1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill... Abstract Background Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. Methods The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time–kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates. Introduction Enterococcus faecium is an important nosocomial pathogen.1 It is highly transmissible and causes a range of infections that are typically difficult to treat.2,E. faecium is highly recombinogenic and promiscuous with regard to horizontal gene transfer, resulting in the emergence of MDR strains.3 The most clinically significant isolates are vancomycin-resistant E. faecium, which are classified as a ‘serious threat’ to human health by the US CDC4 and included on the WHO priority pathogen list for research and development of new antimicrobials.5 Vancomycin is considered an effective first-line therapy against E. faecium. However, the increasing prevalence of vancomycin-resistant E. faecium means that the clinical use of this antibiotic in the treatment of E. faecium infections is becoming increasingly limited, resulting in the use of alternative antimicrobials such as daptomycin and linezolid.6 Recent reports documenting the emergence of E. faecium that display decreased susceptibility to both daptomycin7 and linezolid8 are of serious concern since very few therapeutic options remain for treating infections caused by these isolates.9 There is therefore an urgent need for novel therapeutics that are active against MDR E. faecium isolates, particularly those displaying non-susceptibility to daptomycin and linezolid. Recently, a novel class of antimicrobials known as the 1,2,4-oxadiazoles was discovered.10 While extensive in vitro and in vivo evaluation of these agents against Staphylococcus aureus has been performed,10–12 there are limited data on the activity of the 1,2,4-oxadiazoles against E. faecium. Here we report that this family of antimicrobials has potent activity against an extended panel of clinical E. faecium and acts synergistically with daptomycin against this important human pathogen. Materials and methods Bacterial strains and growth conditions Fifty-three clinical E. faecium isolates (Table S1, available as Supplementary data at JAC Online) were obtained from the Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and Doherty Applied Microbial Genomics culture collections. All isolates were maintained on horse blood agar and grown at 37°C using standard laboratory conditions. Antimicrobials JRH-24-1 was synthesized according to the previously published procedure for ‘compound 2’ in O’Daniel et al.10 Daptomycin was obtained from Novartis under the brand name Cubicin. Antimicrobial susceptibility testing Phenotypic susceptibility testing was performed using CLSI methods.13 A 2-fold dilution series (from 32 to 0.5 mg/L) of JRH-24-1 or daptomycin was made in 100 μL volumes of cation-adjusted Mueller–Hinton w/TES broth (CAMHB w/TES) purchased from Thermo Fisher (additionally supplemented with 50 mg/L Ca2+ for daptomycin assays) in a 96-well plate (Corning) and an inoculum of 100 μL of overnight broth culture adjusted to 5 × 105 cfu/mL was added to each well. After 24 h incubation, the MIC was defined as the lowest antimicrobial concentration that inhibited visible growth. To determine the MBC, 10 μL from each well with no visible growth was plated onto non-selective Mueller–Hinton agar plates. The MBC was defined as the lowest concentration with no growth on subculture after 48 h. All MIC and MBC testing was performed in triplicate. Time–kill assays Time–kill assays were performed with E. faecium strains Aus0085 and DMG1700661, using DMSO-containing (untreated) CAMHB w/TES or equivalent broths supplemented with 1× MIC (2 mg/L), 2× MIC (4 mg/L), 4× MIC (8 mg/L) and 8× MIC (16 mg/L) JRH-24-1 or daptomycin (16 mg/L) (additionally supplemented with 50 mg/L Ca2+ for daptomycin assays). Broths were inoculated with overnight bacterial broth cultures and adjusted to 5 × 105 cfu/mL. Samples withdrawn at 0, 1, 3, 24 and 48 h post-inoculation were centrifuged at 14 000 g to pellet the bacterial cells. The pellets were then washed in PBS and resuspended in an equal volume of PBS. The samples were then serially diluted in PBS and plated onto brain heart infusion (BHI) agar (Oxoid). Plates were then incubated overnight at 37°C before colony enumeration was performed. All assays were performed in biological triplicate. JRH-24-1 and daptomycin combination MIC testing MICs for the combination of JRH-24-1 and daptomycin were determined using a modification of the CLSI protocol described above. A 2-fold dilution series (from 32 to 0.5 mg/L) of daptomycin, with or without a final concentration of 0.5× MIC (1 mg/L) JRH-24-1, was inoculated into 100 μL volumes of CAMHB w/TES supplemented with 50 mg/L Ca2+ in a 96-well plate (Corning), along with a 100 μL overnight bacterial suspension adjusted to 5 × 105 cfu/mL. As before, the MIC was defined as the lowest daptomycin concentration that inhibited visible growth after 24 h incubation. JRH-24-1 and daptomycin synergy testing Synergy testing was performed using E. faecium strains Aus0085 and DMG1700661 in CAMHB w/TES supplemented with 50 mg/L Ca2+. For Aus0085, broths were supplemented with 0.0625× MIC (0.25 mg/L), 0.125× MIC (0.5 mg/L), 0.25× MIC (1 mg/L), 0.5× MIC (2 mg/L) and 1× MIC (4 mg/L) daptomycin with or without either 0.25× MIC (0.5 mg/L) or 0.5× MIC (1 mg/L) JRH-24-1. For DMG1700661, broths were supplemented with 0.125× MIC (1 mg/L), 0.25× MIC (2 mg/L), 0.5× MIC (4 mg/L) and 1× MIC (8 mg/L) daptomycin with or without either 0.25× MIC (0.5 mg/L) or 0.5× MIC (1 mg/L) JRH-24-1. Additionally, DMSO-containing (untreated) broths and broths supplemented with 0.5× MIC JRH-24-1 alone were used as controls. All broths were inoculated with overnight bacterial broth cultures and adjusted to 5 × 105 cfu/mL. Samples withdrawn at 0 and 24 h post-inoculation were centrifuged at 14 000 g to pellet the bacterial cells. The pellets were then washed in PBS and resuspended in an equal volume of PBS. The samples were then serially diluted in PBS and plated onto BHI agar. Plates were then incubated overnight at 37°C before colony enumeration was performed. Synergy was defined as a ≥2 log10 reduction in viability compared with the most active single agent used.14 All assays were performed in biological triplicate. HepG2 cellular toxicity testing HepG2 cells were maintained in MEM (Gibco) supplemented with 10% (v/v) FCS, 2 mM glutamine (Gibco) and Antibiotic-Antimycotic (Gibco) at 37°C in 5% CO2. Cells were seeded at 1 × 104 cells per well in 96-well tissue culture treated plates (Corning). Following overnight growth, the medium was removed and the cells were exposed to JRH-24-1 (0.125–128 mg/L) in MEM lacking phenol red (Gibco) supplemented with 10% (v/v) FCS, 2 mM glutamine (Gibco), Non-Essential Amino Acids (Gibco) and Antibiotic-Antimycotic (Gibco) for 24 h. Cellular viability was then assessed using XTT reagent (Sigma–Aldrich) with PMS (N-methyl dibenzopyrazine methyl sulfate) (Thermo Fisher) according to the XTT cell viability assay protocol (Thermo Fisher). Cells treated with 2.5% (v/v) DMSO (corresponding to the amount of DMSO in the lowest dilution of JRH-24-1) were the negative (vehicle) control. Cells were incubated for 3 h before absorbance was measured at 475 and 660 nm (non-specific reading) using an Ensight Multimode plate reader (PerkinElmer). All assays were performed in biological triplicate. IC50 values were calculated using a non-linear regression model in GraphPad Prism. Haemolysis assay The assay was modified from Evans et al.15 Fresh whole human blood was collected into K2-EDTA-coated Vacutainer tubes and pelleted at 500 g for 5 min. The red blood cell pellet was washed twice in saline solution followed by a third wash in PBS before being diluted 1:25 in PBS. JRH-24-1 was made to 20× concentrations in DMSO and a 10 μL aliquot of each concentration was added to 190 μL of diluted blood in a 96-well U-bottom plate (Corning) giving final JRH-24-1 concentrations of 0.125 to 128 mg/L. The plate was then incubated at 37°C for 1 h. Negative (vehicle) controls were incubated with DMSO and positive controls were incubated with 2% (v/v) Triton X-100. Cell suspensions were centrifuged at 500 g and 100 μL of supernatants collected. The absorbance of each supernatant was then measured at 451 nm using an Ensight Multimode plate reader (PerkinElmer). All assays were performed in biological triplicate. Results JRH-24-1 displays