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Quality Control Evaluation of BMS-284756 (T-3811ME) 5-g Disk Diffusion (DD) and MIC Testing
Poster #169
Background: BMS-284756 (BMSQ) is a novel des-fluoro(6)-quinolone with
antimicrobial activity similar to recently developed fluoroquinolones. The purpose
of this investigation was to determine the appropriate DD and MIC quality control
(QC) ranges for BMSQ against commonly used ATCC QC strains.
Methods: BMSQ 5-g disk content was tested against E. coli (EC) ATCC 25922,
P. aeruginosa (PA) ATCC 27853, H. influenzae (HI) ATCC 49247, S. pneumoniae
(SP) ATCC 49619 and S. aureus (SA) ATCC 25923. MIC QC was determined for
all the above except SA 25923. In addition, E. faecalis (EF) ATCC 29212 and SA
ATCC 29213 were evaluated by broth microdilution. The study design followed
NCCLS M23 guidelines and utilized NCCLS methods and appropriate media
(HTM, Mueller-Hinton  LHB). Seven laboratories tested 2 disk lots on 3 agar
media or 3 or 4 broth lots over a 10 day period generating 420 total disk values
and 210 or 280 MIC values per organism. Internal QC using levofloxacin (LEVO)
as the control agent and daily colony counts were performed.
Results: Modal MIC values were within 2 log2 dilutions among centers for all
tested strains and the range spanned 4 dilution steps. Median DD values were
within 4-6 mm between centers with ranges of 9-12 mm. Internal QC showed 96.4
and 99.6% of LEVO results were with published guidelines for DD and MIC,
respectively. Colony counts ranged 8 x 104 to 8.8 x 105 CFU/ml.
Conclusions: The following DD/MIC QC ranges are suggested for BMSQ and 7
QC strains: EC ATCC 25922 (28-35 mm/0.004-0.016 g/ml), EF ATCC 29212
(0.03-0.25 g/ml), SA ATCC 25923 (30-36 mm), SA ATCC 29213 (0.004-0.016
g/ml), PA ATCC 27853 (19-25 mm/0.5-2 g/ml), HI ATCC 49247 (33-41
mm/0.002-0.008 g/ml), SP ATCC (26-33 mm/0.016-0.06 g/ml). The percent of
participant results in the proposed ranges was 97.4-98.8% and 94.6-100% for DD
and MIC, respectively.
INTRODUCTION
BMS-284756 (formerly T-3811ME) is a novel des-fluoro(6) quinolone that differs
from recent clinically approved quinolones in that the BMS-284756 molecule lacks
a fluorine at the C-6 position (Figure 1). BMS-284756 has antibacterial activity and
pharmacokinetic properties similar to those of fluorinated quinolones, but the desF(6) derivatives were less acutely toxic in mice. Among the most active quinolones
tested against Gram-positive bacteria, BMS-284756 has been particularly potent
against oxacillin-resistant Staphylococcus aureus, Staphylococcus epidermidis
and ciprofloxacin-resistant Streptococcus pneumoniae. The documented BMS284756 activity against the common Gram-negative pathogens associated with
comunity-acquired pneumonia (Haemophilus influenzae, Moraxella catarrhalis)
was also comparable to other quinolones in current clinical use. BMS-284756 has
one of the broadest anti-anaerobic bacterial coverages documented for a
quinolone, inhibiting almost all of the anaerobic bacterial strains tested. This desF(6) compound has demonstrated potency against “atypical” and fastidious
microbes such as mycoplasmas, ureaplasmas, chlamydiae, Campylobacter spp.,
Helicobacter spp., Legionella spp., and ciprofloxacin-nonsusceptible Neisseria
gonorrhoeae. Finally, BMS-284756 has activity against the Enterobacteriaceae
and most non-fermentative Gram-negative bacilli at levels comparable to that of
marketed, true fluoroquinolones.
With the recorded wide spectrum of activity, the clinical trials require accurate
quality control (QC) guidelines for BMS-284756 laboratory testing versus a wide
variety of QC strains. This study summarizes the results of a National Committee
for Clinical Laboratory Standards (NCCLS) M23-A2 qualifying study to establish
QC limits for BMS-284756.
D.J. Biedenbach, R.N. Jones, The Quality Control Study Group.
