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1 AAC Accepts, published online ahead of print on 31 October 2012 Antimicrob. Agents Chemother. doi: /aac Copyright 2012, American Society for Microbiology. All Rights Reserved The Target of Daptomycin is Absent from Escherichia coli and other Gram-Negative Pathogens 4 5 Christopher P. Randall, Katherine R. Mariner, Ian Chopra and Alex J. O Neill* Antimicrobial Research Centre and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom *Corresponding author. Mailing address: Antimicrobial Research Centre and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone +44 (0) , Fax +44 (0) , a.j.oneill@leeds.ac.uk Running title: Activity of daptomycin against Gram-negative bacteria Downloaded from on October 1, 2018 by guest

2 Abstract Antistaphylococcal agents commonly lack activity against Gram-negative bacteria like Escherichia coli owing to the permeability barrier presented by the outer membrane and/or the action of efflux transporters. When these intrinsic resistance mechanisms are artificially compromised, such agents almost invariably demonstrate antibacterial activity against Gram-negatives. Here we show that this is not the case for the antibiotic daptomycin, whose target appears to be absent from E. coli and other Gram-negative pathogens. Downloaded from on October 1, 2018 by guest

3 Text Antibiotic resistance is a growing problem that negatively impacts our ability to treat bacterial infection. The situation is becoming particularly grave in the case of Gram-negative pathogens (1), which in contrast to Gram-positive bacteria are intrinsically insusceptible to many classes of antibacterial agents owing to the permeability barrier presented by the outer membrane (OM) and/or the action of efflux transporters (2). One approach for developing novel treatments against Gramnegative bacteria involves the identification of compounds capable of disrupting these intrinsic resistance mechanisms, with a view to rendering these organisms susceptible to existing anti-gram positive drugs. The concept of combining such potentiator or adjuvant compounds with established antibacterial drugs has shown considerable promise in in vitro studies (3). For example, colistin potentiates the activity of glycopeptides against a range of Gram negative-pathogens, presumably by disrupting the OM (4), and high-throughput screening has identified several compounds which enhance the activity of novobiocin against E. coli by increasing cell permeability (5). Furthermore, inhibitors of efflux sensitise Gram-negative organisms to a variety of antistaphylococcal agents, including linezolid and macrolides (6-7). In this study we show that whilst most antistaphylococcal agents are active against Gram-negative bacteria provided they are allowed to reach their target, this is not true of the lipopeptide antibiotic, daptomycin. Antibacterial agents and chemicals used in this study were from Sigma Aldrich (Poole, UK), with the exception of vancomycin (LEK pharmaceuticals; Ljubljana, Slovenia), nisin (NBS Biologicals Ltd; Huntingdon, UK), flucloxacillin (CP pharmaceuticals Ltd; Wrexham, UK), indolmycin and linezolid (Pfizer, Kalamazoo [MI] USA), and daptomycin (Cubist Pharmaceuticals, Lexington [MA] USA). Susceptibility determinations were performed by broth microdilution in Mueller-Hinton broth according to CLSI guidelines (8), supplemented in the case of daptomycin with 50 μg/ml Ca 2+. Against the standard laboratory E. coli strain 1411 (9), with its intrinsic resistance mechanisms intact, most of the antistaphylococcal agents tested had little or no detectable antibacterial activity (Table 1). However, upon permeabilising the OM of this strain with polymyxin B nonapeptide (PMBN; 4 μg/ml), the antibacterial activity increased considerably for the majority of compounds (Table 1). These results are consistent with previous observations that the OM acts as a barrier to hydrophobic compounds, or those with a molecular weight >600 Da (2). Insertional inactivation of acrab in strain SM1411 (9) also increased the antibacterial activity of many of the agents tested, implying that they are ordinarily substrates for the AcrAB-TolC efflux transporter (Table 1). Some antistaphylococcal agents lack activity against E. coli exclusively owing to OM impermeability (e.g. clofazimine), others primarily as a consequence of AcrAB-TolC-mediated efflux (e.g. linezolid), and yet others as a result of both intrinsic resistance mechanisms (e.g. mupirocin) (Table 1). The observed inability to sensitise E. coli to the antistaphylococcal agents clindamycin, daptomycin, streptomycin and vancomycin suggested that either the OM is not a primary barrier to activity for these compounds, or that sufficient OM remained after PMBN treatment to restrict access of these