bactericidal activity against an extended panel of vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium To expand on previous studies10,12 and evaluate the potential utility of the 1,2,4-oxadiazole antimicrobials for treating E. faecium infections we determined the MIC and MBC values of JRH-24-1 for a panel of 53 clinical E. faecium isolates (Table S1). The panel contained 14 vancomycin-susceptible isolates and 39 vancomycin-resistant isolates. The vancomycin-resistant isolates comprised 24 strains that harboured vanB and 15 that carried vanA. A number of clinically relevant STs (Table S1) were included, as were isolates displaying a daptomycin-non-susceptible phenotype, with 10 strains being considered non-susceptible to daptomycin (MIC = 8–16 mg/L). JRH-24-1 was found to be equally active against vancomycin-susceptible E. faecium, vancomycin-resistant E. faecium (vanA), vancomycin-resistant E. faecium (vanB) and daptomycin-non-susceptible isolates, with an MIC90 of 2 mg/L for each. See Table 1. Table 1. Antimicrobial activity of JRH-24-1 tested against 53 clinical isolates of E. faecium E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  Table 1. Antimicrobial activity of JRH-24-1 tested against 53 clinical isolates of E. faecium E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  To evaluate whether the 1,2,4-oxadiazoles were bactericidal against E. faecium we next determined the MBC of JRH-24-1 against our panel of isolates. Overall, the MBC90 for the isolates was 4 mg/L indicating that the compound was bactericidal against E. faecium. MBC90 values for both vancomycin-susceptible E. faecium and vanB vancomycin-resistant E. faecium were 4 mg/L, while the vanA vancomycin-resistant E. faecium and daptomycin-non-susceptible E. faecium isolates had higher MBC90 values of 16 mg/L. JRH-24-1 displays rapid time–kill kinetics against both daptomycin-susceptible and -non-susceptible vancomycin-resistant E. faecium isolates The time–kill kinetics of 1,2,4-oxadiazole inhibitory activity had not previously been evaluated against any bacterial species. As such, we explored the killing kinetics of our compound against both a daptomycin-susceptible isolate, Aus008516 (MIC = 4 mg/L) and a non-susceptible isolate, DMG1700661 (MIC = 8–16 mg/L). Time–kill assays with varying concentrations (2–16 mg/L) of JRH-24-1 were performed. As controls, the viability of untreated cultures and cultures supplemented with 16 mg/L daptomycin were also evaluated. Killing kinetics were dependent on the concentration of JRH-24-1 used (Figure 1). For example, at 2 mg/L (1× MIC), the compound was not bactericidal (defined here as causing ≥3 log10 reduction in cfu/mL) against either strain. Instead, growth was inhibited for 24 h before an increase in viable counts was observed between 24 h and 48 h. At 4 mg/L (2× MIC), an approximately 4 log10 reduction in colony counts for both E. faecium strains was observed after just 1 h, indicating that the 1,2,4-oxadizoles cause rapid cell death in this organism. By 24 h the number of viable cells was below the detectable limit. After 48 h, however, both strains (Aus0085 and DMG1700661) appeared to rebound with visible growth in all cultures exposed to 4 mg/L (2× MIC) JRH-24-1 and viable E. faecium colonies were isolated following plating. At 8 mg/L (4× MIC) and 16 mg/L (8× MIC) JRH-24-1 appeared to have a rapid and sterilizing effect on the cultures of both Aus0085 and DMG1700661 (Figure 1). After 1 h of exposure, no viable cells could be isolated for either strain and there was no evidence of rebound for the duration of the experiment. Figure 1. View largeDownload slide Time–kill analysis of JRH-24-1 (Oxd) against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, and (b) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate. Blue circles are DMSO-containing (untreated) cultures, red squares are cultures treated with 2 mg/L JRH-24-1, green diamonds are cultures treated with 4 mg/L JRH-24-1, purple circles are cultures treated with 8 mg/L JRH-24-1, orange triangles are cultures treated with 16 mg/L JRH-24-1 and black triangles are cultures treated with 16 mg/L daptomycin. Note that the orange line has been displaced by 0.5 units to the right on the x-axis for visualization purposes. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. DAP, daptomycin. Figure 1. View largeDownload slide Time–kill analysis of JRH-24-1 (Oxd) against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, and (b) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate. Blue circles are DMSO-containing (untreated) cultures, red squares are cultures treated with 2 mg/L JRH-24-1, green diamonds are cultures treated with 4 mg/L JRH-24-1, purple circles are cultures treated with 8 mg/L JRH-24-1, orange triangles are cultures treated with 16 mg/L JRH-24-1 and black triangles are cultures treated with 16 mg/L daptomycin. Note that the orange line has been displaced by 0.5 units to the right on the x-axis for visualization purposes. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. DAP, daptomycin. In comparison, we found that daptomycin, even at a concentration as high as 16 mg/L did not always completely eradicate viable E. faecium cells. In the case of Aus0085, we observed between a 3 log10 and 6 log10 reduction in viability within 3 h, with a low level of viable cells then persisting for the duration of the experiment in some biological replicates. As expected, daptomycin did not inhibit the growth of DMG1700661, which displays a daptomycin-non-susceptible phenotype. These data therefore demonstrate that under the conditions tested the 1,2,4-oxadiazoles are more rapidly bactericidal than daptomycin against both daptomycin-susceptible E. faecium and daptomycin-non-susceptible E. faecium isolates and importantly shows that JRH-24-1 maintains bactericidal activity against daptomycin-non-susceptible E. faecium isolates at concentrations below that of daptomycin. Given our observation of rebound growth after 48 h of exposure to 4 mg/L (2× MIC) of JRH-24-1 for both Aus0085 and DMG1700661, we wished to determine whether 1,2,4-oxadiazole-tolerant mutants had emerged. To do this we sub-cultured colonies isolated from JRH-24-1-exposed cultures onto growth medium supplemented with 2 mg/L (1× MIC) JRH-24-1. In addition, we used a 10% inoculum from the rebound cultures to re-inoculate fresh medium supplemented with 2 mg/L (1× MIC) JRH-24-1 and then monitored growth. None of the isolates grew on medium supplemented with JRH-24-1 and the re-inoculated broths displayed no visible growth, suggesting that resistant mutants had not arisen and that JRH-24-1 is perhaps unstable following prolonged incubation at 37°C. Sub-MIC concentrations of JRH-24-1 reduce the daptomycin MIC for daptomycin-non-susceptible E. faecium to below the clinical breakpoint Since the molecular targets for the 1,2,4-oxadiazoles are thought to be the high molecular weight penicillin binding proteins (PBPs)10 and previous studies have shown that β-lactams such as ampicillin, which also target PBPs, are able to synergize with daptomycin against E. faecium,17 we hypothesized that JRH-24-1 might also act synergistically with daptomycin. To test this hypothesis, we determined the MICs for 12 vancomycin-resistant E. faecium isolates including both vanA- and vanB-containing strains, as well as isolates that were daptomycin non-susceptible (MIC = 8–16 mg/L), following exposure to 1 mg/L (0.5× MIC) JRH-24-1 and varying concentrations of daptomycin. As shown in Figure 2, the addition of this subinhibitory concentration of JRH-24-1 resulted in a 4–16-fold reduction in the MIC of daptomycin for all vancomycin-resistant E. faecium isolates tested. For all daptomycin-non-susceptible E. faecium strains, the addition of JRH-24-1 resulted in a reduction of the MIC to below the clinical breakpoint of daptomycin (≤4 mg/L), thus rendering these isolates daptomycin susceptible. Figure 2. View largeDownload slide MIC of daptomycin alone (DAP) or in combination with 1 mg/L (0.5× MIC) JRH-24-1 (DAP + Oxd) tested against 12 clinical isolates of E. faecium (all), including nine vanA vancomycin-resistant E. faecium isolates (vanA) and three vanB vancomycin-resistant E. faecium isolates (vanB). Eight of the isolates displayed daptomycin non-susceptibility (DNS) (MIC = 8–16 mg/L). The medians and IQRs are shown. Figure 2. View largeDownload slide MIC of daptomycin alone (DAP) or in combination with 1 mg/L (0.5× MIC) JRH-24-1 (DAP + Oxd) tested against 12 clinical isolates of E. faecium (all), including nine vanA