Wild-type Cutoff And Resistance Mechanism Of Apramycin Against Swine-derived Escherichia Coli

The wild-type cutoff(CO_WT)is the interpretive criteria of antimicrobial susceptibility test,which is a crucial technical standard for detecting the susceptibility of bacteria to antimicrobial agents and to detect the rate and level of resistance.There are two authoritative organizations that have set the breakpoint in the world: Clinical and Laboratory Standards Institute?CLSI?and European Committee on Antimicrobial Susceptibility Testing?EUCAST?.There is no institute to set up a breakpoint in our country.Apramycin,an antibacterial drug,is widely used for veterinary clinics,play an important role in the treatment and prevention of diease caused by Escherichia coli?E.coli?.However,to date,there is no susceptibility breakpoint of apramycin against E.coli at home and abroad lead to that there is no uniform scale for judgment.At the same time,as antibacterial agents are used unreasonably for a long time,the drug resistance of E.coli is becoming more and more serious.In response to the above problems,this study refers to the standard processes and methods for establishing breakpoints in CLSI,combined with the breeding situation in different regions of China,collecting anal swabs from pigs in different regions of China.The CO_WT of apramycin against E.coli was established on the basis of isolation,purification,identification of E.coli and determination of apramycin on the minimum inhibitory concentration?MIC?of E.coli,the effects of E.coli on apramycin resistance were investigated from the aspects of inactivation enzyme gene and efflux pump gene.In this study,pig anal swabs from 7 large-scale pig farms in different provinces and municipalities were collected,1230 isolates of swine-derived E.coli were obtained by isolation,purification and identification.MIC of apramycin against the above swine-derived E.coli was determined by microbroth dilution method.The histogram of the MIC value distribution of the strains of apramycin was determined by statistical method and the CO_WT of apramycin against swine-derived E.coli was set to 32 ?g/m L.The MIC₅₀ and MIC₉₀ is 16 ?g/m L and 64 ?g/m L,respectively.The minimum bactericidal concentration?MBC?and the mutant prevention concentration?MPC?of apramycin against the swine-derived E.coli clinical isolates were determined.It showed that the MBC of apramycin against swine-derived E.coli was 1⁴MIC and its mutation selective window?MSW?was wide.A total of 310 swine-derived E.coli clinical isolates with different MICs?0.5²56 ?g/m L?were selected for subsequent resistance research.PCR amplification was conducted to detect aac?3?-IV,npm A and apm A gene.The results showed that only the aac?3?-IV inactivation enzyme gene was detected,and mainly concentrated in MIC ? 64 ?g/m L.This indicates that the aac?3?-IVinactivation enzyme gene is one of the main reasons for the resistance to apramycin,and it also indicates that the CO_WT established in this study is reliable.The above 25 strains of apramycin-resistant strains were selected and their resistance mechanisms were further studied.Firstly,the drug-resistant paper method was used to determine the resistant phenotype of E.coli clinical isolates against 11 antimicrobial agents.The micro-broth dilution method and the disc method were used to detect the resistance of the above strains to the apramycin and other antibiotics before and after the active efflux inhibitor carbonylcyanide-m-chlorophenylhydrazone?CCCP?.The results showed that the combination of CCCP and antibiotics can effectively reduce the resistance of E.coli to antibiotics such as apramycin,doxycycline and neomycin.It is indicated that the active efflux system may be one of the causes of strain resistance to apramycin and multi-drug resistance.It is suggested that the active efflux system may be one of the causes of strain resistance to apramycin and multi-drug resistance.The PCR and real-time PCR methods were used to detect the carrying rate of Acr AB-Tol C active efflux genes in the clinical isolates of apramycin-resistant E.coli positively expressed as active efflux pumps and the relative expression level of the efflux pump gene The correlation between the relative expression levels of the active efflux genes acr A,acr B and aac?3?-IV inactivating enzyme genes of E.coli and the degree of resistance to apramycin in clinical isolates of E.coli were analyzed.The results showed that the carrying rates of three active efflux genes of acr A,acr B and tol C were higher in 25 resistant E.coli.The relative expression levels of acr A and acr B genes in some clinical isolates of E.coli with high resistance to apramycin were higher,but there was no significant correlation between the expression level and the degree of drug resistance.The relative expression of aac?3?-IV inactivation enzyme gene was not significantly correlated with the degree of apramycin resistance in clinical isolates of E.coli.Real-time quantitative PCR was used to detect the relative expression levels of the active efflux genes acr A and acr B and their regulatory genes mar A,sox S and rob A in the multi-drug resistant strains with different drug resistance.The relationship between the number of multidrug resistant strains and m RNA levels were analyzed.The results showed that the expression levels of acr A and acr B efflux pumps and mar A and sox S were positively correlated with the number of multidrug resistance,and the expression of rob A gene was not correlated with the number of drug resistance.In summary,we have initially determined the CO_WT of apramycin against swine-derived E.coli.The mechanism of resistance of apramycin against part swine-derived E.coli clinical isolates was studied.It provides a theoretical basis for the development of susceptibility breakpoints and provides interpretive criteria of antimicrobial susceptibility test of apramycin against swine-derived E.coli.