Study On The Spread Mechanism Of Bla_CTX-M-27 In E.coli

Bacterial resistance to cephalosporin antibiotics is mainly caused by bacteria producing extended spectrum?-lactamases(ESBLs)to hydrolyze the?-lactam ring in cephalosporin drugs,in which CTX-M is inactivated type ESBL is the most popular.In recent years,the isolation rate of bla _CTX-M-27 subtypes has increased gradually in domestic and foreign research reports,and the isolation rate of bla _CTX-M-27 gene in Salmonella is higher than that in E.coli.Some studies have explored the transmission mechanism of bla _CTX-M-27 gene in Salmonella,and the transmission characteristics of bla _CTX-M-27 gene in E.coli is still unclear.In this study,the prevalence and transmission mechanism of bla _CTX-M-27 gene in E.coli from farm animals were investigated.The research results provide a theoretical basis for the prevention and control of cephalosporin antibiotic resistance.In 2014 and 2016,2510 strains of E.coli from food samples from Henan,Guangdong,Shandong,Hubei,and Guangxi provinces were isolated.The bla _CTX-M-9G positive and bla _CTX-M-27subtype E.coli were screened by PCR sequencing and sequence alignment.The PCR results showed that the number of bla _CTX-M-9G positive E.coli was 596,of which 58strains were bla _CTX-M-27 subtype.The other drug resistance genes of the strain were detected by PCR sequencing.The antibiotic susceptibility test of bla _CTX-M-27 positive E.coli was carried out by agar double dilution method.Eight of the 58 strains of bla _CTX-M-27 positive E.coli carried bla _CTX-M-1Ggene(13.79%),and the number of flo R gene was 28(46.55%).The number of fos A3 gene was 14(24.14%),and the number of mcr-1 gene was 11(18.97%).20 strains(34.48%),11strains(18.97%)and 1 strain(1.72%)were detected in oqx AB,qnr S and aac(6')-Ib-cr,respectively.The detected strains showed amino acid mutations at positions 83 and 87 in the QRDR region of the gyr A gene.The strain producing CTX-M-27 showed multidrug resistance to most antibiotics,especially the resistance rate of these strains to tetracycline,nalidixic acid and ampicillin were over 90%and were over 80%to fluoroquinolones,the resistance rate to olaquindox,amide alcohols and aminoglycosides were 40%to 50%,and the resistance rate of all strains to fosfomycin and amikacin were low.Pulsed filed gel electrophoresis(PFGE),MLST typing,conjugative transfer,replicon typing,S1-PFGE,Southern-blot hybridization and plasmid Illumina Hiseq sequencing were also performed to study the dissemination and characterize bla _CTX-M-27-harboring plasmids.The results showed that 58 strains could be divided into 31 clusters,and the bla _CTX-M-27gene was mainly transferred horizontally.38 strains of bla _CTX-M-27 gene could be conjugative transfer,and 7 of them had oqx A,oqx B and fos A3 gene co-transfer with bla _CTX-M-27 gene.The bla _CTX-M-27 gene is all located on the plasmid.The plasmid sizes were about40 kb,104.5 kb,110 kb,138.9 kb and 150 kb.The plasmid type of 23 conjugants was Inc FIB type,3 conjugants were FIB type and N type coexistence,two conjugants existed FIB type,N type and I1 type plasmid coexistence,plasmid type of two strains were Inc HI2type,plasmid type of one strain was Inc I1 type,and plasmid type of seven strains were not separable.The bla _CTX-M-27 harboring plasmid was sequenced and analyzed,the result showed that the upstream of bla _CTX-M-27 was the ISEcp1 sequence,but was truncated by IS26,and the downstream was the IS903B,belonging to the IS26-?ISEcp1-bla_CTX-M-27-IS903B transfer structure.The stability of the plasmid was tested on 3 strains,and the test was maintained for 10days.The results showed that the bla _CTX-M-27 harboring plasmids could be stably present in the host strains under the pressure of no antibiotic,and the loss rate of the plasmid was zero.This study is the first to explore the propagation mechanism of bla _CTX-M-27 in E.coli from animal sources.Studies have shown that bla _CTX-M-27 is mainly horizontally transfer,and there is a phenomenon of clonal propagation in a small range,the multidrug resistance of bla _CTX-M-27 positive isolates was serious,and the prevalence of bla _CTX-M-27 in farmsposes a threat to public health,and reminds us that we need to strictly guard against the abuse of cephalosporin antibiotics.