Ceftaroline Restores Daptomycin Activi ty Against...
Transcript of Ceftaroline Restores Daptomycin Activi ty Against...
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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
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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
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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
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References 331
332
1. Kelesidis T, Humphries, Uslan DZ, Pegues DA. Daptomycin nonsusceptible enterococci: 333
an emerging challenge for clinicians. Clin Infect Dis 2011; 52 (2): 228-234. 334
2. Orloff SL, Busch AM, Olyaei AJ, et al. Vancomycin-resistant Enterococcus in liver 335
transplant patients. Am J Surg 1999;177:418-22. 336
3. Kamboj M, Chung D, Seo SK, et al. The changing epidemiology of vancomycin-resistant 337
Enterococcus (VRE) bacteremia in allogeneic hematopoietic stem cell transplant (HSCT) 338
recipients. Biol Blood Marrow Transplant 2010;16:1576-81. 339
4. Arias CA, Contreras GA, Murray BE. Management of multidrug-resistant enterococcal 340
infections. Clin Microbiol Infect 2010;16:555-62. 341
5. Sakoulas G, Bayer AS, Pogliano J, Tsuji BT, Yang SJ, Mishra NN, Nizet V, Yeaman 342
MR, Moise PA. Ampicillin enhances daptomycin- and cationic host defense peptide-343
mediate killing of ampicillin and vancomycin-resistant Enterococcus faecium. 344
Antimicrob Agents Chemother 2012; 56 (2): 838-844. 345
6. Humphries RM, Kelesidis T, Tewhey R, Rose WE, Schork N, Nizet N, Sakoulas. G. 346
Genotypic and phenotypic evaluation of the evolution of high-level daptomycin non-347
susceptibility in vancomycin-resistant Enterococcus faecium. Antimicrob Agents 348
Chemother 2012; 56: 6051-6053. 349
7. Rose WE, Leonard SN, Sakoulas G, Kaatz GW, Zervos MM, Sheth A, Carpenter CF, 350
Rybak MJ. Daptomycin activity against Staphylococcus aureus following vancomycin 351
exposure in an in vitro pharmacodynamic model with simulated endocardial vegetations. 352
Antimicrob Agents Chemother 2008; 52: 831-836 353
on May 30, 2018 by guest
http://aac.asm.org/
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nloaded from
Page 17 of 21
8. CLSI. 2013. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-354
Third Informational Supplement. CLSI document M100-S23. Clinical and Laboratory 355
Standards Institute, Wayne, PA. 356
9. Forest Laboratories, Inc. 2012. Ceftaroline fosamil package insert. http: 357
//www.accessdata.fda.gov/drugsatfda_docs/label/2010/200327s000lbl.pdf. Accessed 10 358
October 2012. 359
10. Dvorchik BH, Brazier D, DeBruin MF, Arbeit RD. 2003. Daptomycin pharmacokinetics 360
and safety following administration of escalating doses once daily to healthy subjects. 361
Antimicrob. Agents Chemother. 47:1318 –1323. 362
11. Foulds G. Pharmacokinetics of sulbactam/ampicillin in humans: a re- view. Rev. Infect. 363
Dis. 8(Suppl 5):S503–S511. 364
12. Patel IH, Chen S, Parsonnet Hackman MR, Brooks MA, Konikoff J, Kaplan SA. 1981. 365
Pharmacokinetics of ceftriaxone in humans. Antimi- crob. Agents Chemother. 20:634 – 366
641. 367
13. Dryden MS. Linezold pharmacokinetics and pharmacodynamics in clinical treatment. 368
2011. J Antimicrob Chemother. 66 (Suppl 4): iv7-iv15. 369
14. Dhand A, Bayer AS, Pogliano J, Yang SJ, Bolaris M, Nizet, Wang G. Sakoulas G. Use of 370
antistaphylococcal beta-lactams to increase daptomycin activity in eradicating persistent 371
bacteremia due to methicillin-resistant Staphylococcus aureus: role of daptomycin 372
binding. Clin Infect Dis 2011; 53: 158-163. 373
15. Kawada-Matsuo M, Yoshida Y, Nakamura N, Konatsuzawa H. Role of two-component 374
systems in the resistance of Staphylococcus aureus to antibacterial agents. Virulence 375
2011, 2: 427-430. 376
on May 30, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
Page 18 of 21
16. Rice LB, Carias LL, Rudin S, Hutton R, Marshall S, Hassan M, Josseaume N, Dubost L, 377
Marie A, Arthur M. Role of class A penicillin-binding proteins in the expression of beta-378
lactam resistance in Enterococcus faecium. J Bacteriol 2009; 191: 3649. 379
17. Zhang X, Paganelli FL, Bierschenk D, Kulper A, Bonten MJM, Willems RJL, van Schalk 380
W. Genome-wide identification of ampicillin resistance determinants in Enterococcus 381
faecum. PLoS Genetics 2012; 8 (6): e1002804). 382
18. Sakoulas G, Okumura CY, Thienphrapa, Olson J, Nonejuie P, Dam Q, Dhand A, 383
Pogliano J, Yeaman MR, Hensler ME, Bayer AS, Nizet V. Nafcillin enhances innate 384
immune-mediated killing of methicillin-resistant Staphylococcus aureus. J Mol Med 385
2013, in press. 386
19. Berti A, Sakoulas G, Nizet V, Tewhey R, Rose WE. β-lactam antibiotics targeting PBP1 387
selectively enhance daptomycin activity against methicillin-resistant Staphylococcus 388
aureus. Antimicrob Agents Chemother 2013, in press. 389
20. Mishra NN, Bayer AS, Tran TT, Shamoo Y, Mileykovskaya E, et al. (2012) Daptomycin 390
resistance in enterococci is associated with distinct alterations of cell membrane 391
phospholipid content. PLoS ONE 7(8): e43958. doi:10.1371/journal.pone.0043958. 392
21. Munita JM, Panesso D, Diaz L, Tran TT, Reyes J, Wanger A, Murray BE, Arias CA. 393
Correlation between mutations in liaFSR of Enterococcus faecium and MIC of 394
daptomycin: revisiting daptomycin breakpoints. 2012. Antimicrob Agents Chemother. 395
56(8):4354-9. 396
22. Arias CA, Panesso D, McGrath DM, Qin X, Mojica MF, Miller C, Diaz L, Tran TT, 397
Rincon S, Barbu EM, Reyes J, Roh JH, Lobos E, Sodergren E, Pasqualini R, Arap W, 398
on May 30, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
Page 19 of 21
Quinn JP, Shamoo Y, Murray BE, Weinstock GM. 2011. Genetic basis for in vivo 399
daptomycin resistance in enterococci. N Engl J Med. 8;365(10):892-900. 400
23. Xavier H, Amoroso A, Coyette J, Joris B. Interaction of ceftobiprole with the low-affinity 401
PBP 5 of Enterococcus faecium. Antimicrob Agents Chemother 2010; 54: 953-955. 402
24. Sakoulas G, Noneluie P, Nizet V, Pogliano J, Crum-Cianflone N, Haddad F. Treatment 403
of high-level gentamicin-resistant Enterococcus faecalis endocarditis with daptomycin 404
plus ceftaroline. Antimicrob Agents Chemother 2013, May 20 {Epub ahead of print]. 405
25. King EA, McCoy D, Desai S, Nyirenda T, Bicking K. Vancomycin-resistant 406
Enterococcal bacteremia and daptomycin: are higher doses necessary? J Antimicrob 407
Chemother 2011; 66: 2112-8. 408
409
410
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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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