vancomycin-resistant E. faecium isolates (vanA) and three vanB vancomycin-resistant E. faecium isolates (vanB). Eight of the isolates displayed daptomycin non-susceptibility (DNS) (MIC = 8–16 mg/L). The medians and IQRs are shown. Sub-MIC concentrations of JRH-24-1 enhance the killing efficiency of daptomycin against vancomycin-resistant E. faecium Since the addition of JRH-24-1 resulted in a reduction in the MIC of daptomycin for vancomycin-resistant E. faecium, we next explored whether the compound could also enhance the killing efficiency of daptomycin against E. faecium. As demonstrated in Figure 3(a and c), at 1 mg/L (0.5× MIC) JRH-24-1 displayed a slight inhibitory activity against E. faecium with lower cell counts in cultures of both strains treated with the 1,2,4-oxadiazole compared with untreated cultures. Nevertheless, both strains were able to grow in the presence of this concentration of JRH-24-1 as demonstrated by the increased colony counts of both strains after 24 h in comparison with the starting inoculum. As expected, daptomycin alone did not affect the viability of cultures until the concentration approached the MIC (2–4 mg/L for Aus0085 and 8 mg/L for DMG1700661), after which the viable counts of daptomycin-treated cultures dropped in comparison with the starting inoculum. After 24 h of exposure, the combination of daptomycin and JRH-24-1 resulted in significantly lower levels of viability in both Aus0085 and DMG1700661 cultures than did daptomycin alone at every concentration tested. In the case of Aus0085, synergy, as defined by a ≥2 log10 reduction in cell numbers compared with the most active single agent, was observed for the combination of JRH-24-1 and 0.5 mg/L (0.125× MIC), 1 mg/L (0.25× MIC), 2 mg/L (0.5× MIC) and 4 mg/L (1× MIC) of daptomycin (Figure 3a). Similarly, for DMG1700661 synergy was observed when JRH-24-1 was combined with 1 mg/L (0.0125× MIC), 2 mg/L (0.25× MIC), 4 mg/L (0.5× MIC) and 8 mg/L (1× MIC) of daptomycin (Figure 3c). Enhanced killing was also evident at 0.25 mg/L (0.0625× MIC) of daptomycin with Aus0085, but the difference between daptomycin alone and the combination of agents was less than 2 log10 and was therefore not considered synergistic. Figure 3. View largeDownload slide Synergy assays of daptomycin and JRH-24-1 against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1, (b) E. faecium strain Aus0085 using 0.5 mg/L (0.25× MIC) JRH-24-1, (c) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1 and (d) E. faecium DMG1700661 using 0.5 mg/L (0.25× MIC) JRH-24-1. Black bars are DMSO-containing cultures (untreated), grey bars are cultures treated with either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (Oxd) as specified above, white bars with horizontal lines are cultures treated with daptomycin (DAP) alone and chequered bars are cultures treated with daptomycin and either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (DAP + Oxd) as specified above. Note that the concentration of daptomycin used (mg/L) is shown in the x-axis labels. For example, DAP-0.25 denotes that 0.25 mg/L daptomycin was used in these cultures, while DAP-0.25 + Oxd denotes that 0.25 mg/L daptomycin in addition to either 1 or 0.5 mg/L JRH-24-1 was used in these cultures. The cfu/mL values relative to the starting inoculum, which is taken to be zero, are shown. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. Figure 3. View largeDownload slide Synergy assays of daptomycin and JRH-24-1 against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1, (b) E. faecium strain Aus0085 using 0.5 mg/L (0.25× MIC) JRH-24-1, (c) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1 and (d) E. faecium DMG1700661 using 0.5 mg/L (0.25× MIC) JRH-24-1. Black bars are DMSO-containing cultures (untreated), grey bars are cultures treated with either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (Oxd) as specified above, white bars with horizontal lines are cultures treated with daptomycin (DAP) alone and chequered bars are cultures treated with daptomycin and either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (DAP + Oxd) as specified above. Note that the concentration of daptomycin used (mg/L) is shown in the x-axis labels. For example, DAP-0.25 denotes that 0.25 mg/L daptomycin was used in these cultures, while DAP-0.25 + Oxd denotes that 0.25 mg/L daptomycin in addition to either 1 or 0.5 mg/L JRH-24-1 was used in these cultures. The cfu/mL values relative to the starting inoculum, which is taken to be zero, are shown. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. To substantiate the synergistic killing effects observed between daptomycin and 0.5× MIC JRH-24-1, we also tested the ability of JRH-24-1 to synergize with daptomycin at 0.5 mg/L (0.25× MIC) (Figure 3b and d). At this concentration, a more modest synergistic killing effect was observed. As above, the combination of daptomycin and JRH-24-1 (0.25× MIC) resulted in lower levels of viability in both Aus0085 and DMG1700661 cultures than did daptomycin alone at every concentration tested. However, at this concentration, synergy was only observed at a daptomycin concentration of 2 mg/L and 4 mg/L for Aus0085 (Figure 3b) and at 4 mg/L and 8 mg/L for DMG1700661 (Figure 3d). At the other concentrations tested the difference between daptomycin alone and the combination of agents was less than 2 log10 and was therefore not considered synergistic. JRH-24-1 displays minimal toxicity against human cells at the E. faecium MIC90 We next tested the toxicity of JRH-24-1 against HepG2 cells and human erythrocytes. As shown in Figure 4(a), JRH-24-1 was not toxic to HepG2 cells at the MIC90 for E. faecium (2 mg/L), with 100% viability observed. However, a reduction in viability was observed at higher concentrations in a dose-dependent manner (Figure 4a). The IC50 of JRH-24-1 was determined to be 24.37 ± 1.09 mg/L. Limited haemolysis was observed against human erythrocytes following exposure to 2 mg/L JRH-24-1 (the E. faecium MIC90) as well as at the HepG2 IC50 concentration, with only 2%–5% of erythrocytes undergoing lysis. In contrast to O’Daniel et al.,10 however, we found that higher concentrations of JRH-24-1 caused increasing levels of haemolysis in a dose-dependent manner (Figure 4b). Figure 4. View largeDownload slide Toxicity testing of JRH-24-1 against (a) HepG2 cells and (b) human erythrocytes. Doubling dilutions of JRH-24-1 concentration (128–0.125 mg/L) are shown on a log10 scale in both graphs. HepG2 assays were performed with technical duplicates and biological triplicates. Haemolysis assays were performed in biological triplicate. The data shown represent the mean ± SEM. Figure 4. View largeDownload slide Toxicity testing of JRH-24-1 against (a) HepG2 cells and (b) human erythrocytes. Doubling dilutions of JRH-24-1 concentration (128–0.125 mg/L) are shown on a log10 scale in both graphs. HepG2 assays were performed with technical duplicates and biological triplicates. Haemolysis assays were performed in biological triplicate. The data shown represent the mean ± SEM. Discussion The prevalence of vancomycin-resistant E. faecium continues to rise globally18 with linezolid and daptomycin increasingly being needed as last-line therapeutic agents to treat infections caused by these isolates.19 Recent reports documenting the emergence of linezolid8 and daptomycin7 non-susceptibility in E. faecium are therefore a significant cause for concern and have led the WHO to recognize the development of new treatments for E. faecium as a global research priority. In this respect, the recently described 1,2,4-oxadiazoles10 might offer an alternative for the treatment of newly emerged MDR E. faecium. The activity of these compounds has been studied extensively against S. aureus.10,12,20 However, little is known about the activity of these antimicrobials against E. faecium. Our finding that JRH-24-1 is active against a broad range of clinical E. faecium STs, including daptomycin-non-susceptible isolates, greatly expands on previous studies10,12,20 and suggests that the 1,2,4-oxadiazoles might represent a broadly applicable therapeutic group for use against E. faecium. Although the 1,2,4-oxadiazoles were bactericidal against S. aureus,12 this activity was not explored with E. faecium. Furthermore, the kinetics of 1,2,4-oxadiazole-mediated killing have not been described for any bacterial species. Our finding that this antimicrobial displays rapid bactericidal activity against daptomycin-susceptible and -non-susceptible isolates is therefore novel. In