CN110157773A - Drug resistance criterion test method of the chicken virus mycoplasma to tylosin - Google Patents
Drug resistance criterion test method of the chicken virus mycoplasma to tylosin Download PDFInfo
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Abstract
The invention discloses chicken virus mycoplasmas to the drug resistance criterion test method of tylosin, flow chart including establishing the break formulation that tylosin announces the clinical threshold and reference CLSI of chicken virus mycoplasma the pharmacodynamics critical value of chicken virus mycoplasma, formulation tylosin the wild type critical value of chicken virus mycoplasma, formulation tylosin the Drug Resistance Detection method of chicken virus mycoplasma, formulation tylosin, meets COWT> COPD> COCL, corresponding break value should select wild type critical value, obtain drug resistance criterion of the chicken virus mycoplasma to tylosin；By the method for the invention it can be concluded that drug resistance criterion of the chicken virus mycoplasma to tylosin; stable administration data can be provided for scientific culture to support; it more scientific can carry out instructing clinical application; it is highly-safe; chicken virus mycoplasma can effectively be slowed down to generate the drug resistance of tylosin, protection and the validity for maintaining tylosin.
Description
Technical field
Drug resistance criterion the present invention relates to drug resistance judgment technology field more particularly to chicken virus mycoplasma to tylosin
Test method.
Background technique
The Mycoplasma of infection chicken is broadly divided into two kinds of mycoplasma gallisepticum and chicken synovial membrane Mycoplasma, wherein the mould shape of chicken sepsis
Body is also known as chicken virus mycoplasma, main infection chicken and turkey, causes the chronic respiratory systematic infection disease of chicken, and synovial membrane Mycoplasma is then
Infection chicken makes it the symptoms such as arthritis occur, and tylosin has the respiratory disease of livestock and poultry good as animal specific medicine
Good therapeutic effect, and it is not likely to produce medicament residue, toxic effect is weak, has high safety, further promotes it
Application in veterinary clinic.
The unreasonable generation for already leading to drug resistance phenomenon of the current aquaculture medication in China, Drug Resistance Detection have necessity and
Urgency, generates to slow down chicken virus mycoplasma to the drug resistance of tylosin, and protection and the validity for maintaining tylosin need
To carry out drug resistance surveillance and control measure to chicken virus mycoplasma, drug resistance criterion be to carry out the section of effective drug resistance monitoring to bacterium to learn to do
Section, is the scientific principles for judging bacterial drug resistance, generates to monitor drug resistance, analyzes drug resistance status and the rule of development, more section
That learns instructs clinical application, and the present invention proposes chicken virus mycoplasma to the drug resistance criterion test method of tylosin, to solve
Shortcoming in the prior art.
Summary of the invention
In view of the above-mentioned problems, the present invention proposes that chicken virus mycoplasma to the drug resistance criterion test method of tylosin, leads to
The method of the present invention is crossed it can be concluded that drug resistance criterion of the chicken virus mycoplasma to tylosin, can provide stabilization for scientific culture
Administration data support, more scientific can carry out instructing clinical application, it is highly-safe, can effectively slow down chicken virus mycoplasma to Thailand
The drug resistance of happy rhzomorph generates, protection and the validity for maintaining tylosin.