4 drugs to their targets. To examine the antibacterial activity of these agents in the absence of the E. coli OM, we generated protoplasts of E. coli 1411 (10). A protoplasting efficiency of ~99% was achieved, and protoplast preparations were diluted 1:10,000 in MHB containing 0.5M sucrose to ensure removal by dilution of cells retaining an intact OM. Susceptibility determinations with protoplasts were conducted as for whole cells, although MHB was supplemented with 0.5M sucrose to maintain protoplast integrity. The activity of clindamycin, streptomycin and vancomycin against protoplasts increased 8-fold over PMBN-treated cells (Table 1), demonstrating that the OM does ordinarily act to limit the activity of these antibiotics against E. coli. In contrast, daptomycin remained inactive against E. coli protoplasts (Table 1). The antibacterial target of daptomycin in S. aureus and other Gram-positive bacteria is the cytoplasmic membrane (CM) (11). Daptomycin inserts into the CM in a Ca 2+ -dependent manner, resulting in membrane depolarisation and subsequent loss of intracellular components, including K +, Mg 2+ and ATP (11). The presence of Ca 2+ has been proposed to permit the interaction between the anionic daptomycin and the negatively-charged phospholipids within the Gram-positive CM (12). The content of negatively-charged phospholipid in the CM of E. coli is substantially lower (30%) than that found in Gram-positive organisms such as S. aureus (100%) (13). In consequence, daptomycin may lack activity against permeabilised E. coli simply because it cannot interact with, or does not disrupt, the Gram-negative CM. To confirm this, we assessed the ability of daptomycin to compromise the integrity of carboxyfluorescein-filled liposomes (14) made of E. coli CM (approximately 70% [w/w] phosphatidylethanol amine, 20% phosphatidylglycerol, 10% cardiolipin), or with a phospholipid content matching that of the S. aureus CM (60% [w/w] phosphatidylglycerol, 40% cardiolipin) (13). Phospholipids were from Avanti polar lipids (Birmingham, AL, USA). To assess liposome damage, 50 μm of S. aureus or E. coli liposomes were challenged for 10 min with doubling dilutions of daptomycin or clofazimine across a concentration range from 4 μg/ml to 64 μg/ml; clofazimine was employed as a positive control for liposome damage in these experiments since its mode of action involves direct damage to the CM (15). Leakage of carboxyfluorescein from liposomes was monitored at 485 nm, and percentage liposome integrity calculated relative to liposomes challenged with 0.5% Triton X-100 (corresponding to 100% liposome damage [0% liposome integrity]). Exposure to clofazimine led to a comparable loss of integrity for both S. aureus and E. coli liposomes (>60% loss of integrity at concentrations 8 μg clofazimine/ml) (Figure 1). Liposomes consisting of E. coli CM exhibited only minor loss of integrity (up to 6 %) when exposed to concentrations of daptomycin up to 64 μg/ ml in the presence of 50 μg/ml Ca 2+ (Figure 1), and comparable results were obtained in the absence of Ca 2+ (data not shown). In contrast, staphylococcal liposomes underwent a loss of 38% integrity at 8 μg daptomycin/ml, increasing to 97% loss of integrity at 64 μg daptomycin/ml (Figure 1), an effect which was dependent on the presence of Ca 2+ (data not shown). Thus, the limited ability of daptomycin to damage the E. coli CM compared with that of S. aureus appears to be directly attributable to the differences in phosholipid composition.

5 Since the CMs of other Gram-negative pathogens contain similar proportions of anionic phospholipid to that of E. coli (16), we reasoned that daptomycin would be equally ineffective against the CMs of these organisms. To assess this, we determined the susceptibility to daptomycin and clofazimine of both whole cells and protoplasts of Enterobacter cloacae ATCC 13047, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, Moraxella catarrhalis and Salmonella Typhimurium (clinical isolates from the Leeds General Infirmary [LGI], Leeds, UK). As for E. coli, clofazimine only demonstrated activity against these Gram-negative species once the OM had been compromised (Table 2). Daptomycin did not demonstrate detectable antibacterial activity against any of these species, even after removal of the OM (Table 2). In conclusion, whilst we have not ruled out the possibility that the OM and/or efflux transporters may act to restrict the accumulation of daptomycin in Gram-negative bacteria, it is clear that these intrinsic resistance mechanisms are not responsible for the observed lack of anti-gram negative activity of this antibiotic. Instead, the CM of Gram-negative bacteria is insusceptible to daptomycin. This is likely due to the lower proportion of anionic phospholipids in the Gram-negative CM compared to S. aureus, resulting in insufficient sites for Ca 2+ -mediated insertion of daptomycin to prompt an antibacterial effect. Daptomycin is therefore unusual amongst antistaphylococcal agents in that its target appears to be absent from Gram-negative bacteria: it cannot therefore be considered as a potential chemotherapeutic agent against Gram-negative pathogens, even in combination with potentiator compounds that act to compromise the intrinsic resistance mechanisms of these organisms. Acknowledgements We thank L. Wetherill for technical advice and assistance in generating liposomes, and J. Wright for provision of clinical isolates.

6 Antibacterial agent Molecular weight S. aureus SH1000 (17) MIC (μg/ml) PMBN E. coli SM1411 (acrab) 1411 protoplasts Actinonin > ND Clindamycin >256 >256 > Clofazimine >128 2 >128 1 Daptomycin >256 >256 >256 >256 Erythromycin ND Flucloxacillin > ND Fusidic acid > ND Indolmycin ND Linezolid > ND Mupirocin ND Nisin >64 16 >64 8 Novobiocin ND Rifampin ND Streptomycin >256 >256 >256 1 Vancomycin Table 1: Susceptibility of laboratory strains of S. aureus and E. coli to various antibacterial agents. ND not determined. % Liposome integrity Daptomycin concentration (μg/ml) Figure 1: The effect of daptomycin (+ 50 μg/ml Ca 2+ ) and clofazimine on S. aureus and E. coli liposome integrity after a 10 minute challenge. Error bars represent standard deviation from the mean for three independent experiments.

7 Bacterium MIC (μg/ml) Daptomycin Clofazimine Whole cells Protoplasts Whole cells Protoplasts P. aeruginosa PAO1 >256 >256 >128 1 E. cloacae ATCC >256 >256 >128 4 K. pneumoniae >256 >256 >128 4 M. catarrhalis >256 >256 >128 1 S. Typhimurium >256 >256 >128 8 Table 2: Antibacterial activity of clofazimine and daptomycin against Gram-negative pathogens. Downloaded from on October 1, 2018 by guest

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