Study On The CO_WT And In Vitro Pharmacodynamics For Colistin Against Swine-derived Escherichia Coli

The interpretive criteria or breakpoint for determining antimicrobial susceptibility can provide a key standard to monitor drug resistance of pathogenic bacteria and to detect the rate and level of resistance,and the breakpoint contains Wild type cut-off(CO_WT),Clinical cut-off(CO_CL)and PK-PD cut-off(CO_PD).The American CLSI and European EUCAST are authoritative organizations to set the breakpoint in the world.Due to we don't have our own organization to set breakpoint,we can only employ the breakpoint setted by other countries.However,the use of veterinary medicine in clinical and the resistance level or the degree of bacteria in domestic are not completely consistent with other countries,so it's urgently necessary for us to establish a criterion that meets our own actual situation.Escherichia coli?E.coli?is one of the target bacteria of colistin,and the pharmacodynamic data of colistin against E.coli in vitro is relatively lacking,which play an important role in determining the breakpoint of antimicrobial against the target bacteria.CO_WT or epidemiological breakpoint?Ecoff?are important judgement basis for the monitoring of bacterial resistance phenotypes,and play a role in early warning of the emergence and development of bacterial resistance when the final breakpoint was not established.The existence of mcr-1,a plasmid-mediated gene encoding myxin resistance,has been found in recent years,ISApl1 can facilitate the transfer of mcr-1 and capture of other movable elements,therefore,analysis of the role of mcr-1 in colistin resistance is of great significance for further study of its molecular resistance mechanism and its prevention and control.In this study,we collected anal swabs of swine from several large scale swine farms in major provinces and cities of China,through culture,isolation,purification and identification in the laboratory,1222 strains of porcine E.coli were isolated successfully.In vitro efficacy of colistin was studied including minimum inhibitory concentration?MIC?,minimum bactericidal concentration?MBC?,mutant prevention concentration?MPC?and post-antimicrobial effect?PAE?.It showed that colistin was a bactericidal antibacterial agent against E.coli derived-from swine?MBC=1⁴MIC?,which had an in vitro PAE and its mutation selective window?MSW?was wide.The results of antimicrobial susceptibility testing showed that the sensitivity of porcine E.coli to colistin is low and the problem of antimicrobial resistance is serious currently.By plotting the histogram of MIC distribution for colistin against porcine E.coli,and the CO_WT of colistin against porcine E.coli was determined to be 16?g/mL by statistical method.56 strains of colistin-resistant strains isolated from Henan region were selected?MIC?8?g/mL?,and the possibility of drug resistance generation and metastasis were further analyzed.PCR amplification was conducted to detect mcr-1 gene.The result showed that the percentage of mcr-1was 62.5%?35/56?.We used PCR-mapping to determine the genetic context around the mcr-1 gene,and the ability to transfer and the possibility of capturing other removable elements are discussed,and found three kinds of genetic environment of mcr-1.The results showed that the mcr-1 gene was surrounded by two ISApl1sequence in twenty mcr-1 positive strains,ISApl1was only found in the upstream of mcr-1 gene in seven isolates,but we didn't found any ISApl1in the rest of eight mcr-1positive strains.The drug-resistant phenotypes of mcr-1 positive E.coli were detected by drug sensitive disk method.The results showed that these strains had a wide spectrum of drug resistance.PCR amplification was also conducted to detect the presence of other drug-resistant genes in mcr-1 positive strains,the results showed that NDM-1,CTX-M-15,AAC?3?-IV,Ant?3??-I and AAC?3?-II were detected respectively,which is basically consistent with its drug resistance phenotype.In summary,the susceptibility rate of porcine E.coli to colistin is low,and the problem of colistin resistance is serious in domestic.The detection rate of mcr-1 was high in colistin-resistant strains,the discovery of co-existence of other drug resistance genes in mcr-1 positive strains,and we initially determined the CO_WT/Ecoff of colistin against porcine E.coli isolated from swine is 16?g/mL.