ABSTRACT

1. BMS-284756 (T-3811ME) Susceptibility Test Comparisons and Development for More Than 30 Bacterial Species Using 3,328 Recent Clinical Isolates Organism (no. tested) Occurrances at Etest/AD MIC ratio:  0.125 0.25 0.5 1 2 4 8 Organism (no. tested) Etest MIC/reference MIC 4 2 1 0.5 0.25 0.12 P.R. Rhomberg, D.J. Biedenbach, R.N. Jones. The JONES Group/JMI Laboratories, North Liberty, IA Poster #717 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 FIGURE 4: Scattergram comparing the reference broth microdilution BMS-284756 MICs and zone diameters around 5-g disks for staphylococci (300 strains; r = 0.91). Suggested breakpoints are shown as solid vertical and horizontal lines. More conservative criteria separating two distinct organism populations are noted as broken lines. FIGURE 8: Comparison of BMS-284756 MIC results obtained with the Etest and the reference NCCLS method for all streptococci tested (668 strains). Only two of 674 strains varied beyond the  one log2 dilution range (99.7% agreement between methods). ABSTRACT Background: BMS-284756 (BMS) is a novel des-F(6)-quinolone with activity against many Enterobacteriaceae, non-fermentative Gram-negative bacilli, staphylococci, streptococci, and fastidious or anaerobic species. In this report we compare BMS results between various in vitro susceptibility testing methods and propose breakpoint criteria. Methods: BMS activity was evaluated by NCCLS broth microdilution (BMD), agar dilution (AD), 5-g disk diffusion (DD) and Etest methods against 3,328 bacterial strains from various organism collections including the SENTRY Program (1997-2000). Results: The correlation coefficients (r) between BMD and Etest MIC values for the 11 species of Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. ranged 0.92-0.98. BMD vs. DD results were also excellent (r=0.94-0.99). The nonfermenter group Burkholderia/Stenotrophomonas spp. had a lower (r) for both BMD vs. Etest and BMD vs. DD (0.81-0.84). Staphylococci and enterococci were only compared for BMD vs. DD with an overall (r) of 0.94 (400 strains). Streptococci had a reduced correlation for both BMD vs. Etest and vs. DD, ranging from 0.44 to 0.80, but the Etest MIC value  1 log2 dilution was 99.4%. The BMD vs. Etest MIC (r) was consistently better than BMD vs. DD. H. influenzae (292 strains) had higher Etest BMS MICs with low (r) of 0.12 and 0.29, yet perfect categorical agreement. AD results were compared to DD and Etest values (r=0.78-87) against N. gonorrhoeae (137 strains). Etest results were consistently 4-fold lower than the AD MICs. C. jejuni and anaerobes when tested by AD and Etest showed 93-98% of results  2 log2 dilutions. Conclusions: The inter-method correlation (r) for BMS results were greater than 0.90 for nearly all common species evaluated and without serious categorical error using a proposed BMS breakpoint of 4 g/ml. Both Etest and DD approximations were considered to be highly accurate alternatives for testing BMS potency in clinical laboratories. MATERIALS AND METHODS – CON’T TABLE 2: Comparison of Etest and agar dilution (AD) test results for BMS284756 versus strains of C. jejuni,Gram-negative anaerobes and Gram-positive anaerobes Testing was performed according to reference National Committee for Clinical Laboratory Standards (NCCLS) recommended procedures for broth microdilution (BMD), agar dilution (AD) and disk diffusion (DD) for each specific species. The Etest (AB BIODISK, Solna, Sweden) was performed according to the method described in the manufacturer’s product package insert. BMS-284756 laboratory standard powder was supplied by Bristol-Myers Squibb (Princeton, NJ) and the comparison drugs by their domestic manufacturers. The BMS-284756 5-g disks and Etest strips were made by BD Microbiology Systems (Cockeysville, MD) and AB BIODISK, respectively. Results of AD, Etest and BMD MICs when available