Analysis of Solithromycin Bactericidal Activity Against Vancomycin-Susceptible and
Vancomycin-Resistant Enterococci
P 1593
Contact info:
Jennifer Deane, B.S.
Tel: 703-480-2608
Email: jenniferdeane@eurofins.com
J Deane1, C Opiela1, D Shah1, D Sahm1, K Keedy2, A Sheets2, P Fernandes2
1Eurofins, Chantilly, VA, USA 2Cempra Inc., Chapel Hill, NC
Results
Abstract (Amended)
Objectives: Solithromycin (SOL) is a novel fluoroketolide in late clinical development that has
an in vitro activity spectrum different, and frequently more potent, than currently available
macrolides. Against both Enterococcus faecalis (EFA) and Enterococcus faecium (EFM),
including vancomycin-resistant strains, SOL has an MIC90 of 2 mg/L. To further investigate the in
vitro potency of SOL against enterococci, SOL's bactericidal activity was analyzed by time-kill
kinetic analysis relative to that of linezolid (LZD).
MIC (mg/L)
Organism SOL
Methods: Two strains each of vancomycin-susceptible
vancomycin susceptible (VS) and vancomycin-resistant
vancomycin resistant (VR)
EFA and EFM were analyzed. Broth microdilution testing according to CLSI M7 guidelines was
used to obtain the drugs' MICs for each strain. Each strain was challenged with each drug at
2X, 4X, and 8X the MIC with CFU/mL determined at 0, 2, 4 and 24 hours. Bactericidal activity
was defined as a 3 log10 decrease in CFU/mL after 24 hours incubation relative to the
concentration of the starting inoculum.
Results: The following table provides the log10 CFU/mL reductions of initial inocula after 24
hours1 at 2X, 4X, and 8X the SOL and LZD MICs.
0.06
2
EFM VR
0.06
2
0.81
EFM VS
0.06
2
2.92
1 Negative
2
EFM VR
0.06
2
0.81
EFM VS
0.06
2
2.92
0.68
1.13
1.3
1.06 1.25
0 05 0.02
0.05
0 02
0 14 0.18
0.14
0 18
0.23
1.83 0.58
1.83 1.13
1.71
2.76 1.21
2.92 1.71
-0.45
0 45 -0.76
0 76
10 10
10 10
A. 2X MIC
B. 4X MIC
10 10
4X MIC
8X MIC
10 9
10 9
10 9
SOL LZD
10 8
10 8
10 8
10 7
10
7
10 7
10
6
-0.45 -0.76
1.13
3
1.3
3
1.06
06 1.25
5
0.05 0.02
0.14 0.18
0.23
1.83 0.58
1.83 1.13
1.71
2.76 1.21
2.92 1.71
10 6
10 5
numbers indicate growth greater than the initial inoculum
10 5
10 4
SOL (0.5 mg/L)
10 3
LZD (2 mg/L)
Introduction
2
4
6
8
The SOL and LZD MICs were established for each strain following CLSI M7 and M100
guidelines and the appropriate Eurofins SOPs. These MIC results were used for selection of
the concentration of each drug that was used in the time-kill assays.
The time-kill assay was done in accordance with Eurofins SOPs and the CLSI guidance
document M26-A.
o 5 x 105 - 5 x 106 CFU/mL log phased bacteria were inoculated in the presence of the
antibiotic at 2X, 4X, and 8X the organisms' MIC as determined by broth microdilution
testing.
o A growth control, containing no antibiotic, was included for each strain tested.
6
8
10 2
10 12 14 16 18 20 22 24
10 10
B. 4X MIC
10 8
10 7
10 7
CFU/mL
10 9
10 8
10 6
10 5
10 6
10 5
10 4
10 2
0
2
4
6
SOL (0.25 mg/L)
LZD (8 mg/L)
Growth Control
10 3
10 2
8 10 12 14 16 18 20 22 24
0
2
4
6
8
Time (hours)
2
4
6
8
10 12 14 16 18 20 22 24
Time (hours)
Few, if any, single drugs are bactericidal against EFA or EFM
strains. Although the MICs for the enterococcal strains showed
SOL was more potent than LZD,
LZD neither SOL nor LZD
demonstrated bactericidal activity at any of the MIC multiples
tested barring one notable exception (EFM-VS).
10 7
10 6
10 5
SOL (0.5 mg/L)
LZD (16 mg/L)
Growth Control
10 3
10 2
0
10 12 14 16 18 20 22 24
Time (hours)
10 10
A. 2X MIC
2
4
6
8
10 12 14 16 18 20 22 24
Time (hours)
10 10
B. 4X MIC
10 9
10 9
10 8
10 8
10 8
10 7
10 7
CFU/mL
10 9
10 6
10 5
SOL (0.12 mg/L)
LZD (4 mg/L)
Growth Control
10 3
10 2
0
2
4
6
8 10 12 14 16 18 20 22 24
10 6
10 5
SOL (0.25 mg/L)
LZD (8 mg/L)
Growth Control
10 3
10 2
0
Time (hours)
C. 8X MIC
2
4
6
8
10 6
A. 2X MIC
SOL (0.5 mg/L)
LZD (16 mg/L)
Growth Control
10 3
10 2
0
10 12 14 16 18 20 22 24
Time (hours)
2
4
6
8 10 12 14 16 18 20 22 24
Time (hours)
10 9
10 9
10 8
10 8
10 8
10 7
10 7
10 7
10 5
o At 0, 2, 4, and 24 hours post-inoculation, viable bacteria were quantified by serial
dilution plating.