light of these observations, time–kill assays to determine the kill kinetics of the 1,2,4-oxadiazoles against other clinically important Gram-positive pathogens should be performed to determine whether this activity is specific to E. faecium or more general for Gram-positive bacteria. Linezolid and quinupristin/dalfopristin are the only FDA-approved drugs for the treatment of vancomycin-resistant E. faecium. Despite this, daptomycin is often used since it is bactericidal. Our finding that JRH-24-1 is superior to daptomycin in its ability to kill E. faecium is therefore potentially important. Although we did not compare JRH-24-1 with linezolid, a direct comparison between these agents should be fully assessed under appropriate laboratory conditions since linezolid is one of the only FDA-approved drugs for treating vancomycin-resistant E. faecium infections. The 1,2,4-oxadiazoles were previously shown to act synergistically with oxacillin against S. aureus,21 although their ability to potentiate the activity of daptomycin was not assessed. Our finding that the 1,2,4-oxadiazoles can enhance the activity of daptomycin against E. faecium is therefore again novel. There is now substantial research describing the use of antimicrobial combinations against E. faecium.17,22–24 The most well-documented is perhaps the combination of daptomycin and β-lactams,12,24 with the latter family of antimicrobials, particularly ampicillin and ceftaroline, reportedly able to potentiate the activity of daptomycin against E. faecium.25,26 Given the similar predicted molecular targets for β-lactams and the 1,2,4-oxadiazoles, namely the high molecular weight PBPs,10 it is perhaps not surprising to find that JRH-24-1 was able to effectively synergize with daptomycin, reducing the MIC and enhancing the killing efficiency of this antimicrobial against E. faecium. Importantly, combination therapies have been used with effect for the treatment of patients with MDR E. faecium.22 Although the molecular mechanism by which the 1,2,4-oxadiazoles enhance the activity of daptomycin remains to be determined, our observation that subinhibitory concentrations of JRH-24-1 could revert the daptomycin-non-susceptible phenotype in E. faecium to susceptible is clinically important given the limited therapeutic options that are available for treating daptomycin-non-susceptible E. faecium. Our data therefore suggest that the combination of 1,2,4-oxadiazole and daptomycin might find utility in the clinical setting, especially in the treatment of clinically challenging infections caused by daptomycin-non-susceptible isolates. JRH-24-1 was found to have a fairly narrow therapeutic window, with the IC50 against HepG2 cells being approximately 12.5-fold greater than the MIC90 for E. faecium. Nevertheless, these antimicrobials have previously been used to treat S. aureus-mediated disease in murine models of infection, with no reported signs of systemic toxicity.10,12 This suggests that the effective dose might be below the level that would cause toxic side-effects. Importantly, Spink et al.12 also recently reported the development of new 1,2,4-oxadiazoles for S. aureus with reduced toxicity against HepG2 cells, suggesting that similarly designed analogues can be developed that have a wider therapeutic window but maintain activity against MDR E. faecium. In summary, we have shown that JRH-24-1 displays potent antimicrobial activity against a broad range of clinical E. faecium isolates. Furthermore, we demonstrate that the 1,2,4-oxadiazoles display rapid and more complete bactericidal activity than daptomycin against both daptomycin-susceptible and -non-susceptible E. faecium. Finally, we show that JRH-24-1 acts synergistically with daptomycin, with subinhibitory concentrations of the compound lowering the MIC of daptomycin for non-susceptible strains to below the clinical breakpoint. The 1,2,4-oxadiazole antimicrobials therefore hold great promise for the treatment of infections caused by MDR E. faecium. Acknowledgements We would like to thank staff and students at MDU PHL, Department of Microbiology and Immunology at the University of Melbourne (DMI) and Monash Institute of Pharmaceutical Sciences (MIPS) for help with technical aspects of the work. We thank Peter Revill and Tina Sozzi at the Victorian Infectious Diseases Reference Laboratory (VIDRL) for providing HepG2 cells and we would also like to thank Helen Heffernan at the Institute of Environmental Science and Research for help in collecting and characterizing isolates used in the study. Also acknowledged is Australian Federal Government Education Investment Fund Super Science Initiative and the Victorian State Government, Victoria Science Agenda Investment Fund for infrastructure support, and the facilities, and the scientific and technical assistance of the Australian Translational Medicinal Chemistry Facility (ATMCF), Monash Institute of Pharmaceutical Sciences (MIPS). ATMCF is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. Funding This study was funded by a Therapeutic Innovation Australia Grant and internal DMI funding. T. P. S. (GNT1008549), J. B. B. (1020411 & 1117602) and B. P. H. (GNT1105905) are supported by the National Health and Medical Research Council (NHMRC) of Australia. The MDU PHL is funded by the Victorian Government, Australia. Transparency declarations None to declare. Supplementary data Table S1 is available as Supplementary data at JAC Online. References 1 Carter GP, Buultjens AH, Ballard SA et al.   Emergence of endemic MLST non-typeable vancomycin-resistant Enterococcus faecium. J Antimicrob Chemother  2016; 71: 3367– 71. Google Scholar CrossRef Search ADS PubMed  2 Courvalin P. Vancomycin resistance in Gram-positive cocci. Clin Infect Dis  2006; 42 Suppl 1: S25– 34. Google Scholar CrossRef Search ADS PubMed  3 Hegstad K, Mikalsen T, Coque TM et al.   Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium. Clin Microbiol Infect  2010; 16: 541– 54. Google Scholar CrossRef Search ADS PubMed  4 CDC. Antibiotic Resistance Threats in the United States, 2013 . Atlanta, GA, USA: CDC, 2013. PubMed PubMed  5 WHO. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics . Geneva, Switzerland: WHO, 2017. http://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf?ua=1. 6 O’Driscoll T, Crank CW. Vancomycin-resistant enterococcal infections: epidemiology, clinical manifestations, and optimal management. Infect Drug Resist  2015; 8: 217– 30. Google Scholar PubMed  7 Lellek H, Franke GC, Ruckert C et al.   Emergence of daptomycin non-susceptibility in colonizing vancomycin-resistant Enterococcus faecium isolates during daptomycin therapy. Int J Med Microbiol  2015; 305: 902– 9. Google Scholar CrossRef Search ADS PubMed  8 Deshpande LM, Ashcraft DS, Kahn HP et al.   Detection of a new cfr-like gene, cfr(B), in Enterococcus faecium isolates recovered from human specimens in the United States as part of the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother  2015; 59: 6256– 61. Google Scholar CrossRef Search ADS PubMed  9 Cattoir V, Leclercq R. Twenty-five years of shared life with vancomycin-resistant enterococci: is it time to divorce? J Antimicrob Chemother  2013; 68: 731– 42. Google Scholar CrossRef Search ADS PubMed  10 O’Daniel PI, Peng Z, Pi H et al.   Discovery of a new class of non-β-lactam inhibitors of penicillin-binding proteins with Gram-positive antibacterial activity. J Am Chem Soc  2014; 136: 3664– 72. Google Scholar CrossRef Search ADS PubMed  11 Xiao Q, Vakulenko S, Chang M et al.   Mutations in mmpL and in the cell wall stress stimulon contribute to resistance to oxadiazole antibiotics in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother  2014; 58: 5841– 7. Google Scholar CrossRef Search ADS PubMed  12 Spink E, Ding D, Peng Z et al.   Structure-activity relationship for the oxadiazole class of antibiotics. J Med Chem  2015; 58: 1380– 9. Google Scholar CrossRef Search ADS PubMed  13 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement. M100-S26 . CLSI, Wayne, PA, USA, 2015. 14 Kastoris AC, Rafailidis PI, Vouloumanou EK et al.   Synergy of fosfomycin with other antibiotics for Gram-positive and Gram-negative bacteria. Eur J Clin Pharmacol  2010; 66: 359– 68. Google Scholar CrossRef Search ADS PubMed  15 Evans BC, Nelson CE, Yu SS et al.   Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs. J Vis Exp  2013: e50166. 16 Lam MM, Seemann T, Tobias NJ et al.   Comparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium. BMC Genomics  2013; 14: 595. Google Scholar CrossRef Search ADS PubMed  17 Hindler JA, Wong-Beringer A, Charlton CL et al.   