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Ceftaroline Restores Daptomycin Activity Against Daptomycin Nonsusceptible 1

Vancomycin Resistant Enterococcus faecium 2

3

George Sakoulas1, Warren Rose2, Poochit Nonejuie1, Joshua Olson1, Joseph Pogliano1, 4

Romney Humphries3, and Victor Nizet1 5

6

1. University of California San Diego School of Medicine, La Jolla, CA, USA 7

2. University of Wisconsin-Madison School of Pharmacy, Pharmacy Practice Division, 8

Madison, WI, USA 9

3. David Geffen School of Medicine at the University of California, Los Angeles, CA, USA 10

11

Running Title: Ceftaroline plus daptomycin synergy against VRE 12

13

Corresponding author address 14

George Sakoulas, M.D., Division of Pediatric Pharmacology & Drug Discovery 15

University of California San Diego School of Medicine 16

9500 Gilman Drive, Mail Code 0687, La Jolla, CA 92093-0687 17

Phone: (858) 534-2325 ; Fax: (858) 534-5661 ; Email: [email protected] 18

19

20

AAC Accepts, published online ahead of print on 23 December 2013Antimicrob. Agents Chemother. doi:10.1128/AAC.02274-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 21

Background: Daptomycin-nonsusceptible, vancomycin-resistant Enterococcus faecium (VRE) 22

are a formidable emerging threat to patients with comorbidities, leaving few therapeutic options 23

in cases of severe invasive infections. 24

Methods: Using a previously characterized isogenic pair of VRE from the same patient differing 25

in their daptomycin susceptibility (Etest MICs 0.38 mg/L and 10 mg/L), we examined the effect 26

of ceftaroline, ceftriaxone, and ampicillin on membrane fluidity and susceptibility of VRE to 27

surface binding and killing by daptomycin and human cathelicidin antimicrobial peptide LL37. 28

Results: Synergy was noted in vitro between daptomycin and ampicillin and ceftaroline for 29

daptomycin-susceptible VRE strain but only ceftaroline showed synergy against the daptomycin-30

nonsusceptible VRE strain (~ 2 log10 CFU reduction at 24 hours). Ceftaroline co-treatment 31

increased daptomycin surface binding with an associated increase in membrane fluidity and an 32

increase in the net negative surface charge of the bacteria as evidenced by increased poly-L-33

lysine binding. Consistent with the observed biophysical changes, ceftaroline resulted in 34

increased binding and killing of daptomycin-nonsusceptible VRE by human cathelicidin LL37. 35

Conclusions: Using a pair of daptomycin susceptible/nonsusceptible pair of VRE, we noted that 36

VRE is ceftaroline resistant, yet ceftaroline confers significant effects on growth rate as well as 37

biophysical changes on the cell surface of VRE that can potentiate the activity of daptomycin 38

and innate cationic host defense peptides such as cathelicidin. Although limited to just 2 strains, 39

these finding suggest that additional in vivo and in vitro studies need to be done to explore the 40

possibility of using ceftaroline as adjunctive anti-VRE therapy.41


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INTRODUCTION 42

Loss of susceptibility to daptomycin is an increasing concern among vancomycin-43

resistant Enterococcus faecium (VRE) (1). When faced with invasive infections by daptomycin 44

nonsusceptible VRE, clinicians have limited therapeutic options. Of great concern are the lack of 45

a bactericidal agent, antibiotic-associated side effects such as linezolid-induced 46

thrombocytopenia and quinupristin-dalfopristin (QD)-associated myalgias, and drug-drug 47

interactions such as microsomal P450 effects of QD and serotonin syndrome concerns with 48

linezolid and concomitant serotonin reuptake inhibitors. Therefore, a great need exists for 49

infectious disease physicians practicing in tertiary medical centers with patients at high risk for 50

