AAC Accepted Manuscript Posted Online 9 March 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00053-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Daptomycin combinations with beta-lactams are synergistic against vancomycin-
2
resistant Enterococcus faecalis and Enterococcus faecium in in vitro
3
pharmacokinetic/pharmacodynamic models
4 5
Drafted: 1/5/2015
6
Revised: 2/23/2015
7 8
Jordan R. Smith1
9
Katie E. Barber1
10
Animesh Raut1
11
Michael J. Rybak1, 2#
12
1
13
Applebaum College of Pharmacy and Health Sciences, 2School of Medicine Detroit, MI
14
48201
Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene
15 16
#Address correspondence to Michael J. Rybak
17
Telephone: (313)-577-4376
18
Fax: (313)-577-8915
19
Email: [email protected]
20 21
Keywords: Bacteria, combination therapy, infection, in vitro, models
22 23
Funding: This project was supported by internal funding
24 25 26 27 28 29 30 31 32 33 34
1
35
Abstract
36
Background: Enterococcus faecalis (Efc) and Enterococcus faecium (Efm) are
37
frequently resistant to vancomycin and beta-lactams (BL). In enterococcal infections with
38
reduced glycopeptide susceptibility, combination therapy is often administered. Our
39
objective was to conduct pharmacokinetic/pharmacodynamic (PK/PD) models to
40
evaluate BL synergy with daptomycin (DAP) against resistant enterococci. Methods:
41
One Efc (R6981) and two Efm strains (R6370 and 8019) were evaluated. DAP MICs
42
were obtained. All strains were evaluated for response to LL37 in the presence and
43
absence of ceftaroline (CPT), ertapenem (ERT), and ampicillin (AMP). Ninety-six hour,
44
in vitro models were run simulating DAP 10 mg/kg/day, CPT 600 mg q8h, AMP 2 g q4h,
45
and ERT 1 g q24h both alone and in combination against all strains. Results: DAP MICs
46
were 2, 4, and 4 µg/ml for strains R6981, R6370, and 8019, respectively. PK/PD models
47
demonstrated bactericidal activity with DAP and CPT, AMP, or ERT against strain 8019
48
(p2 compared to any single agent). Against strains
49
R6981 and R6370, DAP and AMP demonstrated enhancement against R6370 but not
50
R6981, while the combinations of DAP and CPT or ERT were bactericidal, demonstrated
51
enhancement, and were statistically superior to all other regimens at 96 hours (p 3 log10 CFU/ml decrease in colony count from the initial
160
inoculum. Bacteriostatic activity was defined as a < 3-log10 CFU/ml reduction in colony
161
count form the initial inoculum. Enhancement of combinations was defined as > 2 log10
162
CFU/ml reduction over the most active single agent.
163
Pharmacokinetic analysis
164
CPT, ERT, and AMP concentrations were determined by bioassay using Kocuria
165
rhizophila (formerly Micrococcus luteus) ATCC 9341. Each standard was tested in
166
duplicate by placing the disk on agar plates (antibiotic medium number 11) and
167
inoculated with a 0.5 McFarland suspension of the test organism. DAP concentrations
168
were determined using standard high-performance liquid chromatography (HPLC).(36)
169
The half-lives, areas under the curve (AUC), and peak concentrations of the antibiotics
5
170
were determined by the trapezoidal method using PK analyst software (version 1.10,
171
MicroMath Scientific Software, Salt Lake City, UT).
172
Resistance
173
Emergence of resistance was evaluated at 96 hours by plating 100 µl samples from the
174
model on BHIA plates supplemented with DAP at a concentration 3 times the MIC of the
175
tested antibiotic (e.g. a concentration of 1.5 µg/ml for an organism with an MIC of 0.5
176
µg/ml). Resistant colonies growing on screening plates were evaluated by broth
177
microdilution method to determine the MIC.
178
Cathelicidin LL37 microbicidal assay
179
Strains R6981, R6370, and 8019 were grown to stationary phase (16 to 20 hours) in
180
lysogeny broth (LB) in either the presence or absence of 17 µg/ml CPT, 15.5 µg/ml ERT,
181
or 70 µg/ml AMP, pelleted, washed with PBS, and exposed at an inoculum of 105
182
CFU/ml to 32 µM, 32 µM, and 16 µM LL37, respectively, in RPMI-5% LB. The
183
percentage of surviving bacteria (+ SD) after 2 hours of incubation at 35° C was
184
calculated by plating on BHIA plates.
