In Vitro and In Vivo Synergy of the Oxadiazole Class of Antibacterials with β-Lactams

24 Pages·2016·0.76 MB·English

AAC Accepted Manuscript Posted Online 11 July 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.00787-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 In Vitro and In Vivo Synergy of the Oxadiazole Class of Antibacterials with β-Lactams
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3 Jeshina Janardhanan,a Jayda E. Meisel,a Derong Ding,a Valerie A. Schroeder,b William
4 R. Wolter,b Shahriar Mobashery,a and Mayland Changa, #
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6 aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN D
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7 46556, USA; bFreimann Life Science Center and Department of Biological Sciences, n
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8 University of Notre Dame, Notre Dame, IN 46556, USA d
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10 Running title: Oxadiazole Antibacterial Synergy with β-Lactams

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11 # Corresponding author p
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12 a
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13 Abstract s
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14 The oxadiazole antibacterials target bacterial cell wall and are bactericidal. We investigated o
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15 the synergism of ND-421 with the commonly used β-lactams and non-β-lactam antibiotics by o
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16 the checkerboard method and by time-kill assays. ND-421 synergizes well with β-lactam p
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17 antibiotics and it also exhibits a long post-antibiotic effect (4.7 h). We also evaluated the in il 3
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18 vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in 0
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19 combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant b
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20 infection model (1.49 log10 bacterial reduction for ND-321 versus 0.36 log10 for linezolid u
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21 with NRS119) and acts synergistically with β-lactam antibiotics in vitro and in vivo, and the t
22 combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh
23 methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log bacterial
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24 reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain
25 would have been resistant to oxacillin otherwise.
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26 Introduction
27 Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen associated with
28 serious community-acquired infections and is one of the leading causes of nosocomial
29 infections in the United States and around the world (1). MRSA harbors the mecA gene that
30 encodes penicillin-binding protein 2a (PBP2a), which confers resistance essentially to all β-
31 lactam antibiotics (2). The currently available treatment options for MRSA are glycopeptides D
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32 (vancomycin and telavancin), oxazolidinones (linezolid and tedizolid), daptomycin, and n
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33 ceftaroline, of which only the oxazolidinones are orally bioavailable drugs. Linezolid- and d
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34 vancomycin-resistant strains have already been reported (3-6); mutations leading to
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35 daptomycin resistance have also been observed (7). An increased vancomycin MIC has also

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36 been linked to a possible cross-resistance to daptomycin (8). Ceftaroline was approved in tp
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37 2010 for treatment of community-acquired pneumonia and acute bacterial skin infections, a
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38 owing to its ability to bind penicillin-binding proteins (PBPs). The binding in the case of the s
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39 MRSA PBP2a is both at the allosteric and active sites, imparting an interesting angle to the .o
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40 mechanism of action of this antibiotic (9, 10). Recently, ceftaroline heteroresistance among S. o
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41 aureus has also been reported (11), and ceftaroline-resistant MRSA strains have been isolated A
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42 (12, 13). Tedizolid was approved in 2014 for skin and soft-tissue infections; resistance to it il
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43 has been described in vitro (14). 0
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44 The oxadiazoles are a new class of non-β-lactam antibacterials targeting cell-wall
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45 biosynthesis with excellent in vitro and in vivo activity against MRSA and other Gram- g
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46 positive bacteria (15). ND-421 (Figure 1) is a lead oxadiazole and was also found to be t
47 bactericidal against vancomycin- and linezolid-resistant MRSA (16). This compound
48 exhibits efficacy comparable to linezolid in a mouse peritonitis model of infection, and has
49 low clearance, long half-life, and 97% oral bioavailability (16). As resistance to new
50 antibiotics emerges upon their introduction into the clinic and, combination therapy can
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51 minimize the development of resistance (17), we investigated the possibility of synergism of
52 ND-421 with the commonly used β-lactams and non-β-lactam antibiotics and found that ND-
53 421 synergizes well with β-lactam antibiotics. We also evaluated the in vivo efficacy of ND-
54 421 in a murine neutropenic thigh infection model alone and in combination with oxacillin
55 and demonstrated that the combination decreased bacterial load significantly compared to
56 single agent treatment. D
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57 n
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58 MATERIALS AND METHODS d
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60 Reagents. The antimicrobial agents used in the study included cefepime (Sigma-Aldrich, St.

