wherein the wild type critical value is COWTRepresents: firstly, collecting samples for 20 times from Hubei, Hunan and Henan, collecting 4000 parts of samples in total, separating to obtain 107 suspected mycoplasma gallisepticum strains, and combining with 4 mycoplasma gallisepticum reference strains BG44T, HS, F and PG31 strains stored in a laboratory to form 111 mycoplasma gallisepticum strains, wherein the separation and acquisition processes of the mycoplasma gallisepticum comprise strain separation, strain purification, strain identification and strain storage, then measuring the MIC value of tylosin to the 111 mycoplasma gallisepticum strains by a broth dilution method, introducing the obtained MIC data into Ecoffinder software, firstly, carrying out normal distribution test on the wild strains by logarithm conversion, selecting the MIC range of the wild strains belonging to normal distribution, carrying out nonlinear regression analysis after determining that the MIC distribution belongs to normal distribution, fitting the MIC data converted into logarithmic distribution for many times by a nonlinear regression method, firstly, carrying out fitting from minimum MIC to MIC with highest distribution frequency, then, gradually increasing an MIC gradient in each fitting until the difference between the number of the fitted strains and the actual number of the strains is minimum, then determining that the best fitting range is from the minimum MIC to the minimum MIC of the difference between the number of the strains, then using an NORMINV function in an Excel formula to verify the maximum value upper limit of the wild strains, determining the probability of verifying the maximum upper limit through an NORMDST function, finally determining the critical value at least comprising 95% of the wild strains, integrating the statistical analysis steps by using the final CLSI, and directly analyzing the MIC distribution data of the wild strains through an Excel software ECOFFinder analysis software to obtain the wild critical value of tylosin to mycoplasma gallisepticum;
substituting MIC distribution of tylosin to mycoplasma gallisepticum into RAWCount by using Ecoffiner software, calculating cumulative distribution, fitting the cumulative distribution by using nonlinear regression to obtain a data fitting result of the tylosin to the mycoplasma gallisepticum, and automatically simulating upper limits of distribution of wild-type strains in different confidence intervals of 95.0%, 97.5%, 99.0%, 99.5% and 99.9% by using the software; taking the upper limit of the MIC distribution of the wild mycoplasma gallisepticum within a 95.0% confidence interval as a final wild critical value, namely 2 mug/mL;
step three: establishing pharmacodynamic critical value of tylosin to mycoplasma gallisepticum, wherein CO is used as the pharmacodynamic critical valuePDRepresents: selects MIC90The strain is subjected to chick embryo virulence test, SPF-level chick embryos are selected to be subjected to chick embryo infection test, the chick embryos are incubated to 7 days old, the chick embryos are found under strong light and the positions of the chick embryos and the air chamber are marked, a yolk sac inoculation method is selected, and the inoculation dose is 106CCU, observing chick embryo conditions every day, recording death number of chick embryos every day, finally selecting an M17 strain to perform a PK-PD test, and establishing a pharmacodynamic critical value, wherein the specific process of the chick embryo virulence test is as follows: selecting SPF-level chick embryo to perform chick embryo infection test, incubating the chick embryo to 7 days old, finding out the chick embryo under strong light and marking the position of the chick embryo and an air chamber, performing test on an aseptic operating platform, sterilizing the surface of the egg with about 75% of medical alcohol, wherein the marks of the chick embryo and the air chamber cannot be wiped off, and selecting yolk sac for inoculation in the test, wherein the inoculation dose is 106CCU, the inoculation method is as follows: after an eggshell at the end of the air chamber is disinfected, punching a small hole in the center of the air chamber, vertically puncturing the eggshell by about 3cm by using a No. 7 syringe needle, injecting about 0.2mL of inoculated bacteria liquid, sealing the opening by paraffin, continuously hatching at 37 ℃, and turning the eggshell 2 times every day;
performing in vitro pharmacodynamic study on M17 selected from the chick embryo virulence test to obtain pharmacodynamic data; performing pharmacokinetic study on chicken gavage tylosin to obtain pharmacokinetic data; simulating pharmacodynamic and pharmacokinetic data by utilizing WinNonlin software to obtain pharmacokinetic-pharmacodynamic parameters; selecting an inhibitory model SigmoidEmax model to predict the relationship between pharmacokinetic-pharmacodynamic parameters and antibacterial effects to obtain pharmacodynamic target values under different antibacterial effects;
