Evolving oxazolidinone resistance mechanisms in a worldwide collection of enterococcal clinical isolates: results from the SENTRY Antimicrobial Surveillance Program

Evolving oxazolidinone resistance mechanisms in a worldwide collection of enterococcal clinical... Abstract Objectives This study evaluated the oxazolidinone resistance mechanisms among a global collection of enterococcal clinical isolates. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. Methods Enterococcal isolates (26 648) from the SENTRY Antimicrobial Surveillance Program (2008–16) were identified by MALDI-TOF MS and MICs were determined by broth microdilution. Isolates with linezolid MICs of ≥4 mg/L were screened for resistance mechanisms. Isolates carrying optrA had their genome sequenced for genetic context and epidemiology information. Results Thirty-six Enterococcus faecalis and 66 Enterococcus faecium had linezolid MICs of ≥4 mg/L (0.38% of surveillance enterococci). E. faecalis had a linezolid MIC range of 4–16 mg/L, while E. faecium displayed higher values (4–64 mg/L). Nine E. faecalis had G2576T mutations and optrA was detected in 26 (72.2%) isolates from the Asia-Pacific region, North America, Latin America and Europe; 3 isolates also produced Cfr [Thailand (1)] or Cfr(B) [Panama (2)]. All E. faecium isolates had G2576T alterations, while three isolates from the USA had concomitant presence of cfr(B). The optrA gene was plasmid- and chromosome-located in 22 and 3 E. faecalis, respectively. One isolate signalled hybridization on plasmid and chromosome. The genetic context of optrA varied. E. faecalis belonging to the same clonal complex were detected in distinct geographical regions. Also, genetically distinct isolates from Ireland had an identical optrA context, indicating plasmid dissemination. Conclusions Alterations in 23S rRNA remained the main oxazolidinone resistance mechanism in E. faecium, while optrA prevailed in E. faecalis. These results demonstrate global dissemination of optrA and warrant surveillance for monitoring. Introduction Enterococcus species are considered part of the normal flora of the human gut microbiome. However, these isolates are also opportunistic pathogens broadly implicated in urinary tract infections (UTIs), wound and transplant infections, infections of the heart valve and other prosthetic implants, and septicaemia.1 Risk factors for developing these infections include age-related colonization, prolonged hospital stay, previous antibiotic use and immunosuppression. Enterococcal infections are second only to staphylococci as a cause of Gram-positive nosocomial infections.2 These organisms can acquire antimicrobial resistance and VRE remain a challenge, especially Enterococcus faecium.3 Anti-Gram-positive agents belonging to the oxazolidinone class possess potent activity against clinical pathogens causing infections worldwide, as determined by large surveillance studies.4–7 These agents are effective and widely used to treat VRE infections and only a small percentage of linezolid-resistant enterococcal isolates have been reported during surveillance studies.4,5,7 Resistance occurs especially after prolonged administration and local investigations have reported sporadic outbreaks and dissemination of linezolid-dependent Staphylococcus epidermidis isolates in Greece and Germany.8–15 Ribosomal mutations, including alterations in oxazolidinone binding sites (23S rRNA and L3 and L4 ribosomal proteins) remain the most common mechanisms of resistance among staphylococci and enterococci. Specifically, G2576T mutation in the V domain of the 23S rRNA gene has been responsible for a resistance phenotype among enterococci.16 Additional and transferable resistance determinants, such as cfr, cfr(B), cfr(C) and optrA, have been detected as newer mechanisms responsible for decreased susceptibility to linezolid and/or tedizolid.17–20 These plasmid-borne resistance genes have been documented in numerous human clinical isolate species in several regions worldwide.2,4,21–29 In addition, studies have reported on the concomitant detection of cfr and optrA genes in Staphylococcus sciuri and E. faecium.24 Thus, although oxazolidinones show potent activity (>99% susceptible) against enterococci and Enterococcus faecalis remain susceptible to several agents, the dissemination of optrA in E. faecalis could compromise the clinical utility of the oxazolidinones. In addition, the presence of optrA in the gene pool could further increase its potential to disseminate to other less susceptible species, including E. faecium, which are often only susceptible to oxazolidinones and daptomycin,30–34 and already reported by several local studies. This study was conducted to evaluate the oxazolidinone resistance mechanisms among a global collection of enterococcal clinical isolates from the SENTRY Antimicrobial Surveillance Program. Moreover, the epidemiology and genetic context of optrA-carrying isolates were further investigated. Materials and methods Bacterial strains A total of 17 614 E. faecalis and 9034 E. faecium clinical isolates were submitted to the coordinating monitoring laboratory (JMI Laboratories, North Liberty, IA, USA), as part of the SENTRY Antimicrobial Surveillance Program from 2008 to 2016. This programme monitors antimicrobial resistance and prevalence of pathogens causing bloodstream infections, community-acquired pneumonia, pneumonia in hospitalized patients, skin and skin structure infections, UTIs and intra-abdominal infections (six main study protocols). Participating sites follow instructions specific for each protocol to select and include consecutive and unique (one per patient) isolates that are deemed clinically relevant based on local criteria. Antimicrobial susceptibility testing Susceptibility testing was performed by broth microdilution methods, according to CLSI recommendations.35 In addition, the inoculum density was monitored by colony counts to ensure an adequate number of cells for each testing event. Concurrently testing CLSI-recommended quality control reference strains (Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 29212) validated the MICs.36 MIC interpretations were based on the CLSI M100-S27 and EUCAST documents, when available.36,37 Enterococci exhibiting linezolid MIC results of ≥4 mg/L were selected for further molecular analysis and are presented in this study. Screening for oxazolidinone resistance mechanisms Isolates meeting the pre-established screening criteria (linezolid MIC, ≥4 mg/L) were subjected to the detection of cfr, cfr(B), cfr(C) and optrA and mutations in the 23S rRNA-, L3- and L4-encoding genes by PCR, restriction digests and sequencing as described previously.18,38 Amplicons were sequenced on both strands and amino acid sequences compared with those from E. faecalis and E. faecium reference strains. Characterization of optrA gene context Genomic DNA of enterococcal isolates carrying optrA was extracted using the Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor (Cleveland, OH, USA). Total genomic DNA was used as input material for library construction. DNA libraries were prepared using