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61 Louis, MO), piperacillin (TCI, Portland, OR), linezolid (AmplaChem Inc, Carmel, IN), and tp
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62 imipenem, meropenem, vancomycin, oxacillin, gentamicin, azithromycin and doxycycline a
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63 (all from Sigma-Aldrich). The oxadiazole ND-421 and the internal standard were synthesized s
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64 in our laboratory using methodology reported earlier (16). High performance liquid .o
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65 chromatography grade acetonitrile (Sigma-Aldrich) and formic acid (Sigma-Aldrich) were o
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66 used for mass spectrometry experiments. Distilled water was purified on a MilliQ system A
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67 (Millipore, Billerica, MA). il
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68 0
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69 Microorganisms. MRSA strains NRS70 (N315), NRS123 (MW2), NRS100 (COL),
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70 NRS119, and methicillin-sensitive S. aureus (MSSA) strain NRS128 were obtained through g
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71 the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA). ATCC29213 t
72 and MRSA 252 were purchased from American Type Culture Collection (ATCC, Manassas,
73 VA).
74 MIC determination. The MIC values of ND-421 against these organisms were determined
75 in triplicates in cation adjusted Mueller-Hinton II broth (CAMHB-II, Becton Dickinson and
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76 Co, Sparks, MD) using the microdilution technique according to the Clinical Laboratory
77 Standards Institute (CLSI) guidelines (18).
78 Inoculum effect. The inoculum effect was determined in CAMHB-II according to the CLSI
79 guidelines for broth microdilution (18) using final bacterial concentrations of 104, 105, 106,
80 107, and 108 CFU/mL in 96-well plates containing 2–fold serial dilutions of ND-421.
81 Checkerboard assay. The synergistic interaction of ND-421 with the panel of β-lactams and D
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82 non-β-lactams was tested on four MRSA (NRS70, NRS123, NRS100 and MRSA 252) and n
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83 one MSSA (NRS128) strain, using the checkerboard assay in 96-well plates. The final d
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84 inoculum in each well was 5x105 CFU/mL and the results were read after 18-h incubation at
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85 37 °C. The Fractional-Inhibitory Concentration (FIC) index was calculated for each

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86 combination as reported previously (19). An FIC index of ≤ 0.5 was considered as tp
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87 synergistic, > 0.5 to < 2 as indifferent, and > 2 as antagonistic. The experiments were done in a
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88 triplicates. The synergistic combinations were further validated with time-kill assays. s
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89 Time-kill assay. Time-kill assays were performed in triplicates in CAMHB-II. The assay was .o
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90 done in 5-mL tubes using ¼ MIC of ND-421 alone and in combination with ½ to 1/64 MICs o
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91 of the β-lactams. A control tube with no antibiotic was also included. Bacteria were grown to A
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92 a density of approximately 108 CFU/mL, and diluted 100-fold in fresh MHB. This bacterial il
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93 suspension was added to the MHB tubes to a final concentration of 5x105 CFU/mL and 0
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94 incubated at 37 °C for 24 h. A 100-μL aliquot was obtained from each tube at 0, 3, 6, 12 and
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95 24 h, serially diluted in saline, and plated on antibiotic-free agar (LB agar) in duplicate to g
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96 determine colony counts. Synergy was defined as ≥2 log10 decrease in colony counts at 24 h t
97 with the combination, compared to the most active single drug alone (19), and the final
98 inoculum in the combination was ≥2 log CFU/mL below the starting inoculum.
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99 Post antibiotic effect (PAE). NRS70 was grown in MHB to an OD of 0.1 and diluted 1:10
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100 with fresh MHB. A 2.5-mL aliquot of this culture was mixed with 2.5 mL of antibiotic
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101 solutions to a final concentration of ~106 CFU/mL. The cultures were incubated with ND-
102 421, linezolid and vancomycin at 2× and 4× MIC for 2 h. A growth control in the absence of
103 antibiotic was included. The antibiotics were diluted 1:1000 in fresh pre-warmed (37 °C)
104 MHB and the regrowth rate was measured by viable colony counting. The growth control in
105 the absence of antibiotic was also treated similarly. Samples were drawn at 0 h, 2 h (before
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106 and after dilution), and every hour thereafter for 7 h and plated on LB agar plates. The PAE
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107 was calculated as PAE = T – C, where T is time (h) required for the CFU in the test culture to n
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108 increase by 1 log above the count immediately after dilution and C is time (h) required for d
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109 the CFU in the control culture to increase by 1 log10 above the count immediately after fr
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110 dilution (20). The experiment was performed in duplicates.