The present invention proposes chicken virus mycoplasma to the drug resistance criterion test method of tylosin, comprising the following steps:
Step 1: tylosin is established to the Drug Resistance Detection method of chicken virus mycoplasma: with staphylococcus aureus
For ATCC29213 as Quality-control strains, tylosin measures tylosin to being clinically separated chicken virus mycoplasma as Quality Control drug
MIC；
Step 2: formulating tylosin to the wild type critical value of chicken virus mycoplasma, wherein wild type critical value COWTTable
Show: separation obtains out 107 plants of chicken virus mycoplasma first from clinical infection chicken, the 4 plants of groups of reference culture saved with laboratory
At totally 111 plants of chicken virus mycoplasmas, broth dilution method determination MIC value of the tylosin to 111 plants of chicken virus mycoplasmas is then used,
It brings obtained MIC data into Ecoffinder software, carries out linear regression simulation, obtain the wildness under different confidence intervals
Critical value；
Step 3: pharmacodynamics critical value of the tylosin to chicken virus mycoplasma, pharmacodynamics critical value CO are formulatedPDIt indicates:
Have chosen MIC90Bacterial strain carry out chicken embryo virulence experiment, the progress egg infectious experiments of SPF grade chicken embryos are selected, by hatching to 7
Age in days finds the embryo of chicken embryo under intense light irradiation and marks its position with gas chamber, selects yolk sac inoculation method, dosage of inoculation
It is 106CCU, it is daily to observe chicken embryo situation, daily chicken embryo death quantity is recorded, final choice M17 bacterial strain carries out PK-PD test,
Pharmacodynamics critical value is formulated, obtained PK-PD data are subjected to Monte Carlo simulation using Crystalball7 software, selection reaches
Mark rate is pharmacodynamics critical value in 90% or more corresponding maximum MIC value；
Step 4: clinical threshold of the tylosin to chicken virus mycoplasma, clinical threshold CO are formulatedCLIt indicates: choosing
The bacterial strain of 5 different MIC carries out chicken embryo virulence experiment, and SPF grade chicken embryos is selected identify the virulence of chicken virus mycoplasma,
By hatching to 7 ages in days, the embryo of chicken embryo is found under intense light irradiation and marks its position with gas chamber, experimental selection yolk bag
Inoculation method, dosage of inoculation 106CCU, it is daily to observe chicken embryo situation, record daily chicken embryo death quantity, final choice M1,
M11, M17, M23 and M24 bacterial strain carry out Clinical Treatment Test, formulate tylosin to the clinical threshold of chicken virus mycoplasma, face
The clinical threshold that bed test is formulated can be calculated by " WindoW ", nonlinear regression and binary tree (CART) analysis mode carry out
Verifying；
Step 5: according to tylosin to the wild type critical value of chicken virus mycoplasma, tylosin to chicken virus mycoplasma
It is final to formulate to formulate flow chart using break to the clinical threshold of chicken virus mycoplasma for pharmacodynamics critical value and tylosin
Break obtains drug resistance criterion of the chicken virus mycoplasma to tylosin.
Further improvement lies in that: when establishing tylosin in the step 1 to chicken virus mycoplasma Drug Resistance Detection method, by
Acid is produced in the metabolizable glucose of chicken virus mycoplasma, which can cause fluid nutrient medium that pH variation occurs, and need to become using color
Change unit titer test to test, then reapply in the content of indirect determination culture.
Further improvement lies in that: the separation acquisition process of chicken virus mycoplasma includes strain separating, strain in the step 2
Purifying, strain idenfication and fungi preservation.
Further improvement lies in that: the distribution of wild-type strain normality is carried out by Logarithm conversion first in the step 2
It examines, selection belongs to the wild-type strain MIC range of normal distribution, after determining that MIC distribution belongs to normal distribution, carries out non-linear
Regression analysis is repeatedly fitted the MIC data for being converted to log series model with non-linear regression method, first from minimum
MIC is fitted to the highest MIC of distribution frequency, is then fitted every time and is incremented by a MIC gradient, until the number of strains of fitting
It is minimum with practical number of strains difference, then determine that best fit range is that the smallest MIC is the smallest to number of strains difference
MIC reuses NORMINV function in Excel formula, verifies the maximum value upper limit of wild-type strain, then pass through NORMDST letter
Number determines the probability of verifying maximum upper limit, and finally determining critical value should include at least 95% wild-type strain, using most
CLSI integrates these statistical analysis steps afterwards, analyzes software directly to open country by an Excel software ECOFFinder
Raw type bacterial strain MIC distributed data is analyzed, and obtains tylosin to the wild type critical value of chicken virus mycoplasma.
Further improvement lies in that: chicken embryo virulence experiment detailed process in the step 3 are as follows: select SPF grades of chicken embryos and carry out chicken embryo
Hatching to 7 ages in days is found the embryo of chicken embryo under intense light irradiation and marks its position with gas chamber, in nothing by infection experiment
Bacterium station is tested, and is sterilized egg surface with 75% or so medicinal alcohol, can not be wiped embryo and air bag mark in the process
Note, experimental selection yolk sac inoculation, dosage of inoculation 106CCU, inoculation method are as follows: after the disinfection of gas chamber end eggshell, in gas chamber center
An aperture is made a call to, is vertically pierced into about 3cm, injection inoculation bacterium solution about 0.2mL with No. 7 syringe needles, paraffin sealing continues at 37 DEG C
Hatching, daily egg-turning 2 times.