VRE infections, e.g. bone marrow and liver transplant recipients, to develop innovative 51

pharmacotherapies to treat such patients (2,3). The clinical dilemma faced by physicians treating 52

these patients is further compounded by the facts that novel therapeutics targeting VRE are 53

lacking and that single or combination antibiotics with in vitro activity against VRE have not 54

been clinically validated by appropriate trials (4). 55

Our group has previously shown a perhaps counterintuitive effect of ampicillin in 56

converting daptomycin from a bacteriostatic to a bactericidal antibiotic against an ampicillin-57

resistant bloodstream VRE, with successful clearance of refractory bacteremia by this organism 58

using daptomycin plus ampicillin combination therapy (5). In this study, ampicillin also 59

conferred increased susceptibility of VRE to killing by innate cationic host defense peptides 60

(HDPs) such as cathelicidin LL37. To further explore the possible benefit of beta-lactams against 61

VRE, we examined ampicillin and ceftaroline each in combination with daptomycin against a 62

previously characterized daptomycin-nonsusceptible VRE and its isogenic parent strain (6). 63

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METHODS 65

66

Bacterial Strains and Antimicrobial Susceptibility Testing. Daptomycin-susceptible VRE 67

strain 8019 and the isogenic daptomycin-nonsusceptible strain 5938 later isolated from the same 68

patient were previously characterized phenotypically and genotypically (6). VRE strain 5938 was 69

previously reported as having daptomycin MIC 192 mg/L by Etest. However, upon retrieval of 70

this isolate from the freezer for these studies, the daptomycin MIC by Etest was decreased to 10 71

mg/L, consistent with the instability of daptomycin nonsusceptibility previously reported (7). 72

Antimicrobial susceptibility testing was performed using using CLSI methods or Epsilometer 73

test according to manufacturer instructions (BioMerieux, Durham, NC) (8). Time-kill assays 74

were performed in duplicate using an initial bacterial inoculum of 106 cfu/mL in LB (Luria-75

Bertani Broth) supplemented to calcium 50 mg/L with antibiotic concentrations chosen to 76

encompass readily achievable serum levels of each agent during clinical treatment regimens (9-77

13). Quantitative bacterial counts were determined at 0, 6, and 24 hours incubation at 37oC. LB 78

was chosen because it yielded more consistent results than when using Mueller-Hinton broth. 79

80

Cell surface characterization studies. The cell envelopes of 8019 and 5938 were evaluated 81

morphologically and structurally following overnight cultures with study strains in the presence 82

and absence of antibiotics ampicillin 50 mg/L or ceftaroline 1 mg/L. Transmission electron 83

microscopy (TEM) was used to determine cell wall thickness and septation, and membrane 84

fluidity was analyzed by fluorescence polarization using 6-diphenyl-1,3,5-hexatriene as 85

previously described (6). Fluorescein isothiocyanate (FITC)-labeled poly-L-lysine (PLL) binding 86

assays were performed using flow cytometry methods previously described (5). PLL is a 87


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polycationic molecule used to study the interactions of cationic peptides with charged bacterial 88

envelopes. In this analysis, the extent of bacterial-bound FITC-labeled PLL reflects the relative 89

net negative surface charge. A total of 10,000 events were counted and analyzed using a BD 90

FACSCalibur® system (Becton Dickinson Labwere, San Jose, CA). Data are expressed as mean 91

relative fluorescent units (± SD). At least two independent experiments of triplicate samples 92

were performed. 93

94

Daptomycin Binding Assays. To determine if various beta-lactam antibiotics were able to 95

impact the ability of daptomycin to bind to the VRE membrane, the organism was grown in LB 96

at 37°C to OD600 0.6 in the presence or absence of ampicillin 40 mg/L, ceftaroline 5 mg/L or 97

ceftriaxone 20 mg/L, then incubated for 20 min with Bodipy-fluorescein-labeled daptomycin 98

(Bo-DAP, supplied courtesy of Cubist Pharmaceuticals, Lexington, MA). Concentrations of Bo-99

DAP used were 8 mg/L for staining of the daptomycin susceptible VRE and 16 mg/L of the 100

daptomycin nonsusceptible VRE. These concentration of labeled daptomycin were established 101

by pilot studies as optimal for fluorescence microscopy (data not shown). The activity of Bo-102

