Characterization Of The Prevalence And Distribution Of Cfr In Escherichia Coli Isolated From Livestock And Environment

Encoding a methyltransferase that modifies 23S rRNA at A2503,the cfr gene confers resistance to five chemically distinct classes of antimicrobials,including oxazolidinones,pleuromutilins,streptogramin A,lincosamides and phenicols antibiotics.It is worth noting that the cfr gene is the first resistance mechanism that mediates oxazolidinone and pleuromutilin resistance.Up to now,the cfr gene has been widely reported in strains isolated from animals.However,little is known about the cfr gene in strains that isolated from environment.In this study,we intended to determine the prevalence of cfr genes in E.coli isolated from livestock and environment of the livestock farms,to study the transmission mechanism of cfr gene,in order to help us to understand about the prevalence of cfr in E.coli and provide ideas for the treatment of the infection causd by resistant strains.In 2017,a total of 6152 samples were selected from livestock and environment of the livestock farms in 16 provinces of China.The 11196 E.coli isolates were isolated from these samples.There are 30 E.coli isolates carrying cfr gene.Among the 11196 E.coli isolates,the cfr gene were detected in 0.268%?30/11196?,suggesting a low prevalence in E.coli.Among the 30 cfr-positive strains,22 strains isolated from livestock,and 8 strains isolated from the environment of the livestock farms.In this study,we aim to study the 30cfr-positive strains and realize the characterization of the prevalence and distribution of cfr in E.coli isolated from livestock and environment.The susceptibilities of the 30 cfr-positive strains to 14 antimicrobials were determined by agar dilution method.The results show that all of the 30 cfr-positive strains are resistant to florfenicol and tetracycline,and are sensitive to tigecycline.There was a very high frequency??90.0%?of phenicols,azithromycin,cefotaxime,and trimethoprim-sulfamethoxazole.The resistance rates to ciprofloxacin,polymyxin,gentamicin and ampicillin were between 70%and 90%.The resistance rates to meropenem,amikacin and fosfomycin does not exceed 50%.The PCR method and gene sequencing were used for the detection of commom resistance gene.The results show that,the detection rate of bla_TEMEM is the highest,which is60%.The resistance genes oqxA,oqxB,qnrS,mphA were detected in 53.33%,50%,43.33%,43.33%respectively.And there was detected 2 strains of bla_OXA,1 strains of bla_DHA,1 strains of fosA3.But qnrA?qnrB?qnrC?qnrD?rmtB?bla_CTX-M?fosA?fosC2 gene were not detected.The cfr-positive strains were typed by pulsed-field gel electrophoresis?PFGE?.The results shows that 30 cfr-positive E.coli strains were classified into 17 different subtypes according to the PFGE profiles,implying the genetic background of these stains is relatively diverse.It is worth mentioning that the PFGE profiles of two strains isolated from the same farm animals and environment respectively is the same.And the PFGE profile of another strains isolated from the same farm but in different stages also have a high degree of similarity.The existing of strains with the same PFGE profiles indicating that the clone transmission of cfr gene is excising between livestrocks and the environment as well as in livestocks.10 transconjugants/transformants were gained by conjugation/transformation.The susceptibility test found that all of the 10 transconjugants/transformants are resistant to florfenicol.The MIC of a trasconjugants to FFC is 256?g/mL.Compared with the original bacteria,the MIC of the remaining 9 strains to FFC was reduced by 4-8 times.Southern blotting showed that the cfr gene was located on a³3 kbp plasmids.In summary,there are a low prevalence of cfr gene in E.coli isolated from livestock and the surrounding environment.However,the level of clinical antibiotics resistance is relatively high,the distribution of resistant gene is more extensive.PFGE showed that the cfr-positive E.coli showed a variety of genetic background,but there were also clonal transmission of cfr-positive strains in farm animals and the surrounding environment.The clonal transmission of cfr gene is excising between livestrocks and the environment as well as in livestocks.The prevalence of cfr may be caused by both clonal and horizontal transmission.