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111 Animals: Out-bred, pathogen-free female ICR mice (Harlan Laboratories Inc., Indianapolis, p
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112 IN) aged 6-8 weeks old and weighing 24-26 g were used. Mice were housed in polycarbonate a
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113 shoebox cages with ¼ inch corncob (The Andersons Inc., Maumee, OH) and Alpha-dri s
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114 (Sheperd Specialty Papers, Inc., Richland, MI) bedding with 12-h light/dark cycle at 72 °F. o
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115 The animals were given food (Teklad 2918 Irradiated Extruded Rodent Diet) and water ad o
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116 libitum. All procedures were performed in accordance with and approval by the University of
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117 Notre Dame Institutional Animal Care and Use Committee. il 3
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118 Neutropenic thigh infection model. The mouse neutropenic thigh infection model described 0
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119 by Craig et. al. (21) was used to assess the efficacy of ND-421. Briefly, the mice (n = 10 b
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120 mice per group) were rendered neutropenic by intraperitoneal treatment with 100 µL u
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121 cyclophosphamide (Alfa Aesar, Haverhill, MA) at 200 mg/kg, 4 days and 1 day prior to the t
122 infection. MRSA strains NRS70 and NRS119 were grown in brain heart infusion broth to an
123 OD of 0.5, then diluted to a final concentration of approximately 106 CFU/mL in fresh
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124 brain heart infusion broth. A 0.1-mL aliquot of the bacterial inoculum was injected
125 intramuscularly into the right thigh. One hour after infection the animals were administered a
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126 single oral dose of 40 mg/kg of ND-421, linezolid or vehicle (10% DMSO, 25% Tween-80,
127 65% water) by oral gavage. The animals were sacrificed by CO asphyxiation 48 h after
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128 infection. Terminal plasma was collected by cardiac puncture, followed by aseptic removal
129 of both thigh muscles (uninfected and infected). The infected thigh was weighed,
130 homogenized in phosphate buffered saline using a Bullet blender (Next Advance, Inc.,
131 Averill Park, NY), serially diluted, and plated on LB agar plates (Fisher Scientific, Waltham, D
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132 MA) to determine colony counts. The plates were incubated at 37 °C for 24 h. The colony n
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133 counts were expressed as log CFU/g of tissue. Groups were compared using the Mann- d
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134 Whitney U test.
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135 In vivo synergy: The in vivo efficacy of ND-421 in combination with oxacillin was evaluated

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136 in the neutropenic thigh infection model, as described above. The study included four groups tp
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137 (n = 8 mice/group): control (vehicle = 10% DMSO/25% Tween-80/65% water), ND-421 (40 a
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138 mg/kg orally once a day for 2 days), oxacillin (20 mg/kg subcutaneously three times a day for s
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139 2 days), and combination of ND-421 plus oxacillin (40 mg/kg ND-421 orally once a day for 2 .o
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140 days and 20 mg/kg oxacillin subcutaneously three times a day for 2 days). The animals were o
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141 sacrificed at 48 h. The infected thighs were processed as described above for bacterial counts, A
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142 and the uninfected thighs and plasma were analyzed as described below for determination of il
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143 drug levels. 0
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144 Statistical analysis. Statistical analysis was done using Mann Whitney U test on Graphpad
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145 Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). g
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146 Dose preparation. Compounds were dissolved in 10% DMSO/25% Tween-80/65% water t
147 (vehicle) at a concentration of 10 mg/mL (equivalent to 40 mg/kg) or 5 mg/mL (20 mg/kg).
148 For subcutaneous injections of oxacillin, the doses were filter-sterilized using nylon
149 membranes (0.2 µm, Pall Life Sciences, Port Washington, NY). Mice were given 100 µL of
150 compound orally (ND-421, linezolid, or vehicle) or subcutaneously (oxacillin).
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151 Drug levels in plasma and thigh. Terminal blood collected from the neutropenic thigh
152 infection model was centrifuged at 1500 g for 10 minutes to obtain plasma and stored at -80
153 °C until analysis. Uninfected thighs were weighed and were stored at -80 °C until analysis.
154 A 50-µL aliquot of plasma was quenched with 150 µL of internal standard in acetonitrile to
155 precipitate protein. Standards of each compound ranging from 0 µg/mL to 20 µg/mL were
156 spiked in 50 µL of mouse plasma (Bioreclamation IVT, Baltimore, MD) and quenched with D
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157 150 µL of internal standard (1 µg/mL) in acetonitrile. Samples and standards were then n
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158 centrifuged for 10 minutes at 10,000 g, and the supernatants were analyzed using a Waters d
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159 Acquity Ultra Performance Liquid Chromatograph (UPLC, Waters Corp., Milford, MA)
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160 coupled with a triple quadrupole mass spectrometer (TQD, Waters, Milford, MA) with

