Novel lnu(G) gene conferring resistance to lincomycin by nucleotidylation, located on Tn6260 from Enterococcus faecalis E531

Objectives: To identify a novel putative lincosamide resistance gene determinant in a swine Enterococcus faecalis E531 exhibiting a lincosamide resistance/macrolide susceptibility (L R M S ) phenotype and to determine its location and genetic environment.

Methods: The whole genomic DNA of E. faecalis E531, which tested negative for the known lincosamide nucleotidyltransferase genes, was sequenced. A putative lincosamide resistance gene determinant was cloned into an Escherichia coli - E. faecalis shuttle vector (pAM401) and transformed into E. faecalis JH2-2. The MICs were determined by the microbroth dilution method. Inactivity of lincomycin was examined by UPLC-MS/MS. Inverse PCR and primer walking were used to explore the genetic environment based on the assembled sequence.

Results: A novel resistance gene, designated lnu (G), which encodes a putative lincosamide nucleotidyltransferase, was found in E. faecalis E531. The deduced Lnu(G) amino acid sequence displayed 76.0% identity to Lnu(B) in Enterococcus faecium . Both E. faecalis E531 and E. faecalis JH2-2 harbouring pAM401- lnu (G) showed a 4-fold increase in the MICs of lincomycin, compared with E. faecalis JH2-2 or E. faecalis JH2-2 harbouring empty vector pAM401 only. UPLC-MS/MS demonstrated that the Lnu(G) enzyme catalysed adenylylation of lincomycin. The genetic environment analysis revealed that the lnu (G) gene was embedded into a novel putative transposon, designated Tn 6260 , which was active.

Conclusions: A novel lincosamide nucleotidyltransferase gene lnu (G) was identified in E. faecalis . The location of the lnu (G) gene on a mobile element Tn 6260 makes it easy to disseminate.