601. TelA and XpaC Are Novel Mediators of Daptomycin Resistance in Enterococcus faecium

BACKGROUND: The YycFG system is an essential two-component regulatory system involved in cell wall homeostasis associated with the development of daptomycin (DAP) resistance in E. faecium. Importantly, the standard combination of DAP plus β-lactam is ineffective against strains harboring mutations in yycFG. Transcriptional profiling identified a cluster of two genes (xpaC and telA) that is upregulated in the presence of a YycG(S333L) substitution. xpaC and telA are annotated as 5-bromo-4-chloroindolyl phosphate hydrolysis and tellurite resistance proteins, respectively. Here, we aimed to determine the contribution of xpaC and telA in DAP resistance. METHODS: Non-polar in-frame deletions of xpaC/telA and complementation of xpaC were performed in clinical strain E. faecium R446(RIF). All mutants were characterized by PFGE and sequencing of the open reading frames to confirm the deletion. DAP MIC determination was performed by Etest on Mueller–Hinton agar. Binding of DAP was evaluated using BODIPY-labeled DAP (BDP-DAP). Cell membrane phospholipid microdomains were visualized using 10-N-nonyl acridine orange. All assays were compared with a DAP-susceptible clinical E. faecium strain S447. RESULTS: R446(RIF)Δ (telA) and R446(RIF)Δ (xpaCtelA) did not alter DAP MICs in R446(RIF) (24–32 μg/mL). However, deletion of xpaC alone (R446(RIF)Δ (xpaC)) markedly decreased DAP MIC 8 fold (to 4 μg/mL). R446(RIF)Δ (telA) and R446(RIF)Δ (xpaCtelA) exhibited similar binding of BDP-DAP compared with parental R446(RIF). In contrast, R446(RIF)Δ (xpaC) exhibited increased binding of the antibiotic molecule to the cell membrane, similar to that of DAP-susceptible S447. Complementation of xpaC restored DAP MIC to 32–48 µg/mL and decrease binding of DAP. NAO staining of S447, R446(RIF), R446(RIF)Δ (telA), R446(RIF)Δ (xpaCtelA), and R446(RIF)Δ (xpaC):: (xpaC) displayed septal and polar distribution. In stark contrast, R446(RIF)Δ (xpaC) showed a redistribution of phospholipid microdomains away from the septa. CONCLUSION: XpaC is a key contributor to DAP binding and phospholipid architecture of E. faecium but only in the presence of an intact TelA. The xpaC and telA gene cluster is a novel mediator of DAP-resistance in E. faecium via theYycFG system and independent of the LiaFSR system [Image: see text] DISCLOSURES: All authors: No reported disclosures.