CN110060784B - Drug resistance judgment standard test method for mycoplasma gallisepticum to danofloxacin - Google Patents
Drug resistance judgment standard test method for mycoplasma gallisepticum to danofloxacin Download PDFInfo
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Abstract
The invention discloses a drug resistance judgment standard test method of mycoplasma gallisepticum for danofloxacin, which comprises the following steps: establishing a wild critical value and a pharmacodynamic critical value of danofloxacin to the mycoplasma gallisepticum, establishing a dosing scheme of the danofloxacin to the mycoplasma gallisepticum, establishing a clinical critical value of the danofloxacin to the mycoplasma gallisepticum, and obtaining a drug resistance judgment standard of the danofloxacin to the mycoplasma gallisepticum through a dendrogram established by a CLSI sensitivity break point; the method can be used for preparing the drug resistance judgment standard of the mycoplasma gallisepticum to the danofloxacin, can provide stable drug data support for scientific breeding, can scientifically guide clinical drug administration, effectively treat chronic respiratory diseases such as nasosinusitis, tracheitis and air sacculitis of chickens, provides reference for clinical drug administration, can effectively relieve the drug resistance of the mycoplasma gallisepticum to the danofloxacin to a certain extent, and protects and maintains the effectiveness of the danofloxacin.
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 on danofloxacin.
Background
Mycoplasma gallisepticum, which has high pathogenicity to chickens and turkeys, has the greatest influence on poultry farming, and mainly causes chronic respiratory diseases to chickens, mainly sinusitis, tracheitis and cystitis.
With increasingly serious antibiotic resistance of mycoplasma gallisepticum, more and more drugs cannot be used for clinical treatment of mycoplasma gallisepticum, so that the generation and propagation of drug resistance are controlled and slowed down by strict scientific means, danofloxacin is widely applied to clinical treatment of mycoplasma gallisepticum diseases by veterinarians due to the characteristics of wide antibacterial spectrum, strong activity and the like, but with wide and unreasonable clinical medication of danofloxacin, drug resistance appears to a great extent, and in order to scientifically standardize the application of danofloxacin in the treatment of mycoplasma gallisepticum diseases, slow down the generation of drug resistance of danofloxacin and help veterinarians in clinical process, the invention provides a standard test method for judging drug resistance of mycoplasma gallisepticum to danofloxacin, so as to solve the defects in the prior art.
Disclosure of Invention
Aiming at the problems, the method can be used for preparing the drug resistance judgment standard of the mycoplasma gallisepticum to the danofloxacin, can provide stable drug data support for scientific breeding, can more scientifically guide clinical drug administration, can effectively treat chronic respiratory diseases such as nasosinusitis, tracheitis, bursitis and the like of chickens, has high safety, can effectively reduce the generation of drug resistance of the mycoplasma gallisepticum to the danofloxacin, protects and maintains the effectiveness of the danofloxacin, and improves the stable development and the economic benefit of the breeding industry.
The invention provides a standard test method for drug resistance judgment of mycoplasma gallisepticum on danofloxacin, which is characterized by comprising the following steps:
the method comprises the following steps: establishing wild type critical value of danofloxacin to mycoplasma gallisepticum, wherein CO is used as the wild type critical value WT Representing that 107 mycoplasma gallisepticum strains and 4 mycoplasma gallisepticum strains collected in a laboratory are separated and identified from a chicken larynx swab to form a mycoplasma gallisepticum strain sample with the total strain number of 111 strains, determining the drug sensitivity of danofloxacin to the 111 mycoplasma gallisepticum strains clinically separated by adopting a trace broth dilution method, counting the Minimum Inhibitory Concentration (MIC) result, analyzing MIC distribution by using ECOFFinder software, performing nonlinear regression analysis to obtain wild critical values under different confidence intervals, and taking the MIC under 95% or 97.5% confidence intervals as the final wild critical value of danofloxacin to mycoplasma gallisepticum;
step two: establishing pharmacodynamic critical value of danofloxacin to mycoplasma gallisepticum, wherein CO is used for the pharmacodynamic critical value PD Showing that MIC is selected according to MIC distribution and experimental requirements 90 Performing chick embryo virulence experiment on the treated strain, and screening out MIC with strong pathogenicity by the chick embryo virulence experiment 90 In vitro and in vivo pharmacodynamics of strain M19The experiment comprises the steps of determining MIC, MBC, MPC, PAE of danofloxacin to the M19 strain of the mycoplasma gallisepticum, and pharmacokinetics experiments of the danofloxacin in chicken, researching pharmacodynamics of the danofloxacin to the M19 strain and pharmacokinetics in vivo, selecting proper PK-PD parameters, and establishing pharmacodynamics critical value of the danofloxacin to the mycoplasma gallisepticum through software simulation;