the NexteraXT™ library construction protocol (Illumina, San Diego, CA, USA) following the manufacturer’s instructions and sequenced on a MiSeq Sequencer (JMI Laboratories). The coding and surrounding regions of optrA, as well as other antimicrobial resistance determinants and MLST information, were extracted from assembled genomes (SPAdes 9.3.0). The optrA gene cluster sequences of all distinct isolates in this study have been deposited in GenBank (accession numbers MF443367 to MF443388). The optrA gene arrays were analysed and are presented in Figure 1. Figure 1. View largeDownload slide Schematic representation of optrA genetic background (array) in plasmid and chromosomal context. Arrows indicate direction of transcription; black arrows indicate miscellaneous genes with no known functions (listed as hypothetical proteins or ORFs). Figure 1. View largeDownload slide Schematic representation of optrA genetic background (array) in plasmid and chromosomal context. Arrows indicate direction of transcription; black arrows indicate miscellaneous genes with no known functions (listed as hypothetical proteins or ORFs). Southern hybridization Whole genomic DNA was prepared in 1% agarose blocks. DNA was digested by S1 nuclease and I-CeuI enzymes and resolved with PFGE on CHEF DRII (Bio-Rad, Hercules, CA, USA). DNA fragments were transferred to nylon membranes by Southern blotting, probed with a DIG-labelled-optrA-specific probe and developed as per manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). Results A total of 36 E. faecalis and 66 E. faecium met the study screening criteria of a linezolid MIC of ≥4 mg/L, which constituted 0.38% of surveillance enterococci received between 2008 and 2016 (Table 1). E. faecalis isolates had a linezolid MIC range of 4–16 mg/L (MIC50/90, 4/8 mg/L), while E. faecium isolates displayed slightly higher values (4–64 mg/L; MIC50/90, 8/32 mg/L; Table 1). Most E. faecalis isolates exhibited a susceptible phenotype to ampicillin (97.2%–100.0% susceptible), tigecycline (100.0% susceptible) and daptomycin (100.0% susceptible), while marginal activities were observed for the glycopeptides (88.9% susceptible). E. faecium showed high susceptibility rates for tigecycline (95.9% susceptible) and daptomycin (98.6% susceptible; Table 1). Table 1. Activity of antimicrobial agents tested against an enterococcal collection that exhibited linezolid MIC values of ≥4 mg/L Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  a Breakpoint criteria according to CLSI (M100-S27, 2017)36 and EUCAST,37 as available. CLSI and EUCAST linezolid susceptible breakpoint is ≤2 mg/L. b Uncomplicated UTIs only. Table 1. Activity of antimicrobial agents tested against an enterococcal collection that exhibited linezolid MIC values of ≥4 mg/L Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  a Breakpoint criteria according to CLSI (M100-S27, 2017)36 and EUCAST,37 as available. CLSI and EUCAST linezolid susceptible breakpoint is ≤2 mg/L. b Uncomplicated UTIs only. The optrA gene was detected in 26 (72.2%) E. faecalis [Tables 1–3 and Figure S1 (available as Supplementary data at JAC Online)]. Among these isolates, one from Thailand and two clonal strains from Panama also carried cfr and cfr(B), respectively (Tables 2 and 3). Nine other (25.0%) E. faecalis isolates had G2576T mutations in the 23S rRNA. Six E. faecalis isolates had an alteration in L4 (F101L) in addition to either optrA (two from China and one each from Sweden, Ireland and Thailand) or G2576T (USA). A linezolid resistance mechanism was not identified in one E. faecalis from Ireland (linezolid MIC, 4 mg/L; Table 2). All E. faecium isolates showed G2576T mutations, while two isolates from New Orleans (USA)18 and one from Atlanta (USA) had concomitant presence of cfr(B). One E. faecium isolate showed alteration in L3 (K95T) in addition to G2576T mutation (Table 2). Table 2. Summary of oxazolidinone resistance mechanisms detected among isolates Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  a Mutations were investigated in the gene encoding 23S rRNA and amino acid alterations in the L3 and L4 ribosomal proteins. b Represents isolates with the cfr(B) variant. c A linezolid resistance mechanism was not identified in this isolate (Ireland). Table 2. Summary of oxazolidinone resistance mechanisms detected among isolates Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  a Mutations were investigated in the gene encoding 23S rRNA and amino acid alterations in the L3 and L4 ribosomal proteins. b Represents isolates with the cfr(B) variant. c A linezolid resistance mechanism was not identified in this isolate (Ireland). Table 3. Characteristics of OptrA-producing E. faecalis isolates Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  a OptrA protein alterations compared with KP399637. b Gene array as depicted in Figure 1. c Macrolide, lincosamide and streptogramin B agents. Table 3. Characteristics of OptrA-producing E. faecalis isolates Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  a OptrA protein alterations compared with KP399637. b Gene array as depicted in Figure 1. c Macrolide, lincosamide and streptogramin B agents. Genetic characteristics of OptrA-producing isolates, other resistance determinants and optrA genetic context information are detailed in Table 3 and Figure 1. Based on the S1 nuclease/I-CeuI Southern blot analysis, optrA was plasmid-located in 22 isolates, while 3 isolates carried this gene in the chromosome (Table 3). Hybridization signals were obtained on chromosomal DNA and plasmid in one isolate from France. E. faecalis isolates carrying optrA were detected in the SENTRY program as early as 2008 in China and Sweden and in 10 additional countries through 2016 (Table 3 and Figure S1). Isolates belonging to the same clonal complex (CC) were observed in distinct regions, such as CC16 isolates that were detected in the USA and Thailand, and CC116 isolates that were detected in China and Ireland (Table 3). The CC116 isolates from China and CC86 from Sweden (both from 2008) showed a very similar optrA genetic context (array A; Figure 1). Similarly, three E. faecalis isolates from a medical centre in Ireland and two from Taiwan27 exhibited distinct lineages based on MLST, but a similar optrA genetic context (array A1 and array H, respectively) (Table 3 and Figure 1). One isolate from Ireland yielded a small contiguous sequence (array E). Other isolates from China yielded smaller contiguous sequences and different gene arrangements around optrA (arrays B–D and F). Array F was also observed in an isolate (CC16) from the USA; however, the CC16 isolates from Thailand had a very distinct optrA array (G). A CC59 isolate from Malaysia showed an optrA gene context (array I) similar to F (China/USA), K (USA) and N (Guatemala). Isolates from Taiwan and France showed optrA genes associated with Tn554, but these isolates showed distinct gene arrangements (array H and J, respectively). Clonally related OptrA-producing isolates were detected in Panama in 2011 (CC103) and were detected in Mexico (CC480) and Guatemala (CC256) in 2016 (Table 3). The optrA context observed