In vitro activity of daptomycin in combination with β-lactams, gentamicin, rifampin, and tigecycline against daptomycin-nonsusceptible enterococci. Antimicrob Agents Chemother  2015; 59: 4279– 88. Google Scholar CrossRef Search ADS PubMed  18 Agudelo Higuita NI, Huycke MM. Enterococcal disease, epidemiology, and implications for treatment. In: Gilmore MS, Clewell DB, Ike Y et al.  , eds. Enterococci: From Commensals to Leading Causes of Drug Resistant Infection . Boston, MA, USA: Massachusetts Eye and Ear Infirmary, 2014. 19 Zhao M, Liang L, Ji L et al.   Similar efficacy and safety of daptomycin versus linezolid for treatment of vancomycin-resistant enterococcal bloodstream infections: a meta-analysis. Int J Antimicrob Agents  2016; 48: 231– 8. Google Scholar CrossRef Search ADS PubMed  20 Ding D, Boudreau MA, Leemans E et al.   Exploration of the structure-activity relationship of 1,2,4-oxadiazole antibiotics. Bioorg Med Chem Lett  2015; 25: 4854– 7. Google Scholar CrossRef Search ADS PubMed  21 Janardhanan J, Meisel JE, Ding D et al.   In vitro and in vivo synergy of the oxadiazole class of antibacterials with β-lactams. Antimicrob Agents Chemother  2016; 60: 5581– 8. Google Scholar CrossRef Search ADS PubMed  22 Desai H, Wong R, Pasha AK. A novel way of treating multidrug-resistant enterococci. N Am J Med Sci  2016; 8: 229– 31. Google Scholar CrossRef Search ADS PubMed  23 Hall Snyder A, Werth BJ, Barber KE et al.   Evaluation of the novel combination of daptomycin plus ceftriaxone against vancomycin-resistant enterococci in an in vitro pharmacokinetic/pharmacodynamic simulated endocardial vegetation model. J Antimicrob Chemother  2014; 69: 2148– 54. Google Scholar CrossRef Search ADS PubMed  24 Smith JR, Barber KE, Raut A et al.   β-Lactam combinations with daptomycin provide synergy against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium. J Antimicrob Chemother  2015; 70: 1738– 43. Google Scholar PubMed  25 Werth BJ, Barber KE, Tran KN et al.   Ceftobiprole and ampicillin increase daptomycin susceptibility of daptomycin-susceptible and -resistant VRE. J Antimicrob Chemother  2015; 70: 489– 93. Google Scholar CrossRef Search ADS PubMed  26 Sakoulas G, Rose W, Nonejuie P et al.   Ceftaroline restores daptomycin activity against daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium. Antimicrob Agents Chemother  2014; 58: 1494– 500. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium

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Abstract

Abstract Background Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. Methods The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time–kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates. Introduction Enterococcus faecium is an important nosocomial pathogen.1 It is highly transmissible and causes a range of infections that are typically difficult to treat.2,E. faecium is highly recombinogenic and promiscuous with regard to horizontal gene transfer, resulting in the emergence of MDR strains.3 The most clinically significant isolates are vancomycin-resistant E. faecium, which are classified as a ‘serious threat’ to human health by the US CDC4 and included on the WHO priority pathogen list for research and development of new antimicrobials.5 Vancomycin is considered an effective first-line therapy against E. faecium. However, the increasing prevalence of vancomycin-resistant E. faecium means that the clinical use of this antibiotic in the treatment of E. faecium infections is becoming increasingly limited, resulting in the use of alternative antimicrobials such as daptomycin and linezolid.6 Recent reports documenting the emergence of E. faecium that display decreased susceptibility to both daptomycin7 and linezolid8 are of serious concern since very few therapeutic options remain for treating infections caused by these isolates.9 There is therefore an urgent need for novel therapeutics that are active against MDR E. faecium isolates, particularly those displaying non-susceptibility to daptomycin and linezolid. Recently, a novel class of antimicrobials known as the 1,2,4-oxadiazoles was discovered.10 While extensive in vitro and in vivo evaluation of these agents against Staphylococcus aureus has been performed,10–12 there are limited data on the activity of the 1,2,4-oxadiazoles against E. faecium. Here we report that this family of antimicrobials has potent activity against an extended panel of clinical E. faecium and acts synergistically with daptomycin against this important human pathogen. Materials and methods Bacterial strains and growth conditions Fifty-three clinical E. faecium isolates (Table S1, available as Supplementary data at JAC Online) were obtained from the Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and Doherty Applied Microbial Genomics culture collections. All isolates were maintained on horse blood agar and grown at 37°C using standard laboratory conditions. Antimicrobials JRH-24-1 was synthesized according to the previously published procedure for ‘compound 2’ in O’Daniel et al.10 Daptomycin was obtained from Novartis under the brand name Cubicin. Antimicrobial susceptibility testing Phenotypic susceptibility testing was performed using CLSI methods.13 A 2-fold dilution series (from 32 to 0.5 mg/L) of JRH-24-1 or daptomycin was made in 100 μL volumes of cation-adjusted Mueller–Hinton w/TES broth (CAMHB w/TES) purchased from Thermo Fisher (additionally supplemented with 50 mg/L Ca2+ for daptomycin assays) in a 96-well plate (Corning) and an inoculum of 100 μL of overnight broth culture adjusted to 5 × 105 cfu/mL was added to each well. After 24 h incubation, the MIC was defined as the lowest antimicrobial concentration that inhibited visible growth. To determine the MBC, 10 μL from each well with no visible growth was plated onto non-selective Mueller–Hinton agar plates. The MBC was defined as the lowest concentration with no growth on subculture after 48 h. All MIC and MBC testing was performed in triplicate. Time–kill assays Time–kill assays were performed with E. faecium strains Aus0085 and DMG1700661, using DMSO-containing (untreated) CAMHB w/TES or equivalent broths supplemented with 1× MIC (2 mg/L), 2× MIC (4 mg/L), 4× MIC (8 mg/L) and 8× MIC (16 mg/L) JRH-24-1 or daptomycin (16 mg/L) (additionally supplemented with 50 mg/L Ca2+ for daptomycin assays). Broths were inoculated with overnight bacterial broth cultures and adjusted to 5 × 105 cfu/mL. Samples withdrawn at 0, 1, 3, 24 and 48 h post-inoculation were centrifuged at 14 000 g to pellet the bacterial cells. The pellets were then washed in PBS and resuspended in an equal volume of PBS. The samples were then serially diluted in PBS and plated onto brain heart infusion (BHI) agar (Oxoid). Plates were then incubated overnight at 37°C before colony enumeration was performed. All assays were performed in biological triplicate. JRH-24-1 and daptomycin combination MIC testing MICs for the combination of JRH-24-1 and daptomycin were determined using a modification of the CLSI protocol described above. A 2-fold dilution series (from 32 to 0.5 mg/L) of daptomycin, with or without a final concentration of 0.5× MIC (1 mg/L) JRH-24-1, was inoculated into 100 μL volumes of CAMHB w/TES supplemented with 50 mg/L Ca2+ in a 96-well plate (Corning), along with a 100 μL overnight bacterial suspension adjusted to 5 × 105 cfu/mL. As before, the MIC was defined as the lowest daptomycin concentration that inhibited visible growth after 24 h incubation. JRH-24-1 and daptomycin synergy testing Synergy testing was performed using E. faecium strains Aus0085 and DMG1700661 in CAMHB w/TES supplemented with 50 mg/L Ca2+. For Aus0085, broths were supplemented with 0.0625× MIC (0.25 mg/L), 0.125× MIC (0.5 mg/L), 0.25× MIC (1 mg/L), 0.5× MIC (2 mg/L) and 1× MIC (4 mg/L) daptomycin with or without either 0.25× MIC (0.5 mg/L) or 0.5× MIC (1 mg/L) JRH-24-1. For DMG1700661, broths were supplemented with 0.125× MIC (1 mg/L), 0.25× MIC (2 mg/L), 0.5× MIC (4 mg/L) and 1× MIC (8 mg/L) daptomycin with or without either 0.25× MIC (0.5 mg/L) or 0.5× MIC (1 mg/L) JRH-24-1. Additionally, DMSO-containing (untreated) broths and broths supplemented with 0.5× MIC JRH-24-1 alone were used as controls. All broths were inoculated with overnight bacterial broth cultures and adjusted to 5 × 105 cfu/mL. Samples withdrawn at 0 and 24 h post-inoculation were centrifuged at 14 000 g to pellet the bacterial cells. The pellets were then washed in PBS and