Download In Vitro Activity of Lefamulin Against Macrolide

In Vitro Activity of Lefamulin Against Macrolide-Susceptible (MSMP) and Macrolide-Resistant (MRMP)
Mycoplasma pneumoniae from the United States, Europe, and China
Abstract 3972
1Dept.
K. B. Waites1, D. M. Crabb1, L. B. Duffy1, Y. Liu2, and S. Paukner3
of Pathology, University of Alabama at Birmingham, Birmingham, AL, 2Fudan University, Shanghai, China, 3Nabriva Therapeutics AG, Vienna, Austria
ABSTRACT (amended)
MATERIALS & METHODS
Background: Pleuromutilin (PM) antibiotics bind to the peptidyl transferase
center of the 50S ribosomal subunit, blocking protein synthesis. Lefamulin
(LMU, Nabriva Therapeutics) is a novel semi-synthetic PM with potent activity
against gram-positive, gram-negative and atypical bacteria, including multi-drug
resistant strains. LMU is formulated for IV and oral use and is in Phase 3 trials
for the treatment of community-acquired bacterial pneumonia. Although
macrolide-resistance in Mycoplasma pneumoniae (MPN) has become
increasingly prevalent globally, in previous in vitro surveillance studies crossresistance between LMU and commonly used respiratory antibiotics has not
been observed. We performed an in vitro evaluation of pediatric respiratory
isolates of MSMP and MRMP from the US, Europe, and China.
Methods: The broth microdilution MIC assay per CLSI guidelines (2011) was
used to test strains against LMU and 4 comparators (see Table).
Results: LMU was highly active against all strains, with MICs < 0.004 µg/ml.
The LMU MIC90 for MRMP (0.002 µg/ml) was the lowest among all 5 drugs
tested. Additionally, LMU minimum bactericidal concentrations (MBCs) were
determined for 2 macrolide-susceptible and 6 macrolide-resistant isolates and
all MBCs were 2 to 4 times the MIC values, indicating a bactericidal effect.
Conclusions: LMU has potent in vitro activity against MPN, including macrolide
resistant strains. LMU may be a promising therapeutic option for MPN
infections.
Lefamulin
(LMU)
Azithromycin
(AZI)
Erythromycin
(ERY)
Tetracycline
(TET)
Moxifloxacin
(MOX)
<0.00025-0.001
<0.00025-0.001
0.004-0.008
0.25-1
0.063-0.25
MIC50
<0.00025
0.0005
0.008
0.5
0.125
MIC90
0.001
0.001
0.008
1
0.25
0.0005-0.004
16 - >32
>32
0.25-1
0.125-0.25
MIC50
0.002
32
>32
0.5
0.125
MIC90
0.002
>32
>32
1
0.25
MSMP (n = 14)
Range [µg/ml]
MRMP (n = 36)
Range [µg/ml]
BACKGROUND & OBJECTIVES
•
•
•
•
•
Mycoplasma pneumoniae is an important cause of community-acquired
respiratory infections in persons of all ages. Historically, macrolides have
been the treatments of choice for these infections, especially in children
for whom other drug classes such as tetracyclines and fluoroquinolones
are less desirable because of potential adverse effects.
Acquired macrolide resistance occurs in M. pneumoniae as a result of
target modification caused by mutations in 23S rRNA. High level, clinically
significant resistance began in Asia in 2000 and has spread globally.
Recent surveillance from several states in the USA has indicated 13.2%
of clinical isolates are macrolide-resistant.
Pleuromutilins inhibit bacterial growth by binding to a highly conserved
region of the peptidyl transferase center of the 50S ribosomal subunit via
multiple non-covalent bonds and central positioning of their tricyclic core,
as well as steric interference with nucleotides created by side chain that
hinder tRNA rotary motion.
Lefamulin (LMU) is a semi-synthetic pleuromutilin with potent activity
against gram-positive, gram-negative and atypical bacteria, including
multi-drug resistant strains, in Phase 3 clinical trials for treatment of
community-acquired bacterial pneumonia.
The objective of this study was to assess the in vitro activity of lefamulin
against 14 MSMP and 36 MRMP pediatric clinical isolates and reference
strains obtained from several countries to determine the potential utility of
lefamulin in the treatment MSPP and MRMP infections.
