| Chicken colibacillosis is a general term for bacterial infections caused by certain serotypes of Escherichia coli(E.coli),often presenting secondary local or systemic infections,and has now become a flock.One of the most serious bacterial diseases.Because of the serotype of E.coli disease,the use of vaccine is limited,so drug treatment is still the main prevention and treatment of the disease.However,with the long-term application of antibacterial drugs,the emergence of drugresistant strains has made the antibacterial drug prevention and treatment effects increasingly weak.In recent years,the incidence of multi-drug resistant Gram-negative infections has increased,and colistin has been used as the last line of defense for multi-drug resistant Gram-negative bacteria.In order to slow the production of colistin resistance in chicken Escherichia coli,it is urgent to monitor its drug resistance.The criteria for determining the sensitivity of antimicrobial agents(breakpoints)are important technical standards for detecting the resistance of pathogenic bacteria and determining their resistance rate and drug resistance,They include epidemiological cut-off value(ECV)and clinical folds?Clinical cut-off value(COCL)and pharmacokinetics-pharmaceuticals(COPD).ECV is an epidemiological surveillance of the resistance of pathogens,and it plays an early warning role in the discovery and development of bacterial resistance before the final breakpoint is established.This experiment was based on the establishment of the Clinical and Laboratory Standards Institute(CLSI)breakpoint establishment process,established the ECV of colistin on chicken E.coli,and studied the multi-drug resistance of chicken E.coli by drug sensitivity test.From the molecular point of view,the prevalence of the drug resistance gene mcr(1~5)was studied,and the screening of colistin was used to provide a reference for clinical rational combination therapy.In this study,from September 2016 to the beginning of January 2018,from the major largescale chicken farms in China's top ten provinces,the use of sterile cotton swabs to collect fresh faeces from chickens,a total of 1470 samples were collected.Through MH solid Medium,Mc Kay(MAC),Eosin Blue(EMB)selection medium for initial separation,gram staining,16 S r RNA PCR,biochemical identification and other methods successfully isolated and identified 1250(85%)strains of E.coli.The clinical isolation of mucin was tested by micro-broth dilution method.The minimum bacteriostatic concentration(MIC)of E.coli,MIC50 and MIC90 were 2 ?g/m L and 4 ?g/m L,respectively.The MIC value is transformed into the log2 MIC value into the Ecoffinder analysis software,and the nonlinear regression simulation is carried out to obtain the ECV of the slime hormone on the E.coli of chicken as 8 ?g/m L.The colistin-resistant strains were screened according to the breakpoints,and the sensitivity of the strains to the seven 17 antibiotics was detected by the drug sensitive paper diffusion method(Kirby-Bauer,K-B).The results of drug susceptibility test showed that the resistance rate of E.coli to 14 antibiotics was higher than 50%,of which 100% resistance to amoxicillin,sulfisoxazole and oxymethazine;to amikacin(5.88%),spectinomycin(17.65%)and fosfomycin(41.18%)have lower resistance rates.At the same time,the E.coli detected in this test are all multi-drug resistant E.coli,mainly showing 8-16 resistance,multi-drug resistance is serious,and the drug resistance spectrum is wide.To understand the prevalence of mcr(1~5)in colistin-sensitive strains,95 strains of E.coli isolated from different regions were screened according to MIC.The detection rate of mcr-3 and mcr-5 was zero by PCR.The detection rates of mcr-1,mcr-2 and mcr-4 in chromosomal DNA were 23.2 %,7.3 % and 2.1 %,respectively.The carrying rate of mcr-1 in plasmid was 41.1 %,followed by 7.3 % and 3.2 % respectively.E.coli carries mcr-1 in 8(8/18)strains of colistin-sensitive strains and carries multilocus sequencing typing(MLST).The eight strains are ST10,ST48,ST57,ST155,ST359,ST744 and ST2458,respectively,Two of them are ST48.New Delhi Metal-betalactamase-1(NDM-1)was detected in 37.5 %(3/8)of the 8 mcr-1 positive strains.The combination of amikacin,doxycycline,gentamicin with colistin was tested by in vitro susceptibility test by broth dilution board method.The combined effect of colistin and these three drugs increased in turn,belonging to unrelated,additive and synergistic effects.In conclusion,at present,E.coli of chicken has low sensitivity to colistin,serious multi-drug resistance,wide drug resistance spectrum,and the prevalence of drug resistance gene mcr(1~5)in E.coli from China.Mcr-1 is a dominant gene mediated low-level drug resistance;mcr-4 is only detected in drug-resistant strains,presumably it may mediate high-level drug resistance,and coccinresistant strains carrying Mcr-1 in E.coli Some of the strains produced NDM-1 at the same time.The multi-site sequence was ST-type,and the combined antibacterial effect of colistin and gentamicin was found to be synergistic,which provided a reference for clinical rational drug use. |
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