step three: substituting PK-PD data of danofloxacin to mycoplasma gallisepticum into a dose equation, calculating to obtain the administration dose under bacteriostasis, sterilization and eradication, and making a reasonable administration scheme;
step four: establishing clinical critical value of danofloxacin to mycoplasma gallisepticum, wherein the clinical critical value is CO CL The method comprises the steps of (1) screening mycoplasma gallisepticum with strong pathogenicity under different MICs through a chick embryo virulence experiment, establishing a diseased model artificially infecting different MIC mycoplasma gallisepticum strains, treating through a danofloxacin treatment dose group, counting cure rates corresponding to the different MIC strain groups after infection administration, and taking the MIC with the cure rate more than or equal to 90% as a final clinical critical value through a statistical analysis method;
step five: and obtaining the drug resistance judgment standard of the danofloxacin to the mycoplasma gallisepticum through a dendrogram established by a CLSI sensitivity break point according to the wild critical value of the danofloxacin to the mycoplasma gallisepticum, the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum and the clinical critical value of the danofloxacin to the mycoplasma gallisepticum.
The further improvement is that: the specific process in the first step is as follows: separating and identifying 107 mycoplasma gallisepticum strains and 4 mycoplasma gallisepticum strains collected in a laboratory from a swab of the chicken larynx to form a mycoplasma gallisepticum strain sample with the total strain number of 111 strains, determining MIC data of danofloxacin to the mycoplasma gallisepticum by adopting a trace broth dilution method, analyzing the MIC distribution by using ECOFFinder software to obtain a distribution diagram, and respectively obtaining the MICs of the danofloxacin and the 4 mycoplasma gallisepticum strains 50 And MIC 90 Performing normal distribution test on MIC data by a nonlinear regression analysis method, converting MIC into logarithm with base 2, simulating to obtain an optimal fitting range, and verifying the maximum value upper limit of the wild strain by using an NORMINV function in an Excel formulaAnd determining the probability obtained by taking the maximum upper limit through a NORMDST function, wherein the finally determined critical value comprises at least 95 percent of wild strains to obtain the final wild critical value.
The further improvement is that: the chick embryo virulence experiment in the step two comprises the following specific processes: selecting 110 SPF healthy chick embryos, each strain is a group, each group comprises 10 strains, and injecting 0.5mL1 multiplied by 10 strains through yolk sac in a sterile manner 9 CCU/mL bacterial solution is dripped and sealed by paraffin, the survival state of chick embryos is observed regularly every day, the process needs to be carried out in a dark room for 7 days, the survival condition of each group of chick embryos is recorded every day, the chick embryos killed by the challenge strain are observed for pathological changes and then are dissected, and the infected parts are subjected to bacteria division culture, and the chick embryos are identified as positive mycoplasma gallisepticum by PCR, so that the infection is considered to be successful.
The further improvement lies in that: when the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum is established in the second step, an artificial infection model is established, 5mg/kgb.w of danofloxacin mesylate soluble powder is orally gavaged for healthy chickens and sick chickens respectively, the drug concentration in blood plasma and lung tissues at different time points is measured through high performance liquid chromatography, the pharmacokinetics in the blood plasma and the lung tissues is researched, an appropriate PK-PD parameter is selected through an intra-half-body sterilization curve, then the Winnolin software is used for simulating a Sigmoid Emax PK-PD model equation to calculate the pharmacodynamic target value of the danofloxacin to the mycoplasma gallisepticum under different antibacterial effects, and the Monte Carlo software is used for simulating and analyzing to obtain the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum.
The further improvement lies in that: the concrete process in the third step is as follows: corresponding to different antibacterial effect of danofloxacin (AUC) 24h /MIC) ex Substituting the values into a dosage calculation equation (1) to obtain the required dosage of the danofloxacin to achieve different antibacterial effects:
in formula (1), dose represents the administered Dose; (AUC/MIC) ex Represents a pharmacokinetic parameter in a half body; CL/F for biolistic(iv) volume clearance corrected by degree; MIC is the MIC value of the bacterium; fu denotes the protein binding rate.