Further improvement lies in that: Meng Teka is carried out to pharmacokinetic data available using Crystalball7 software in the step 3
Lip river simulates to obtain compliance rate of the pharmacokinetics target value at different MIC, selects compliance rate in 90% or more corresponding maximum MIC value
For pharmacodynamics critical value.
Further improvement lies in that: when formulating clinical threshold of the tylosin to chicken virus mycoplasma in the step 4, root
According to MIC distribution, the chicken virus mycoplasma with pathogenicity, selection gist: peak-peak MIC, peak-peak MIC or so are selected
, wild type critical value, MIC50、MIC90MIC bacterial strain, according to specific MIC distribution comprehensively consider selection 5 MIC bacterial strains.
Further improvement lies in that: the Virulence detection of bacterial strain is carried out by egg infectious model test in the step 4
Verifying, corresponds to bacterium solution for logarithmic growth phase and is inoculated in chicken embryo by yolk sac injection, each same concentration of egg inoculation 0.3mL
Bacterium solution, while chicken embryo is injected intraperitoneally as blank control group using sterile blank meat soup, chicken embryo death situation is observed, the death rate is selected
High bacterial strain.
Further improvement lies in that: the clinical threshold that clinical test obtains in the step 4 can bring " WindoW " calculation into
The binary tree (CART) of method, the nonlinear regression in SPSS software and SalfordPredictiveModeler software, which is analyzed, to carry out
Verifying, is first analyzed with result of " WindoW " method to clinical efficacy first, measures its parameter value MaxDiff and CAR, root
Clinical threshold range is determined according to parameter value, and the formula between POC and MIC proposed further according to EUCAST utilizes SPSS software
In nonlinear regression its data obtained is fitted, with Log2MIC is independent variable, and POC is dependent variable, obtains related coefficient
Highest model expression determines clinical threshold range, finally again brings clinical testing data into
In SalfordPredictiveModeler software, using MIC as predictive variable, POC is target variable, is carried out binary tree (CART)
Analyze the range to determine clinical threshold.
Further improvement lies in that: it is first that wild type critical value, pharmacodynamics critical value and clinic is critical in the step 5
Value is compared, and when there is wild type critical value equal to clinical threshold situation, which is break value；It is unequal when occurring
When situation, then compare the size of three critical values, determines that final break value is drug resistance of the chicken virus mycoplasma to tylosin
Criterion.
Beneficial effects of the present invention: by the method for the invention it can be concluded that chicken virus mycoplasma determines the drug resistance of tylosin
Standard can provide stable administration data for scientific culture and support, can be more scientific carry out instructs clinical application, safety
Height can effectively slow down chicken virus mycoplasma and generate to the drug resistance of tylosin, protection and the validity for maintaining tylosin.
Detailed description of the invention
Fig. 1 is the flow diagram for formulating break in the method for the present invention according to CLSI.
Fig. 2 is to carry out the result that wild type critical value is formulated in linear regression using Ecoffinder software in the method for the present invention
Schematic diagram.
Fig. 3 is to carry out Monte Carlo simulation compliance rate using Crystalball7 software in the method for the present invention to formulate pharmacodynamics
The result schematic diagram of critical value.