185
Statistical analysis
186
Changes in CFU/ml at 96 hours were compared by one-way analysis of variance
187
(ANOVA) for time kills and in vitro models, respectively. A p-value of 128
638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
18
655
Table 2. MIC Values of DAP in the Presence of Beta-lactams CPT (17 µg/ml), ERT
656
(15.5 µg/ml), and AMP (70 µg/ml) MIC in µg/ml Strain
DAP
DAP + CPT
DAP + ERT
DAP + AMP
R6981
2
0.25
0.5
0.5
R6370
4
0.25
1
1
8019
4
0.13
1
0.5
657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
19
675
Table 3. In vitro activities of regimens against R6981, R6370 and 8019 at 96 hours Log10 CFU/ml at 96 hours (+/- SD) R6981
R6370
8019
DAP
6.43 + 0.26
6.65 + 0.21
7.75 + 0.52
CPT
8.58 + 0.05
8.02 + 0.05
7.38 + 0.28
ERT
9.42 + 0.47
8.15 + 0.07
8.18 + 0.14
AMP
7.53 + 0.11
7.60 + 0.14
7.63 + 0.15
DAP + CPT
2.00 + 0.00a, b
2.25 + 0.35a, b
2.00 + 0.00a, b
DAP + ERT
2.34 + 0.26a, b
2.00 + 0.00a, b
2.14 + 0.08a, b
DAP + AMP
4.75 + 0.21a
4.63 + 0.01a, b
2.09 + 0.10a, b
Growth Control
8.94 + 0.13
8.50 + 0.14
8.42 + 0.08
Regimen
676 677 678
a b
Significantly different from any single agent regimen Enhancement from any single agent regimen
679 680 681 682 683 684 685 686 687 688 689
20
690
Table 4. DAP fAUC0-24:MIC ratios observed in PK/PD regimens based on reduced
691
DAP MIC values in the presence of beta-lactams DAP fAUC0-24:MIC R6981
R6370
8019
DAP
60.8
30.4
30.4
DAP + CPT
486
486
973
DAP + ERT
243
121.5
121.5
DAP + AMP
243
121.5
243
Regimen
692 693 694
fAUC0-24:MIC corresponds to initial isolate DAP MIC for DAP, DAP MIC in the presence of CPT for DAP + CPT, DAP MIC in the presence of ERT for DAP + ERT, and DAP MIC in the presence of AMP for DAP + AMP
21
695 22
696
Figure 1. 96-hour, one-compartment PK/PD model. Solid circles, growth control;
697
solid upward triangles, DAP 10 mg/kg/day; solid downward triangles, CPT 600 mg
698
q12h; solid squares, ERT 1 g q24h; solid diamonds, AMP 2 g q4h; dashed
699
downward triangles, DAP + CPT; dashed squares, DAP + ERT; dashed diamonds,
700
DAP + AMP. A) E. faecalis R6981; B) E. faecium R6370; C) E. faecium 8019.
23
701 24
702
Figure 2. % Survival of A) E. faecalis R6981 against 32 µM LL37, B) E. faecium
703
R6370 against 16 µM LL37, and C) E. faecium 8019 against 16 µM LL37 compared
704
to untreated growth control at 2 hours. Whiskers indicate standard deviation.
25
1
Daptomycin combinations with beta-lactams are synergistic against vancomycin-
2
resistant Enterococcus faecalis and Enterococcus faecium in in vitro
3
pharmacokinetic/pharmacodynamic models
4 5
Drafted: 1/5/2015
6
Revised: 2/23/2015
7 8
Jordan R. Smith1
9
Katie E. Barber1
10
Animesh Raut1
11
Michael J. Rybak1, 2#
12
1
13
Applebaum College of Pharmacy and Health Sciences, 2School of Medicine Detroit, MI
14
48201
Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene
15 16
#Address correspondence to Michael J. Rybak
17
Telephone: (313)-577-4376
18
Fax: (313)-577-8915
19
Email: [email protected]
20 21
Keywords: Bacteria, combination therapy, infection, in vitro, models
22 23
Funding: This project was supported by internal funding
24 25 26 27 28 29 30 31 32 33 34
1
35
Abstract
36
Background: Enterococcus faecalis (Efc) and Enterococcus faecium (Efm) are
37
frequently resistant to vancomycin and beta-lactams (BL). In enterococcal infections with
38
reduced glycopeptide susceptibility, combination therapy is often administered. Our
39
objective was to conduct pharmacokinetic/pharmacodynamic (PK/PD) models to
40
evaluate BL synergy with daptomycin (DAP) against resistant enterococci. Methods:
41
One Efc (R6981) and two Efm strains (R6370 and 8019) were evaluated. DAP MICs
42
were obtained. All strains were evaluated for response to LL37 in the presence and
43
absence of ceftaroline (CPT), ertapenem (ERT), and ampicillin (AMP). Ninety-six hour,
44
in vitro models were run simulating DAP 10 mg/kg/day, CPT 600 mg q8h, AMP 2 g q4h,
45
and ERT 1 g q24h both alone and in combination against all strains. Results: DAP MICs
46
were 2, 4, and 4 µg/ml for strains R6981, R6370, and 8019, respectively. PK/PD models
47
demonstrated bactericidal activity with DAP and CPT, AMP, or ERT against strain 8019
48
(p2 compared to any single agent). Against strains
49
R6981 and R6370, DAP and AMP demonstrated enhancement against R6370 but not
50
R6981, while the combinations of DAP and CPT or ERT were bactericidal, demonstrated
51
enhancement, and were statistically superior to all other regimens at 96 hours (p 3 log10 CFU/ml decrease in colony count from the initial
160
inoculum. Bacteriostatic activity was defined as a < 3-log10 CFU/ml reduction in colony
161
count form the initial inoculum. Enhancement of combinations was defined as > 2 log10
162
CFU/ml reduction over the most active single agent.