The further improvement lies in that: the selection basis for screening different MICs in the fourth step is as follows: selecting the highest peak MIC, wild type critical value, most sensitive strain and MIC 50 、MIC 90 The MIC strains of (a), if duplicated, can be screened for different MIC strains based on a particular MIC profile.
The further improvement lies in that: after screening different MICs in the fourth step, infecting test group chickens with bacterial liquid corresponding to logarithmic growth phase in a trachea perfusion mode for 1 time/day for 1 week, observing the state of the chickens every day after infection, treating according to a treatment administration scheme obtained by PK-PD research after more than 80% of the chickens have mycoplasma gallisepticum typical symptoms, counting the cure rate of each group during treatment, analyzing and calculating MaxDeff and CAR by using a WindoW method according to the counted cure rate to obtain a selection WindoW of clinical critical values, and analyzing and reducing the range of the clinical critical values of the ofloxacin to the mycoplasma gallisepticum by using a CART regression tree.
The further improvement lies in that: in the fourth step, a selection WindoW of clinical critical values is obtained by analyzing and calculating Maxdiff and CAR by using a Window method, and the specific process of analyzing the range of the clinical critical values of danofloxacin to mycoplasma gallisepticum by using a CART regression tree is as follows: firstly, analyzing the clinical treatment result by using 'Window', calculating parameters MaxDiff and CAR, obtaining a selection WindoW of a clinical critical value formed by the MaxDiff and the CAR, fitting the obtained data by using nonlinear regression analysis in span software, obtaining a corresponding model expression by using MIC as an independent variable and POC as a dependent variable, then calculating a corresponding MIC value when the cure rate is 90%, finally, analyzing the obtained data by using a CART regression tree, segmenting the MIC to obtain cure rates of different MIC intervals, and taking the MIC value corresponding to the cure rate more than or equal to 90% as the clinical critical value of the danofloxacin to the mycoplasma gallisepticum to be formulated.
The beneficial effects of the invention are as follows: the method can be used for preparing the drug resistance judgment standard of the mycoplasma gallisepticum to the danofloxacin, can provide stable drug data support for scientific breeding, can scientifically guide clinical drug administration, and effectively treat chronic respiratory diseases such as nasosinusitis, tracheitis and air sacculitis of chickens, has high safety, can effectively slow down the drug resistance generation of the mycoplasma gallisepticum to the danofloxacin, provides reference for clinical drug administration, protects and maintains the effectiveness of the danofloxacin, and improves the stable development and economic benefit of breeding industry.
Drawings
FIG. 1 is a diagram showing the results of fitting by Ecoffinder software in the method of the present invention.
FIG. 2 is a first schematic of the achievement rates of danofloxacin in lung tissue for Mycoplasma gallisepticum at different MICs in accordance with the methods of the present invention.
FIG. 3 is a second graphical representation of the achievement rates of danofloxacin in lung tissue versus Mycoplasma gallisepticum for different MICs in a method of the invention.
FIG. 4 is a third graphical representation of the achievement rates of danofloxacin in lung tissue versus Mycoplasma gallisepticum at different MICs in accordance with the methods of the present invention.
Fig. 5 is a schematic diagram illustrating the MaxDiff method in the method of the present invention.
FIG. 6 is a schematic diagram illustrating the CAR method in the method of the present invention.
FIG. 7 is a graph showing the results of the CART model in the method of the present invention.
FIG. 8 is a Log of the method of the present invention 2 Nonlinear regression analysis simulation of MIC and cure rate (POC).
FIG. 9 is a schematic flow chart of the method for creating a break point according to CLSI.