Fig. 4 is to carry out binary tree (CART) using SalfordPredictiveModeler software in the method for the present invention to analyze
Formulate the result schematic diagram of clinical threshold.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
According to Fig. 1,2,3,4, the present embodiment proposes that chicken virus mycoplasma tests the drug resistance criterion of tylosin
Method, comprising the following steps:
Step 1: tylosin is established to chicken virus mycoplasma Drug Resistance Detection method: with staphylococcus aureus ATCC29213
As Quality-control strains, tylosin measures tylosin to the MIC for being clinically separated chicken virus mycoplasma, establishes as Quality Control drug
When tylosin is to chicken virus mycoplasma Drug Resistance Detection method, acid is produced due to the metabolizable glucose of chicken virus mycoplasma, which can
It causes fluid nutrient medium that pH variation occurs, needs to test using the test of color changing units titre, then reapply in indirect
Measure the content of culture；
Step 2: wild type critical value of the tylosin to chicken virus mycoplasma, wild type critical value CO are formulatedWTIt indicates:
It carries out more than 20 sample acquisitions altogether from Hubei, Hunan and Henan first, amounts to acquisition more than 4000 part of sample, it is isolated doubtful
107 plants of chicken virus mycoplasma, 4 plants of chicken virus mycoplasma reference strain BG44T, HS, F and PG31 bacterial strains saved with laboratory, composition
Totally 111 plants of chicken virus mycoplasmas, the separation acquisition process of chicken virus mycoplasma include strain separating, purifying agaric, strain idenfication and bacterium
Kind saves, then using broth dilution method determination MIC value of the tylosin to 111 plants of chicken virus mycoplasmas, the MIC number that will be obtained
According to Ecoffinder software is brought into, first by Logarithm conversion, wild-type strain normality distribution inspection is carried out, selection belongs to just
The wild-type strain MIC range of state distribution carries out nonlinear regression analysis, utilization is non-after determining that MIC distribution belongs to normal distribution
Linear regression method is repeatedly fitted the MIC data for being converted to log series model, first from minimum MIC to distribution frequency highest
MIC be fitted, then every time be fitted be incremented by a MIC gradient, until fitting number of strains and practical number of strains it is poor
Then different minimum determines that best fit range is the smallest MIC to the smallest MIC of number of strains difference, reuses Excel formula
Middle NORMINV function verifies the maximum value upper limit of wild-type strain, then determines verifying maximum upper limit by NORMDST function
Probability, finally determining critical value includes at least 95% wild-type strain, is walked these statistical analysis using last CLSI
Suddenly it is integrated, software is analyzed by an Excel software ECOFFinder, directly wild-type strain MIC distributed data is carried out
Analysis, obtains tylosin to the wild type critical value of chicken virus mycoplasma；
Using Ecoffinder software, tylosin is substituted into RAWCount to the MIC distribution of chicken virus mycoplasma, is calculated
Cumulative distribution obtains tylosin to the data fitting result of chicken virus mycoplasma using nonlinear regression and fitting cumulative distribution, soft
Part automatic imitation wild-type strain in different confidence intervals 95.0%, 97.5%, 99.0%, 99.5% and 99.9% is distributed
The upper limit；The upper limit for taking the wild type chicken virus mycoplasma MIC in 95.0% confidence interval to be distributed is final wild type critical value,
As 2 μ g/mL；
Step 3: pharmacodynamics critical value of the tylosin to chicken virus mycoplasma, pharmacodynamics critical value CO are formulatedPDIt indicates:
Have chosen MIC90Bacterial strain carry out chicken embryo virulence experiment, the progress egg infectious experiments of SPF grade chicken embryos are selected, by hatching to 7
Age in days finds the embryo of chicken embryo under intense light irradiation and marks its position with gas chamber, selects yolk sac inoculation method, dosage of inoculation
It is 106CCU, it is daily to observe chicken embryo situation, daily chicken embryo death quantity is recorded, final choice M17 bacterial strain carries out PK-PD test,
Formulate pharmacodynamics critical value, chicken embryo virulence experiment detailed process are as follows: select SPF grades of chicken embryos and carry out egg infectious experiment, chicken embryo is incubated
Change to 7 ages in days, the embryo of chicken embryo found under intense light irradiation and marks its position with gas chamber, is tested in aseptic operating platform,
Egg surface is sterilized with 75% or so medicinal alcohol, embryo and balloon marker, experimental selection yolk bag can not be wiped in the process