163
Pharmacokinetic analysis
164
CPT, ERT, and AMP concentrations were determined by bioassay using Kocuria
165
rhizophila (formerly Micrococcus luteus) ATCC 9341. Each standard was tested in
166
duplicate by placing the disk on agar plates (antibiotic medium number 11) and
167
inoculated with a 0.5 McFarland suspension of the test organism. DAP concentrations
168
were determined using standard high-performance liquid chromatography (HPLC).(36)
169
The half-lives, areas under the curve (AUC), and peak concentrations of the antibiotics
5
170
were determined by the trapezoidal method using PK analyst software (version 1.10,
171
MicroMath Scientific Software, Salt Lake City, UT).
172
Resistance
173
Emergence of resistance was evaluated at 96 hours by plating 100 µl samples from the
174
model on BHIA plates supplemented with DAP at a concentration 3 times the MIC of the
175
tested antibiotic (e.g. a concentration of 1.5 µg/ml for an organism with an MIC of 0.5
176
µg/ml). Resistant colonies growing on screening plates were evaluated by broth
177
microdilution method to determine the MIC.
178
Cathelicidin LL37 microbicidal assay
179
Strains R6981, R6370, and 8019 were grown to stationary phase (16 to 20 hours) in
180
lysogeny broth (LB) in either the presence or absence of 17 µg/ml CPT, 15.5 µg/ml ERT,
181
or 70 µg/ml AMP, pelleted, washed with PBS, and exposed at an inoculum of 105
182
CFU/ml to 32 µM, 32 µM, and 16 µM LL37, respectively, in RPMI-5% LB. The
183
percentage of surviving bacteria (+ SD) after 2 hours of incubation at 35° C was
184
calculated by plating on BHIA plates.
185
Statistical analysis
186
Changes in CFU/ml at 96 hours were compared by one-way analysis of variance
187
(ANOVA) for time kills and in vitro models, respectively. A p-value of 128
638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
18
655
Table 2. MIC Values of DAP in the Presence of Beta-lactams CPT (17 µg/ml), ERT
656
(15.5 µg/ml), and AMP (70 µg/ml) MIC in µg/ml Strain
DAP
DAP + CPT
DAP + ERT
DAP + AMP
R6981
2
0.25
0.5
0.5
R6370
4
0.25
1
1
8019
4
0.13
1
0.5
657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
19
675
Table 3. In vitro activities of regimens against R6981, R6370 and 8019 at 96 hours Log10 CFU/ml at 96 hours (+/- SD) R6981
R6370
8019
DAP
6.43 + 0.26
6.65 + 0.21
7.75 + 0.52
CPT
8.58 + 0.05
8.02 + 0.05
7.38 + 0.28
ERT
9.42 + 0.47
8.15 + 0.07
8.18 + 0.14
AMP
7.53 + 0.11
7.60 + 0.14
7.63 + 0.15
DAP + CPT
2.00 + 0.00a, b
2.25 + 0.35a, b
2.00 + 0.00a, b
DAP + ERT
2.34 + 0.26a, b
2.00 + 0.00a, b
2.14 + 0.08a, b
DAP + AMP
4.75 + 0.21a
4.63 + 0.01a, b
2.09 + 0.10a, b
Growth Control
8.94 + 0.13
8.50 + 0.14
8.42 + 0.08
Regimen
676 677 678
a b
Significantly different from any single agent regimen Enhancement from any single agent regimen
679 680 681 682 683 684 685 686 687 688 689
20
690
Table 4. DAP fAUC0-24:MIC ratios observed in PK/PD regimens based on reduced
691
DAP MIC values in the presence of beta-lactams DAP fAUC0-24:MIC R6981
R6370
8019
DAP
60.8
30.4
30.4
DAP + CPT
486
486
973
DAP + ERT
243
121.5
121.5
DAP + AMP
243
121.5
243
Regimen
692 693 694
fAUC0-24:MIC corresponds to initial isolate DAP MIC for DAP, DAP MIC in the presence of CPT for DAP + CPT, DAP MIC in the presence of ERT for DAP + ERT, and DAP MIC in the presence of AMP for DAP + AMP
21
695 22
696
Figure 1. 96-hour, one-compartment PK/PD model. Solid circles, growth control;
697
solid upward triangles, DAP 10 mg/kg/day; solid downward triangles, CPT 600 mg
698
q12h; solid squares, ERT 1 g q24h; solid diamonds, AMP 2 g q4h; dashed
699
downward triangles, DAP + CPT; dashed squares, DAP + ERT; dashed diamonds,
700
DAP + AMP. A) E. faecalis R6981; B) E. faecium R6370; C) E. faecium 8019.
23
701 24
702
Figure 2. % Survival of A) E. faecalis R6981 against 32 µM LL37, B) E. faecium
703
R6370 against 16 µM LL37, and C) E. faecium 8019 against 16 µM LL37 compared
704
to untreated growth control at 2 hours. Whiskers indicate standard deviation.
25
