Sentinel Surveillance Reveals Emerging Daptomycin-Resistant ST736 Enterococcus faecium and Multiple Mechanisms of Linezolid Resistance in Enterococci in the United States

Abstract

Enterococcus faecalis and faecium with resistance to daptomycin and/or linezolid are emerging globally. We present the genomic characterization of daptomycin- and linezolid-resistant E. faecalis and E. faecium surveillance isolates from the United States, 2013-2016. Daptomycin resistance was low among E. faecalis (2/364, 0.5%) and E. faecium (17/344, 5%). The majority (71%, 12/17) of daptomycin-resistant E. faecium isolates belonged to the emerging ST736 clone and contained mutations in liaFSR and cls previously associated with resistance. However, 1/2 E. faecalis and 3/17 E. faecium did not contain these mutations previously associated with daptomycin resistance. Linezolid resistance was rare among E. faecalis (1/364, 0.3%) and E. faecium (2/344, 0.6%). These two E. faecium isolates, one of which was also resistant to daptomycin and vancomycin, contained the 23S rRNA nucleotide mutation (G2576T) associated with linezolid resistance. Long-read sequencing revealed the linezolid-resistant E. faecalis isolate contained chromosomal- and plasmid-encoded copies of optrA. The chromosomal optrA was located on the recently described Tn6674 multiresistance transposon. The second copy of optrA was encoded on an ∼65 kb mosaic plasmid, with component regions sharing high sequence identity to optrA-encoding multiresistance plasmids of animal origin. The optrA-encoding plasmid contained open reading frames predicted to encode proteins associated with a pheromone-responsive plasmid transfer system, and filter mating experiments confirmed the plasmid was conjugative. Continued surveillance of enterococci is necessary to assess the prevalence and trends of daptomycin and linezolid resistance in the United States, characterize resistance mechanisms and how they transfer, and monitor for emerging sequence types associated with resistance.

Keywords: Enterococcus faecalis; Enterococcus faecium; conjugation; daptomycin; linezolid; optrA; pheromone responsive plasmid; transmission.

FIGURE 1

Whole genome multi-locus sequence typing of daptomycin-resistant ST736 E. faecium isolates. Vancomycin-resistant (VAN-R), linezolid-resistant (LZD-R), and daptomycin-resistant (DAP-R) profiles are indicated (X: resistant). Amino acid mutations in LiaR, LiaS, and Cls are designated (Cls mutation not previously reported as associated with DAP resistance is underlined and in bold). States from which the isolates were collected are indicated (colored boxes).

FIGURE 2

Diagram of AR-0780’s chromosomal transposon, Tn6674 (12,933 bp). optrA along with the resistance genes spc, erm(A), and fexA, are shown in red. Transposase genes, tnpA, tnpB, and tnpC, are shown in blue. The left and right hexanucleotide sequences marking the left and right junction of the Tn6674 radC integration site are shown in boxes. The disrupted radC (ΔradC) is shown in gray.

FIGURE 3

Comparative analysis of pAR-0780 with similar optrA-encoding plasmids. pAR-0780 contained a near identical copy (100% query coverage, 99.95% identity) of a 14,349 bp optrA gene cluster flanked on the left- and right-hand sides by IS1216R elements (black) previously described on plasmid p10-2-2 from an E. faecalis isolate (ST59) recovered from a pig in China in 2015. The remaining 51,543 bp of pAR-0780 was nearly identical (100% coverage, 99.88% identity) to a 51,543 region of the 84,468 bp plasmid, pE508, a pheromone-responsive conjugative multiresistance plasmid from E. faecalis strain E508 (ST256), isolated from a swine fecal sample in 2015.