The JONES Group/JMI Laboratories, North Liberty, Iowa
METHODS
The design of this QC study followed the NCCLS [2001] M23-A2 criteria and
included seven participant laboratories. Each laboratory tested two disk lots of
BMS-284756 (BBL) and one disk lot of levofloxacin (REMEL) as a control, on
three different agar lots of Haemophilus Test Medium or Mueller-Hinton
supplemented with or without 5% sheep blood depending on the species tested
(BBL, Remel, Difco, and Accumedia). Three or four lots of broth media for MIC
testing were supplied by Difco, BBL or Criterion. Reference broth microdilution
and disk diffusion standards were used by all seven laboratories. Each laboratory
tested two disk lots on three media lots over a 10 day period yielding a total of 420
values for the disk diffusion phase and each QC organism tested. Each participant
also tested three broth medium lots over 10 days for all non-fastidious species,
and four medium lots were used for H. influenzae and S. pneumoniae which
resulted in 210 or 280 total test values for each QC strain, respectively. In
addition, internal QC using levofloxacin established NCCLS [2001b] range criteria
resulted in a total of 1,050 and 560 results for disk and MIC tests, respectively.
Colony counts of the inoculum were performed from the broth microdilution trays
by quantitative subculturing onto drug-free plates, and the same undiluted 0.5
McFarland standard inoculum was applied to the agar plates in the disk diffusion
phase of this study which further validated participant results.
RESULTS- CONT’D
 Table 3 lists all proposed QC ranges for the seven ATCC strains which are commonly
used by clinical laboratories as quality assurance of susceptibility testing methods. The
proposed BMS284756 ranges for the five QC strains used for the disk diffusion phase
spanned 7 to 9 mm. The larger range was for H. influenzae ATCC 49247 which was
heavily influenced by the conservative reading of Laboratory F. This same laboratory
produced results representing nearly all of the results at 28 or 29 mm for E. coli ATCC
25922 (proposed range, 28 to 35 mm). Overall, between 97.4 - 98.8% of participant
results were within the proposed ranges for the disk diffusion component of this trial.
 Six QC strains were tested against BMS-284756 by the broth microdilution method
(Table 3). A three dilution range was proposed for all strains except E. faecalis ATCC
29212, which has a four dilution range recommendation (broad mode at 0.06 and 0.12
g/ml). Nearly all laboratory MIC QC results fell within the proposed ranges ( 99.5%),
except for H. influenzae ATCC 49247 (94.6%). Laboratory D accounted for two-thirds of
the results outside the range for the H. influenzae QC strain.
FIGURE 1: Chemical structure of BMS-284756
Using the study design described, this study proposes disk diffusion zone
diameter and MIC broth microdilution ranges for seven commonly used American
Type Culture Collection (ATCC) strains. These strains included Escherichia coli
ATCC 25922, Enterococcus faecalis ATCC 29212 (MIC only), S. aureus ATCC
25923 (disk diffusion only) and 29213 (MIC only), Pseudomonas aeruginosa
ATCC 27853, H. influenzae ATCC 49247 and S. pneumoniae ATCC 49616. The
proposed ranges were optimized, where possible, to contain  95% of all recorded
results as recommended by NCCLS M23-A2 guidelines.



4
2
0.008
21
24
0.016
5
4
F
5
10
20
24
1
25
5
0.03
9
20
G
17 (8.1)a
20
133 (63.3)a
4
Mode (g/ml)
0.008 0.008 0.016 0.008 0.008 0.008 0.008 0.008
Range (dilutions)
3
3
30
2
30
1 (0.5)
30
2
30
59
(28.1)a
Total
3
30
Total (%)
6
1
30
Disk diffusion
3
30
3
210
4
Disk diffusion
MIC tests
Proposed
range (mm)
%
in range
Proposed
range (g/ml)
%
in range
28-35
97.9
0.004-0.016a
100.0
E.faecalis ATCC 29212
-
-
0.03-0.25
100.0
S.aureus ATCC 25923
30-36
97.4
-
-
S.aureus ATCC 29213
-
-
0.004-0.016a
99.5
P.aeruginosa ATCC 27853
19-25
97.9
0.5-2
99.5
H.influenzae ATCC 49247
33-41
97.4
0.002-0.008
94.6a
S.pneumoniae ATCC 49616
26-33
98.8
0.016-0.06
E.coli ATCC 25922
a
b
100.0
A wider range of 0.004-0.03 g/ml was selected by the NCCLS following initial results from
commercial panels including BMS284756.