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 obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Referring to fig. 1, 2, 3, 4, 5, 6, 7, 8, and 9, this example proposes a standard test method for drug resistance determination of mycoplasma gallisepticum to danofloxacin, which comprises the following steps:
the method comprises the following steps: establishing wild type critical value of danofloxacin to mycoplasma gallisepticum, wherein CO is used as the wild type critical value WT Showing that 107 mycoplasma gallisepticum strains and 4 mycoplasma gallisepticum strains collected in a laboratory are separated and identified from a chicken larynx swab to form a mycoplasma gallisepticum strain sample with the total strain number of 111 strains, measuring the MIC data of danofloxacin to the mycoplasma gallisepticum by adopting a trace broth dilution method, analyzing the MIC distribution by ECOFFinder software to obtain a distribution diagram, and respectively obtaining the MICs of the danofloxacin and the mycoplasma gallisepticum strains 50 And MIC 90 Performing normal distribution test on MIC data by a nonlinear regression analysis method, converting the MIC into a logarithm with the base of 2, simulating to obtain an optimal fitting range, verifying the maximum value upper limit of the wild strain by using an NORMINV function in an Excel formula, determining the probability obtained by taking the maximum upper limit by using an NORMDST function, finally determining the critical value to include at least 95% of the wild strain, analyzing and integrating by using CLSI, and directly analyzing and counting the MIC distribution data of the wild strain by using Excel software ECOFFinder analysis software to obtain the final wild critical value;
and (3) performing a normal distribution test on the sensitivity result of 111 strains of mycoplasma gallisepticum, and knowing that MIC distribution accords with normal distribution. Then, logarithmic transformation is carried out on the MIC distribution, numerical values after ECOFFinder simulation are shown in figures 1 and 2, nonlinear regression analysis simulation is carried out on the cumulative distribution to obtain a result shown in figure 3, the nonlinear regression analysis starts to fit from the distribution with the highest MIC, namely fitting is started from the MIC of 8 mug/mL, the difference value between the fitting value and the real value in different fitting ranges is shown as 'ABS (N-Nest') in figure 2, when the MIC is less than or equal to 1 mug/mL, the difference value between the real value and the fitting value is 0, the difference value is the smallest, therefore, the selected optimal fitting range is less than or equal to 1 mug/mL, and NORMINV function simulation is carried out to obtain the upper limit of distribution of different confidence intervals of 95%,97.5%,99.0% and 99.9% endogenetic strains, and the fitting result is shown in Table 1:
TABLE 1 results of the EcofFinder fitting of danofloxacin to Mycoplasma gallisepticum
Selecting MIC with 95% confidence interval as wild critical value, that is, the wild critical value of danofloxacin to mycoplasma gallisepticum is 1 mug/mL;
step two: establishing pharmacodynamic critical value of danofloxacin to mycoplasma gallisepticum, wherein CO is used for the pharmacodynamic critical value PD Showing that MIC is selected according to MIC distribution and experimental requirements 90 Performing chick embryo virulence experiment on the strains, selecting 110 SPF healthy chick embryos, taking one group of each strain and 10 strains in each group, and performing yolk sac sterile injection on the strains to obtain the strain with the density of 0.5mL1 multiplied by 10 9 CCU/mL bacterial solution is dripped and sealed by paraffin, the survival state of chick embryos is observed at regular time every day, the process is carried out in a dark room for 7 days, the survival condition of each group of chick embryos is recorded every day, the chick embryos killed by the challenge strain are observed for pathological change and then dissected, the infected parts are subjected to bacteria division culture, mycoplasma gallisepticum is identified as positive by PCR, the infection is considered to be successful, and the MIC with stronger pathogenicity is screened out by a chick embryo virulence experiment 90 Performing in vitro and in vivo pharmacodynamic experiments on a strain M19 at the department, determining MIC, MBC, MPC, PAE and in vivo pharmacokinetics experiments of danofloxacin on a strain M19 of mycoplasma gallisepticum, researching pharmacodynamics of danofloxacin on the strain M19, selecting proper PK-PD parameters, preparing pharmacodynamic critical values of danofloxacin on mycoplasma gallisepticum, establishing an artificial infection model when the pharmacodynamic critical values of danofloxacin on mycoplasma gallisepticum are established, orally irrigating 5mg/kgb.w of danofloxacin mesylate soluble powder for healthy chickens and sick chickens respectively, determining drug concentrations in plasma and lung tissues at different time points through high performance liquid chromatography, researching the pharmacokinetics in the plasma and lung tissues, selecting proper PK-PD parameters through an in vivo sterilization curve, simulating a pharmaceutical efficacy simulation EmaxPK-PD model equation by using Winnolin software to simulate a pharmaceutical efficacy target software of the danofloxacin model under different antibiotic effects of danofloxacin on mycoplasma gallisepticum, and simulating pharmacodynamic target software of Mongolian in vivo pharmacodynamics on mycoplasma gallisepticum to obtain pharmacodynamics target values of danofloxacin on mycoplasma gallisepticum by using a Troxiloid model software to analyze pharmacodynamicsA cutoff value;