Inoculation, dosage of inoculation 106CCU, inoculation method are as follows: after the disinfection of gas chamber end eggshell, make a call to an aperture in gas chamber center, infused with No. 7
Emitter syringe needle is vertically pierced into about 3cm, injection inoculation bacterium solution about 0.2mL, and paraffin sealing continues to hatch at 37 DEG C, daily egg-turning 2
It is secondary；
Pharmacodynamics in vitro research is carried out to the M17 that chicken embryo virulence experiment is elected, obtains Pharmacodynamic Data；It is safe to chicken stomach-filling
Happy rhzomorph carries out pharmacokinetic studies, obtains pharmacokinetic data available；Pharmacodynamics and pharmacokinetic data available are simulated using WinNonlin software,
It obtains medicine and moves-pharmacodynamic parameter；Choose inhibition model SigmoidEmax model prediction medicine it is dynamic-pharmacodynamic parameter and antimicrobial effect
Between relationship, obtain the pharmacodynamics target value under different antibacterial effects；
Using Crystalball7 software in illness group lung tissue pharmacokinetic data available AUC mean value and standard deviation respectively into
Row Monte Carlo simulation, the pharmacokinetic data available for 10000 chickens simulated are simulated to obtain illness group E using Winnonlin
Corresponding pharmacokinetics target value is 51.19 when=- 3, so that the results are shown in Table 1 for compliance rate of the calculating at different MIC, works as MIC
When for 2 μ g/mL, compliance rate 0%；When MIC is 1 μ g/mL, compliance rate 93.68% is higher than 90%；Faced according to pharmacodynamics
Dividing value lays down a regulation, and compliance rate is pharmacodynamics critical value in 90% or more corresponding maximum MIC value, due to using infection target
The analog result at position is more meaningful to the formulation of critical value, and the tylosin finally formulated faces the pharmacodynamics of chicken virus mycoplasma
Dividing value is 1 μ g/mL；
Compliance rate of the 1 Monte Carlo simulation pharmacokinetic parameters of table under different MIC values
Step 4: clinical threshold of the tylosin to chicken virus mycoplasma, clinical threshold CO are formulatedCLIt indicates: according to
MIC distribution selects the chicken virus mycoplasma with pathogenicity, comprehensively considers selection 5 different tops according to specific MIC distribution
Value MIC, peak-peak MIC or so, wild type critical value, MIC50、MIC90MIC bacterial strain carry out chicken embryo virulence experiment, choosing
It selects SPF grades of chicken embryos progress egg infectious experiments and hatching to 7 ages in days is found into the embryo of chicken embryo and label under intense light irradiation
The position of itself and gas chamber, experimental selection yolk sac inoculation method, dosage of inoculation 106CCU, it is daily to observe chicken embryo situation, record
Daily chicken embryo death quantity, final choice M1, M11, M17, M23 and M24 bacterial strain carry out Clinical Treatment Test, formulate tylosin
To the clinical threshold of chicken virus mycoplasma, wherein the Virulence detection of bacterial strain is verified by egg infectious model test,
Logarithmic growth phase is corresponded into bacterium solution, chicken embryo is inoculated in by yolk sac injection, the bacterium solution of each same concentration of egg inoculation 0.3mL,
Chicken embryo is injected intraperitoneally as blank control group using sterile blank meat soup simultaneously, chicken embryo death situation is observed, selects the death rate high
Bacterial strain；
Dosage is calculated using PK-PD parameter break value, calculation formula is as follows:
Wherein, CL refers to tylosin clearance rate in chicken lung tissue；(AUC/MIC)BPRefer to the PK/ of corresponding different response to treatment
PD parameter break value；MIC is the MIC of clinical chicken virus mycoplasma；F is bioavilability；Fu is free drug concentration ratio；
Tylosin is reached into AUC corresponding to different antimicrobial effect purposes24h/ MIC value substitutes into Rapid Dose Calculation equation
The day dosage that tylosin reaches required when different antimicrobial effects is found out, when the MIC value for the aimed strain being clinically separated is 2
When μ g/mL, each dosage obtained the results are shown in Table 2:
Dosage under the different medication purposes of table 2
By under three dosages of Mlxplore software simulation and forecast (prevent, treat, remove) and different dosing interval
The growing state of bacterium, to obtain optimal dosage regimen and dosage regimen that dosing interval is finally formulated is between administration for 24 hours
Every, the dosage of 45.88mg/kgb.w, successive administration 3 days；
Test chicken is passed through 7 days before the experiments and is adapted to, during adaptation, no chicken is dead, and feeding drinking-water situation is just
Often, health status is good, behavior without exception, and also without other bacterium infection phenomenons, the laundering period carries out artificial challenge to chicken later, adopts