Bacterial Strains & Inoculum Preparation
50 unique strains of M. pneumoniae tested included 14 macrolide-susceptible clinical
isolates obtained between 1980 and 2013 from several states in the USA, Europe,
and China, and one reference strain (MAC). An additional 36 clinical isolates were
obtained between 2009 and 2013 from several states in the USA and from Shanghai,
China that had been shown previously to be macrolide-resistant (MIC > 32 µg/ml).
Both P1 subtypes 1 and 2 were represented. Organism sources included nasal
swabs, throat swabs, sputum, bronchoalveolar lavage fluids, and 1 cerebrospinal fluid.
Most specimens were obtained from children. Organism stock cultures were previously
tested to assess purity and to determine numbers present and then stored frozen at 80oC until tested. Organisms were thawed to room temperature and diluted in SP4
broth in 50 ml conical tubes to yield a final inoculum of approximately 104 CFU/ml.
Broths were incubated aerobically at 37oC for 2 hours prior to use to allow
mycoplasmas to become metabolically active prior to inoculating microtiter plates.
Antimicrobial Agents
Antimicrobial agents were obtained in powder form of known purity from their
respective manufacturers or from commercial sources. Drugs were dissolved
according to each manufacturer’s instructions and in accordance with CLSI guidelines.
An appropriate amount of each powdered drug was weighed to prepare 10 mls of a
stock solution, allowing for the percentage purity of each agent. If not used
immediately, stock solutions were frozen at -80oC.
Broth Microdilution Assay (CLSI Method)
Each drug was tested in duplicate along with solvent, media, drug, and growth controls
in accordance with CLSI guidelines. Microdilution trays were incubated aerobically at
37oC and examined after 18-24 hours and then daily for color change in the growth
control wells. Endpoint MICs were usually measurable after 4-5 days of incubation.
MIC Determination
MICs were recorded as the lowest concentration of antimicrobial inhibiting color
change in SP4 broth at the time the organism growth control well demonstrated a color
change from pink to yellow indicative of glucose metabolism.
Table 2. Mycoplasmacidal concentration of lefamulin
MBC Determination
The mycoplasmacidal concentration for lefamulin was determined for 2 macrolidesusceptible and 6 macrolide-resistant isolates by subculturing 0.30 ml of fluid from all
wells of the MIC microtiter plate that did not show color change and from the growth
control well at time of the initial color change into 2.97mls of SP4 broth to dilute the
antibiotic beyond the MIC (1:100 dilution). A subculture onto SP4 agar was prepared
from the lowest concentration showing color change in the MIC microtiter plate to
verify organism viability. Subcultures were incubated aerobically at 37oC and agar
plates were incubated at 37o C in air plus 5% CO2 until the growth control showed
color change and the date was noted.
The MBC was recorded as the last tube without color change after twice the length of
incubation time for the MIC well to show initial color change. MBC controls included
tetracycline (non-cidal) and levofloxacin (cidal) set up and monitored in the same
manner as the investigational agent. Drugs were considered bactericidal if the MBC is
< 4-fold (2 dilutions) of the MIC.
Quality Control (QC)
American Type Culture Collection (ATCC) strain 29342 (M129-B7) was included with
each assay on every day of performance. MIC reference ranges for several
antimicrobial agents have been established for this ATCC QC type strain. This QC
strain performed as expected for all MIC assays for which data are presented. Solvent,
drug, media, and growth controls also performed as expected. A final determination of
the CFU/mL of the working dilution used to inoculate each microtiter plate was made
by preparing 6 serial dilutions of the inoculum (0.1 ml inoculum in 0.9 ml of SP4 broth)
and pipetting 20 µL of each dilution onto SP4 agar to verify that a proper dilution was
made and that the inoculum contained 104-105 CFU/mL. Agar plates were incubated
at 37o C in air plus 5% CO2 until colonies were visible and could be counted using a
stereomicroscope. Results were considered valid if the control agar plate for organism
concentration indicated that there were between 104 and 105 CFU/mL.
• Lefamulin had potent activity against all M. pneumoniae isolates
(n=50) with all MIC values ≤0.004 µg/mL. MIC50/90 of MS and MR
subsets were ≤0.00025/0.001 µg/mL and 0.002/0.002 µg/mL
(Table 1).