substituting the pharmacokinetic data of danofloxacin in lung tissues and the pharmacodynamic data in a half body into Crystalball software to carry out Monte Carlo simulation to obtain the pharmacokinetic data of 10000 feather chickens, calculating the probability that AUC/MIC reaches pharmacodynamic targets under different MICs, and obtaining the critical values under different simulated pharmacodynamic targets as shown in Table 2:
TABLE 2 Standard Rate of Danofloxacin to Mycoplasma gallisepticum at different MICs (PTA) in diseased lung tissue of chickens
When E =0, the corresponding pharmacodynamic target value is 20.09, and the corresponding pharmacodynamic critical value is 1 mug/mL; when E = -3, the corresponding pharmacodynamic target value is 37.34, and the corresponding pharmacodynamic critical value is 0.5 mu g/mL; when E = -4, the corresponding pharmacodynamic target value is 46.67, the corresponding pharmacodynamic critical value is 0.25 mu g/mL, according to the pharmacodynamic target value when E = -3, 99.9% of bacteria are killed to reach the clinical treatment effect, the drug resistance standard is made, and the pharmacodynamic critical value of danofloxacin to the mycoplasma gallisepticum is 0.5 mu g/mL;
step three: formulating a drug administration scheme of danofloxacin to mycoplasma gallisepticum, substituting PK-PD data of danofloxacin to mycoplasma gallisepticum into a dose equation according to the pharmacodynamic critical value of danofloxacin to mycoplasma gallisepticum, calculating to obtain the drug administration dose under bacteriostasis, sterilization and eradication, and formulating a reasonable drug administration scheme;
corresponding to different purposes of antibacterial effect of danofloxacin (AUC) 24h /MIC) ex Substituting the values into a dosage calculation equation (1) to obtain the required dosage of the danofloxacin to achieve different antibacterial effects:
in the formula (1), dose represents the administration Dose; (AUC/MIC) ex Represents a pharmacokinetic parameter in a half body; CL/F stands for rawBioavailability-corrected body clearance; MIC is the MIC value of the bacterium; fu represents the protein binding rate;
step four: establishing clinical critical value of danofloxacin to mycoplasma gallisepticum, wherein the clinical critical value is CO CL Showing that 5 MICs covering the whole drug sensitivity experiment distribution are selected according to the following selection criteria: selecting the highest peak MIC, wild type critical value, most sensitive strain and MIC 50 、MIC 90 If the MIC strain is repeated, different MIC strains can be comprehensively considered and screened according to specific MIC distribution, mycoplasma gallisepticum with stronger pathogenicity under different MICs is screened through an embryotoxicity experiment, a chicken strain diseased model for artificially infecting different MIC mycoplasma gallisepticum is established, a danofloxacin treatment dose group is used for treatment, the cure rates corresponding to different MIC strain groups after infection administration are counted, the MIC when the cure rate is more than or equal to 90% is taken as a final clinical critical value, a bacterial liquid corresponding to the logarithmic growth phase is infected into a test group chicken in a mode of tracheal perfusion, 1 time/day and 1 week of infection, the broiler chicken state is observed every day after infection, after more than 80% of mycoplasma gallisepticum typical symptoms appear, treatment is carried out according to a treatment administration scheme obtained through PK-PD research, the cure rate of each group is counted during treatment, the obtained clinical cure rate is analyzed and calculated through a WindoW method, a selection WindoW of the clinical critical value is obtained, a CARxWF regression tree is used for obtaining a CARxWF, the CAR, the clinical analysis range of the CARxWF is obtained, and the clinical analysis range of the obtained through a WindoW method, and the WindoW method, the clinical analysis of the carrier regression WindoW of the obtained clinical mycoplasma gallisepticum is taken as a clinical analysis range of a CARxWF, and the clinical analysis WindoW, the clinical analysis range of the method, the clinical Mycoplasma gallisepticum is obtained, the clinical Mycoplasma gallicin is obtained, and the clinical analysis range of a clinical Mycoplasma gallicin, the clinical Mycoplasma gallicin threshold value, the clinical Mycoplasma gallicin is obtained: firstly, analyzing the clinical treatment result by using 'Window', calculating parameters MaxDiff and CAR, obtaining a selection WindoW of a clinical critical value formed by the MaxDiff and the CAR, fitting the obtained data by using nonlinear regression analysis in span software, taking MIC as an independent variable and POC as a dependent variable to obtain a corresponding model expression, then calculating a corresponding MIC value when the cure rate is 90%, finally analyzing the obtained data by using a CART regression tree, segmenting the MIC to obtain cure rates of different MIC intervals, and taking the cure rate which is more than or equal to 90% and corresponds toThe MIC value of (a) is the clinical critical value of the danofloxacin to the mycoplasma gallisepticum to be established;
