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...

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The development of chronic and recurrent Staphylococcus aureus infections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure of S. aureus to sublethal concentrations of H2O2 leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H2O2 exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within the S. aureus population. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H2O2 also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H2O2 than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2. Taken together, these data reveal a mechanism by which S. aureus adapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production.

35Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain 36 and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. 37Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic 38 devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and 39 resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To 40 address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains 41 high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of 42 leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone 43 biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in human blood 44 or the presence of purified neutrophils. Supplementation of the culture medium of the heme-auxotrophic 45 SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity 46 to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream 47 infection, we explored whether restoration of the electron transport chain in this organism also affected 48 survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but 49 resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, 50 the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced 51 growth rate but provides resistance to a key immune defence mechanism. 52 53

RcsB interacts with GadE to mediate acid resistance in stationary-phase Escherichia coli K-12. We show here that RcsB is also required for inducible acid resistance in exponential phase and that it acts on promoters that are not GadE regulated. It is also required for acid resistance in E. coli O157:H7.The glutamate-dependent acid resistance system AR2 of Escherichia coli is regulated by a regulatory network that responds to general stress, via the alternative sigma factor RpoS, and to low pH (8,13,17,18,20,22,25). AR2 requires several regulators, including the central regulator GadE, to integrate signals from the EvgAS and PhoPQ two-component systems (3,(10)(11)(12)(13)17). RcsB, a regulator with a wide range of roles in many enteric bacteria (5), is essential for survival during extreme acid challenge (pH 2.5 or below) during stationary phase and regulates transcription of some AR2 genes by forming a heterodimer with GadE (2, 4, 15, 16). RcsB also forms heterodimers with RcsA, TviA, and BglJ to regulate colanic acid synthesis, antigen VI expression, and sugar transport, respectively (26-28). Here we show that the inducible acid resistance of exponential-phase E. coli is also completely dependent on RcsB and that this resistance correlates with dependence of activation of the AR2 network on RcsB. We show that several AR2 genes that are not GadE regulated require RcsB and that RcsB must interact downstream of the sensor kinase EvgS but upstream of the first regulator, YdeO. These results suggest an additional role for RcsB in the activation of acid resistance and show that there is cross talk between the Rcs and EvgAS systems. We show that the role of RcsB extends to the pathogenic strain E. coli O157:H7 (Sakai).Induced acid resistance is rcsB dependent. A ⌬rcsB derivative of E. coli K-12 MG1655 was constructed as previously described (7). We determined the survival of this strain and the wild-type parent to extreme acid challenge in exponential phase, with and without induction by mild acidification (pH 5.7). Both strains grew at the same rate. Cells were grown, from overnight cultures diluted at least 500-fold, to an optical density at 600 nm (OD 600 ) of 0.2 in M9 with glucose (22.2 mM) and Casamino Acids (0.2% [wt/vol]), buffered with morpholinepropanesulfonic acid (MOPS) and morpholineethanesulfonic acid (MES) as described previously (M9supp) (1), and then incubated at pH 5.7 or pH 7 for 70 min before acid challenge at pH 2.4 for 2 h. We determined that under these conditions there are no detectable carryover effects from stationary phase on gene expression or cell survival. Cultures were always checked to ensure that the adjusted pH values remained constant for the entire experiment (data not shown). Survival was measured as previously described (1). Induction at pH 5.7 caused a significant increase in resistance in the wild-type strain (Fig. 1a). Survival of the ⌬rcsB strain was below the level of detection in both induced and uninduced cultures (Fig. 1a). To complement the rcsB deletion, we constructed p...