DAP has consistently shown an approximately a 2-fold increased MIC compared to the 103

unlabeled daptomycin molecule (MIC 1 mg/L for 8019, 16-32 mg/L for 5938). Excess 104

unincorporated label was removed by washing the cells three times in LB. The cells were 105

counterstained with DAPI 2 mg/L in the final LB wash to visualize the nucleoid, and then 106

imaged using a Delta Vision Deconvolution microscope (Applied Precision, Inc. Issaquah, WA) 107

as previously described (5). 108

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Human Cathelicidin LL37 Killing Assays. Human cathelicidin LL37 (net charge +6 at pH 7.5) 110

was purchased from AnaSpec, Inc. (Fremont, CA). The VRE strain was grown to stationary 111

phase in LB either in the absence or presence of the different antibiotics, pelleted and washed in 112

PBS, and resuspended to OD600nm 0.5 in PBS. Bacteria were diluted to 103 cfu/ml in RPMI+5% 113

LB containing 1X MIC concentrations of LL37 (2 μM for 8019 and 4 μM for 5938) and 114

incubated at 37oC. Aliquots (10 μl) were plated on blood agar after 2 hr incubation, and colonies 115

were enumerated after 24 hrs to quantify the percent of surviving bacteria (± SD). Results 116

represent three separate experiments performed in duplicate. 117

118

LL37 Binding Assays. The VRE strains were grown in the presence or absence of antibiotics as 119

in the Bo-DAP preparation steps described above, diluted 1:1 in phenol red-free RPMI1640 120

media (Invitrogen, Grand Island, NY) to a final volume of 200 μL, with TAMRA-labeled LL37 121

(American Peptide, Sunnyvale, CA) to a final concentration of 4 μM, and incubated for 45 122

minutes at 37oC with shaking. Bacteria were pelleted, washed 3 times with RPMI, counterstained 123

with DAPI 2 mg/L in the final wash, and visualized using a Delta Vision Deconvolution 124

microscope (Applied Precision, Inc. Issaquah, WA). 125

126 Statistics. Statistical evaluations of the differences in survival in the presence of various cationic 127

peptides, and differences in PLL binding were performed via Mann-Whitney U (GraphPad Prism 128

5.0 (GraphPad Software, Inc. San Diego, CA). Differences in cell wall thickness, membrane 129

fluidity, and Bo-DAP or LL37 binding studies were evaluated by non-paired t-test. “P” values of 130

< 0.05 were considered as statistically significant. 131

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RESULTS 133

Daptomycin, vancomycin, and LL37 MIC activity against VRE, in combination with 134

ceftaroline. Daptomycin and vancomycin MICs were determined for VRE 8019 (daptomycin-135

susceptible) and 5938 (daptomycin-nonsusceptible) in the presence of increasing concentrations 136

of ampicillin or ceftaroline, as well as selected concentrations of other beta-lactams. Both strains 137

had MICs (mg/L) in Mueller-Hinton broth of ceftaroline >32, ampicillin >64, piperacillin >64, 138

ceftriaxone >64, cefazolin >64, linezolid 2.. Of interest however, was that ceftaroline had 139

appreciable effects on growth on both VRE strains, most notable of which was a dose-dependent 140

decrease in maximal bacterial density (Supplemental Figure 1). Morphologically, bacteria grown 141

in the presence of ceftaroline also showed increased chain length (data not shown). Results in 142

Table 1 show a decreased daptomycin MIC of VRE 5938 in the presence of ceftaroline across 143

the concentration range expected in vivo after dosing 600 mg q12 hr. Minimal effects were seen 144

for other beta-lactam agents at much higher concentrations. 145

MICs to cathelicidin LL37 in RPMI/5% LB for 8019 and 5938 were 2 μM and 4 μM, 146

respectively, consistent with tracking of the susceptibility of daptomycin to cationic 147

antimicrobial peptides (14). No change in LL37 MIC was observed for strain 8019 in the 148

presence of ceftaroline 2 mg/L or ampicillin 50 mg/L, or for 5938 in ampicillin 50 mg/L. In 149

ceftaroline 2 mg/L, the LL37 MIC decreased consistently (n=4) to 2 μM for strain 5938. 150

151

Ceftaroline enhanced daptomycin killing of both daptomycin susceptible and nonsuscepible 152

VRE. In vitro kill curve studies were performed for VRE 8019 (Figure 1, left panel) and 5938 153

