IS Ec69-Mediated Mobilization of the Colistin Resistance Gene mcr-2 in Escherichia coli

Abstract

Objectives: The emergence of mobile colistin resistance genes has compromised the efficacy of the last resort antibiotic, colistin, in clinical treatment. The mcr-2 gene was first identified in Belgium in association with the insertion sequence ISEc69. However, the molecular mechanisms of mcr-2 mobilization are not well understood.

Methods: To further explore the mobilization of mcr-2 gene via ISEc69, we constructed a conjugative plasmid that carries an intact composite transposon Tn7052. Transposition assays were performed by conjugation, the transposition sites were characterized by arbitrary primed PCR and DNA sequencing.

Results: In this study, we experimentally demonstrated that mcr-2 could be mobilized as a composite transposon Tn7052 and its transposition generated 8-bp AT-rich duplications in the host genome.

Conclusion: These results indicate that mcr-2 gene could be mobilized by ISEc69, the current investigations provide mechanistic insights in the transposition of mcr-2.

Keywords: ISEc69; colistin resistance; mcr-2 gene; transposition mechanism; transposition sites.

FIGURE 1

Target site analyses of Tn7052 transposons. (A) Molecular characterization of 23 transposition events of Tn7052 transposons in E. coli MG1655 (recA::Km). The duplicated 8-bp target sites are underlined in the context of the surrounding 42 nucleotides upstream and downstream from the target sites. (B) Statistical analyses of the 23 transposition sites. The percentage of AT and GC at each position from 42 nucleotides upstream to 42 nucleotides downstream of the target site are shown. The 8-bp duplicated target site (C1 to C8) are indicated by black bars. The AT and GC percentages of regions spanning positions –42 to –2 and positions +2 to +42 and that of the region spanning positions –2 to 2 are indicated in the upper and lower graphs, respectively. Relative nucleotide frequencies at each target site deduced from the 23 experimental transposition events shown in (A,C).