resuspended in an equal volume of PBS. The samples were then serially diluted in PBS and plated onto BHI agar. Plates were then incubated overnight at 37°C before colony enumeration was performed. Synergy was defined as a ≥2 log10 reduction in viability compared with the most active single agent used.14 All assays were performed in biological triplicate. HepG2 cellular toxicity testing HepG2 cells were maintained in MEM (Gibco) supplemented with 10% (v/v) FCS, 2 mM glutamine (Gibco) and Antibiotic-Antimycotic (Gibco) at 37°C in 5% CO2. Cells were seeded at 1 × 104 cells per well in 96-well tissue culture treated plates (Corning). Following overnight growth, the medium was removed and the cells were exposed to JRH-24-1 (0.125–128 mg/L) in MEM lacking phenol red (Gibco) supplemented with 10% (v/v) FCS, 2 mM glutamine (Gibco), Non-Essential Amino Acids (Gibco) and Antibiotic-Antimycotic (Gibco) for 24 h. Cellular viability was then assessed using XTT reagent (Sigma–Aldrich) with PMS (N-methyl dibenzopyrazine methyl sulfate) (Thermo Fisher) according to the XTT cell viability assay protocol (Thermo Fisher). Cells treated with 2.5% (v/v) DMSO (corresponding to the amount of DMSO in the lowest dilution of JRH-24-1) were the negative (vehicle) control. Cells were incubated for 3 h before absorbance was measured at 475 and 660 nm (non-specific reading) using an Ensight Multimode plate reader (PerkinElmer). All assays were performed in biological triplicate. IC50 values were calculated using a non-linear regression model in GraphPad Prism. Haemolysis assay The assay was modified from Evans et al.15 Fresh whole human blood was collected into K2-EDTA-coated Vacutainer tubes and pelleted at 500 g for 5 min. The red blood cell pellet was washed twice in saline solution followed by a third wash in PBS before being diluted 1:25 in PBS. JRH-24-1 was made to 20× concentrations in DMSO and a 10 μL aliquot of each concentration was added to 190 μL of diluted blood in a 96-well U-bottom plate (Corning) giving final JRH-24-1 concentrations of 0.125 to 128 mg/L. The plate was then incubated at 37°C for 1 h. Negative (vehicle) controls were incubated with DMSO and positive controls were incubated with 2% (v/v) Triton X-100. Cell suspensions were centrifuged at 500 g and 100 μL of supernatants collected. The absorbance of each supernatant was then measured at 451 nm using an Ensight Multimode plate reader (PerkinElmer). All assays were performed in biological triplicate. Results JRH-24-1 displays bactericidal activity against an extended panel of vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium To expand on previous studies10,12 and evaluate the potential utility of the 1,2,4-oxadiazole antimicrobials for treating E. faecium infections we determined the MIC and MBC values of JRH-24-1 for a panel of 53 clinical E. faecium isolates (Table S1). The panel contained 14 vancomycin-susceptible isolates and 39 vancomycin-resistant isolates. The vancomycin-resistant isolates comprised 24 strains that harboured vanB and 15 that carried vanA. A number of clinically relevant STs (Table S1) were included, as were isolates displaying a daptomycin-non-susceptible phenotype, with 10 strains being considered non-susceptible to daptomycin (MIC = 8–16 mg/L). JRH-24-1 was found to be equally active against vancomycin-susceptible E. faecium, vancomycin-resistant E. faecium (vanA), vancomycin-resistant E. faecium (vanB) and daptomycin-non-susceptible isolates, with an MIC90 of 2 mg/L for each. See Table 1. Table 1. Antimicrobial activity of JRH-24-1 tested against 53 clinical isolates of E. faecium E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  Table 1. Antimicrobial activity of JRH-24-1 tested against 53 clinical isolates of E. faecium E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  E. faecium strain type  n  MIC/MBC (mg/L)   MIC range  MIC50  MIC90  MBC90  All  53  1–2  2  2  4  Vancomycin-susceptible E. faecium  14  2  2  2  4  Vancomycin-resistant E. faecium (vanA)  15  2  2  2  16  Vancomycin-resistant E. faecium (vanB)  24  1–2  2  2  4  Daptomycin-non-susceptible E. faecium  10  2  2  2  16  To evaluate whether the 1,2,4-oxadiazoles were bactericidal against E. faecium we next determined the MBC of JRH-24-1 against our panel of isolates. Overall, the MBC90 for the isolates was 4 mg/L indicating that the compound was bactericidal against E. faecium. MBC90 values for both vancomycin-susceptible E. faecium and vanB vancomycin-resistant E. faecium were 4 mg/L, while the vanA vancomycin-resistant E. faecium and daptomycin-non-susceptible E. faecium isolates had higher MBC90 values of 16 mg/L. JRH-24-1 displays rapid time–kill kinetics against both daptomycin-susceptible and -non-susceptible vancomycin-resistant E. faecium isolates The time–kill kinetics of 1,2,4-oxadiazole inhibitory activity had not previously been evaluated against any bacterial species. As such, we explored the killing kinetics of our compound against both a daptomycin-susceptible isolate, Aus008516 (MIC = 4 mg/L) and a non-susceptible isolate, DMG1700661 (MIC = 8–16 mg/L). Time–kill assays with varying concentrations (2–16 mg/L) of JRH-24-1 were performed. As controls, the viability of untreated cultures and cultures supplemented with 16 mg/L daptomycin were also evaluated. Killing kinetics were dependent on the concentration of JRH-24-1 used (Figure 1). For example, at 2 mg/L (1× MIC), the compound was not bactericidal (defined here as causing ≥3 log10 reduction in cfu/mL) against either strain. Instead, growth was inhibited for 24 h before an increase in viable counts was observed between 24 h and 48 h. At 4 mg/L (2× MIC), an approximately 4 log10 reduction in colony counts for both E. faecium strains was observed after just 1 h, indicating that the 1,2,4-oxadizoles cause rapid cell death in this organism. By 24 h the number of viable cells was below the detectable limit. After 48 h, however, both strains (Aus0085 and DMG1700661) appeared to rebound with visible growth in all cultures exposed to 4 mg/L (2× MIC) JRH-24-1 and viable E. faecium colonies were isolated following plating. At 8 mg/L (4× MIC) and 16 mg/L (8× MIC) JRH-24-1 appeared to have a rapid and sterilizing effect on the cultures of both Aus0085 and DMG1700661 (Figure 1). After 1 h of exposure, no viable cells could be isolated for either strain and there was no evidence of rebound for the duration of the experiment. Figure 1. View largeDownload slide Time–kill analysis of JRH-24-1 (Oxd) against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, and (b) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate. Blue circles are DMSO-containing (untreated) cultures, red squares are cultures treated with 2 mg/L JRH-24-1, green diamonds are cultures treated with 4 mg/L JRH-24-1, purple circles are cultures treated with 8 mg/L JRH-24-1, orange triangles are cultures treated with 16 mg/L JRH-24-1 and black triangles are cultures treated with 16 mg/L daptomycin. Note that the orange line has been displaced by 0.5 units to the right on the x-axis for visualization purposes. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. DAP, daptomycin. Figure 1. View largeDownload slide Time–kill analysis of JRH-24-1 (Oxd) against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, and (b) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate. Blue circles are DMSO-containing (untreated) cultures, red squares are cultures treated with 2 mg/L JRH-24-1, green diamonds are cultures treated with 4 mg/L JRH-24-1, purple circles are cultures treated with 8 mg/L JRH-24-1, orange triangles are cultures treated with 16 mg/L JRH-24-1 and black triangles are cultures treated with 16 mg/L daptomycin. Note that the orange line has been displaced by 0.5 units to the right on the x-axis for visualization purposes. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. DAP, daptomycin. In comparison, we found that daptomycin, even at a concentration as high as 16 mg/L did not always completely eradicate viable E. faecium cells. In the case of Aus0085, we observed between a 3 log10 and 6 log10 reduction in viability within 3 h, with a low level of viable cells then persisting for the duration of the experiment in some biological replicates. As expected, daptomycin did not inhibit the growth of DMG1700661, which displays a daptomycin-non-susceptible phenotype. These data therefore demonstrate that under the conditions tested the 1,2,4-oxadiazoles are more rapidly bactericidal than daptomycin against both daptomycin-susceptible E. faecium and daptomycin-non-susceptible E. faecium isolates and importantly shows that JRH-24-1 maintains bactericidal activity against daptomycin-non-susceptible E. faecium isolates at concentrations below that of daptomycin. Given our observation of rebound growth after 48 h of exposure to 4 mg/L (2× MIC) of JRH-24-1 for both Aus0085 and DMG1700661, we wished to determine whether 1,2,4-oxadiazole-tolerant mutants had emerged. To do this we sub-cultured colonies isolated from JRH-24-1-exposed cultures onto growth medium supplemented with 2 mg/L (1× MIC) JRH-24-1. In addition, we used a 10% inoculum from the rebound cultures to re-inoculate fresh medium supplemented with 2 mg/L (1× MIC) JRH-24-1 and then monitored growth. None of the isolates grew on medium supplemented with JRH-24-1 and the re-inoculated broths displayed no visible growth, suggesting that resistant mutants had not arisen and that JRH-24-1 is perhaps unstable following prolonged incubation at 37°C. Sub-MIC concentrations of JRH-24-1 reduce the daptomycin MIC for daptomycin-non-susceptible E. faecium to below the clinical breakpoint Since the molecular targets for the 1,2,4-oxadiazoles are thought to be the high molecular weight penicillin binding proteins (PBPs)10 and previous studies have shown that β-lactams such as ampicillin, which also target PBPs, are able to synergize with daptomycin against E. faecium,17 we hypothesized that JRH-24-1 might also act synergistically with daptomycin. To test this hypothesis, we determined the MICs for 12 vancomycin-resistant E. faecium isolates including both vanA- and vanB-containing strains, as well as isolates that were daptomycin non-susceptible (MIC = 8–16 mg/L), following exposure to 1 mg/L (0.5× MIC) JRH-24-1 and varying concentrations of daptomycin. As shown in Figure 2, the addition of this subinhibitory concentration of JRH-24-1 resulted in a 4–16-fold reduction in the MIC of daptomycin for all vancomycin-resistant E. faecium isolates tested. For all daptomycin-non-susceptible E. faecium strains, the addition of JRH-24-1 resulted in a reduction of the MIC to below the clinical breakpoint of daptomycin (≤4 mg/L), thus rendering these isolates daptomycin susceptible. Figure 2. View largeDownload slide MIC of daptomycin alone (DAP) or in combination with 1 mg/L (0.5× MIC) JRH-24-1 (DAP + Oxd) tested against 12 clinical isolates of E. faecium (all), including nine vanA vancomycin-resistant E. faecium isolates (vanA) and three vanB vancomycin-resistant E. faecium isolates (vanB). Eight of the isolates displayed daptomycin non-susceptibility (DNS) (MIC = 8–16 mg/L). The medians and IQRs are shown. Figure 2. View largeDownload slide MIC of daptomycin alone (DAP) or in combination with 1 mg/L (0.5× MIC) JRH-24-1 (DAP + Oxd) tested against 12 clinical isolates of E. faecium (all), including nine vanA vancomycin-resistant E. faecium isolates (vanA) and three vanB vancomycin-resistant E. faecium isolates (vanB). Eight of the isolates displayed daptomycin non-susceptibility (DNS) (MIC = 8–16 mg/L). The medians and IQRs are shown. Sub-MIC concentrations of JRH-24-1 enhance the killing efficiency of daptomycin against vancomycin-resistant E. faecium Since the addition of JRH-24-1 resulted in a reduction in the MIC of daptomycin for vancomycin-resistant E. faecium, we next explored whether the compound could also enhance the killing efficiency of daptomycin against E. faecium. As demonstrated in Figure 3(a and c), at 1 mg/L (0.5× MIC) JRH-24-1 displayed a slight inhibitory activity against E. faecium with lower cell counts in cultures of both strains treated with the 1,2,4-oxadiazole compared with untreated cultures. Nevertheless, both strains were able to grow in the presence of this concentration of JRH-24-1 as demonstrated by the increased colony counts of both strains after 24 h in comparison with the starting inoculum. As expected, daptomycin alone did not affect the viability of cultures until the concentration approached the MIC (2–4 mg/L for Aus0085 and 8 mg/L for DMG1700661), after which the viable counts of daptomycin-treated cultures dropped in comparison with the starting inoculum. After 24 h of exposure, the combination of daptomycin and JRH-24-1 resulted in significantly lower levels of viability in both Aus0085 and DMG1700661 cultures than did daptomycin alone at every concentration tested. In the case of Aus0085, synergy, as defined by a ≥2 log10 reduction in cell numbers compared with the most active single agent, was observed for the combination of JRH-24-1 and 0.5 mg/L (0.125× MIC), 1 mg/L (0.25× MIC), 2 mg/L (0.5× MIC) and 4 mg/L (1× MIC) of daptomycin (Figure 3a). Similarly, for DMG1700661 synergy was observed when JRH-24-1 was combined with 1 mg/L (0.0125× MIC), 2 mg/L (0.25× MIC), 4 mg/L (0.5× MIC) and 8 mg/L (1× MIC) of daptomycin (Figure 3c). Enhanced killing was also evident at 0.25 mg/L (0.0625× MIC) of daptomycin with Aus0085, but the difference between daptomycin alone and the combination of agents was less than 2 log10 and was therefore not considered synergistic. Figure 3. View largeDownload slide Synergy assays of daptomycin and JRH-24-1 against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1, (b) E. faecium strain Aus0085 using 0.5 mg/L (0.25× MIC) JRH-24-1, (c) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1 and (d) E. faecium DMG1700661 using 0.5 mg/L (0.25× MIC) JRH-24-1. Black bars are DMSO-containing cultures (untreated), grey bars are cultures treated with either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (Oxd) as specified above, white bars with horizontal lines are cultures treated with daptomycin (DAP) alone and chequered bars are cultures treated with daptomycin and either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (DAP + Oxd) as specified above. Note that the concentration of daptomycin used (mg/L) is shown in the x-axis labels. For example, DAP-0.25 denotes that 0.25 mg/L daptomycin was used in these cultures, while DAP-0.25 + Oxd denotes that 0.25 mg/L daptomycin in addition to either 1 or 0.5 mg/L JRH-24-1 was used in these cultures. The cfu/mL values relative to the starting inoculum, which is taken to be zero, are shown. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. Figure 3. View largeDownload slide Synergy assays of daptomycin and JRH-24-1 against (a) E. faecium strain Aus0085, a vanB vancomycin-resistant E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1, (b) E. faecium strain Aus0085 using 0.5 mg/L (0.25× MIC) JRH-24-1, (c) E. faecium DMG1700661, a vanA daptomycin-non-susceptible E. faecium isolate, using 1 mg/L (0.5× MIC) JRH-24-1 and (d) E. faecium DMG1700661 using 0.5 mg/L (0.25× MIC) JRH-24-1. Black bars are DMSO-containing cultures (untreated), grey bars are cultures treated with either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (Oxd) as specified above, white bars with horizontal lines are cultures treated with daptomycin (DAP) alone and chequered bars are cultures treated with daptomycin and either 1 mg/L (0.5× MIC) or 0.5 mg/L (0.25× MIC) JRH-24-1 (DAP + Oxd) as specified above. Note that the concentration of daptomycin used (mg/L) is shown in the x-axis labels. For example, DAP-0.25 denotes that 0.25 mg/L daptomycin was used in these cultures, while DAP-0.25 + Oxd denotes that 0.25 mg/L daptomycin in addition to either 1 or 0.5 mg/L JRH-24-1 was used in these cultures. The cfu/mL values relative to the starting inoculum, which is taken to be zero, are shown. All conditions were tested in triplicate, with the data shown representing the mean ± SEM. The limit of detection in these assays is denoted by the broken line and ‘LoD’. To substantiate the synergistic killing effects observed between daptomycin and 0.5× MIC JRH-24-1, we also tested the ability of JRH-24-1 to synergize with daptomycin at 0.5 mg/L (0.25× MIC) (Figure 3b and d). At this concentration, a more modest synergistic killing effect was observed. As above, the combination of daptomycin and JRH-24-1 (0.25× MIC) resulted in lower levels of viability in both Aus0085 and DMG1700661 cultures than did daptomycin alone at every concentration tested. However, at this concentration, synergy was only observed at a daptomycin concentration of 2 mg/L and 4 mg/L for Aus0085 (Figure 3b) and at 4 mg/L and 8 mg/L for DMG1700661 (Figure 3d). At the other concentrations tested the difference between daptomycin alone and the combination of agents was less than 2 log10 and was therefore not considered synergistic. JRH-24-1 displays minimal toxicity against human cells at the E. faecium