monte Carlo simulation is respectively carried out on the mean value and the standard deviation of pharmacokinetic data AUC (acquired by central analysis) in lung tissues of an affected group by using Crystalball7 software to obtain simulated pharmacokinetic data of 10000 chickens, and the pharmacokinetic target value corresponding to the affected group E-3 is 51.19 by using Winnolin simulation, so that the standard reaching rate results under different MICs are calculated and shown in Table 1, and when the MIC is 2 mug/mL, the standard reaching rate is 0%; when the MIC is 1 mug/mL, the standard reaching rate is 93.68 percent and is higher than 90 percent; according to a pharmacodynamic critical value formulation rule, the maximum MIC value corresponding to the standard reaching rate of more than 90 percent is the pharmacodynamic critical value, and because the formulation of the critical value by adopting a simulation result of an infection target part is more meaningful, the finally formulated pharmacodynamic critical value of the tylosin to the mycoplasma gallisepticum is 1 microgram/mL;
TABLE 1 Monte Carlo simulated pharmacokinetic parameters achievement Rate at different MIC values
Step four: establishing a clinical critical value of tylosin to mycoplasma gallisepticum, wherein the clinical critical value is COCLRepresents: selecting pathogenic mycoplasma gallisepticum according to MIC distribution range, and selecting 5 different maximum peak MICs, about maximum peak MIC, wild critical value, MIC according to specific MIC distribution50、MIC90Performing chick embryo virulence test on the MIC strain, selecting SPF (specific pathogen free) chick embryos to perform chick embryo infection test, incubating the chick embryos to 7 days old, finding out the chick embryo embryos under strong light, marking the positions of the chick embryo embryos and an air chamber, and selecting a yolk sac inoculation method in the test, wherein the inoculation dose is 106CCU, observing the chick embryo condition every day, recording the death number of the chick embryos every day, finally selecting M1, M11, M17, M23 and M24 strains to carry out clinical treatment tests, and making clinical critical values of tylosin on mycoplasma gallisepticum, wherein the pathogenicity test of the strains is verified through a chick embryo infection model test, inoculating a bacterial liquid corresponding to a logarithmic growth phase into the chick embryos through yolk sac injection, inoculating 0.3mL of a bacterial liquid with the same concentration into each chick embryo, and simultaneously taking sterile blank broth abdominal injection of the chick embryos as a blank control group, observing the death condition of the chick embryos and selecting strains with high mortality;
calculating the drug delivery amount by using the PK-PD parameter breakpoint value, wherein the calculation formula is as follows:
wherein CL refers to the clearance rate of tylosin in lung tissues of chickens; (AUC/MIC)BPRefers to PK/PD parameter break values for different therapeutic effects; MIC is MIC of clinical mycoplasma gallisepticum; f is bioavailability; fu is the concentration ratio of the free drug;
AUC corresponding to tylosin achieving different antibacterial effects24hSubstituting the MIC value into a dose calculation equation to calculate the daily dose required by the tylosin to achieve different antibacterial effects, and when the MIC value of a clinically isolated target strain is 2 mug/mL, obtaining the results of each dose as shown in Table 2:
TABLE 2 dosage for different purposes
Three dosing doses (prevention, treatment, clearance) and bacterial growth at different dosing intervals were predicted by simulation with the mlxpore software, so that the optimal dosing schedule and dosing interval were finally established as 24h dosing intervals, 45.88mg/kgb.w dosing dose, 3 days of continuous dosing;
adapting test chicken for 7 days before test, wherein no chicken dies, normal drinking water and food, good health status, no abnormal behavior and no other bacterial infection phenomenon, artificially infecting chicken after adaptation period, injecting 1ml mycoplasma gallisepticum into trachea for infection, continuously infecting for 7 days, and obtaining mycoplasma gallisepticum titer of 1 × 109CCU, blank control group inoculated with blank FM-4 broth by the same tracheal injection;