among isolates from Mexico included TnMERI1 native to Bacillus spp. (array L), while those from Guatemala were associated with Tn551 (array N), with erm(B) and erm(A)-like genes upstream and downstream of optrA, respectively. The optrA genetic context (array O) noted in isolates from Mexico was similar to arrays K and F detected in isolates from the USA and China, except for the number and orientation of ORFs of unknown functions. A very short optrA contiguous sequence was obtained for isolate 751258 from Ecuador with only araC upstream of optrA (array M). IS1216, fex(A) and erm(A)-like were often present with optrA. The fex(A) gene was present in all plasmid backgrounds, except for one isolate (ST69) recovered from China in 2009 and two isolates (ST103) from Panama (2011). A total of 11 isolates (11/23 plasmid optrA) carried the erm(A)-like gene, as previously described by He et al.39 (Table 3). The fer gene encoding ferredoxin-NADP reductase (4Fe–2S ferredoxin iron–sulphur binding domain protein) was present downstream of optrA in 13 isolates. OptrA sequences in 18/26 isolates showed minor differences compared with the index sequence (plasmid pE349 from E. faecalis19 GenBank accession number KP399637); eight variants, including those described by Cui et al.,34 were noted (Table 3). Three alteration combinations not described by Cui et al.34 were noted in Sweden/France (Y176D; identical to Streptococcus suis; GenBank accession number WP_099810410), Thailand (G40D, T481P; identical to GenBank accession number WP_002415370) and Panama (K3E, Y176D, G393D, I622M; novel combination). All but one OptrA-producing E. faecalis from Sweden carried erm(B); dfr(G) was present in 18/26 and dfr(D) in 1/26 isolates. The majority of isolates (16/26) carried tet(L) and tet(M) and 5/26 isolates had tet(L) (2 isolates) or tet(M) (3 isolates) alone (Table 3). The cfr gene in the E. faecalis isolate from Thailand (2010) was embedded in an IS256-like structure with a chromosomal genetic context (tnp-aacA/aphD-IS256 like-orf-cfr) identical to S. epidermidis isolated in Tempe (Arizona) (GenBank accession number JX910899). cfr(B) in isolates from Panama showed a genetic context that was 98% identical to the isolate reported in E. faecium from New Orleans18 (GenBank accession number KR610408). Discussion This study shows a low prevalence of enterococcal isolates non-susceptible to linezolid (0.38%) and resistance mechanisms to oxazolidinones seem to have become distinct between E. faecalis and E. faecium. A previous review reported 23S rRNA alterations (G2576T) as the main resistance mechanism in both E. faecalis and E. faecium, especially in the USA.16 However, this review described several E. faecalis isolates, mostly from countries other than the USA, where mechanisms of oxazolidinone resistance were not detected. Further investigations detected the presence of optrA in these isolates, which are described here. The study shows that optrA has now become more prevalent than the presence of 23S rRNA alterations in E. faecalis and the sole oxazolidinone resistance mechanism among isolates from 2014 to 2016. Alterations in L3 and L4 proteins remained rare in Enterococcus spp.; however, six E. faecalis isolates in this set showed an L4 (F101L) alteration and one E. faecium displayed an L3 (K95T) alteration. The significance of these mutations remains unknown as these were always associated with another established linezolid resistance mechanism (Table 2) and also observed in linezolid-susceptible isolates.34 Cfr(B) seems to be emerging in Enterococcus spp. and this study reports it from E. faecalis isolates for the first time. The cfr or cfr(B) genes were always associated with another linezolid resistance mechanism in E. faecalis (optrA) and E. faecium (G2576T) (Table 2), so it is not clear if cfr confers linezolid resistance in Enterococcus spp. We showed that cfr(B) confers a resistance phenotype similar to cfr in an S. aureus background;18 however, its relevance in Enterococcus spp. has not been clearly defined and 23S rRNA alterations remain the main linezolid resistance mechanism in E. faecium.22,40 Isolates from China, Sweden, Ireland and the USA showed an optrA gene context similar to the plasmid structures described from China,19,39 with 452115 (China) and 441341 (Sweden) sequences showing 99% identity to pE349. These genetic contexts were also similar to those reported in isolates from Poland and Colombia.25,26 The fexA gene was upstream of optrA in 13 isolates, albeit with variations between the arrays. E. faecalis isolates 739884 (China) and 912300 (USA) showed an identical optrA genetic context (array F), which was previously described in an isolate recovered from a pig (ST59; CC59) carrying p10-2-2.39 Isolate 529360 (China; array B) showed IS1216 tnp bracketing optrA in a configuration most similar to that observed for pFX13 documented in an isolate from a pig in China. The optrA gene context beyond the IS1216 element for isolates from Guatemala was distinct compared with any reports thus far, which included Tn551. Isolates with chromosomally located optrA showed distinct array structures: G (Thailand), H (Taiwan) and J (France). E. faecalis 599799 from Thailand had optrA flanking regions displaying high homology with those noted for human isolates E016 and E079 previously reported from China.39 The optrA array for isolate 973450 from France was identical to isolate G20 (human isolate) from China and was flanked by Tn558 (He et al.39). While this isolate showed hybridization signals on plasmid and chromosomal DNA bands, distinct optrA gene arrays could not be identified. All chromosomal optrA had flanking sequences containing the putative transcriptional regulator araC.27 Finally, this study reports the global dissemination of optrA-carrying E. faecalis recovered from patients in countries beyond the Asia-Pacific region. E. faecalis isolates carrying optrA showed a very diverse genetic background. Interestingly, isolates belonging to the same CC were detected in distinct geographical regions. Also, isolates from Ireland with distinct genetic backgrounds had a similar optrA context, indicating plasmid dissemination. From the gene array analysis, it was evident that a great degree of genetic rearrangement is taking place as optrA spreads across the globe; the core genetic elements remaining similar, their positions in the array are divergent in isolates from different geographical locations. These results indicate the potential for dissemination and warrant constant surveillance for monitoring purposes. Although the majority of optrA genes were found in E. faecalis so far, this gene has also been documented in S. sciuri, E. faecium and other Gram-positive organisms,21–24,34 and in in vitro transfer to different species.25 Therefore, it is important to monitor the emergence and spread of this resistance determinant at a local and regional level, especially due to the potential for E. faecalis to serve as a reservoir for spreading optrA to MDR pathogens (i.e. E. faecium). Acknowledgements Part of this study was presented at the American Society for