(Figure 1, right panel) using daptomycin alone or in combination with ceftriaxone, ampicillin, 154

or ceftaroline. The concentration of daptomycin utilized was below MIC whereas the 155


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concentration of the beta-lactam antibiotic was chosen to reflect concentrations achievable with 156

standard dosing. Mean linezolid concentrations of 8 mg/L expected with standard doses and 4X 157

MIC were performed in parallel for comparison (13). At the concentrations chosen, growth was 158

seen for daptomycin alone. Synergy was seen with ampicillin and ceftaroline against 159

daptomycin-susceptible VRE 8019. However, for daptomycin nonsusceptible 5938, synergy (eg. 160

2 log10 reduction in CFU) was seen only with ceftaroline plus daptomycin. Ceftriaxone did not 161

enhance daptomycin killing of either strain. Linezolid showed bacteriostatic activity at 8 mg/L, 162

with nearly identical CFU counts at 6 and 24 hr as in the starting inoculum. 163

164

Ceftaroline alters cell wall thickness. Growth of daptomycin susceptible and nonsusceptible 165

VRE resulted in differences in cell wall thickness with subinhibitory antibiotic exposures, as 166

shown in Figure 2A. There were no differences in cell wall thickness between the 2 strains 167

grown in the absence of antibiotic. Against daptomycin-susceptible 8019, ampicillin and 168

ceftaroline resulted in decreases in cell wall thickness while the opposite was seen for 169

daptomycin-resistant 5938. We had previously noted significant increases in cell division septum 170

formation with 5938 compared to 8019 (6). Ceftaroline markedly reduced septum formation in 171

strain 8019 (36% vs. 47%, p<0.05, Chi-square). Against 5938, no significant changes in septum 172

formation were seen with ceftaroline (54%) compared to growth control (58%). 173

174

Ceftaroline increases cell membrane fluidity. Previous work has shown that daptomycin 175

resistance in Enterococcus faecium and Enterococcus faecalis may be accompanied by decreases 176

in membrane fluidity (6,16). Interestingly, our previous characterization of these 2 strains 177

showed no changes in this property between the strains under standard growth conditions (6). 178


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Growth of 5938 in ceftaroline 1 mg/L showed significant increases in membrane fluidity, as 179

evidenced by mean polarization index changes by ceftaroline. For 8019, membrane fluidity was 180

not significantly different (Figure 2B). 181

182

Ceftaroline Decreases Net Surface Charge. Strains 8019 and 5938 were grown in antibiotic-183

free, ceftaroline- or ampicillin-supplemented media and subjected to PLL binding studies. As 184

seen in Figure 3, 5938 exhibited significantly less binding to PLL compared to 8019, as can be 185

expected upon loss of daptomycin susceptibility. Against 8019, only ceftaroline but not 186

ampicillin resulted in an increase in net negative surface charge. Against 5938, both ampicillin 187

and ceftaroline resulted in increased PLL binding, reflective of increased negative surface charge 188

on the bacterial surface. 189

190

Ceftaroline increases cell-surface daptomycin binding, VRE 8019 and 5938 were labeled with 191

Bo-DAP 8 mg/L, demonstrating reduced relative binding of DAP for the non-susceptible strain 192

5938 compared to the susceptible strain 8019 (Supplemental Figure 2). To measure the effect of 193

various antibiotics on Bo-DAP binding, VRE 8019 and 5938 were labeled with 8 mg/L or 16 194

mg/L Bo-DAP, respectively, after growth in LB containing no antibiotic, ampicillin 40 mg/L, 195

ceftriaxone 20 mg/L, or ceftaroline 5 mg/L (Figure 4). Daptomycin binding on the cell surface 196

was significantly increased by ceftaroline for both strains as evidenced by the number of binding 197

foci/cell, without differences in intensity of the foci. Ampicillin increased the number of 198

daptomycin binding foci only for 5938, but not 8019. We tested ceftriaxone as many clinicians 199

might consider the convenience of once daily dosing, but found no effect to increase binding of 200

daptomycin. 201


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202

Ceftaroline increases killing of and binding to human cathelicidin LL37. Given that 203

daptomycin bound to calcium is a de facto antimicrobial peptide (17), examination of similarities 204

