channels encoded by lsa genes in E. faecalis and msrC gene in E. faecium (described above). VgaD has been described only in E. faecium. vgaD was found on a plasmid with vatH, both of which confer only streptogramin A resistance.92 To date, no other acquired streptogramin efflux pumps have been described. Perhaps the best understood mechanism of streptogramin resistance is dimethylation of the 23S rRNA.25 This resistance mechanism, which confers the MLSA or MLSB phenotype occurs through acquisition of either the ermA or ermB genes on broad host range plasmids such as pAMβ1. If these plasmids also contain vatE or vatD genes, then they confer resistance to quinupristin- dalfopristin when acquired by a recipient cell. Linezolid. Prior to FDA approval in 2000, reports of linezolid resistance in enterococci existed but were rare. The emergence of linezolid resistance occurred slowly and only in sporadic cases associated with prolonged exposure.93 The industry-sponsored LEADER trial has monitored linezolid efficacy from 2004 to 2009 and has found yearly resistance rates between 0.49 and 1.83%.56 In contrast, Pogue et al.94 found linezolid resistance in 20% of GRE samples from the University of Pittsburgh Medical Center. Only 25% of isolates in their study were associated with prior linezolid exposure, suggesting clonal spread. Linezolid is a first-in-class oxazolidinone, an entirely synthetic class of antibiotics that binds to the initiation complex and inhibits protein synthesis. Most bacteria, including the enter- ococci, have multiple copies of the genes encoding 23S rRNA. E. faecalis have four copies of the gene95 and E. faecium six copies.96 In theory, the presence of multiple gene copies makes resistance from sporadic mutations less likely because the unaffected gene copies would mask the effect of the mutated gene. However, recombination between susceptible and resistant copies (referred to as “gene conversion”) will yield strains with multiple mutated copies under persistent linezolid selective pressure. In clinical isolates, a mutation in one E. faecium rRNA gene conferred an MIC of 8–16 mg/ml. The same mutation in . 3 rRNA genes conferred an MIC between 64–128 mg/ml.96 A variety of point mutations that confer linezolid resistance have been identified, the most common of which is G2576T. In the most recent LEADER study results (2009), the G2576T mutation was identified in all eight of the linezolid-resistant enterococci strains isolated in the United States. Four of the eight strains found in this study were isolated in Louisville, KY and appeared clonally related.56,97 Other sporadic point mutations have been associated with linezolid resistance, including G2505A and L4 (F101L).98-100 In 2006, the transferable cfr gene was identified in S. aureus as the source of resistance to linezolid, lincosamides and strepto- gramin A compounds, among others.101 Cfr encodes an rRNA methyltransferase that modifies an adenosine in the linezolid- binding region on the 23S rRNA, preventing antibiotic binding. It is hypothesized that the cfr gene emerged from animal strains of bacteria that were exposed to natural compounds with an rRNA binding site similar to linezolid.101,102 In 2011, cfr was identified in an E. faecalis strain (designated EF-01) from a cattle farm in China.103 In this strain, the gene was located on a plasmid (pEF-01) and flanked by IS1216, suggesting transposability. This was the first enterococcus harboring cfr to be reported in the literature, although human isolates of E. faecalis and E. faecium with cfr were reported in a 2010 abstract.103 Overall, linezolid resistance remains rare in enterococci. Daptomycin. Daptomycin is a lipoprotein with bactericidal activity against enterococci. While not FDA approved for treatment of GRE, it is often used by clinicians for this purpose.104-106 The epidemiology of daptomycin resistance in enterococcus (defined as MIC . 4 mg/ml) was recently reviewed by Kelesidis et al.107 Rates of daptomycin resistance in this study were approximately 0.6% (111 daptomycin resistant isolates/ 17,084 enterococcus isolates total). In general, E. faecium is more likely than E. faecalis to express daptomycin resistance, although resistance has been reported in both species. The increased prevalence of daptomyicin resistance in E. faecium may reflect increased use of daptomycin with this species compared with E. faecalis, which is usually susceptible to penicillins. Daptomycin resistance appears to be less common in North America than in Asia or Europe.107 Daptomycin incorporates itself into the cell membrane of Gram-positive organisms in the presence of physiologic calcium concentrations and promotes leakage of intracellular potassium into the extracellular space, resulting in cell death by destruction of the transcellular potassium gradient.108-110 Normal cell membrane polarity is required for daptomycin intercalation. In staphylococci, alteration of the cell membrane charge by virtue of modification of cell membrane lipoproteins has been associated with reduced daptomycin susceptibility.111 A number of genes have been described in staphylococci that contribute to daptomycin resistance,112 none of which have been identified in enterococcus to date. While the mechanism of daptomycin resistance in enterococcus remains unresolved, several reports have elucidated gene mutations associated with enterococcus dapto- mycin resistance.113,114 Palmer et al.113 created three daptomycin-resistant strains by exposing E. faecalis to increasing daptomycin concentrations until stable resistance was identified. They then performed complete genome sequencing of the strains before and after emergence of daptomycin resistance and identified seven gene mutations. Of the seven mutations observed in this study, only EF1797 and EF0631 gene mutations were identified in all three resistant strains. EF1797 encodes a putative membrane protein that may be involved in phosphatidylserine and sphingolipid synthesis, but its function has yet to be determined. EF0631 encodes a putative cardiolipin sythetase (cls), a transphosphatidylase involved in the synthesis of the cell membrane protein cardiolipin. One specific mutation in this gene, R218Q, was found in two of the resistant strains and occurs in the presumed active domain of the EF0631 enzyme. Through comparison with a DNA sequence database, the authors identified one other E. faecalis strain with an EF0631 frameshift mutation, but this isolate had a daptomycin-susceptible phenotype. In a similar study, Arias et al.114 compared the nucleotide sequence and cell membrane proteins of E. faecalis isolates before and after the development of daptomycin resistance in a patient with enterococcus bacteremia. Genome sequencing of the www.landesbioscience.com Virulence 427