after drug treatment, the cure rates of the sensitive strains M1 and M11 can reach 100%, the cure rate of the strain with relatively large MIC is lower than that of the sensitive strain, and the higher the MIC value is, the lower the cure rate is, which shows that the treatment effect of the drug has negative correlation with the MIC value of the infected strain, and when the MIC of the infected strain is 0.03 mu g/mL, the cure rate is 100%; when the MIC of the infected strain is 0.5 mu g/mL, the cure rate is 93.3 percent; when the MIC of the infected strain is 1 mug/mL, the cure rate is 80%, and the maximum MIC value when the cure rate is more than or equal to 90% is selected as the clinical critical value according to the clinical critical value, so that the clinical critical value of the tylosin to the mycoplasma gallisepticum is 0.5 mug/mL;
analyzing the obtained data on the result of clinical curative effect by using a Window method, measuring the parameter values Maxdiff and CAR of the data, and determining the clinical critical value range according to the parameter values; the number of the strain number corresponding to the Maxdiff and CAR is more than 4; in the research, the probabilities obtained by the CAR calculation method are respectively 1, 0.97, 0.92 and 0.84, but the condition that CAR falls in the minimum MIC distribution and the maximum MIC distribution needs to be excluded, and finally, the MIC value which is one gradient smaller than the maximum MIC can be selected as the upper limit of a critical value and is 1 mu g/mL; the corresponding numerical values obtained through the calculation of the Maxdiff algorithm are respectively 11, 13, 11, 3 and 0, and then the MIC distribution corresponding to the maximum Maxdiff value is selected as the lower limit of a critical value, namely 0.03 mu g/mL; the results obtained by the two algorithms are inconsistent, so the distribution range of the clinical critical value obtained by the method is 0.03-1 mu g/mL;
fitting the obtained data by nonlinear regression in SPSS software according to a formula between POC and MIC proposed by EUCAST, and using Log2MIC is independent variable, POC is dependent variable, and the model expression with the highest correlation coefficient with the test data is selected as y-82.992-9.364 x-1.628x2-0.91x3The resulting simulated R2A value of 0.975; according to the result expression, calculating the independent variable Log corresponding to the cure rate of 90 percent2MIC is-0.82, MIC is 0.57. mu.g/mL, so the clinical cutoff value is less than 0.57. mu.g/mL;
finally, clinical test data are brought into SalfordpredictiveModler software, MIC is taken as a prediction variable, POC is taken as a target variable, and binary tree (CART) analysis is carried out; from the regression tree, when MIC is 0.75 μ g/mL, the cure rate is 97.8%; when MIC is more than 0.75 μ g/mL, the cure rate is 76.7%; when the MIC is close to 0.75 mu g/mL, the cure rate is 89.3 percent; the clinical cutoff obtained by the CART assay should therefore be the value closest to and less than 0.75 μ g/mL;
by integrating the analysis method, the clinical critical value selection WindoW obtained by the Window method is 0.03-1 mug/mL; nonlinear regression analysis shows that the clinical critical value is less than 0.57 mu g/mL; CART regression tree analysis indicates that the clinical cutoff value should be a value close to and less than 0.75 μ g/mL. The clinical critical value of 0.5 mug/mL obtained by the clinical treatment test meets the above conditions, so the clinical critical value of tylosin to mycoplasma gallisepticum finally formulated by the test is 0.5 mug/mL;
step five: according to a wild critical value of the tylosin to the mycoplasma gallisepticum, a pharmacodynamic critical value of the tylosin to the mycoplasma gallisepticum and a clinical critical value of the tylosin to the mycoplasma gallisepticum, a tree diagram is made by using a breakpoint to make a final breakpoint, the wild critical value, the pharmacodynamic critical value and the clinical critical value are compared, and when the wild critical value is equal to the clinical critical value, the final breakpoint is a breakpoint value; when the situation is unequal, comparing the three critical values, and determining the final breakpoint value as the drug resistance judgment standard of the gallinaceous body to tylosin;
the wild-type Cutoff (CO) for Mycoplasma gallisepticum is known for tylosinWT) At 2. mu.g/mL, the pharmacodynamic Cutoff (CO) of tylosin for Mycoplasma gallisepticumPD) Is 1 ug/mL and the clinical Cutoff (CO) for tylosin against Mycoplasma gallisepticumCL) Is 0.5 mu g/mL, and the three critical values are brought into a flow chart established by a break point published by CLSI, which conforms to the COWT＞COPD＞COCLAnd selecting a wild critical value for the corresponding breakpoint value, and obtaining the drug resistance judgment standard of the mycoplasma gallisepticum to tylosin, wherein the drug resistance judgment standard is 2 mu g/mL.