or differences on the effects of ceftaroline or ampicillin on human cathelicidin LL37 killing of 205

the two strains was of interest. Strains 8019 and 5938 were grown to stationary phase in LB 206

media without antibiotic or with ampicillin 40 mg/L, ceftriaxone 20 mg/L, or ceftaroline 5 mg/L 207

and exposed to LL37 killing assays at MIC for 2 hours. Ceftaroline-treated VRE 8019 (Figure 5, 208

left) and 5938 (Figure 5, right) were significantly more susceptible to LL37 killing than 209

untreated controls. The concentrations of ampicillin required to achieve comparable 210

enhancement of LL37 killing was approximately 10-fold higher, at 50 mg/L. Given that linezolid 211

is the only approved therapy for VRE bloodstream infections, we also examined its ability at 8 212

mg/L to sensitize VRE to LL37 and found that it also provided sensitization of 8019 and 5938 to 213

LL37 killing (Figure 5). Figure 6 shows that growth of VRE 5938 in ampicillin 40 mg/L or 214

ceftaroline resulted in increased LL37 (4μM) binding compared to cells grown in antibiotic-free 215

media. Quantitation shows significantly increasing intensity of binding/cell. 216

217

218


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DISCUSSION 219

The lack of available therapies against daptomycin-nonsusceptible VRE requires 220

innovative strategies to fulfill this need. We used a pair of previously characterized VRE strains 221

that differ by their daptomycin susceptibility. We noted a very unstable phenotype in the 222

daptomycin nonsusceptible strain, consistent with what has been previously reported. 223

Daptomycin susceptibility is driven in large part by differential expressions of various two-224

component regulatory systems influencing surface charge and other properties of the bacterial 225

surface that are modulated based on environmental conditions. (15) Consistent with this 226

property, the daptomycin MIC can be readily increased in 5938 upon serial passage in 227

daptomycin-containing media (unpublished observations). 228

The cephalosporin class of antibiotics has traditionally been considered inactive against 229

E. faecium, which represents the majority of VRE species in which daptomycin nonsusceptibility 230

has emerged. Of interest, however, we noted that ceftaroline was not inert against these 2 strains, 231

as shown by considerable effects on growth, especially on the maximal bacterial density 232

achieved after 24 hours (Supplemental Figure 1). We had also noted microscopically that 233

ceftaroline results in significant chaining of the bacteria, with resultant lower CFU/OD600 nm 234

compared to bacteria grown in antibiotic-free media (data not shown). These phenotypes are part 235

of other studies in our lab on the effects of beta-lactams on bacterial autolysins. We are currently 236

investigating the differential effect of class A PBP inactivation on susceptibility to daptomycin 237

and antimicrobial peptides and whether ceftaroline and/or ampicillin exert their effects on VRE 238

through these targets (16). Additional evidence by others suggests that low molecular weight 239

PBPs, such as ddcP, which influences D-alanine-D-alanine carboxypeptidase activity, warrants 240


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study as well, particularly with the possible tangential effects on vancomycin MIC by ceftaroline 241

(17). 242

For daptomycin-nonsusceptible VRE 5938, daptomycin at 8 mg/L, which would be close 243

to the maximum free drug concentration in vivo (10), allows bacterial growth that is not different 244

from growth control. Ceftriaxone showed no appreciable synergy with daptomycin against both 245

daptomycin susceptible and nonsusceptible strains 8019 and 5938, respectively. Consistent with 246

our prior data (5), ampicillin resulted in synergy with daptomycin against 8019, but this synergy 247

was lost upon emergence of daptomycin resistance in 5938. Of great interest, however, 248

ceftaroline showed synergy with daptomycin against both strains, achieving > 2 log10 CFU 249

killing at 24 hours. This combination regimen was considerably more potent than linezolid, the 250

only drug currently approved for VRE bacteremia (13), which was entirely bacteriostatic against 251

both strains. Nevertheless, we did find that linezolid did sensitize VRE to killing by cathelicidin 252

LL37, suggesting that in vivo the drug may yield greater activity than kill curves might suggest. 253

