Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-34resistant Staphylococcus aureus (MRSA) 1,2 . Although resistance is rare, treatment failure can occur 35 in >20% of cases 3,4 and so there is a pressing need to identify and mitigate factors that contribute 36 to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which 37 frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. 38Wild-type S. aureus was killed rapidly by daptomycin but Agr-defective mutants survived 39 antibiotic exposure by releasing membrane phospholipid, which bound and inactivated the 40 antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-41 triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented 42 antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was 43 inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and 44 enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that 45 protects against daptomycin, which can be compromised by Agr-triggered toxin production or an 46 existing therapeutic antibiotic. 47 S. aureus encodes multiple virulence factors, many of which are controlled by Agr 5,6 , a 48 quorum-sensing system encoded by a 4 gene operon (agrBDCA) and a gene encoding a regulatory 49 RNA (RNAIII). However, invasive S. aureus infections often give rise to Agr-defective mutants, 50 typically involving agrA or agrC, hypothesised to provide a selective advantage in the presence of 51 antibiotics [7][8][9][10][11][12][13][14] . To test this hypothesis, we determined the killing kinetics of wild-type S. aureus or agr 52 mutants by clinically-relevant antibiotics. 53Agr status did not affect the rate of staphylococcal killing by vancomycin, oxacillin or 54 gentamicin ( Supplementary Fig. 1, 2). By contrast, whilst wild-type S. aureus was killed by 55 daptomycin, loss of quorum-sensing components of Agr (AgrA or AgrC) enabled S. aureus strains 56 USA300 or SH1000 to survive in the presence of daptomycin during the first 8 hours of exposure (Fig. 57 1a,b). A mutant lacking the regulatory RNAIII component of agr was killed as efficiently as the wild-58 type (Fig. 1a), as were agrA or agrC mutants complemented with the relevant genes on plasmids 59 3 ( Supplementary Fig. 3). After the initial period of killing, CFU counts of both wild-type and agr-60 mutant S. aureus recovered to similar levels by 24 h, without the acquisition of resistance, explaining 61 why all strains had identical daptomycin MIC and MBC values (Fig. 1c, Supplementary Supplementary Fig. 4). This biphasic killing and subsequent recovery profile is similar to several 63 previously reported daptomycin killing assays, although the contribution of Agr to this phenomenon 64 was unknown [15][16][17] . 65In addition to agr-deletion mutants, clinical isolat...