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161 multiple reaction monitoring (MRM). The uninfected thighs were homogenized in an tp
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162 equivalent volume of acetonitrile and were centrifuged at 10,000 g for 10 minutes. A 50-µL a
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163 aliquot of the thigh supernatant was diluted with 150 µL of internal standard solution in s
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164 acetonitrile (1 µg/mL). The thigh samples containing ND-471 were diluted 10-fold with .o
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165 acetonitrile prior to internal standard addition. Samples were analyzed using UPLC-MRM. o
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166 Acquisition parameters were as follows: Supelco Ascentis C18 column (3 µm particle size, A
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167 10 cm x 2.1 mm, Sigma-Aldrich, St. Louis, MO), electrospray ionization positive mode il
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168 (ESI+), flow rate 0.5 mL/min, capillary voltage 4 kV, cone voltage 30 V, collision voltage 25 0
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169 V. The solvent program was as follows: 95% A/5% B for 0.25 min, 0.75-min linear gradient
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170 to 5% A/95% B, hold for 4 minutes, where A = 0.1% formic acid/water and B = 0.1% formic g
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171 acid/acetonitrile. Retention times were 1.76 min for linezolid, 1.77 min for oxacillin, 2.60 t
172 min for ND-421, and 2.85 min for the internal standard. The methods were linear between 0
173 µg/mL and 20 µg/mL (R2 values > 0.98). MRM transitions for each compound were 338.2 
174 296.1 for linezolid, 422.0  143.7 for ND-421, 401.9  160.1 for oxacillin, and 401.1 
175 122.8 for the internal standard. Peak areas for each compound were calculated relative to
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176 those for the internal standard using Waters MassLynx software. A standard curve of peak
177 area ratios plotted against standard concentration was generated from which drug
178 concentrations were determined using regression parameters and dilution factor.
179 Concentrations are reported in µg/mL for plasma and in µg/g tissue for thigh.
180
181 RESULTS AND DISCUSSION D
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182 MIC and inoculum effect. We determined the MIC values of ND-421 for the S. aureus n
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183 strains used in the study, including NRS119, a linezolid-resistant strain; the values ranged d
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184 from 2 to 4 µg/mL (Table 1), indicating that ND-421 was efficacious by itself. The MICs of
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185 vancomycin for the same strains ranged from 1 to 2 µg/mL and those of linezolid were from

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186 1 to 32 µg/mL. tp
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187 As the inoculum effect can reduce the potency of an antibiotic with increasing bacterial a
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188 concentration, we investigated the effect of high MRSA density (up to 108 CFU/mL) on the as
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189 activity of ND-421. An inoculum effect is defined as 8-fold or greater increase in MIC with a .o
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190 higher inoculum. Thus, ND-421 showed no inoculum effect (Table 2). o
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192 Checkerboard assay. The checkerboard assay was used to evaluate the in vitro synergy il
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193 between ND-421 and six cell-wall agents: five β-lactams (oxacillin, piperacillin, imipenem, 0
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194 meropenem, and cefepime) and a non-β-lactam (vancomycin), as well as four additional
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195 antibiotics targeting protein synthesis (the oxazolidinone linezolid, the aminoglycoside g
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196 gentamycin, the tetracycline doxycycline, and the macrolide azithromycin). We used four t
197 MRSA strains: NRS70, NRS123, NRS100, and MRSA252, and an MSSA strain (NRS128).
198 Results are shown as isobolograms in Figure 1, where FIC of ND-421 is plotted on the x-axis
199 and FIC of antibiotic is plotted on the y-axis; a concave curve is observed for synergy, a
200 linear curve represents additivity, and a convex curve indicates antagonism (22). With
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201 NRS100, ND-421 synergized with oxacillin, piperacillin, and imipenem; with MRSA252
202 synergy was observed with oxacillin, imipenem, cefepime and doxycycline. However ND-
203 421 synergized with all the β-lactams tested using MRSA strains NRS70 and NRS123. With
204 the NRS128 strain, synergy was observed in combination with oxacillin and piperacillin.
205 Cassat el al. compared the genomes of several S. aureus strains, including those of NRS70,
206 NRS123, NRS100, MRSA252, and NRS128 (23). A difference between these strains is the D
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207 expression of the MHC class II analog protein Map in S. aureus strains that do not show n
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208 synergy, namely NRS100, MRSA252, and NRS128, and its absence in NRS70 and NRS123, d
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209 strains that display synergy. Map is an immunomodulator that potentiates the survival of S.
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210 aureus by interfering with T cell-mediated host immunity (24). Mice infected with S. aureus