Although not ideal, linezolid has been used to treat VRE bacteremia successfully and is approved 254

for this indication (13). 255

In support of the in vitro findings of killing of daptomycin plus ceftaroline against VRE, 256

we have shown that ceftaroline i) increases daptomycin surface binding against daptomycin 257

susceptible and nonsusceptible VRE, with parallel increases in poly-L-lysine binding suggesting 258

increase in net negative charge; ii) increases membrane fluidity for daptomycin nonsusceptible 259

VRE, a property that would be expected to increase susceptibility to daptomycin given that 260

daptomycin nonsusceptible strains show a decreased membrane fluidity phenotype; iii) sensitizes 261

VRE to killing by the cationic antimicrobial peptide human cathelicidin LL37, via increased 262

LL37 binding to the surface of VRE. A summary of the changes are presented in Table 2. Note 263


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that ampicillin and ceftaroline resulted in decreases in cell wall thickness in the daptomycin 264

susceptible strain and increases in the nonsusceptible strain (Table 2). Collectively, these data 265

provide an interesting pattern on analysis. First, appears that enhancement of daptomycin binding 266

by ampicillin may not be consistent across VRE as the daptomycin susceptible strain in this 267

study did not behave like the strain in our prior study (5) . Nevertheless, it appears that marked 268

decreases in cell wall thickness induced by ampicillin on this strain may somehow render the 269

organism more daptomycin susceptible independent of daptomycin binding. Secondly, in the 270

setting of increased cell wall thickness that occurs with both ceftaroline and ampicillin, a 271

concomitant increase in membrane fluidity may be necessary to result in daptomycin synergy. 272

Ampicllin, which failed to increase membrane fluidity, failed to show synergy with daptomycin 273

against the daptomycin nonsusceptible strain. Finally, increased daptomycin binding may not be 274

necessary or sufficient for enhanced daptomycin activity. Daptomycin susceptible VRE showed 275

synergy with ampicillin plus daptomycin without an improvement in bo-DAP or poly-L-lysine 276

binding. On the other hand, increased Bo-DAP and poly-L-lysine by ampicillin in the 277

daptomycin nonsusceptible strain did not result in synergy. 278

These data are built upon an increasing line of evidence demonstrating that the in vitro 279

effects of antibiotics are not fully appreciated by current methods of susceptibility testing. 280

Specifically, beta-lactam antibiotics appear to have profound effects on beta-lactam resistant 281

Gram-positive organisms. Similar to the effects on VRE by ampicillin that we previously 282

discussed (5), we have shown that anti-staphylococcal beta-lactams increase susceptibility of 283

MRSA to daptomycin and innate immunity (14,18). Not all beta-lactams are equal in this effect, 284

as PBP-1 binding beta-lactams are better at potentiating daptomycin activity than non-PBP-1 285

specific beta-lactams (19). 286


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The development of daptomycin nonsusceptibility appears to be multifactorial and 287

complex and, therefore, effects of drugs that restore its susceptibility would be expected to be 288

equally complex (5,20-22). Daptomycin-nonsusceptible enterococci have differences from their 289

susceptible counterparts in sensing cell wall stress (via liaFSR) (16-18), phospholipid 290

composition (via cls) (20-22), and increased proclivity to septation (via ezrA) (6). While we are 291

not equiped to perform analysis of phospholipid content with ceftaroline therapy, we noticed 292

changes in septation and cell wall thickness induced by ceftaroline. Perhaps ceftaroline, similar 293

to what has been observed with ceftobiprole (23), binds PBP5 and alters the surface physiology 294

to allow enhanced daptomycin and HDP activity. 295

It is important to point out that these data are limited to a single daptomycin 296

susceptible/nonsusceptible pair of VRE. As it is well-appreciated that clinical strains are quite 297

heterogeneous, additional in vitro and in vivo studies are needed to explore the effects of 298

ceftaroline on VRE as potential adjunctive therapy and to determine whether all the changes we 299

observed in these strains are generalizable to other clinical VRE strains. Several of us currently 300

exploring in vitro PK/PD modeling of ceftaroline combination therapy against other VRE strains, 301

while others are completing checkerboard studies of daptomycin plus various beta-lactams, 302

including ceftaroline, against a larger collection of VRE strains, some of which are daptomycin 303

