Persistent, toxin-antitoxin system-independent, tetracycline resistance-encoding plasmid from a dairy Enterococcus faecium isolate

Abstract

A tetracycline-resistant (Tet(r)) dairy Enterococcus faecium isolate designated M7M2 was found to carry both tet(M) and tet(L) genes on a 19.6-kb plasmid. After consecutive transfer in the absence of tetracycline, the resistance-encoding plasmid persisted in 99% of the progenies. DNA sequence analysis revealed that the 19.6-kb plasmid contained 28 open reading frames (ORFs), including a tet(M)-tet(L)-mob gene cluster, as well as a 10.6-kb backbone highly homologous (99.9%) to the reported plasmid pRE25, but without an identified toxin-antitoxin (TA) plasmid stabilization system. The derived backbone plasmid without the Tet(r) determinants exhibited a 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits still to be elucidated. The tet(M)-tet(L) gene cluster from M7M2 was functional and transmissible and led to acquired resistance in Enterococcus faecalis OG1RF by electroporation and in Streptococcus mutans UA159 by natural transformation. Southern hybridization showed that both the tet(M) and tet(L) genes were integrated into the chromosome of S. mutans UA159, while the whole plasmid was transferred to and retained in E. faecalis OG1RF. Quantitative real-time reverse transcription-PCR (RT-PCR) indicated tetracycline-induced transcription of both the tet(M) and tet(L) genes of pM7M2. The results indicated that multiple mechanisms might have contributed to the persistence of antibiotic resistance-encoding genes and that the plasmids pM7M2, pIP816, and pRE25 are likely correlated evolutionarily.

Fig. 1.

Circular map of plasmid pM7M2 (see Table 1 for further details). The putative open reading frames are numbered. The coding regions are represented by arrows indicating the direction of transcription, and putative proteins encoded by them correspond to the proteins from the database with the highest amino acid homology. Black solid ring, 99.5% nucleotide sequence identity (positions 1 to 7097) with S. gallolyticus UCN34 (GenBank accession no. FN597254.1) genome, which has an additional 32 bp behind position 6493 of pM7M2, as indicated by the black solid triangle; gray solid ring, 99% to 100% nucleotide sequence identity (positions 7098 to 9438, 10500 to 14353, 17014 to 18706, and 18749 to 19557) with E. faecium plasmid pIP816 (GenBank accession no. AM932524.1); open ring, 99.9% nucleotide sequence identity (positions 8744 to 19557) with E. faecalis plasmid pRE25 (GenBank accession no. X92945.2) and E. faecium plasmid p5753cB (GenBank accession no. GQ900487.1).

Fig. 2.

Relative quantification of tet(M) and tet(L) transcripts with 8, 16, and 32 μg/ml tetracycline compared to 0 μg/ml tetracycline. Bars represent means ± standard errors of the means for three independent biological replicates.