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211 deficient in Map have significantly reduced arthritis, osteomyelitis, and abscesses compared tp
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212 to controls, and mice treated with recombinant Map had reduced T cell-mediated responses a
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213 and significantly reduced hypersensitivity, when challenged with antigen (24). Therefore, s
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214 Map plays a role in S. aureus infections by interfering with cellular immunity. It is not known .o
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215 if this factor also influences the lack of in vitro synergy between ND-421 and β-lactams in S. o
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216 aureus strains expressing Map. With non-β-lactams, the only synergy observed with ND-421 A
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217 was in the case of strain NRS123 for gentamycin and with strains NRS123 and MRSA252 for il
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218 doxycycline combinations. 0
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219 Among the β-lactam antibiotics, the highest synergy is with oxacillin (ΣFIC values of 0.31
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220 and 0.37 against NRS70 and NRS123, respectively), which inhibits PBP1, 2, and 3; and the g
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221 least synergy is with piperacillin (ΣFIC values of 0.41 against NRS70 and 0.50 against t
222 NRS123), which inhibits PBP3. Synergy is found with imipenem (preferentially inhibits
223 PBP2; ΣFIC values of 0.41 and 0.30 against NRS70 and NRS123, respectively), meropenem
224 (preferentially inhibits PBP1, but also inhibits PBP2 and PBP4; ΣFIC values of 0.35 against
225 NRS70 and 0.31 against NRS123), as well as with cefepime (inhibits PBP2 and PBP3; ΣFIC
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226 values of 0.35 and 0.33 against NRS70 and NRS123, respectively). As the oxadiazoles inhibit
227 PBP2a (15) in MRSA and cooperation between the transpeptidase domain of PBP2a and the
228 transglycosylase domain of PBP2 is required for cell-wall synthesis in the presence of β-
229 lactam antibiotics (25), we hypothesize that this is the basis of synergy between ND-421 and
230 β-lactam antibiotics. Indeed, good synergy is observed between ND-421 and oxacillin,
231 imipenem, meropenem, and cefepime, all of which inhibit PBP2, while the weakest synergy D
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232 is observed with piperacillin, which inhibits preferentially PBP3. n
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233 In support of this mechanism for synergy, while ND-421 synergizes with β-lactam antibiotics d
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234 that inhibit transpeptidases in cell-wall biosynthesis, it does not synergize with vancomycin,
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235 an inhibitor of transglycosylation. Likewise, ND-421 does not synergize with the protein-

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236 synthesis inhibitors linezolid (bacteriostatic), the aminoglycoside gentamycin (bactericidal; tp
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237 synergy observed only with strain NRS123), the tetracycline doxycycline (bacteriostatic; a
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238 mild synergy observed in NRS123), or the macrolide azithromycin (bacteriostatic). s
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239 Time-kill curves. Synergy between ND-421 and the β-lactams (oxacillin, piperacillin, .o
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240 imipenem, cefepime) and doxycycline was confirmed using time-kill assays with MRSA o
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241 strains NRS70 and NRS123, as we had observed synergy by the checkerboard assay with A
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242 these strains. In order to allow for assessment of synergy, ¼ MIC of ND-421 was used, a il
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243 concentration at which a bacteriostatic effect was observed up to 12 h of growth. The 0
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244 concentrations of the β-lactams and doxycyline ranged from ½ to 1/64 MIC. Following 24 h
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245 of incubation, ≥ 2 log reduction in NRS70 colony counts was observed with the g
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246 combination of ND-421 plus ¼ or 1/8 or 1/16 MIC of β-lactams compared to either of the t
247 single agents alone and from the starting inoculum (Figure 2), indicating that ND-421
248 synergizes with β-lactam antibiotics. With the NRS123 strain synergy was observed for
249 oxacillin, imipenem, and cefepime in combination with ND-421, however, the combination
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