Quality Control Evaluation of BMS-284756 (T-3811ME) 5-  g Disk Diffusion (DD) and MIC Testing

1. Quality Control Evaluation of BMS-284756 (T-3811ME) 5-g Disk Diffusion (DD) and MIC Testing D.J. Biedenbach, R.N. Jones, The Quality Control Study Group. The JONES Group/JMI Laboratories, North Liberty, Iowa Poster #169 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 ronald-jones@jmilabs.com ABSTRACT 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. METHODS RESULTS- CONT’D 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 TABLE 3: Proposed quality control ranges from BMS-284756 disk diffusion and MIC dilution tests 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. 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. • 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. Disk diffusion Disk diffusion MIC tests Organism Proposed range (mm) %in range Proposed range (g/ml) %in range Occurrences by laboratory: E.coli ATCC 25922 E.faecalis ATCC 29212 S.aureus ATCC 25923 S.aureus ATCC 29213 P.aeruginosa ATCC 27853 H.influenzae ATCC 49247 S.pneumoniae ATCC 49616 28-35 - 30-36 - 19-25 33-41 26-33 97.9 - 97.4 - 97.9 97.4 98.8 0.004-0.016a 0.03-0.25 - 0.004-0.016a 0.5-2 0.002-0.008 0.016-0.06 100.0 100.0 - 99.5 99.5 94.6a 100.0 MIC (g/ml) A B C D E F G Total (%) 0.004 0.008 0.016 0.03 Total Mode (g/ml) Range (dilutions) 4 21 5 30 0.008 3 2 24 4 30 0.008 3 10 20 30 0.016 2 5 24 1 30 0.008 3 25 5 30 0.008 2 9 20 1 30 0.008 3 6 20 4 30 0.008 3 17 (8.1)a 133 (63.3)a 59 (28.1)a 1 (0.5) 210 0.008 4 a A wider range of 0.004-0.03 g/ml was selected by the NCCLS following initial results from commercial panels including BMS284756. b Only rate less than 95%. All other proposed ranges contained 97.4 - 100.0% of recorded results (average, 98.8%). FIGURE 1: Chemical structure of BMS-284756 aProposed range (0.004 - 0.016 g/ml) includes 99.5% of reported values. CONCLUSIONS • This study documents a standardized NCCLS QC study evaluating 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. 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) A P. aeruginosa ATCC 27853 S. pneumoniae ATCC 49619 E. coli ATCC 25922 S. aureus ATCC 29213 H. influenzae 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 20 a 48 104 132 78 28 1 a 3 2 6 a 43 86 83 62 70 39 26 a 2 0 1 1 14 a 19 42 61 105 96 50 24 a 6 2 1 9 21 a 51 70 98 110 48 11 a 1 8 3 12 a 10 31 55 79 118 65 37 2 a REFERENCES RESULTS INTRODUCTION 1. Fung-Tomc JC, Minassian B, Kolek B, Huczko E, Aleksunes L, Stickle T, Washo T, Gradelski E, Valera L, Bonner DP. (2000). Antibacterial spectrum of a novel des-fluoro(6) quinolone, BMS-284756. Antimicrob Agents Chemother 44; 3351-3356. 2. Hoellman DB, Kelly LM, Jacobs MR, Appelbaum PC. (2001). Comparative antianaerobic activity of BMS-284756. Antimicrob Agents Chemother 45; 589-592. 3. National Committee for Clinical Laboratory Standards. (2000a). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, fifth edition: Approved standard, M7-A5. Wayne, PA:NCCLS. 4. National Committee for Clinical Laboratory Standards. (2000b). Performance standards for antimicrobial disk susceptibility tests, seventh edition: Approved standard, M2-A7. Wayne, PA:NCCLS. 5. National Committee for Clinical Laboratory Standards. (2001). Development of in vitro susceptibility testing criteria and quality control parameters, second edition: Approved guideline M23-A2. Wayne, PA:NCCLS. 6. Rhomberg PR, Biedenbach DJ, Jones RN. (2001). Activity of BMS-284756 (T-3811) Tested Against Anaerobic Bacteria, Campylobacter jejuni, Helicobacter pylori, and Legionella spp. Diagn Microbiol Infect Dis 40:45-49. 7. SENTRY Participants Group (Latin America), Gales A, Sader H, Jones RN. (2001). Antimicrobial activity of BMS-284756 (T-3811) tested against Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae isolates from SENTRY Antimicrobial Surveillance Program medical centers in Latin America (1999). Antimicrob Agents Chemother 45:1463-1466. 8. Takahata M, Mitsuyama J, Yamashiro Y, Yonezawa M, Araki H, Todo Y, Minami S, Watanabe Y, Narita H. (1999). In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)-quinolone. Antimicrob Agents Chemother 43; 1077-1084. • Table 1 illustrates the MIC distribution of S. aureus ATCC 29213 tested against 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. 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 des-F(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 BMS-284756 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 des-F(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. 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 140 120 100 80 Frequency of Occurrence (n) 60 40 20 0 17 18 19 20 21 22 23 24 25 26 27 Zone Diameter (mm) aProposed range limits that included 97.4 to 98.8% of participant results. A156-26