10 4
o Bactericidal activity was defined as a ≥ 3-log10 kill relative to the initial inoculum size.
10 2
SOL (0.12 mg/L)
LZD (4 mg/L)
Growth Control
10 3
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (hours)
10 6
10 5
10 4
SOL (0.25 mg/L)
LZD (8 mg/L)
Growth Control
10 3
10
2
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (hours)
C. 8X MIC
10 10
10 9
10 6
This may be a strain-specific phenomenon,
investigation into this finding is warranted.
but further
CLSI 2012. CLSI document M7-A9.
CLSI 2012. CLSI document M100-S22.
NCCLS (CLSI) 1999. CLSI document M26-A
B. 4X MIC
10 10
Given that enterococci are refractory to the bactericidal activity of
most single agents, and that macrolides are not typically
considered bactericidal, this finding was unexpected.
References
10 5
Figures 4A-C. Time-Kill Kinetics of SOL and LZD Against EFM-VS
10 10
For the EFM-VS strain, each multiple of the SOL MIC closely
approached the strict definition of cidality.
10 7
10 4
10 4
Summary and Conclusions
C. 8X MIC
Figures 3A-C. Time-Kill Kinetics of SOL and LZD Against EFM-VR
10 10
• Interestingly, for the EFM-VS strain, SOL closely approached or
met the strict definition of cidality with each multiple of the MIC
tested. This level of cidality was not observed with LZD (Figure
4A - C).
Growth Control
0
10 4
10 4
SOL ( 0.12 mg/L)
LZD (4 mg/L)
Growth Control
• For the EFM-VR strain, SOL demonstrated some level of cidal
activity at 4X and 8X the MIC, but did not achieve the required
99.9% drop in CFU. LZD did not exhibit cidal activity against this
strain at any of the three concentrations tested (Figure 3A - C).
LZD (8 mg/L)
10 10
10 9
CFU/mL
•
4
10 8
CFU/mL
A VR and VS strain of EFA and of EFM were analyzed.
2
10 9
10 4
•
0
SOL (2 mg/L)
Time (hours)
A. 2X MIC
10 10
10 6
10 3
Growth Control
2
Figures 2A-C. Time-Kill Kinetics of SOL and LZD Against EFA-VS
CFU/mL
Methods
10
C. 8X MIC
10 4
LZD (4 mg/L)
10 3
10 12 14 16 18 20 22 24
Time (hours)
10 3
SOL is a novel fluoroketolide in late clinical development that has an in vitro activity spectrum
different, and frequently more potent, than currently available macrolides. SOL has been
reported to have activity against enterococci, with MIC values ≤2 mg/L against both EFA and
EFM, including VR strains (Putnam 2011). To further investigate the in vitro potency of SOL
against enterococci, SOL's bactericidal activity was analyzed by time-kill kinetic analysis
relative to that of LZD.
LZD
• Similarly, against the EFA-VS strain, neither SOL nor LZD
achieved a cidal level of activity after 24 hours, regardless of the
multiple of the MIC tested (Figure 2A - C).
10 5
SOL (1 mg/L)
10 4
Growth Control
10 2
CFU/mL
Conclusions: Although SOL MICs for each enterococcal strain tested were relatively low (0.06
- 0.25 mg/L), bactericidal activity was not achieved against any of the strains. As a comparator,
LZD also failed to demonstrate bactericidal activity against any of the four strains tested.
However, against the EFM VS strain, SOL activity that approached cidality was noted. This
finding may warrant further analysis of a greater variety of enterococcal strains and indicates
that SOL's spectrum can be quite different from those of older macrolides and ketolides.
• Against the EFA-VR strain, neither SOL nor LZD achieved a cidal
level of activity after 24 hours
hours, regardless of the multiple of the
MIC tested (Figure 1A - C).
numbers indicate growth greater than the initial inoculum
SOL LZD
0.68
0
68
• SOL was 4 to 32-fold more potent than LZD against the EFA and
EFM strains evaluated.
Figures 1A-C. Time-Kill Kinetics of SOL and LZD Against EFA-VR
0
•
0.81
SOL LZD
CFU/mL
L
EFA VS
1
0 06
0.06
SOL LZD
CFU/mL
0.81
0
8
0.25
EFA VS
8X MIC
CFU/mL
1
LZD
LZD
Results Summary:
4X MIC
CFU/mL
0.25
0
5
2X MIC
SOL
SOL
CFU/mL
L
EFA VR
LZD
LZD
2X MIC
EFA VR
1 Negative
CFU/mL
L
MIC (mg/L)
Organism SOL
Results
Table 1. Log10 CFU/mL Reduction After 24 Hours1
Putnam, S
Putnam
S. D
D., H
H. S
S. Sader,
Sader D.
D J.
J Farrell
Farrell, D
D. JJ. Biedenbach,
Biedenbach and M
M.
Castanheira. 2011. Antimicrobial characterisation of solithromycin
(CEM-101), a novel fluoroketolide: activity against staphylococci
and enterococci. Int. J. Antimicrob. Agents 37:39-45.
10 6
10 5
10 4
SOL (0.5 mg/L)
LZD (16 mg/L)
Growth Control
10 3
10 2
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (hours)
rd ECCMID Berlin, Germany – 27 -30 April 2013
23rd23ECCMID
Berlin, Germany – 27 -30 April 2013