Microbiology Microbe Conference, Boston, MA, USA, 2016 (Poster #Saturday 332). We would like to thank all SENTRY participating sites contributing clinical isolates during the 2008–16 study period. Funding This study was supported by JMI Laboratories (North Liberty, IA, USA) through the SENTRY Antimicrobial Surveillance Program. Transparency declarations JMI Laboratories was contracted to perform services in 2017 for Achaogen, Allecra Therapeutics, Allergan, Amplyx Pharmaceuticals, Antabio, API, Astellas Pharma, AstraZeneca, Athelas, Basilea Pharmaceutica, Bayer AG, BD, Becton, Dickinson and Co., Boston, CEM-102 Pharma, Cempra, Cidara Therapeutics, Inc., CorMedix, CSA Biotech, Cutanea Life Sciences, Inc., Entasis Therapeutics, Inc., Geom Therapeutics, Inc., GSK, Iterum Pharma, Medpace, Melinta Therapeutics, Inc., Merck & Co., Inc., MicuRx Pharmaceuticals, Inc., N8 Medical, Inc., Nabriva Therapeutics, Inc., NAEJA-RGM, Novartis, Paratek Pharmaceuticals, Inc., Pfizer, Polyphor, Ra Pharma, Rempex, Riptide Bioscience Inc., Roche, Scynexis, Shionogi, Sinsa Labs Inc., Skyline Antiinfectives, Sonoran Biosciences, Spero Therapeutics, Symbiotica, Synlogic, Synthes Biomaterials, TenNor Therapeutics, Tetraphase, The Medicines Company, Theravance Biopharma, VenatoRx Pharmaceuticals, Inc., Wockhardt, Yukon Pharma, Zai Laboratory and Zavante Therapeutics, Inc. There are no speakers’ bureaus or stock options to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online. References 1 Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol  2012; 10: 266– 78. Google Scholar CrossRef Search ADS PubMed  2 Mendes RE, Castanheira M, Farrell DJ et al.   Longitudinal (2001–14) analysis of enterococci and VRE causing invasive infections in European and US hospitals, including a contemporary (2010–13) analysis of oritavancin in vitro potency. J Antimicrob Chemother  2016; 71: 3453– 8. Google Scholar CrossRef Search ADS PubMed  3 Miller WR, Murray BE, Rice LB et al.   Vancomycin-resistant enterococci: therapeutic challenges in the 21st century. Infect Dis Clin North Am  2016; 30: 415– 39. 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Google Scholar CrossRef Search ADS PubMed  19 Wang Y, Lv Y, Cai J et al.   A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin. J Antimicrob Chemother  2015; 70: 2182– 90. Google Scholar CrossRef Search ADS PubMed  20 Candela T, Marvaud JC, Nguyen TK et al.   A cfr-like gene cfr(C) conferring linezolid resistance is common in Clostridium difficile. Int J Antimicrob Agents  2017; 50: 496– 500. Google Scholar CrossRef Search ADS PubMed  21 Fan R, Li D, Wang Y et al.   Presence of the optrA gene in methicillin-resistant Staphylococcus sciuri of porcine origin. Antimicrob Agents Chemother  2016; 60: 7200– 5. Google Scholar PubMed  22 Brenciani A, Morroni G, Vincenzi C et al.   Detection in Italy of two clinical Enterococcus faecium isolates carrying both the oxazolidinone and phenicol resistance gene optrA and a silent multiresistance gene cfr. 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HPS Weekly Report  2016; 50: 230– 1. 30 Lee SM, Huh HJ, Song DJ et al.   Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: low rate of 23S rRNA mutations in Enterococcus faecium. J Med Microbiol  2017; 66: 1730– 5. Google Scholar CrossRef Search ADS PubMed  31 Lazaris A, Coleman DC, Kearns AM et al.   Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE. J Antimicrob Chemother  2017; 72: 3252– 7. Google Scholar CrossRef Search ADS PubMed  32 Cavaco LM, Korsgaard H, Kaas RS et al.   First detection of linezolid resistance due to the optrA gene in enterococci isolated from food products in Denmark. J Glob Antimicrob Resist  2017; 9: 128– 9. Google Scholar CrossRef Search ADS PubMed  33 Tamang MD, Moon DC, Kim SR et al.   Detection of novel oxazolidinone and phenicol resistance gene optrA in enterococcal isolates from food animals and animal carcasses. Vet Microbiol  2017; 201: 252– 6. Google Scholar CrossRef Search ADS PubMed  34 Cui L, Wang Y, Lv Y et al.   Nationwide surveillance of novel oxazolidinone resistance gene optrA in Enterococcus isolates in China from 2004 to 2014. Antimicrob Agents Chemother  2016; 60: 7490– 3. Google Scholar CrossRef Search ADS PubMed  35 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Tenth Edition: Approved Standard M07-A10 . CLSI, Wayne, PA, USA, 2015. 36 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Seventh Informational Supplement M100-S27 . CLSI, Wayne, PA, USA, 2017. 37 EUCAST. Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, January 2018. http://www.eucast.org/clinical_breakpoints/. 38 Mendes RE, Deshpande LM, Bonilla HF et al.   Dissemination of a pSCFS3-like cfr-carrying plasmid in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates recovered from hospitals in Ohio. Antimicrob Agents Chemother  2013; 57: 2923– 8. Google Scholar CrossRef Search ADS PubMed  39 He T, Shen Y, Schwarz S et al.   Genetic environment of the transferable oxazolidinone/phenicol resistance gene optrA in Enterococcus faecalis isolates of human and animal origin. J Antimicrob Chemother  2016; 71: 1466– 73. Google Scholar CrossRef Search ADS PubMed  40 Bender JK, Fleige C, Klare I et al.   Detection of a cfr(B) variant in German Enterococcus faecium clinical isolates and the impact on linezolid resistance in Enterococcus spp. PLoS One  2016; 11: e0167042. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Evolving oxazolidinone resistance mechanisms in a worldwide collection of enterococcal clinical isolates: results from the SENTRY Antimicrobial Surveillance Program

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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0305-7453
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1460-2091
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10.1093/jac/dky188
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Abstract

Abstract Objectives This study evaluated the oxazolidinone resistance mechanisms among a global collection of enterococcal clinical isolates. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. Methods Enterococcal isolates (26 648) from the SENTRY Antimicrobial Surveillance Program (2008–16) were identified by MALDI-TOF MS and MICs were determined by broth microdilution. Isolates with linezolid MICs of ≥4 mg/L were screened for resistance mechanisms. Isolates carrying optrA had their genome sequenced for genetic context and epidemiology information. Results Thirty-six Enterococcus faecalis and 66 Enterococcus faecium had linezolid MICs of ≥4 mg/L (0.38% of surveillance enterococci). E. faecalis had a linezolid MIC range of 4–16 mg/L, while E. faecium displayed higher values (4–64 mg/L). Nine E. faecalis had G2576T mutations and optrA was detected in 26 (72.2%) isolates from the Asia-Pacific region, North America, Latin America and Europe; 3 isolates also produced Cfr [Thailand (1)] or Cfr(B) [Panama (2)]. All E. faecium isolates had G2576T alterations, while three isolates from the USA had concomitant presence of cfr(B). The optrA gene was plasmid- and chromosome-located in 22 and 3 E. faecalis, respectively. One isolate signalled