It is infected with the chicken virus mycoplasma of Intratracheal instillation 1ml, consecutive infection 7 days, infection chicken virus mycoplasma titre is 1 × 109CCU,
Blank control group is inoculated with blank FM-4 meat soup in such a way that same tracheae is injected；
After drug therapy, sensitive strain M1 bacterial strain and the corresponding cure rate of M11 bacterial strain can achieve 100%, MIC phase
Sensitive strain to be lower than to the cure rate of biggish bacterial strain, and MIC value is bigger, cure rate is lower, illustrates the treatment effect of drug
The MIC value of fruit and infection strain is that have negative correlation, when infection strain MIC is 0.03 μ g/mL, cure rate 100%；Infection
When bacterial strain MIC is 0.5 μ g/mL, cure rate 93.3%；When infection strain MIC is 1 μ g/mL, cure rate 80%, according to facing
It is clinical threshold that bed critical value, which selects maximum MIC value of the cure rate more than or equal to 90% when, obtains tylosin to chicken poison branch
The clinical threshold of substance is 0.5 μ g/mL；
Obtained data are first analyzed with result of " WindoW " method to clinical efficacy, its parameter value is measured
MaxDiff and CAR determines clinical threshold range according to parameter value；Bacterial strain quantity repeat number corresponding to MaxDiff and CAR
It is greater than 4；This research is respectively 1,1,0.97,0.92,0.84 by the probability that CAR calculation method obtains, but needs to exclude
CAR falls in the case where minimum and maximum MIC distribution, and finally the MIC value of an optional gradient smaller than maximum MIC is in critical value
Limit is 1 μ g/mL；And the correspondence numerical value being calculated by MaxDiff algorithm is respectively 11,13,11,3,0, is then selected most
MIC corresponding to big MaxDiff value is distributed as critical value lower limit, i.e. 0.03 μ g/mL；Since the result that two kinds of algorithms obtain is different
It causes, therefore is 0.03-1 μ g/mL by the distribution that this method obtains clinical threshold；
The formula between POC and MIC proposed further according to EUCAST, using the nonlinear regression in SPSS software to its institute
It obtains data to be fitted, using Log2MIC as independent variable, POC is dependent variable, selection and the highest model of experimental data related coefficient
Expression formula is y=82.992-9.364x-1.628x2-0.91x3, obtained simulation R2Value is 0.975；According to result expression,
It is -0.82, MIC is 0.57 μ g/mL that calculate cure rate, which be independent variable Log2MIC corresponding to 90%, so clinical threshold is small
In 0.57 μ g/mL；
It is that prediction becomes with MIC finally, bringing clinical testing data into SalfordPredictiveModeler software again
Amount, POC is target variable, carries out binary tree (CART) analysis；From regression tree it is found that as the μ g/mL of MIC≤0.75, cure rate
It is 97.8%；As the μ g/mL of MIC > 0.75, cure rate 76.7%；When MIC is close to 0.75 μ g/mL, cure rate 89.3%；
Therefore CART, which analyzes obtained clinical threshold, should be value near and less than 0.75 μ g/mL；
The above analysis method, the clinical threshold that " WindoW " method obtains select window for 0.03-1 μ g/mL；It is non-thread
Property regression analysis shows clinical threshold less than 0.57 μ g/mL；CART regression tree analysis show clinical threshold should be it is close and
Less than the value of 0.75 μ g/mL.Obtained 0.5 μ g/mL of clinical threshold, which is tested, by clinical treatment all meets condition above, institute
The tylosin finally formulated with this experiment is to the clinical threshold of chicken virus mycoplasma for 0.5 μ g/mL；
Step 5: according to tylosin to the wild type critical value of chicken virus mycoplasma, tylosin to chicken virus mycoplasma
It is final to formulate to formulate dendrogram using break to the clinical threshold of chicken virus mycoplasma for pharmacodynamics critical value and tylosin
Wild type critical value, pharmacodynamics critical value and clinical threshold are compared by break first, when there is wild type critical value etc.
When clinical threshold situation, which is break value；When there is unequal situation, then compare the size of three critical values,
Determine that final break value is drug resistance criterion of the chicken Mycoplasma to tylosin；
Wild type critical value (CO of the known tylosin to chicken virus mycoplasmaWT) it is 2 μ g/mL, tylosin is to chicken poison branch
Pharmacodynamics critical value (the CO of substancePD) it is the clinical threshold (CO of 1 μ g/mL and tylosin to chicken virus mycoplasmaCL) be
0.5 μ g/mL, the flow chart that the break that these three critical values bring CLSI announcement into is formulated, meets COWT> COPD> COCL, corresponding
Break value should select wild type critical value, obtaining chicken virus mycoplasma is 2 μ g/mL to the drug resistance criterion of tylosin.
