Sentinel Surveillance reveals phylogenetic diversity and detection of linear plasmids harboring vanA and optrA among enterococci collected in the United States

Abstract

Enterococcus faecalis and Enterococcus faecium are frequent causes of healthcare-associated infections. Antimicrobial-resistant enterococci pose a serious public health threat, particularly vancomycin-resistant enterococci (VRE), for which treatment options are limited. The Centers for Disease Control and Prevention's Division of Healthcare Quality Promotion Sentinel Surveillance system conducted surveillance from 2018 to 2019 to evaluate antimicrobial susceptibility profiles and molecular epidemiology of 205 E. faecalis and 180 E. faecium clinical isolates collected from nine geographically diverse sites in the United States. Whole genome sequencing revealed diverse genetic lineages, with no single sequence type accounting for more than 15% of E. faecalis or E. faecium. Phylogenetic analysis distinguished E. faecium from 19 E. lactis (previously known as E. faecium clade B). Resistance to vancomycin was 78.3% among E. faecium, 7.8% among E. faecalis, and did not occur among E. lactis isolates. Resistance to daptomycin and linezolid was rare: E. faecium (5.6%, 0.6%, respectively), E. faecalis (2%, 2%), and E. lactis (5.3%, 0%). All VRE harbored the vanA gene. Three of the seven isolates that were not susceptible to linezolid harbored optrA, one chromosomally located and two on linear plasmids that shared a conserved backbone with other multidrug-resistant conjugative linear plasmids. One of these isolates contained optrA and vanA co-localized on the linear plasmid. By screening all enterococci, 20% of E. faecium were predicted to harbor linear plasmids, whereas none were predicted among E. faecalis or E. lactis. Continued surveillance is needed to assess the future emergence and spread of antimicrobial resistance by linear plasmids and other mechanisms.IMPORTANCEThis work confirms prior reports of E. faecium showing higher levels of resistance to more antibiotics than E. faecalis and identifies that diverse sequence types are contributing to enterococcal infections in the United States. All VRE harbored the vanA gene. We present the first report of the linezolid resistance gene optrA on linear plasmids in the United States, one of which co-carried a vanA cassette. Additional studies integrating epidemiological, antimicrobial susceptibility, and genomic methods to characterize mechanisms of resistance, including the role of linear plasmids, will be critical to understanding the changing landscape of enterococci in the United States.

Keywords: Enterococcus faecalis; Enterococcus faecium; daptomycin; linear plasmid; linezolid; optrA.

Fig 1

E. faecalis maximum likelihood phylogeny based on core gene alignments generated by Roary and RaxML. Annotation rings (from inner to outer) show isolate ID, state of origin, multi-locus sequence type (ST), minimum inhibitory concentration (MIC) interpretations for vancomycin, daptomycin, and linezolid. ST obtained using E. faecalis scheme from PubMLST, MIC interpretations represent susceptible in white; intermediate in gray; and resistant in black. Black circles on branches represent 100% support for the branch out of 100 bootstraps, and the tree is rooted at the midpoint. Abbreviations: LZD, linezolid; DAP, daptomycin; VAN, vancomycin; ST; multi-locus sequence type, CA, California; IA, Iowa; MD, Maryland; MN, Minnesota; NM, New Mexico; NY, New York; NC, North Carolina; PA, Pennsylvania; and WA, Washington.

Fig 2

E. faecium maximum likelihood phylogeny based on core gene alignments generated by Roary and RaxML. Annotation rings (from inner to outer) show isolate ID, state of origin, multi-locus sequence type (ST), minimum inhibitory concentration (MIC) interpretations for vancomycin, daptomycin, and linezolid. ST obtained using E. faecium scheme from PubMLST, MIC interpretations following E. faecium guidance represents susceptible – white; susceptible dose-dependent – gray; and resistant – black. Black circles on branches represent 100% support for the branch out of 100 bootstraps. Abbreviations: LZD, linezolid; DAP, daptomycin; VAN, vancomycin; ST, multi-locus sequence type; CA, California; IA, Iowa; MD, Maryland; MN, Minnesota; NM, New Mexico; NY, New York; NC, North Carolina; PA, Pennsylvania; and WA, Washington.

Fig 3

E. lactis maximum likelihood phylogeny based on core gene alignments generated by Roary and RaxML. Annotation (from left to right) shows isolate ID, state of origin, multi-locus sequence type (ST), MALDI-TOF MS identified species, ANI identified species, minimum inhibitory concentration (MIC) interpretations for vancomycin, daptomycin, and linezolid. ST obtained using E. faecium scheme from PubMLST, MIC interpretations following E. faecium guidance represents susceptible in white; susceptible dose-dependent in gray; and resistant in black. Black circles on branches represent 100% support for the branch out of 100 bootstraps. Abbreviations: LZD, linezolid; DAP, daptomycin; VAN, vancomycin; ST, multi-locus sequence type; CA, California; IA, Iowa; MD, Maryland; MN, Minnesota; NM, New Mexico; NY, New York; NC, North Carolina; PA, Pennsylvania; and WA, Washington.

Fig 4

Confirmation of the presence of linear plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) of optrA-positive enterococci. Lane 1, PFG MidRange Marker; lane 2, 2001206 (undigested); lane 3, 2001206 (digested); lane 4, 2001564 (undigested, ~114 kb linear plasmid present); lane 5, 2001564 (digested, ~114 kb linear plasmid present); lane 6, 2008300 (undigested, ~106 kb plasmid present); and lane 7, 2008300 (digested, ~106 kb plasmid present).

Fig 5

Comparison of this study’s linear plasmids with optrA, the highly similar plasmid pBA17124_P1(34), and the first documented Enterococcal Linear Form plasmids, pELF1 and pELF2 (33, 35). Accession and plasmid length are shown under the plasmid name. Regions of similarity between sequences are connected by vertical blocks when there is a minimum of 500 bp and 80% BLASTN identity. Coding sequences are shown with arrows, and gene functions are color-coded as follows: vancomycin-related resistance genes are in orange, other resistance genes in red, radC in purple, DNA replication in brown, plasmid partitioning machinery in blue, parM in light blue, and IS elements or transposases in yellow.