the method comprises the following steps of carrying out adaptation treatment on experimental chickens, wherein the adaptation period is one week, in the adaptation period, drinking water is freely taken, ventilation and heat preservation measures are good, the drinking water taking condition is normal, the mental state is good, no abnormal behavior appears, after one week of adaptation, 5 strains M57, M19, M73, M24 and M2 with different MICs are selected to infect common broiler chickens between 525g, the infection mode is tracheal perfusion, a blank control group is perfused with FM-4 blank broth in the same mode, during artificial infection, the chickens in the blank control group are good in health state, drinking water and drinking water have no other abnormal behavior, the weight gain is normal compared with a treatment-free group, killing and observing lung tissues, trachea and respiratory tract after the experiment is finished, no eye pathological change appears, most broiler chickens begin to suffer from listlessness on the 4 th day after infection, water drinking is increased, the drinking water intake is reduced, coughing, rhinorrhea, respiration has obvious coughing, sneezing and sneezing, growth is stopped, the chickens do not drink water, the PD in the 4 th day after infection, the PD does not reach the clinical treatment symptom, the PD-PK is judged, and the use score is more recommended by the clinical use standard: 16.60mg/kg once a day, orally irrigating stomach, counting the cure rate, after 24 hours after administration, turning the mental state of most broilers, trying to drink water and eat, obviously reducing rale, slowly and normally breathing, continuing administration and observation until symptoms completely disappear, after the symptoms disappear, continuing to stop administration and observe the state of chicken flocks, killing all the chickens after a negative infection non-administration group is infected for 10 days, observing pathological changes of air bags and scoring, recording the air bag injury reduction rate according to the average scoring, adopting a disposable nasal cavity flocking swab to take a laryngeal swab of 5 groups of chickens before and after infection and before and after administration, immediately extracting DNA, carrying out real-time fluorescence quantitative PCR measurement, then counting indexes such as cure rate, effective rate, average weight gain and air bag average injury meter in each test group, and obtaining a table 3:
TABLE 3 therapeutic Effect of danofloxacin on different MIC Mycoplasma gallisepticum
As can be seen from Table 3, the cure rate of clinical treatment is also significantly reduced as the MIC value of the infecting strain is increased; along with the increase of the MIC value, the degree of air bag damage is more serious, and the weight gain is in a decreasing state;
by integrating the clinical critical value obtained by the CART analysis algorithm, the clinical critical value range of the danofloxacin to the mycoplasma gallisepticum can be known to be between 0.25 mu g/mL and 0.63 mu g/mL. The MIC of 5 strains of bacteria was subjected to base 2 logarithmic transformation, a relationship graph between logarithmic MIC values and cure rates was plotted, and as shown in FIG. 4, nonlinear regression analysis was performed on the graph to obtain an expression: y =71.708-9.463X +0.453X 2 +0.206X 3 Coefficient of correlation R 2 Is 0.98, can be used for describing the corresponding relation between MIC logarithm and cure rate, when the cure rate is 90%, the MIC logarithm value taking 2 as the base is-1.9, the obtained MIC value is 0.27 mu g/mL, therefore, the clinical critical value is 0.4 mu g/mL, when the MIC is 0.5 mu g/mL, the nonlinear regression equation is substituted, the obtained POC is 81.418%, which does not accord with the experimental requirements, therefore, the clinical critical value of the final danofloxacin to the mycoplasma gallisepticum is 0.25 mu g/mL;
step five: according to the wild critical value of danofloxacin to mycoplasma gallisepticum, the pharmacodynamics critical value of danofloxacin to mycoplasma gallisepticum and the clinical critical value of danofloxacin to mycoplasma gallisepticum, the drug resistance judgment standard of danofloxacin to mycoplasma gallisepticum is obtained through a dendrogram made by a CLSI sensitivity break point;
the wild critical value of the danofloxacin to the mycoplasma gallisepticum is 1 mu g/mL, the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum is 0.5 mu g/mL, the clinical critical value of the danofloxacin to the mycoplasma gallisepticum is 0.25 mu g/mL, the comparison of the sizes of the three results shows that the wild critical value of the danofloxacin to the mycoplasma gallisepticum is larger than the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum and larger than the clinical critical value, and the final drug resistance judgment standard of the danofloxacin to the mycoplasma gallisepticum is 1 mu g/mL according to a dendrogram established by CLSI sensitivity break.