By the method for the invention it can be concluded that drug resistance criterion of the chicken virus mycoplasma to tylosin, can be section's scholarship and self-cultivation
It grows and stable administration data is provided supports, can be more scientific carry out instructs clinical application, and it is highly-safe, it can effectively slow down chicken poison
Mycoplasma generates the drug resistance of tylosin, protection and the validity for maintaining tylosin.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements
It all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent circle
It is fixed.
Claims (10)
1. chicken virus mycoplasma is to the drug resistance criterion test method of tylosin, which comprises the following steps:
Step 1: tylosin is established to the Drug Resistance Detection method of chicken virus mycoplasma: with staphylococcus aureus ATCC29213 work
For Quality-control strains, tylosin measures tylosin to the MIC for being clinically separated chicken virus mycoplasma as Quality Control drug；
Step 2: tylosin is formulated to the wild type critical value (CO of chicken virus mycoplasmaWT): divide first from clinical infection chicken
From obtaining out 107 plants of chicken virus mycoplasma, totally 111 plants of chicken virus mycoplasmas are formed with 4 plants of reference culture that laboratory saves, are then adopted
With broth dilution method determination tylosin to the MIC value of 111 plants of chicken virus mycoplasmas, obtained MIC data are brought into
Ecoffinder software carries out linear regression simulation, obtains the wildness critical value under different confidence intervals；
Step 3: tylosin is formulated to the pharmacodynamics critical value (CO of chicken virus mycoplasmaPD): have chosen MIC90Bacterial strain carry out chicken
Embryo virulence experiment selects SPF grades of chicken embryos to carry out egg infectious experiment and hatching to 7 ages in days is found chicken embryo under intense light irradiation
Embryo and mark its position with gas chamber, select yolk sac inoculation method, dosage of inoculation 106CCU, it is daily to observe chicken embryo feelings
Condition records daily chicken embryo death quantity, and final choice M17 bacterial strain carries out PK-PD test, formulates pharmacodynamics critical value, will obtain
PK-PD data carry out Monte Carlo simulation using Crystalball7 software, it is corresponding 90% or more to choose compliance rate
Maximum MIC value is pharmacodynamics critical value；
Step 4: tylosin is formulated to the clinical threshold (CO of chicken virus mycoplasmaCL): have chosen the bacterial strain of 5 different MIC
Chicken embryo virulence experiment is carried out, SPF grade chicken embryos is selected identify the virulence of chicken virus mycoplasma, by hatching to 7 ages in days,
The embryo of chicken embryo is found under intense light irradiation and marks its position with gas chamber, and experimental selection yolk sac inoculation method, dosage of inoculation is
106CCU, it is daily to observe chicken embryo situation, record daily chicken embryo death quantity, final choice M1, M11, M17, M23 and M24 bacterial strain
Clinical Treatment Test is carried out, formulates tylosin to the clinical threshold of chicken virus mycoplasma, the clinic that clinical test is formulated is critical
Value can be calculated by " WindoW ", nonlinear regression and binary tree (CART) analysis mode are verified；
Step 5: according to tylosin to the wild type critical value of chicken virus mycoplasma, tylosin to the drug effect of chicken virus mycoplasma
Critical value and tylosin are learned to the clinical threshold of chicken virus mycoplasma, formulates flow chart using break to formulate final folding
Point obtains drug resistance criterion of the chicken virus mycoplasma to tylosin.
2. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: when establishing tylosin in the step 1 to chicken virus mycoplasma Drug Resistance Detection method, due to the metabolizable Portugal of chicken virus mycoplasma
Grape are sugared and produce acid, which can cause fluid nutrient medium that pH variation occurs, and need that the test of color changing units titre is used to be examined
It tests, then reapplies in the content of indirect determination culture.
3. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: the separation acquisition process of chicken virus mycoplasma includes that strain separating, purifying agaric, strain idenfication and strain are protected in the step 2
It deposits.
4. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: wild-type strain normality distribution inspection is carried out by Logarithm conversion first in the step 2, selection belongs to normal distribution
Wild-type strain MIC range, determine MIC distribution belong to normal distribution after, carry out nonlinear regression analysis, with non-linear time
Method is returned repeatedly to be fitted the MIC data for being converted to log series model, first from minimum MIC to the highest MIC of distribution frequency
It is fitted, is then fitted every time and is incremented by a MIC gradient, until the number of strains and practical number of strains difference of fitting are most
It is small, it then determines that best fit range is the smallest MIC to the smallest MIC of number of strains difference, reuses in Excel formula
NORMINV function verifies the maximum value upper limit of wild-type strain, then determines verifying maximum upper limit by NORMDST function
Probability, finally determining critical value should include at least 95% wild-type strain, be statisticallyd analyze these using last CLSI
Step is integrated, by Excel software ECOFFinder analyze software directly to wild-type strain MIC distributed data into
Row analysis, obtains tylosin to the wild type critical value of chicken virus mycoplasma.
5. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: chicken embryo virulence experiment detailed process in the step 3 are as follows: select SPF grades of chicken embryos and carry out egg infectious experiment, by hatching
To 7 ages in days, the embryo of chicken embryo is found under intense light irradiation and marks its position with gas chamber, is tested in aseptic operating platform, use
75% or so medicinal alcohol sterilizes egg surface, can not wipe embryo and balloon marker in the process, experimental selection yolk bag connects
Kind, dosage of inoculation 106CCU, inoculation method are as follows: after the disinfection of gas chamber end eggshell, make a call to an aperture in gas chamber center, injected with No. 7
Device syringe needle is vertically pierced into about 3cm, injection inoculation bacterium solution about 0.2mL, and paraffin sealing continues to hatch at 37 DEG C, daily egg-turning 2 times.
6. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: Monte Carlo simulation is carried out to pharmacokinetic data available using Crystalball7 software in the step 3 and obtains pharmacokinetics target value
Compliance rate at different MIC, selecting compliance rate in 90% or more corresponding maximum MIC value is pharmacodynamics critical value.
7. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: when formulating clinical threshold of the tylosin to chicken virus mycoplasma in the step 4, according to MIC distribution, selection tool
Have the chicken virus mycoplasma of pathogenicity, selection gist: peak-peak MIC, peak-peak MIC or so, wild type critical value,
MIC50、MIC90MIC bacterial strain, according to specific MIC distribution comprehensively consider selection 5 MIC bacterial strains.
8. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: the Virulence detection of bacterial strain is verified by egg infectious model test in the step 4, by logarithmic growth phase pair
Bacterium solution is answered to be inoculated in chicken embryo by yolk sac injection, the bacterium solution of each same concentration of egg inoculation 0.3mL, while with sterile blank
Chicken embryo is injected intraperitoneally as blank control group in meat soup, observes chicken embryo death situation, the bacterial strain for selecting the death rate high.
9. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
" WindoW " algorithm, non-linear in SPSS software can be brought into: the clinical threshold that clinical test obtains in the step 4
It returns and the analysis of the binary tree (CART) of SalfordPredictiveModeler software is verified, first with the side " WindoW "
Method analyzes the result of clinical efficacy, measures its parameter value MaxDiff and CAR, determines clinical threshold according to parameter value
Range, the formula between POC and MIC proposed further according to EUCAST, using the nonlinear regression in SPSS software to its gained
Data are fitted, with Log2MIC is independent variable, and POC is dependent variable, obtains the highest model expression of related coefficient to determine
Clinical threshold range, finally again brings clinical testing data in SalfordPredictiveModeler software into, is with MIC
Predictive variable, POC are target variable, carry out binary tree (CART) analysis to determine the range of clinical threshold.
10. chicken virus mycoplasma according to claim 1 exists to the drug resistance criterion test method of tylosin, feature
In: wild type critical value, pharmacodynamics critical value and clinical threshold are compared first in the step 5, it is wild when occurring
When type critical value is equal to clinical threshold situation, which is break value；When there is unequal situation, then compare three it is critical
The size of value determines that final break value is drug resistance criterion of the chicken virus mycoplasma to tylosin.
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