hybridization on plasmid and chromosome. The genetic context of optrA varied. E. faecalis belonging to the same clonal complex were detected in distinct geographical regions. Also, genetically distinct isolates from Ireland had an identical optrA context, indicating plasmid dissemination. Conclusions Alterations in 23S rRNA remained the main oxazolidinone resistance mechanism in E. faecium, while optrA prevailed in E. faecalis. These results demonstrate global dissemination of optrA and warrant surveillance for monitoring. Introduction Enterococcus species are considered part of the normal flora of the human gut microbiome. However, these isolates are also opportunistic pathogens broadly implicated in urinary tract infections (UTIs), wound and transplant infections, infections of the heart valve and other prosthetic implants, and septicaemia.1 Risk factors for developing these infections include age-related colonization, prolonged hospital stay, previous antibiotic use and immunosuppression. Enterococcal infections are second only to staphylococci as a cause of Gram-positive nosocomial infections.2 These organisms can acquire antimicrobial resistance and VRE remain a challenge, especially Enterococcus faecium.3 Anti-Gram-positive agents belonging to the oxazolidinone class possess potent activity against clinical pathogens causing infections worldwide, as determined by large surveillance studies.4–7 These agents are effective and widely used to treat VRE infections and only a small percentage of linezolid-resistant enterococcal isolates have been reported during surveillance studies.4,5,7 Resistance occurs especially after prolonged administration and local investigations have reported sporadic outbreaks and dissemination of linezolid-dependent Staphylococcus epidermidis isolates in Greece and Germany.8–15 Ribosomal mutations, including alterations in oxazolidinone binding sites (23S rRNA and L3 and L4 ribosomal proteins) remain the most common mechanisms of resistance among staphylococci and enterococci. Specifically, G2576T mutation in the V domain of the 23S rRNA gene has been responsible for a resistance phenotype among enterococci.16 Additional and transferable resistance determinants, such as cfr, cfr(B), cfr(C) and optrA, have been detected as newer mechanisms responsible for decreased susceptibility to linezolid and/or tedizolid.17–20 These plasmid-borne resistance genes have been documented in numerous human clinical isolate species in several regions worldwide.2,4,21–29 In addition, studies have reported on the concomitant detection of cfr and optrA genes in Staphylococcus sciuri and E. faecium.24 Thus, although oxazolidinones show potent activity (>99% susceptible) against enterococci and Enterococcus faecalis remain susceptible to several agents, the dissemination of optrA in E. faecalis could compromise the clinical utility of the oxazolidinones. In addition, the presence of optrA in the gene pool could further increase its potential to disseminate to other less susceptible species, including E. faecium, which are often only susceptible to oxazolidinones and daptomycin,30–34 and already reported by several local studies. This study was conducted to evaluate the oxazolidinone resistance mechanisms among a global collection of enterococcal clinical isolates from the SENTRY Antimicrobial Surveillance Program. Moreover, the epidemiology and genetic context of optrA-carrying isolates were further investigated. Materials and methods Bacterial strains A total of 17 614 E. faecalis and 9034 E. faecium clinical isolates were submitted to the coordinating monitoring laboratory (JMI Laboratories, North Liberty, IA, USA), as part of the SENTRY Antimicrobial Surveillance Program from 2008 to 2016. This programme monitors antimicrobial resistance and prevalence of pathogens causing bloodstream infections, community-acquired pneumonia, pneumonia in hospitalized patients, skin and skin structure infections, UTIs and intra-abdominal infections (six main study protocols). Participating sites follow instructions specific for each protocol to select and include consecutive and unique (one per patient) isolates that are deemed clinically relevant based on local criteria. Antimicrobial susceptibility testing Susceptibility testing was performed by broth microdilution methods, according to CLSI recommendations.35 In addition, the inoculum density was monitored by colony counts to ensure an adequate number of cells for each testing event. Concurrently testing CLSI-recommended quality control reference strains (Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 29212) validated the MICs.36 MIC interpretations were based on the CLSI M100-S27 and EUCAST documents, when available.36,37 Enterococci exhibiting linezolid MIC results of ≥4 mg/L were selected for further molecular analysis and are presented in this study. Screening for oxazolidinone resistance mechanisms Isolates meeting the pre-established screening criteria (linezolid MIC, ≥4 mg/L) were subjected to the detection of cfr, cfr(B), cfr(C) and optrA and mutations in the 23S rRNA-, L3- and L4-encoding genes by PCR, restriction digests and sequencing as described previously.18,38 Amplicons were sequenced on both strands and amino acid sequences compared with those from E. faecalis and E. faecium reference strains. Characterization of optrA gene context Genomic DNA of enterococcal isolates carrying optrA was extracted using the Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor (Cleveland, OH, USA). Total genomic DNA was used as input material for library construction. DNA libraries were prepared using the NexteraXT™ library construction protocol (Illumina, San Diego, CA, USA) following the manufacturer’s instructions and sequenced on a MiSeq Sequencer (JMI Laboratories). The coding and surrounding regions of optrA, as well as other antimicrobial resistance determinants and MLST information, were extracted from assembled genomes (SPAdes 9.3.0). The optrA gene cluster sequences of all distinct isolates in this study have been deposited in GenBank (accession numbers MF443367 to MF443388). The optrA gene arrays were analysed and are presented in Figure 1. Figure 1. View largeDownload slide Schematic representation of optrA genetic background (array) in plasmid and chromosomal context. Arrows indicate direction of transcription; black arrows indicate miscellaneous genes with no known functions (listed as hypothetical proteins or ORFs). Figure 1. View largeDownload slide Schematic representation of optrA genetic background (array) in plasmid and chromosomal context. Arrows indicate direction of transcription; black arrows indicate miscellaneous genes with no known functions (listed as hypothetical proteins or ORFs). Southern hybridization Whole genomic DNA was prepared in 1% agarose blocks. DNA was digested by S1 nuclease and I-CeuI enzymes and resolved with PFGE on CHEF DRII (Bio-Rad, Hercules, CA, USA). DNA fragments were transferred to nylon membranes by Southern blotting, probed with a DIG-labelled-optrA-specific probe and developed as per manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). Results