MIC90 We next tested the toxicity of JRH-24-1 against HepG2 cells and human erythrocytes. As shown in Figure 4(a), JRH-24-1 was not toxic to HepG2 cells at the MIC90 for E. faecium (2 mg/L), with 100% viability observed. However, a reduction in viability was observed at higher concentrations in a dose-dependent manner (Figure 4a). The IC50 of JRH-24-1 was determined to be 24.37 ± 1.09 mg/L. Limited haemolysis was observed against human erythrocytes following exposure to 2 mg/L JRH-24-1 (the E. faecium MIC90) as well as at the HepG2 IC50 concentration, with only 2%–5% of erythrocytes undergoing lysis. In contrast to O’Daniel et al.,10 however, we found that higher concentrations of JRH-24-1 caused increasing levels of haemolysis in a dose-dependent manner (Figure 4b). Figure 4. View largeDownload slide Toxicity testing of JRH-24-1 against (a) HepG2 cells and (b) human erythrocytes. Doubling dilutions of JRH-24-1 concentration (128–0.125 mg/L) are shown on a log10 scale in both graphs. HepG2 assays were performed with technical duplicates and biological triplicates. Haemolysis assays were performed in biological triplicate. The data shown represent the mean ± SEM. Figure 4. View largeDownload slide Toxicity testing of JRH-24-1 against (a) HepG2 cells and (b) human erythrocytes. Doubling dilutions of JRH-24-1 concentration (128–0.125 mg/L) are shown on a log10 scale in both graphs. HepG2 assays were performed with technical duplicates and biological triplicates. Haemolysis assays were performed in biological triplicate. The data shown represent the mean ± SEM. Discussion The prevalence of vancomycin-resistant E. faecium continues to rise globally18 with linezolid and daptomycin increasingly being needed as last-line therapeutic agents to treat infections caused by these isolates.19 Recent reports documenting the emergence of linezolid8 and daptomycin7 non-susceptibility in E. faecium are therefore a significant cause for concern and have led the WHO to recognize the development of new treatments for E. faecium as a global research priority. In this respect, the recently described 1,2,4-oxadiazoles10 might offer an alternative for the treatment of newly emerged MDR E. faecium. The activity of these compounds has been studied extensively against S. aureus.10,12,20 However, little is known about the activity of these antimicrobials against E. faecium. Our finding that JRH-24-1 is active against a broad range of clinical E. faecium STs, including daptomycin-non-susceptible isolates, greatly expands on previous studies10,12,20 and suggests that the 1,2,4-oxadiazoles might represent a broadly applicable therapeutic group for use against E. faecium. Although the 1,2,4-oxadiazoles were bactericidal against S. aureus,12 this activity was not explored with E. faecium. Furthermore, the kinetics of 1,2,4-oxadiazole-mediated killing have not been described for any bacterial species. Our finding that this antimicrobial displays rapid bactericidal activity against daptomycin-susceptible and -non-susceptible isolates is therefore novel. In light of these observations, time–kill assays to determine the kill kinetics of the 1,2,4-oxadiazoles against other clinically important Gram-positive pathogens should be performed to determine whether this activity is specific to E. faecium or more general for Gram-positive bacteria. Linezolid and quinupristin/dalfopristin are the only FDA-approved drugs for the treatment of vancomycin-resistant E. faecium. Despite this, daptomycin is often used since it is bactericidal. Our finding that JRH-24-1 is superior to daptomycin in its ability to kill E. faecium is therefore potentially important. Although we did not compare JRH-24-1 with linezolid, a direct comparison between these agents should be fully assessed under appropriate laboratory conditions since linezolid is one of the only FDA-approved drugs for treating vancomycin-resistant E. faecium infections. The 1,2,4-oxadiazoles were previously shown to act synergistically with oxacillin against S. aureus,21 although their ability to potentiate the activity of daptomycin was not assessed. Our finding that the 1,2,4-oxadiazoles can enhance the activity of daptomycin against E. faecium is therefore again novel. There is now substantial research describing the use of antimicrobial combinations against E. faecium.17,22–24 The most well-documented is perhaps the combination of daptomycin and β-lactams,12,24 with the latter family of antimicrobials, particularly ampicillin and ceftaroline, reportedly able to potentiate the activity of daptomycin against E. faecium.25,26 Given the similar predicted molecular targets for β-lactams and the 1,2,4-oxadiazoles, namely the high molecular weight PBPs,10 it is perhaps not surprising to find that JRH-24-1 was able to effectively synergize with daptomycin, reducing the MIC and enhancing the killing efficiency of this antimicrobial against E. faecium. Importantly, combination therapies have been used with effect for the treatment of patients with MDR E. faecium.22 Although the molecular mechanism by which the 1,2,4-oxadiazoles enhance the activity of daptomycin remains to be determined, our observation that subinhibitory concentrations of JRH-24-1 could revert the daptomycin-non-susceptible phenotype in E. faecium to susceptible is clinically important given the limited therapeutic options that are available for treating daptomycin-non-susceptible E. faecium. Our data therefore suggest that the combination of 1,2,4-oxadiazole and daptomycin might find utility in the clinical setting, especially in the treatment of clinically challenging infections caused by daptomycin-non-susceptible isolates. JRH-24-1 was found to have a fairly narrow therapeutic window, with the IC50 against HepG2 cells being approximately 12.5-fold greater than the MIC90 for E. faecium. Nevertheless, these antimicrobials have previously been used to treat S. aureus-mediated disease in murine models of infection, with no reported signs of systemic toxicity.10,12 This suggests that the effective dose might be below the level that would cause toxic side-effects. Importantly, Spink et al.12 also recently reported the development of new 1,2,4-oxadiazoles for S. aureus with reduced toxicity against HepG2 cells, suggesting that similarly designed analogues can be developed that have a wider therapeutic window but maintain activity against MDR E. faecium. In summary, we have shown that JRH-24-1 displays potent antimicrobial activity against a broad range of clinical E. faecium isolates. Furthermore, we demonstrate that the 1,2,4-oxadiazoles display rapid and more complete bactericidal activity than daptomycin against both daptomycin-susceptible and -non-susceptible E. faecium. Finally, we show that JRH-24-1 acts synergistically with daptomycin, with subinhibitory concentrations of the compound lowering the MIC of daptomycin for non-susceptible strains to below the clinical breakpoint. The 1,2,4-oxadiazole antimicrobials therefore hold great promise for the treatment of infections caused by MDR E. faecium. Acknowledgements We would like to thank staff and students at MDU PHL, Department of Microbiology and Immunology at the University of Melbourne (DMI) and Monash Institute of Pharmaceutical Sciences (MIPS) for help with technical aspects of the work. We thank Peter Revill and Tina Sozzi at the Victorian Infectious Diseases Reference Laboratory (VIDRL) for providing HepG2 cells and we would also like to thank Helen Heffernan at the Institute of Environmental Science and Research for help in collecting and characterizing isolates used in the study. Also acknowledged is Australian Federal Government Education Investment Fund Super Science Initiative and the Victorian State Government, Victoria Science Agenda Investment Fund for infrastructure support, and the facilities, and the scientific and technical assistance of the Australian Translational Medicinal Chemistry Facility (ATMCF), Monash Institute of Pharmaceutical Sciences (MIPS). ATMCF is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. Funding This study was funded by a Therapeutic Innovation Australia Grant and internal DMI funding. T. P. S. (GNT1008549), J. B. B. (1020411 & 1117602) and B. P. H. (GNT1105905) are supported by the National Health and Medical Research Council (NHMRC) of Australia. The MDU PHL is funded by the Victorian Government, Australia. Transparency declarations None to declare. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 6, 2018

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