nonsusceptible. While we did not have the opportunity to employ this combination clinically, we 304

did recently publish a report showing in vitro synergy using daptomycin plus ceftaroline in the 305

successful treatment of E. faecalis aortic valve endocarditis (24). Optimal dosing of daptomycin 306

or ceftaroline should a clinician choose this combination remains to be determined, although we 307

might suggest 8-10 mg/kg/day of daptomycin given prior studies showing increased efficacy of 308

higher doses compared to < 6 mg/kg/day (25). 309

310


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Acknowledgments 311

312

Ceftaroline powder was obtained from Forest Pharmaceuticals. The research described in the 313

manuscript was conducted at the sole discretion of the authors without the knowledge or support 314

of Forest Laboratories, Inc., Forest Research Institute, Inc., or Cerexa, Inc. 315

316

Conflicts of Interest Statement: G.S. has received speaking honoraria from Cubist, Forest, and 317

Pfizer Pharmaceuticals, consulting fees from Cubist and Forest Pharmaceuticals, and research 318

grant support from Forest Pharmaceuticals; W.R. has received grant funding and speaking 319

honoraria from Cubist and consults for The Medicines Company. R.H. has received research 320

funding from Cubist. V.N. is on the Scientific Advisory Board of Trius Therapeutics. 321

322

Funding: Funding for this research was provided by U54 HD071600-01 09/26/2011-06/30/2016 323

NICHD on Developmental and Translational Pharmacology of Pediatric Antimicrobial Therapy 324

(G.S. and V.N.), the Great Lakes Regional Center for Excellence in Biodefense and Emerging 325

Infectious Disease Research (AI057153, VN) and R01GM073898 (JP). No funding bodies had 326

any role in study design, data collection and analysis, decision to publish, or preparation of the 327

manuscript. 328

329

330


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Table 1. Daptomycin (DAP) and vancomycin (VAN) MICs of strains 8019 (daptomycin-411

susceptible) and 5938 (daptomycin-resistant) in Mueller-Hinton agar plates containing various 412

beta-lactams at selected concentrations by E-test. 413

414

415 Beta-Lactam (mg/L)

8019 DAP MIC (mg/L)

8019 VAN MIC (mg/L)

5938 DAP MIC* (mg/L)

5938 VAN MIC (mg/L)

NONE 0.38 >256 10 >256 CPT 1 0.38 >256 6 >256 CPT 2 0.38 >256 2 >256 CPT 4 0.25 >256 2 >256 CPT 8 0.19 >256 0.75 24 AMP 25 0.25 >256 6 >256 AMP 50 0.25 >256 6 >256 PIP 20 0.38 >256 6 >256 CRO 20 0.38 >256 6 >256 CZL 20 0.38 >256 6 >256 416 *VRE strain 5938 has been previously reported as initially having DAP MIC 192 mg/L by Etest. 417

However, upon retrieval of this isolate from the freezer, the DAP MIC by Etest was decreased to 418

10 mg/L, consistent with the instability of DAP nonsusceptibility previously reported. Mutation 419

differences between isogenic strains 8019 and 5938 have been previously characterized by whole 420

genome sequencing (6). MICs (mg/L) of 8019 and 5938 to various antibiotics are as follows: 421

CPT >32, AMP >64, PIP >64, CRO, >64, CZL >64, LIN 2. 422

423

Abbreviations: CPT ceftaroline, AMP ampicillin, PIP piperacillin, CRO ceftriaxone, CZL 424

cefazolin, LIN linezolid. 425

426


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Table 2: Summary of changes induced on daptomycin (DAP) susceptible and nonsusceptible 427 VRE by ampicillin (AMP) or ceftaroline (CPT). 428 429 DAP S VRE DAP NS VRE 430 431 Synergy With DAP 432

AMP + - 433

CPT + + 434 435 Effects on Cell Wall Thickness 436 AMP 437 CPT 438 439 Effects on Membrane Fluidity 440

AMP - - 441

CPT - 442 443 Poly-L-Lysine Binding 444

AMP - 445 CPT 446 447 Bodipy-DAP Binding 448

AMP - 449 CPT 450

451 LL37 Binding and Activity 452

AMP 453 CPT 454 455 456


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