A total of 36 E. faecalis and 66 E. faecium met the study screening criteria of a linezolid MIC of ≥4 mg/L, which constituted 0.38% of surveillance enterococci received between 2008 and 2016 (Table 1). E. faecalis isolates had a linezolid MIC range of 4–16 mg/L (MIC50/90, 4/8 mg/L), while E. faecium isolates displayed slightly higher values (4–64 mg/L; MIC50/90, 8/32 mg/L; Table 1). Most E. faecalis isolates exhibited a susceptible phenotype to ampicillin (97.2%–100.0% susceptible), tigecycline (100.0% susceptible) and daptomycin (100.0% susceptible), while marginal activities were observed for the glycopeptides (88.9% susceptible). E. faecium showed high susceptibility rates for tigecycline (95.9% susceptible) and daptomycin (98.6% susceptible; Table 1). Table 1. Activity of antimicrobial agents tested against an enterococcal collection that exhibited linezolid MIC values of ≥4 mg/L Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  a Breakpoint criteria according to CLSI (M100-S27, 2017)36 and EUCAST,37 as available. CLSI and EUCAST linezolid susceptible breakpoint is ≤2 mg/L. b Uncomplicated UTIs only. Table 1. Activity of antimicrobial agents tested against an enterococcal collection that exhibited linezolid MIC values of ≥4 mg/L Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  Organism (number tested)/antimicrobial agenta  MIC (mg/L)   % susceptible (S)/% intermediate (I)/% resistant (R)a   range  MIC50  MIC90  CLSI   EUCAST           S  I  R  S  I  R  E. faecalis (36)                     linezolid  4–16  4  8  0.0  52.8  47.2  52.8  —  47.2   ampicillin  ≤1–8  ≤1  2  100.0  —  0.0  97.2  2.8  0.0   clindamycin  8 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  8–128  64  64  12.9  16.1  71.0  —  —  —   retapamulin  0.5 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  ≤1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  >2 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  13.9  2.8  83.3  16.7  —  83.3   doxycycline  ≤0.06 to >8  8  >8  22.2  38.9  38.9  —  —  —   tetracycline  ≤0.25 to >8  >8  >8  19.4  0.0  80.6  —  —  —   tigecycline  ≤0.03–0.25  0.0  0.12  —  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2 to >8  ≤2  >8  88.9  0.0    88.9  —  11.1   vancomycin  ≤0.5 to >16  1  >16  88.9  0.0  11.1  88.9  —  11.1  optrA-positive E. faecalis (26)                     linezolid  4–16  4  8  0.0  61.5  38.5  61.5  —  38.5   ampicillin  ≤1–4  ≤1  2  100.0  —  0.0  100.0  0.0  0.0   clindamycin  32 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  16–128  64  128  0.0  9.1  90.9  —  —  —   retapamulin  1 to >8  >8  >8  —  —  —  —  —  —   virginiamycin  1–32  8  32  —  —  —  —  —  —   quinupristin/dalfopristin  4 to >16  16  >16  —  —  —  —  —  —   levofloxacinb  1 to >4  >4  >4  15.4  3.8  80.8  19.2  —  80.8   doxycycline  8 to >8  >8  >8  0.0  40  60  —  —  —   tetracycline  0.5 to >8  >8  >8  15.4  0.0  84.6  —  —  —   tigecycline  0.03–0.12  0.06  0.25  100.0  —  —  100.0  0.0  0.0   daptomycin  0.12–2  1  2  100.0  —  —  —  —  —   teicoplanin  ≤2  ≤2  ≤2  100.0  0.0  0.0  100.0  —  0.0   vancomycin  0.5–4  1  2  100.0  0.0  0.0  100.0  —  0.0  E. faecium (66)                     linezolid  4–64  8  32  0.0  26.0  74.0  26.0  —  74.0   ampicillin  ≤1 to >8  >8  >8  4.1  —  95.9  2.7  1.4  95.9   clindamycin  ≤0.5 to >64  >64  >64  —  —  —  —  —  —   chloramphenicol  4–64  16  32  41.4  34.5  24.1  —  —  —   retapamulin  ≤0.06 to >8  0.25  1  —  —  —  —  —  —   virginiamycin  ≤1–8  ≤1  2  —  —  —  —  —  —   quinupristin/dalfopristin  ≤0.25–4  1  2  78.1  15.1  6.8  78.1  21.9  0.0   levofloxacinb  >4  >4  >4  2.7  1.4  95.9  4.1  —  95.9   doxycycline  ≤0.12 to >8  1  >8  65.3  14.3  20.4  —  —  —   tetracycline  ≤2 to >8  ≤2  >8  57.5  0.0  42.5  —  —  —   tigecycline  ≤0.03–2  0.06  0.25  —  —  —  95.9  2.7  1.4   daptomycin  0.5 to >8  2  4  98.6  —  —  —  —  —   teicoplanin  ≤2 to >8  >8  >8  27.4  —  —  27.4  —  72.6   vancomycin  0.5 to >16  >16  >16  20.5  1.4  78.1  20.5  —  79.5  a Breakpoint criteria according to CLSI (M100-S27, 2017)36 and EUCAST,37 as available. CLSI and EUCAST linezolid susceptible breakpoint is ≤2 mg/L. b Uncomplicated UTIs only. The optrA gene was detected in 26 (72.2%) E. faecalis [Tables 1–3 and Figure S1 (available as Supplementary data at JAC Online)]. Among these isolates, one from Thailand and two clonal strains from Panama also carried cfr and cfr(B), respectively (Tables 2 and 3). Nine other (25.0%) E. faecalis isolates had G2576T mutations in the 23S rRNA. Six E. faecalis isolates had an alteration in L4 (F101L) in addition to either optrA (two from China and one each from Sweden, Ireland and Thailand) or G2576T (USA). A linezolid resistance mechanism was not identified in one E. faecalis from Ireland (linezolid MIC, 4 mg/L; Table 2). All E. faecium isolates showed G2576T mutations, while two isolates from New Orleans (USA)18 and one from Atlanta (USA) had concomitant presence of cfr(B). One E. faecium isolate showed alteration in L3 (K95T) in addition to G2576T mutation (Table 2). Table 2. Summary of oxazolidinone resistance mechanisms detected among isolates Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  a Mutations were investigated in the gene encoding 23S rRNA and amino acid alterations in the L3 and L4 ribosomal proteins. b Represents isolates with the cfr(B) variant. c A linezolid resistance mechanism was not identified in this isolate (Ireland). Table 2. Summary of oxazolidinone resistance mechanisms detected among isolates Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  Organism  Year  No. of isolates  Linezolid MIC (mg/L)  Resistance mechanism   23S rRNA gene/L3-L4 amino acid alterationsa  ribosomal protection optrA  methyltransferase cfr  E. faecalis (36)  2008  1  8  −  +  −    2008  2  8  G2576T  −  −    2008  1  8  L4 (F101L)  +  −    2009  1  4  −  +  −    2009  2  4  L4 (F101L)  +  −    2010  1  8  −  +  −    2010  1  16  L4 (F101L)  +  +    2010  2  8–16  G2576T  −  −    2011  2  4  −  +  +b    2011  2  8  G2576T  −  −    2012  1  8  L4 (F101L)  +  −    2012  1  4  G2576T, L4 (F101L)  −  −    2012  1  4  G2576T  −  −    2012  2  4  −  +  −    2013  1  16  −  +  −    2013  1  8  G2576T  −  −    2014  3  4–8  −  +  −    2014c  1  4  −  −  −    2015  2  4–8  −  +  −    2016  8  4–8  −  +  −  E. faecium (66)  2008  8  4–64  G2576T  −  −    2009  14  4–32  G2576T  −  −    2010  12  4–64  G2576T  −  −    2011  9  4–16  G2576T  −  −    2012  4  4–8  G2576T  −  −    2012  1  8  G2576T  −  +b    2013  5  8–32  G2576T  −  −    2013  1  8  G2576T  −  +b    2014  4  4–16  G2576T  −  −    2014  1  8  G2576T  −  +b    2015  2  8–16  G2576T  −  −    2015  1  8  G2576T, L3 (K95T)  −  −    2016  4  4–8  G2576T  −  −  a Mutations were investigated in the gene encoding 23S rRNA and amino acid alterations in the L3 and L4 ribosomal proteins. b Represents isolates with the cfr(B) variant. c A linezolid resistance mechanism was not identified in this isolate (Ireland). Table 3. Characteristics of OptrA-producing E. faecalis isolates Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  a OptrA protein alterations compared with