1. No category

AZD0914: Establishment of Preliminary Quality Control Ranges against Bacterial Reference Strains
Sara A. Patey, Linda G. Otterson, Michael D. Huband.
Infection Discovery, AstraZeneca R&D Boston, Waltham, MA USA
P1667
Abstract # ECCMI-0662
Abstract
Results
Objective: The increasing prevalence of multi-drug resistant (MDR) infections including those caused by
Neisseria gonorrhoeae, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae and
vancomycin-R enterococci (VRE) has led to fewer treatment options. AZD0914 is a new bacterial DNA
Gyrase/Topoisomerase IV Inhibitor with activity against fluoroquinolone-resistant and MDR Gram-positive and
fastidious Gram-negative bacterial species including N. gonorrhoeae, MRSA, S. pneumoniae, Enterococcus
faecalis, and Haemophilus influenzae. In this study, we established preliminary Quality Control (QC) ranges for
AZD0914 against Clinical and Laboratory Standards Institute (CLSI) QC reference bacterial strains prior to
initiating an official 8 lab CLSI M23 study.
Methods: MICs of AZD0914 were obtained by using broth microdilution or agar dilution methodology over
multiple test days and employing different lots/manufacturers of cation-adjusted Mueller-Hinton broth (CAMHB,
broth microdilution), Haemophilus Test medium (HTM, broth microdilution) or GC broth + 1% Isovitalex (agar
dilution). MIC values were determined following CLSI guidelines (M07-A9, 2012) with slight modification for broth
microdilution testing. Serially diluted stock compound plates (2-fold dilutions) were spotted (2 µl aliquots) into
96-well daughter plates followed by inoculation with 100 µl of a 5 x 105 CFU/mL inoculum using an automated
liquid handling system. An inoculum of 1 x 105 was utilized from agar dilution susceptibility testing. For both broth
microdilution and agar dilution MIC testing, a new inoculum was used for each test replicate.
Results: In broth microdilution (n=10) and agar dilution (n=9) MIC testing, AZD0914 MICs against CLSI quality
control reference strains were:
Organism
Method
Staphylococcus aureus (ATCC 29213)
MIC (# of replicates at each MIC)
broth microdilution
0.12(2), 0.25(8)
Results (continued)
A summary of MIC testing for AZD0914 and levofloxacin is listed in Table 1.
Table 1: MICs of AZD0914 and Levofloxacin over multiple days of testing (mg/L)
Organism
Method
MIC AZD0914
MIC Levofloxacin
S. aureus (ATCC 29213)
broth microdilution
0.12(2)a, 0.25(8)
0.12 (2), 0.25 (8)
S. pneumoniae (ATCC 49619)
broth microdilution
0.12(3), 0.25(7)
0.5 (4), 1 (6)
E. faecalis (ATCC 29212)
broth microdilution
1(10)
1 (9), 2 (1)
0.12-0.25
broth microdilution
0.12(3), 0.25(7)
0.12-0.25
E. faecalis (ATCC 29212)
broth microdilution
1(10)
1
H. influenzae (ATCC 49247)
broth microdilution
0.25(5), 0.5(5)
0.25-0.5
E. coli (ATCC 25922)
broth microdilution
2(10)
2
E. coli (ATCC 35218)
broth microdilution
2(9), 4(1)
2-4
agar dilution
0.06(3), 0.12(6)
0.06-0.12
Comparator compound MICs for both broth and agar dilution were within published CLSI QC ranges (M100-S23)
for each of the strains tested.
Conclusions: Preliminary CLSI QC ranges for AZD0914 against reference bacterial strains were established
over multiple days of testing employing different lots/manufacturers of media. No more than a 2-fold variability in
AZD0914 broth microdilution and agar dilution MICs was observed.
H. influenzae (ATCC 49247)
broth microdilution
E. coli (ATCC 25922)
A medical need exists for new antibacterial compounds with activity against MDR bacterial
pathogens including MRSA, S. pneumoniae, and Neisseria gonorrhoeae. AZD0914 is a new
spiropyrimidinetrione bacterial DNA Gyrase inhibitor with potent in vitro antibacterial activity
against Gram-positive and fastidious Gram-negative bacteria including fluoroquinolone-resistant
and MDR S. aureus, S. pneumoniae, and N. gonorrhoeae. Preliminary MIC QC ranges for
AZD0914 were determined against CLSI reference strains in replicate testing by broth
microdilution and agar dilution methodologies.
0.25(5), 0.5(5)
broth microdilution
E. coli (ATCC 35218)
0.016 (5), 0.03 (5)
2(10)
broth microdilution
N. gonorrhoeae (ATCC 49226)
Compounds: The structure of AZD0914 is shown in Figure 1.
Bacterial strains: The following CLSI QC reference strains were tested: S. aureus ATCC 29213,
S. pneumoniae ATCC 49619, E. faecalis ATCC 29212, H. influenzae ATCC 49247, E. coli ATCC
25922 and ATCC 35218, and N. gonorrhoeae ATCC 49226.