The method can obtain the drug resistance judgment standard of the mycoplasma gallisepticum to tylosin, can provide stable drug data support for scientific culture, can scientifically guide clinical drug, has high safety, can effectively slow down the generation of drug resistance of the mycoplasma gallisepticum to tylosin, and protects and maintains the effectiveness of tylosin.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. The drug resistance judgment standard test method of mycoplasma gallisepticum to tylosin is characterized by comprising the following steps:
the method comprises the following steps: establishing a drug resistance detection method of tylosin to mycoplasma gallisepticum: the method comprises the following steps of (1) determining the MIC of tylosin to clinically isolated mycoplasma gallisepticum by taking staphylococcus aureus ATCC29213 as a quality control strain and taking tylosin as a quality control drug;
step two: establishing a wild-type cutoff value (CO) for tylosin against Mycoplasma gallisepticumWT): firstly, 107 strains of mycoplasma gallisepticum are separated and obtained from clinically infected chickens, the 107 strains of mycoplasma gallisepticum and 4 strains of standard strains stored in a laboratory form 111 strains of mycoplasma gallisepticum, then the MIC value of tylosin to the 111 strains of mycoplasma gallisepticum is determined by adopting a broth dilution method, the obtained MIC data is introduced into Ecoffinder software, nonlinear regression simulation is carried out, and wild type critical values under different confidence intervals are obtained;
step three: determination of pharmacodynamic Critical values (CO) of tylosin against Mycoplasma gallisepticumPD): selects MIC90The strain is subjected to chick embryo virulence test, SPF-level chick embryos are selected to be subjected to chick embryo infection test, the chick embryos are incubated to 7 days old, the chick embryos are found under strong light and the positions of the chick embryos and the air chamber are marked, a yolk sac inoculation method is selected, and the inoculation dose is 106CCU, observing the chick embryo condition every day, recording the death number of the chick embryos every day, finally selecting an M17 strain to perform a PK-PD test, establishing a pharmacodynamic critical value, performing Monte Carlo simulation on the obtained PK-PD data by using Crystalball7 software to obtain the standard reaching rate of a pharmacokinetic target value under different MICs, and selecting the maximum MIC value corresponding to the standard reaching rate of more than 90% as the pharmacodynamic critical value;
step four: clinical cutoff value (CO) of tylosin against Mycoplasma gallisepticum was establishedCL): 5 different MIC strains are selected for chick embryo virulence test,selecting SPF-level chick embryo for identifying the toxicity of mycoplasma gallisepticum, incubating the chick embryo to 7 days old, finding out the embryo of the chick embryo under strong light and marking the position between the embryo and the air chamber, and selecting a yolk sac inoculation method in an experiment, wherein the inoculation dose is 106CCU, observing chick embryo condition every day, recording death number of chick embryos every day, and finally selecting maximum peak MIC, about maximum MIC, wild critical value, MIC50、MIC90The 5 strains with different MICs are subjected to clinical treatment tests, the clinical critical value of the tylosin to the mycoplasma gallisepticum is established, and the clinical critical value established by the clinical tests can be verified by a 'Window' calculation, nonlinear regression and binary tree (CART) analysis mode;
step five: according to the wild critical value of the tylosin to the mycoplasma gallisepticum, the pharmacodynamics critical value of the tylosin to the mycoplasma gallisepticum and the clinical critical value of the tylosin to the mycoplasma gallisepticum, a final break point is formulated by utilizing a break point formulation flow chart to obtain a drug resistance judgment standard of the mycoplasma gallisepticum to the tylosin;
the chicken embryo virulence test in the third step comprises the following specific processes: selecting SPF-level chick embryo to perform chick embryo infection test, incubating the chick embryo to 7 days old, finding out the chick embryo under strong light and marking the position of the chick embryo and an air chamber, performing test on an aseptic operating platform, sterilizing the surface of the egg with 75% medical alcohol, wherein the marks of the chick embryo and the air chamber cannot be wiped off, and the test selects yolk sac inoculation with the inoculation dose of 106CCU, the inoculation method is as follows: after an eggshell at the end of the air chamber is disinfected, punching a small hole in the center of the air chamber, vertically puncturing the eggshell by a No. 7 syringe needle for 3cm, injecting 0.2mL of inoculated bacteria liquid, sealing the port by paraffin, continuously hatching at 37 ℃ and turning eggs for 2 times every day;