KP399637. b Gene array as depicted in Figure 1. c Macrolide, lincosamide and streptogramin B agents. Table 3. Characteristics of OptrA-producing E. faecalis isolates Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  Isolate ID  Year  Country  MLST (CC)  optrA   Resistance determinants   alterationsa  location  arrayb  linezolid/MLSBc  aminoglycoside  tetracycline  trimethoprim  other  452115  2008  China  ST116 (116)  WT  plasmid  A  erm(B), lsa(A)  2aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  529360  2009  China  ST632 (96)  Y176D, T481P  plasmid  B  erm(A)-like, erm(B), lsa(A)  aac(6 ′)-aph(2′′  tet(L), tet(M)  dfr(G)  fexA, cat  532444  2009  China  ST69 (96)  Y176D, T481P  plasmid  C  erm(B)  aph(3 ′)-III  —  dfr(G)  —  539673  2009  China  ST69 (96)  Y176D, T481P  plasmid  D  erm(B), lsa(A)  —  tet(L), tet(M)  dfr(G)  fexA, cat  570347  2010  China  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  739884  2012  China  ST585 (585)  WT  plasmid  F  erm(A)-like, erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), str  tet(L), tet(M)  dfr(G)  fexA  441341  2008  Sweden  ST86 (86)  Y176D  plasmid  A  lsa(A)  aac(6′)-aph(2′′), aadD, aadE  tet(L)  dfr(G)  cat, fexA  599799  2010  Thailand  ST16 (16)  G40D, T481P  chromosome  G  cfr, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), spc  tet(L), tet(M)  dfr(G)  cat  743142  2012  Taiwan  ST767 (767)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, spc  tet(L), tet(M)  dfr(G)  fexA  981649  2016  Taiwan  ST766 (766)  K3E, Y176D, G393D  chromosome  H  erm(A)-like, erm(B), lsa(A)  spc, str  tet(L), tet(M)  dfr(G)  —  838523  2014  Malaysia  ST59 (59)  WT  plasmid  I  erm(A)-like, erm(B), lsa(A), lnuB  ant(6)-Ia, aph(3′)-III  tet(L)  —  fexA  719171  2012  Ireland  ST116 (116)  WT  plasmid  E  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  839260  2014  Ireland  ST41 (41)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(M)  dfrD  fexA, cat(pC221)  898246  2015  Ireland  ST768 (768)  WT  plasmid  A1  erm(B), lsa(A)  ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA, cat(pC221)  973450  2016  France  ST775 (775)  Y176D  plasmid +  chromosome  J  erm(B), lsa(A), lnuB  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  cat, fexA  824270  2014  USA  ST585 (585)  WT  plasmid  K  erm(A)-like, erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA  912300  2015  USA  ST179 (16)  Y176D, G393D  plasmid  F  erm(A)-like, erm(B), lsa(A), lnu(B)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′), aadE, str  tet(L), tet(M)  dfr(G)  fexA  687669  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  687671  2011  Panama  ST103 (103)  K3E, Y176D, G393D, I622M  plasmid  L  cfr(B), erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(M)  —  —  751258  2013  Ecuador  ST86 (86)  K3E, Y176D  plasmid  M  erm(B), lsa(A)  aph(3′)-III, ant(6)-Ia, aac(6′)-aph(2′′)  tet(L), tet(M)  dfr(G)  fexA, qnrD  956335  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956343  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956349  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  956359  2016  Guatemala  ST256 (256)  I104K, Y176D, E256K  plasmid  N  erm(A)-like, erm(B), lsa(A)  —  —  —  fexA  986223  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lnuB, lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  986247  2016  Mexico  ST480 (480)  Y176D, T481P  plasmid  O  erm(A)-like, erm(B), lsa(A)  aac(6′)-aph(2′′), ant(6)-Ia, aph(3′)-III  tet(L), tet(M)  dfr(G)  fexA  a OptrA protein alterations compared with KP399637. b Gene array as depicted in Figure 1. c Macrolide, lincosamide and streptogramin B agents. Genetic characteristics of OptrA-producing isolates, other resistance determinants and optrA genetic context information are detailed in Table 3 and Figure 1. Based on the S1 nuclease/I-CeuI Southern blot analysis, optrA was plasmid-located in 22 isolates, while 3 isolates carried this gene in the chromosome (Table 3). Hybridization signals were obtained on chromosomal DNA and plasmid in one isolate from France. E. faecalis isolates carrying optrA were detected in the SENTRY program as early as 2008 in China and Sweden and in 10 additional countries through 2016 (Table 3 and Figure S1). Isolates belonging to the same clonal complex (CC) were observed in distinct regions, such as CC16 isolates that were detected in the USA and Thailand, and CC116 isolates that were detected in China and Ireland (Table 3). The CC116 isolates from China and CC86 from Sweden (both from 2008) showed a very similar optrA genetic context (array A; Figure 1). Similarly, three E. faecalis isolates from a medical centre in Ireland and two from Taiwan27 exhibited distinct lineages based on MLST, but a similar optrA genetic context (array A1 and array H, respectively) (Table 3 and Figure 1). One isolate from Ireland yielded a small contiguous sequence (array E). Other isolates from China yielded smaller contiguous sequences and different gene arrangements around optrA (arrays B–D and F). Array F was also observed in an isolate (CC16) from the USA; however, the CC16 isolates from Thailand had a very distinct optrA array (G). A CC59 isolate from Malaysia showed an optrA gene context (array I) similar to F (China/USA), K (USA) and N (Guatemala). Isolates from Taiwan and France showed optrA genes associated with Tn554, but these isolates showed distinct gene arrangements (array H and J, respectively). Clonally related OptrA-producing isolates were detected in Panama in 2011 (CC103) and were detected in Mexico (CC480) and Guatemala (CC256) in 2016 (Table 3). The optrA context observed among isolates from Mexico included TnMERI1 native to Bacillus spp. (array L), while those from Guatemala were associated with Tn551 (array N), with erm(B) and erm(A)-like genes upstream and downstream of optrA, respectively. The optrA genetic context (array O) noted in isolates from Mexico was similar to arrays K and F detected in isolates from the USA and China, except for the number and orientation of ORFs of unknown functions. A very short optrA contiguous sequence was obtained for isolate 751258 from Ecuador with only araC upstream of optrA (array M). IS1216, fex(A) and erm(A)-like were often present with optrA. The fex(A) gene was present in all plasmid backgrounds, except for one isolate (ST69) recovered from China in 2009 and two isolates (ST103) from Panama (2011). A total of 11 isolates (11/23 plasmid optrA) carried the erm(A)-like gene, as previously described by He et al.39 (Table 3). The fer gene encoding ferredoxin-NADP reductase (4Fe–2S ferredoxin iron–sulphur binding domain protein) was present downstream of optrA in 13 isolates. OptrA sequences in 18/26 isolates showed minor differences compared with the index sequence (plasmid pE349 from E. faecalis19 GenBank accession number KP399637); eight variants, including those described by Cui et al.,34 were noted (Table 3). Three alteration combinations not described by Cui et al.34 were noted in Sweden/France (Y176D; identical to Streptococcus suis; GenBank accession number WP_099810410), Thailand (G40D, T481P; identical to GenBank accession