The method can be used for preparing the drug resistance judgment standard of the mycoplasma gallisepticum to the danofloxacin, can provide stable drug data support for scientific breeding, can scientifically guide clinical drug administration, and effectively treat chronic respiratory diseases such as nasosinusitis, tracheitis and air sacculitis of chickens, has high safety, can effectively reduce the drug resistance of the mycoplasma gallisepticum to the danofloxacin, provides reference for clinical drug administration, protects and maintains the effectiveness of the danofloxacin, and improves the stable development and economic benefit of breeding industry.
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 given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The standard test method for drug resistance judgment of mycoplasma gallisepticum on danofloxacin is characterized by comprising the following steps:
the method comprises the following steps: wild-type cutoff values for danofloxacin on mycoplasma gallisepticum were established: separating and identifying 107 mycoplasma gallisepticum strains from a chicken larynx swab and 4 mycoplasma gallisepticum strains collected in a laboratory to form a mycoplasma gallisepticum strain sample with a total strain number of 111 strains, determining the drug sensitivity of danofloxacin to the 111 clinical separated mycoplasma gallisepticum strains by adopting a trace broth dilution method, counting the Minimum Inhibitory Concentration (MIC) result, analyzing MIC distribution by using ECOFFinder software, performing nonlinear regression analysis to obtain wild critical values under different confidence intervals, and taking the MIC under 95% or 97.5% confidence intervals as the final wild critical value of danofloxacin to mycoplasma gallisepticum;
step two: establishing a pharmacodynamic critical value of danofloxacin to mycoplasma gallisepticum: MIC selection based on MIC distribution and experimental requirements 90 Performing chick embryo toxicity test on the obtained strain, and screening by using the chick embryo toxicity testMIC with strong pathogenicity 90 Carrying out in-vitro and in-vivo pharmacodynamic experiments on the strain M19, determining MIC, MBC, MPC, PAE and pharmacokinetics experiments of the danofloxacin on the strain M19 of the mycoplasma gallisepticum in a chicken body, researching the pharmacodynamics and pharmacokinetics of the danofloxacin on the strain M19 in the chicken body, selecting proper PK-PD parameters, and simulating and making a pharmacodynamics critical value of the danofloxacin on the mycoplasma gallisepticum by software;
step three: substituting PK-PD data of danofloxacin to mycoplasma gallisepticum into a dose equation, calculating to obtain the administration dose under bacteriostasis, sterilization and eradication, and making a reasonable administration scheme;
step four: clinical threshold for danofloxacin against mycoplasma gallisepticum was established: screening mycoplasma gallisepticum with strong pathogenicity under different MICs through a chick embryo virulence experiment, establishing a mycoplasma gallisepticum strain disease model for artificially infecting different MICs, treating through a danofloxacin treatment dose group, counting cure rates corresponding to different MIC strain groups after infection and administration, and taking the MIC with the cure rate more than or equal to 90% as a final clinical critical value through a statistical analysis method;
step five: and obtaining the drug resistance judgment standard of the danofloxacin to the mycoplasma gallisepticum through a dendrogram established by a CLSI sensitivity break point according to the wild critical value of the danofloxacin to the mycoplasma gallisepticum, the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum and the clinical critical value of the danofloxacin to the mycoplasma gallisepticum.
2. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 1, wherein the standard test method comprises: the specific process in the first step is as follows: separating and identifying 107 mycoplasma gallisepticum strains and 4 mycoplasma gallisepticum strains collected in a laboratory from a swab of the chicken larynx to form a mycoplasma gallisepticum strain sample with the total strain number of 111 strains, determining MIC data of danofloxacin to the mycoplasma gallisepticum by adopting a trace broth dilution method, analyzing the MIC distribution by using ECOFFinder software to obtain a distribution diagram, and respectively obtaining the MICs of the danofloxacin and the 4 mycoplasma gallisepticum strains 50 And MIC 90 And then carrying out normal distribution on MIC data by a nonlinear regression analysis methodAnd (3) testing, converting MIC into logarithm with base 2, simulating to obtain an optimal fitting range, verifying the maximum value upper limit of the wild type strain by using a NORMINV function in an Excel formula, determining the probability obtained by taking the maximum upper limit through the NORMDST function, and obtaining the final wild type critical value, wherein the finally determined critical value comprises at least 95% of the wild type strain.
3. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 1, wherein the standard test method comprises: the chick embryo virulence experiment in the step two comprises the following specific processes: selecting 110 SPF healthy chick embryos, each strain is a group, each group comprises 10 strains, and injecting 0.5mL1 multiplied by 10 strains through yolk sac in a sterile manner 9 CCU/mL bacterial liquid is dripped and sealed by paraffin, the survival state of the chick embryos is observed regularly every day, the process is carried out in a dark room for 7 days, the survival condition of each group of chick embryos is recorded every day, the chick embryos killed by the challenge strain are observed for pathological changes and then are dissected, the infected parts are subjected to bacteria division culture, and if the chick embryos are identified to be mycoplasma gallisepticum positive through PCR, the infection is considered to be successful.
4. The standard test method for determination of resistance of mycoplasma gallisepticum to danofloxacin according to claim 1, wherein: when the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum is established in the second step, an artificial infection model is established, 5mg/kgb.w of danofloxacin mesylate soluble powder is orally administrated to healthy chickens and sick chickens respectively, the drug concentration in blood plasma and lung tissues at different time points is measured through high performance liquid chromatography, the pharmacokinetics in the blood plasma and the lung tissues is researched, an appropriate PK-PD parameter is selected through an intra-body sterilization curve, then the winolin software is used for simulating a Sigmoid Emax PK-PD model equation to calculate the pharmacodynamic target value of the danofloxacin to the mycoplasma gallisepticum under different antibacterial effects, and the Monte Carlo software is used for simulating and analyzing to obtain the pharmacodynamic critical value of the danofloxacin to the mycoplasma gallisepticum.
5. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 1The method is characterized in that: the concrete process in the third step is as follows: corresponding to different antibacterial effect of danofloxacin (AUC) 24h /MIC) ex Substituting the values into a dosage calculation equation (1) to obtain the required dosage of the danofloxacin to achieve different antibacterial effects:
in formula (1), dose represents the administered Dose; (AUC/MIC) ex Represents a pharmacokinetic parameter in a half body; CL/F represents bioavailability-corrected body clearance; MIC is the MIC value of the bacterium; fu denotes the protein binding rate.
6. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 1, wherein the standard test method comprises: the selection basis for screening different MICs in the fourth step is as follows: selecting the highest peak MIC, wild type critical value, most sensitive strain and MIC 50 、MIC 90 The MIC strains of (a), if duplicated, can be screened for different MIC strains based on a particular MIC profile.
7. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 6, wherein the test method comprises: after different MICs are screened in the fourth step, the corresponding bacteria liquid in the logarithmic growth phase is used for infecting test group chickens in a trachea perfusion mode for 1 time/day and 1 week after infection, the state of the chickens is observed every day, after more than 80% of the chickens have mycoplasma gallisepticum typical symptoms, treatment is carried out according to a treatment administration scheme obtained by PK-PD research, the cure rate of each group is counted during the treatment period, maxdiff and CAR are analyzed and calculated by using a WindoW method according to the counted cure rate, a selection WindoW of clinical critical values is obtained, and then a CART regression tree is used for analyzing and reducing the range of the clinical critical values of the ofloxacin to the mycoplasma gallisepticum.
8. The standard test method for determining resistance of mycoplasma gallisepticum to danofloxacin according to claim 7, wherein the test method comprises: in the fourth step, a selection WindoW of clinical critical values is obtained by analyzing and calculating Maxdiff and CAR by using a Window method, and the specific process of analyzing the range of the clinical critical values of danofloxacin to mycoplasma gallisepticum by using a CART regression tree is as follows: firstly, analyzing clinical treatment results by using 'Window', calculating parameters MaxDeff and CAR, obtaining a selection WindoW of clinical critical values formed by the MaxDeff and the CAR, fitting obtained data by using nonlinear regression analysis in span software, obtaining corresponding model expressions by using MIC as an independent variable and POC as a dependent variable, then calculating the corresponding MIC value when the cure rate is 90%, finally analyzing the obtained data by using a CART regression tree, segmenting the MIC to obtain the cure rates of different MIC intervals, and taking the MIC value corresponding to the cure rate of more than or equal to 90% as the clinical critical value of the danofloxacin to the mycoplasma gallisepticum to be formulated.
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