Study design: MIC determinations for both broth microdilution and agar dilution methodologies
followed CLSI guidelines (CLSI, M07-A09, 2012). Broth microdilution was completed using a
Perkin-Elmer MiniTrak™ MultiPosition liquid handling robot. 2 µl aliquots of serially diluted
compound was spotted into 96-well plates followed by addition of 100 µl of a 5 x 105 CFU/ml
inoculum. Levofloxacin was used as a class comparator to validate the test method.
Experimental procedures: Broth microdilution and agar dilution MIC testing of AZD0914, and
comparators was conducted according to CLSI guidelines (M07-A9, and M100-S23).
O
N
O
N
N
O
O
N
F
H
2(9), 4(1)
0.016 (2), 0.03 (8)
agar dilution
0
•Ciprofloxacin MICs were within CLSI QC ranges for Neisseria gonorrhoeae.
Results (continued)
8
6
6
4
4
2
2
0
0
0.12
0.25
0.5
1
2
4
10
8
8
6
6
4
4
2
2
0
0
0.06
0.12
0.25
0.5
1
2
4
S. aureus (ATCC 29213)
0.25
0.12 (1), 0.25 (1)
S. pneumoniae (ATCC 49619)
0.25
0.12 (1), 0.25 (1)
1
0.5 (1), 1 (1)
0.5
0.25 (1), 0.5 (1)
E. coli (ATCC 25922)
2
2 (2)
E. coli (ATCC 35218)
4
2 (1), 4 (1)
Organism
Conclusions
0.12
0.25
0.5
1
2
4
0.06
0.12
0.25
0.5
1
2
• Preliminary CLSI QC ranges for AZD0914 were established against bacterial
reference strains.
• MIC values were similar over multiple days of testing with no more than a 2-fold
variation in MIC observed.
• AZD0914 broth microdilution and agar dilution methodologies were in agreement
with no more than an occasional 2-fold variation in MIC.
• No more than a 2-fold variation in AZD0914 MICs was observed when various lots of
media were used.
E. faecalis ATCC 29212
10
4
Number in parenthesis is number of replicates at each MIC, Number in bold is modal MIC
0.06
H. influenzae ATCC 49247
2
Agar
Dilution
MIC
H. influenzae (ATCC 49247)
S. pneumoniae ATCC 49619
8
1
Broth
Microdilution
MIC
E. faecalis (ATCC 29212)
Figure 2: Frequency Distribution of AZD0914 MICs (mg/L) against
CLSI QC strains using broth microdilution testing
10
0.5
Table 2: Comparison of AZD0914 MICs utilizing Broth microdilution (BMD)
and Agar dilution methods (mg/L)
• Levofloxacin broth microdilution MICs were within CLSI QC ranges (M100-S23) for each of the
organisms tested.
10
0.25
A comparison of broth and agar MICs for AZD0914 is presented in Table 2. MICs for the two
methods were comparable with all values within 2-fold of one another.
• MIC values remained consistent in the various lots of media tested.
S. aureus ATCC 29213
0.12
≤0.008(5), 0.004 (1)b
0.06(3), 0.12(6)
• MICs for each strain remained within 2-fold during each day of testing.
4
References
E. coli ATCC 25922
E. coli ATCC 35218
10
10
8
8
6
6
4
4
2
2
0
0
0.06
O
4
0.016 (8), 0.03 (2)
Number in parenthesis is number of replicates at each MIC, Number in bold is modal MIC
b Ciprofloxacin was tested by agar dilution against Neisseria gonorrhoeae
Materials and Methods
Figure 1
AZD0914
6
0.06
0.06
N
10
2
Introduction
O
N. gonorrhoeae ATCC 49226
MIC range (mg/L)
Strep. pneumoniae (ATCC 49619)
O
Figure 3: Frequency Distribution of AZD0914 MICs (mg/L) against
N. gonorrhoeae ATCC 49226 employing agar dilution MIC testing
8
a
N. gonorrhoeae (ATCC 49226)
Agar dilution MICs for AZD0914 against N. gonorrhoeae ATCC 49226 are presented in
Figure 3. Overall, AZD0914 MICs for this study ranged 0.06 and 0.12 mg/L over nine
days of testing
0.12
0.25
0.5
1
2
4
0.06
0.12
0.25
0.5
1
2
4
• CLSI, M07-A9, 2012
Clinical and Laboratory Standards Institute. M07-A9. 2012. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically; approved standard. Ninth edition. Wayne,
PA. Volume 32. Number 2.
• CLSI, M100-S23, 2013
Clinical and Laboratory Standards Institute. M100-S23. 2012. Performance standards for
antimicrobial susceptibility testing; twenty-third informational supplement. Wayne, PA. Volume
33. Number 1.