number WP_002415370) and Panama (K3E, Y176D, G393D, I622M; novel combination). All but one OptrA-producing E. faecalis from Sweden carried erm(B); dfr(G) was present in 18/26 and dfr(D) in 1/26 isolates. The majority of isolates (16/26) carried tet(L) and tet(M) and 5/26 isolates had tet(L) (2 isolates) or tet(M) (3 isolates) alone (Table 3). The cfr gene in the E. faecalis isolate from Thailand (2010) was embedded in an IS256-like structure with a chromosomal genetic context (tnp-aacA/aphD-IS256 like-orf-cfr) identical to S. epidermidis isolated in Tempe (Arizona) (GenBank accession number JX910899). cfr(B) in isolates from Panama showed a genetic context that was 98% identical to the isolate reported in E. faecium from New Orleans18 (GenBank accession number KR610408). Discussion This study shows a low prevalence of enterococcal isolates non-susceptible to linezolid (0.38%) and resistance mechanisms to oxazolidinones seem to have become distinct between E. faecalis and E. faecium. A previous review reported 23S rRNA alterations (G2576T) as the main resistance mechanism in both E. faecalis and E. faecium, especially in the USA.16 However, this review described several E. faecalis isolates, mostly from countries other than the USA, where mechanisms of oxazolidinone resistance were not detected. Further investigations detected the presence of optrA in these isolates, which are described here. The study shows that optrA has now become more prevalent than the presence of 23S rRNA alterations in E. faecalis and the sole oxazolidinone resistance mechanism among isolates from 2014 to 2016. Alterations in L3 and L4 proteins remained rare in Enterococcus spp.; however, six E. faecalis isolates in this set showed an L4 (F101L) alteration and one E. faecium displayed an L3 (K95T) alteration. The significance of these mutations remains unknown as these were always associated with another established linezolid resistance mechanism (Table 2) and also observed in linezolid-susceptible isolates.34 Cfr(B) seems to be emerging in Enterococcus spp. and this study reports it from E. faecalis isolates for the first time. The cfr or cfr(B) genes were always associated with another linezolid resistance mechanism in E. faecalis (optrA) and E. faecium (G2576T) (Table 2), so it is not clear if cfr confers linezolid resistance in Enterococcus spp. We showed that cfr(B) confers a resistance phenotype similar to cfr in an S. aureus background;18 however, its relevance in Enterococcus spp. has not been clearly defined and 23S rRNA alterations remain the main linezolid resistance mechanism in E. faecium.22,40 Isolates from China, Sweden, Ireland and the USA showed an optrA gene context similar to the plasmid structures described from China,19,39 with 452115 (China) and 441341 (Sweden) sequences showing 99% identity to pE349. These genetic contexts were also similar to those reported in isolates from Poland and Colombia.25,26 The fexA gene was upstream of optrA in 13 isolates, albeit with variations between the arrays. E. faecalis isolates 739884 (China) and 912300 (USA) showed an identical optrA genetic context (array F), which was previously described in an isolate recovered from a pig (ST59; CC59) carrying p10-2-2.39 Isolate 529360 (China; array B) showed IS1216 tnp bracketing optrA in a configuration most similar to that observed for pFX13 documented in an isolate from a pig in China. The optrA gene context beyond the IS1216 element for isolates from Guatemala was distinct compared with any reports thus far, which included Tn551. Isolates with chromosomally located optrA showed distinct array structures: G (Thailand), H (Taiwan) and J (France). E. faecalis 599799 from Thailand had optrA flanking regions displaying high homology with those noted for human isolates E016 and E079 previously reported from China.39 The optrA array for isolate 973450 from France was identical to isolate G20 (human isolate) from China and was flanked by Tn558 (He et al.39). While this isolate showed hybridization signals on plasmid and chromosomal DNA bands, distinct optrA gene arrays could not be identified. All chromosomal optrA had flanking sequences containing the putative transcriptional regulator araC.27 Finally, this study reports the global dissemination of optrA-carrying E. faecalis recovered from patients in countries beyond the Asia-Pacific region. E. faecalis isolates carrying optrA showed a very diverse genetic background. Interestingly, isolates belonging to the same CC were detected in distinct geographical regions. Also, isolates from Ireland with distinct genetic backgrounds had a similar optrA context, indicating plasmid dissemination. From the gene array analysis, it was evident that a great degree of genetic rearrangement is taking place as optrA spreads across the globe; the core genetic elements remaining similar, their positions in the array are divergent in isolates from different geographical locations. These results indicate the potential for dissemination and warrant constant surveillance for monitoring purposes. Although the majority of optrA genes were found in E. faecalis so far, this gene has also been documented in S. sciuri, E. faecium and other Gram-positive organisms,21–24,34 and in in vitro transfer to different species.25 Therefore, it is important to monitor the emergence and spread of this resistance determinant at a local and regional level, especially due to the potential for E. faecalis to serve as a reservoir for spreading optrA to MDR pathogens (i.e. E. faecium). Acknowledgements Part of this study was presented at the American Society for Microbiology Microbe Conference, Boston, MA, USA, 2016 (Poster #Saturday 332). We would like to thank all SENTRY participating sites contributing clinical isolates during the 2008–16 study period. Funding This study was supported by JMI Laboratories (North Liberty, IA, USA) through the SENTRY Antimicrobial Surveillance Program. Transparency declarations JMI Laboratories was contracted to perform services in 2017 for Achaogen, Allecra Therapeutics, Allergan, Amplyx Pharmaceuticals, Antabio, API, Astellas Pharma, AstraZeneca, Athelas, Basilea Pharmaceutica, Bayer AG, BD, Becton, Dickinson and Co., Boston, CEM-102 Pharma, Cempra, Cidara Therapeutics, Inc., CorMedix, CSA Biotech, Cutanea Life Sciences, Inc., Entasis Therapeutics, Inc., Geom Therapeutics, Inc., GSK, Iterum Pharma, Medpace, Melinta Therapeutics, Inc., Merck & Co., Inc., MicuRx Pharmaceuticals, Inc., N8 Medical, Inc., Nabriva Therapeutics, Inc., NAEJA-RGM, Novartis, Paratek Pharmaceuticals, Inc., Pfizer, Polyphor, Ra Pharma, Rempex, Riptide Bioscience Inc., Roche, Scynexis, Shionogi, Sinsa Labs Inc., Skyline Antiinfectives, Sonoran Biosciences, Spero Therapeutics, Symbiotica, Synlogic, Synthes Biomaterials, TenNor Therapeutics, Tetraphase, The Medicines Company, Theravance Biopharma, VenatoRx Pharmaceuticals, Inc., Wockhardt, Yukon Pharma, Zai Laboratory and Zavante Therapeutics, Inc. 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Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Jun 6, 2018

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