Only rate less than 95%. All other proposed ranges contained 97.4 - 100.0% of recorded
results (average, 98.8%).
range (0.004 - 0.016 g/ml) includes 99.5% of reported values.
 This study documents a standardized NCCLS QC study evaluating
TABLE 2: Distribution of BMS-284756 zone diameter (mm)
results for five ATCC quality control strains in a NCCLS
M23-A2 multicenter evaluation (420 data points per isolate)
CO 2H
N
HN
O
A
CHF2
 Table 1 illustrates the MIC distribution of S. aureus ATCC 29213 tested against

0.004
TABLE 3: Proposed quality control ranges from BMS-284756
disk diffusion and MIC dilution tests
Organism
Occurrences by laboratory:
A
B
C
D
E
MIC (g/ml)
aProposed
O
RESULTS
BMS-284756. Nearly two-thirds of all participant results were 0.008 g/ml, and
this was the modal value for five out of seven participant laboratories. The range
never exceeded three log2 dilutions (mode  one log2 dilution step) for any one
participant and the proposed range of 0.004 - 0.016 g/ml encompassed 99.5%
of reported MICs. Overall, MIC QC results indicated that Laboratory D had a
tendency towards lower values for nearly all the organisms tested (data not
shown).
Figure 2 is a bar graph of all BMS-284756 zone diameter results tested against P.
aeruginosa ATCC 27853. A total of 420 zone diameter results were distributed
over a 9 mm range. The proposed QC range (19 - 25 mm) encompassed 97.9%
of all participant results. The disk diffusion phase of this QC study showed that
Laboratory F consistently read the inhibitory zone diameters more conservatively
(smaller zones), and always contributed the lowest median values among the
participants for all QC strains. The median value of Laboratory F was, however,
no more than three millimeters lower than the all participant median zone
diameter. The proposed ranges for all QC strains produced  97.4% of study
results in the range.
Table 2 shows the zone diameter distributions for all five QC strains.
Internal testing with levofloxacin produced the following percentages within
published guidelines: 96.4 and 99.6% for the disk diffusion (1,050 results) and
MIC (560 results) phases, respectively.
Colony counts of the inoculums showed that an average among all participants
was 2.9 x 105 CFU/ml and the range of inoculum counts was 8 x 104 to 8.8 x 105
CFU/ml.
TABLE 1: Occurrence of MIC values by participant when testing
S. aureus ATCC 29213 against BMS-284756 in a
NCCLS [2001] M23-A2 quality control trial
CONCLUSIONS
FIGURE 2: Frequency of zone diameter occurrences when testing
P. aeruginosa ATCC 27853 against BMS-284756. Proposed QC range
(19-25 mm) includes 97.9% of participant results.
Proposed Range
140
Frequency of Occurrence (n)
ABSTRACT
Ronald N. Jones, M.D.
The JONES Group / JMI Laboratories
345 Beaver Kreek Centre, Suite A, North Liberty, Iowa 52317
Phone: 319-665-3370 Fax: 319-665-3371
[email protected]
120
100
80
60
40
20
0
17
18
19
20
21
22
23
24
25
26
27
P. aeruginosa S. pneumoniae
E. coli
S. aureus H. influenzae
ATCC 27853
ATCC 49619 ATCC 25922 ATCC 29213 ATCC 49247
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
6
20a
48
104
132
78
28
1a
3
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2
6a
43
86
83
62
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39
26a
2
0
1
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1
14a
19
42
61
105
96
50
24a
6
2
1
9
21a
51
70
98
110
48
11a
1
Zone Diameter (mm)
aProposed
BMS-284756, a new des-fluoro(6)-quinolone, against commonly used ATCC
control strains. The proposed ranges presented here are recommendations that
have been modified and approved by the NCCLS Subcommittee on
Antimicrobial Susceptibility Testing (June, 2001; see footnote “a” Table 3); and
should be utilized during the BMS-284756 clinical trials and after approval by
the United States Food and Drug Administration.
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3
12a
10
31
55
79
118
65
37
2a
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