Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids

Abstract

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...

Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing

“…Further, predicted drug failure in a particular host-mimicking media may indicate that certain drugs may be effective against certain types of infections but not others (e.g., systemic vs. localized). Supporting this suggestion, MRSA inactivates daptomycin by releasing membrane phospholipids under certain experimental conditions (Pader et al, 2016); and herein we show that a MRSA isolate was susceptible to daptomycin in tissue culture medium and in a murine model of sepsis, but displayed resistance in other host-mimicking media examined (minimal Lacks medium).…”

Antibiotic Capture by Bacterial Lipocalins Uncovers an Extracellular Mechanism of Intrinsic Antibiotic Resistance

“…BCNs bind a range of antibiotics with diverse chemical structures, increasing antibiotic resistance in vitro and enhancing bacterial survival in vivo . The effect of BCNs on multiple antibiotics and their wide conservation in most bacterial species distinguish this mechanism from the recently discovered effect of membrane-released phospholipids on daptomycin (14), which is restricted to this particular antibiotic and to S. aureus .…”

“…Ineffective microbial killing and exposure to sublethal antibiotic concentrations elicit adaptive bacterial stress responses, enhancing antibiotic resistance and tolerance (2–7). Much has been learned about antibiotic resistance mechanisms at the cellular level (8, 9), but whether microbes subvert the action of antibiotics before they come in contact with bacterial cells has remained largely unexplored, with the exception of β-lactamases, which are often trapped in released membrane vesicles (10–13), and the recent observation that Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids (14). …”

Environment Shapes the Accessible Daptomycin Resistance Mechanisms inEnterococcus faecium

“…We also examined whether the isolates with mutations in yvcRS , bound less bodipy-DAP (BDP:DAP) than the ancestor. BDP:DAP is a conjugation of the fluorophore, bodipy-FL, with DAP (BDP:DAP) and is used as a proxy for DAP binding (27, 28, 37). After incubation with 32 µg/ml BDP:DAP, bound BDP:DAP was quantified using fluorescence microscopy (Fig.…”