Location Of 16S RRNA Methylase Genes Among Escherichia Coli Strains Isolated From Chickens And Genetic Background Of RmtB

To investigate the prevalence and molecular epidemiology of 16S rRNA methylase genesamong 120 Escherichia coli strains isolated from chickens in Henan province in 2008. PCR was used for screening for the presence of 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD and npmA). In addition, ESBL genes (TEM, SHV and CTX-M) were detected among Escherichia coli strains producing 16S rRNA methylase.Consequently, Midi plasmid purification kit was used for purifying the plasmid harboring 16SrRNA methylase.The electraporation transformation experiment was performed by using E. coli DH10B as the recipient strain.The plasmids of the transformants were extracted with Qiagen Midi Plasmid Kit. Then the plasmids from the original strains and transformants were analyzed by the MageBase agarose gel electrophoresis to compare their difference. Southern hybridi- zation was performed to identify whether 16S rRNA methyalse genes were located on the plasmids.The plasmids harboring rmtB were digested, and ligated to the pBC SK+ vector. After confirming, the positive plasmids were sent for sequencing. The sequencing results in the flanking region of the rmtB gene were compared using the Blast procedure to analyze the genetic background of rmtB.The PCR results showed that, of 120 E. coli strains isolated from chickens in 2008, only armA and rmtB were detected and the positive rates were 2.5%(3/120), 7.5%(9/120), respectively. Of 12 16S rRNA methylase-producing E. coli strains, the positive rates for TEM were 100.0%. Besides, there is also each strain harbored blaCTX-M-14 and blaCTX-M-65, respectively.The results of both electroporation transformation and plasmid electroporesis revealed that the plasmids harboring 16S rRNA methylase genes could be transformed into the recipients, E coli DH10B.RmtB 16S rRNA methyalse genes were located on the plasmids,as determined by Southern hybridization. Two genetic maps consisting of the rmtB gene were successfully obtained, including a 4.6k and 7.0k fragment, by plasmid transformation, construction of the recombinant plasmids and sequencing of the flanking region of the rmtB gene. Of them, 4.6k fragment contained rmtB gene with the subsequent inverted ISCR1 downstream, while 7.0k fragment contained rmtB gene with the IS26 recombinase and Tn1721 transposase downstream.In conclusion, this study revealed that rmtB and armA type 16S rRNA methyalse genes were prevalent among E. coli in chickens.Southern hybridization results showed 16S rRNA methyalse genes were located on the plasmids. Although the genetic background of the rmtB genes displayed a disparity, it was associated with the mobile genetic elements. Therefore, it could be inferred that the rmtB gene may be transferred to the different plasmids by integration and transposition. This study also lay a solid basis for the further understanding the molecular mechanism of horizontal dissemination of the rmtB gene.