• This activity was comparable to the activity of azithromycin when
tested against the MS subset of organisms (MIC50/90,
0.0005-0.001 µg/mL).
isolates
were
susceptible
to
moxifloxacin
(MIC50/90,
• All
0.125/0.25 µg/mL) and tetracycline (MIC50/90, 0.5/1 µg/mL) when CLSI
breakpoints were applied.
• Lefamulin MBC values of 0.002-0.008 µg/mL were 2- to 4-fold higher
than the corresponding MICs (Table 2). Thus, lefamulin was
bactericidal against all 8 M. pneumoniae isolates, including macrolideresistant and macrolide-susceptible isolates.
Table 1. In vitro activity of lefamulin and comparators
M. pneumoniae, total
(n = 50)
MSMP
(n = 14)
MRMP
(n = 36)
Strain
Susceptibility
MIC
[µg/mL]
MBC
[µg/ml]
1
Macrolide-susceptible
0.0005
0.002
2
Macrolide-susceptible
0.001
0.002
3
Macrolide-resistant
0.001
0.004
4
Macrolide-resistant
0.002
0.008
5
Macrolide-resistant
0.002
0.008
6
Macrolide-resistant
0.001
0.004
7
Macrolide-resistant
0.002
0.004
8
Macrolide-resistant
0.002
0.008
DISCUSSION & CONCLUSIONS
•
•
•
RESULTS
•
Emergence of clinically significant macrolide resistance and its
spread from Asia to Europe and North America has complicated
empiric treatment for mycoplasmal infections. As a result, agents
with a novel mechanism of action that are unaffected by resistance
to commonly utilized respiratory antibacterial agents are needed.
Lefamulin demonstrated excellent in vitro activity against
M. pneumoniae, regardless of resistance phenotype, inhibiting all 50
strains at concentrations of < 0.004 µg/mL.
Lefamulin demonstrated bactericidal activity against all 8 strains
tested regardless of resistance phenotype.
In conclusion, lefamulin is a promising agent for infections caused
by M. pneumoniae in the respiratory tract.
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MIC
[µg/mL]
Lefamulin
Azithromycin
Erythromycin
Tetracycline
Moxifloxacin
Range
≤0.00025 - 0.004
≤0.00025 - >32
0.004 - >32
0.25 - 1
0.063 - 0.25
MIC50
0.001
>32
>32
0.5
0.125
MIC90
0.002
>32
>32
1
0.25
Range
<0.00025-0.001
<0.00025-0.001
0.004-0.008
0.25-1
0.063-0.25
MIC50
<0.00025
0.0005
0.008
0.5
0.125
MIC90
0.001
0.001
0.008
1
0.25
Range
0.0005-0.004
16 - >32
>32
0.25-1
0.125-0.25
Zheng, X., Lee, S., Selvarangen, R., Qin, X., Tang, Y-W., Stiles, J., Hong, T., Todd, K.,
Ratliff, A., Crabb, D.M., Xiao, L., Atkinson, T.P., Waites,K.B. Multicenter study of macrolideresistant Mycoplasma pneumoniae in the United States. Emerging Infectious Diseases
21:1470-1472, 2015.
MIC50
0.002
32
>32
0.5
0.125
ACKNOWLEDGEMENTS
MIC90
0.002
>32
>32
1
0.25
RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
Contact:
Ken Waites
Univ. AL Birmingham
Tel: (205) 934-4960
Email: [email protected]
ASM Microbe / ICAAC 2016, June 16-20, Boston, MA
Sader, H.S., Paukner, S., Ivezic-Schoenfeld,et al. Antimicrobial activity of the novel
pleuromutilin antibiotic BC-3781 against organisms responsible for community-acquired
respiratory tract infections (CARTIs) J Antimicrob Chemother 67:1170-1175, 2012.
Waites, K.B., Duffy, L.B., Bébéar, C.M., et al. Methods for antimicrobial susceptibility testing
for human mycoplasmas; approved guideline. Clinical and Laboratory Standards Institute,
Wayne, PA. 2011.
Waites, K.B., Lysynyansky, Bébéar, C.M. Antibiotic susceptibility testing and resistance in
mycoplasmas of humans and animals. In: Browning, G., Citti, C., eds. Mollicutes Molecular
Biology and Pathogenesis, Caister Academic Press. pp. 289-322, 2014.
This work was supported by Nabriva Pharmaceuticals AG, Vienna, Austria