and when the clinical critical value of the tylosin to the mycoplasma gallisepticum is formulated in the fourth step, selecting the pathogenic mycoplasma gallisepticum according to the MIC distribution range, wherein the selection basis is as follows: maximum peak MIC, about maximum MIC, wild critical value, MIC50、MIC90Selecting 5 MIC strains according to the specific MIC distribution;
the pathogenicity test of the strain in the fourth step is verified by a chicken embryo infection model test, a bacterial liquid corresponding to a logarithmic phase is inoculated to chicken embryos through yolk sac injection, each chicken embryo is inoculated with 0.3mL of bacterial liquid with the same concentration, meanwhile, the chicken embryos are injected in an abdominal cavity by using sterile blank broth as a blank control group, the death condition of the chicken embryos is observed, and the strain with high mortality rate is selected;
the clinical critical value obtained by the clinical test in the fourth step can be substituted into a 'Window' algorithm, nonlinear regression in SPSS software and binary tree (CART) analysis of SalfordpredictiveModler software for verification, firstly, the result of clinical curative effect is analyzed by a 'Window' method, the parameter values MaxDeff and CAR are measured, the clinical critical value range is determined according to the parameter values, then, the data obtained by the nonlinear regression in SPSS software is fitted according to a formula between POC and MIC proposed by EUCAST, and Log is used2MIC is independent variable, POC is dependent variable, a model expression with the highest correlation coefficient is obtained to determine the clinical critical value range, and finally clinical test data are introduced into SalfordpredictiveModler software, and binary tree analysis (CART) is carried out by taking MIC as a predictive variable and POC as a target variable to determine the clinical critical value range.
2. The standard test method for drug resistance judgment of mycoplasma gallisepticum to tylosin according to claim 1, wherein: when the method for detecting the drug resistance of the tylosin to the mycoplasma gallisepticum is established in the first step, the mycoplasma gallisepticum can metabolize glucose to produce acid, the characteristic can cause the pH of a liquid culture medium to change, a color change unit titer test is required to be adopted for detection, and then the method is applied to indirect determination of the content of the culture.
3. The standard test method for drug resistance judgment of mycoplasma gallisepticum to tylosin according to claim 1, wherein: and the separation and acquisition process of the mycoplasma gallisepticum in the second step comprises strain separation, strain purification, strain identification and strain preservation.
4. The standard test method for drug resistance judgment of mycoplasma gallisepticum to tylosin according to claim 1, wherein: in the second step, firstly, the normal distribution of the wild strains is tested by logarithmic transformation, the MIC range of the wild strains belonging to the normal distribution is selected, the MIC distribution is determined to belong to the normal distribution, then, the nonlinear regression analysis is carried out, the MIC data converted into the logarithmic distribution is subjected to multiple fitting by using the nonlinear regression method, firstly, fitting is carried out from the minimum MIC to the MIC with the highest distribution frequency, then, an MIC gradient is increased progressively for each fitting until the difference between the number of the fitted strains and the number of the actual strains is minimum, then, the optimal fitting range is determined to be the minimum MIC to the MIC with the lowest difference between the number of the strains, then, the NORMINV function in an Excel formula is used for verifying the maximum upper limit value of the wild strains, then, the probability of verifying the maximum upper limit is determined by the NORMDST function, the finally determined critical value at least comprises 95 percent of the wild strains, and the statistical analysis steps are integrated, directly analyzing the MIC distribution data of the wild strain by using Excel software ECOFFinder analysis software to obtain the wild critical value of the tylosin to the mycoplasma gallisepticum.
5. The standard test method for drug resistance judgment of mycoplasma gallisepticum to tylosin according to claim 1, wherein: comparing the wild type critical value, the pharmacodynamic critical value and the clinical critical value, wherein when the wild type critical value is equal to the clinical critical value, the value is a breakpoint value; and when the conditions are not equal, comparing the sizes of the three critical values, and determining a final breakpoint value as a drug resistance judgment standard of the mycoplasma gallisepticum to tylosin.
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