were compared to establish essential agreement between the results of these quantitative methods. MICs and DD zone diamters were analyzed by regression analysis and error-rate bounding to determine correlation between results. C. jejuni (30)% Gram-negative anaerobes (121)% Gram-positive anaerobes (76)% 26.7 21.6 22.6 1033.3a 4335.5b 1215.8c 620.0a 4940.5b 3039.5c 1033.3a  1814.9 3140.8c 26.7a 65.0b 11.3c 00.0a 21.7b 00.0c 00.0 10.8 00.0 aPercentage  two log2 dilutions was 93.3% (60.0% at  one log2 dilution). bPercentage  two log2 dilutions was 97.6% (60.4% at  one log2 dilution). cPercentage  two log2 dilutions was 97.4% (81.6% at  one log2 dilution). FIGURE 5: Scattergram comparing the reference broth dilution BMS-284756 MICs and zone diameters around 5-g disks for enterococci (99 strains; r = 0.93). Suggested breakpoints are shown as solid vertical and horizontal lines. More conservative criteria are noted as broken lines. RESULTS CONCLUSIONS • Overall, excellent inter-method, (Etest, BMD, DD, AD) correlation coefficients (generally r  0.90) were observed for enteric bacilli, pseudomonads, Acinetobacter, staphylococci, enterococci, Campylobacter, and Gram-negative and -positive anaerobes. • The 5-g disk concentration appears to be appropriate for MIC breakpoints up to  2 g/ml. A larger disk content of BMS-284756 may be required for a higher breakpoint ( 4 g/ml). • The Etest, AD, and disk diffusion testing methods can be used to provide accurate susceptibility testing results for BMS-284756 when testing a wide variety of bacteria. • When comparing enteric bacilli reference BMD MIC results to the zone diameters of inhibition an excellent correlation was achieved (r=0.94). • The Etest versus BMD essential agreement was 97.7% within  one log2 dilution and 99.8% when  two log2 dilutions were compared for enteric bacilli. • Inter-method comparison results for BMS-284756 DD versus reference MIC was r=0.95. • The summary of the non-fermentor strains testing BMS-284756 Etest versus reference MIC showed an essential agreement  one log2 dilution of 89.3% (97.0% for  two log2 dilutions). • When comparing BMS-284756 MIC to disk zone diameters for staphylococci (both oxacillin-susceptible and -resistant included) an excellent linear correlation developed, r=0.91. • The correlation of r=0.93 was achieved when comparing BMS-284756 BMD MICs to disk zone diameters for all enterococci tested. • Excellent linear correlation was observed for Etest versus reference BMD results (r=0.74) with 99.7% of results within  one log2 dilution for the streptococci group including S. pneumoniae, viridans group streptococci, and -haemolytic streptococci. • Evaluations and comparisons of BMS-284756 MICs and disk zone diameters showed that only a susceptible breakpoint was necessary and quantitative or categorical agreement was 79.5% and 100.0%, respectively when testing H. influenzae. • Testing penicillin-susceptible or -resistant and ciprofloxacin-resistant strains of gonococci produced an acceptable correlation (r=0.78) between BMS-284756 MICs and disk zone diameter results. • Inter-method comparisons of BMS-284756 Etest MICs and AD MICs were 93.3%, 97.6%, and 97.4% for results with C. jejuni, Gram-negative and -positive anaerobes, respectively when using criteria within  two log2 dilutions. FIGURE 1: Chemical Structure of BMS-284756 FIGURE 6: Scattergram comparison BMS-284756 MIC and zone diameter (5-g disk) results for 327 S. pneumoniae. INTRODUCTION REFERENCES FIGURE 2: Scattergram comparing reference BMS-284756 MIC results to the zone diameters around 5-g disks. Broken vertical line represents interpretive breakpoint suggested for Enterobacteriaceae (susceptible at  15 mm, resistant at  11 mm and a  4 g/ml MIC breakpoint). Fluoroquinolone therapy has been steadily increasing world wide over the last 12 - 14 years due to a wide spectrum of activity, low toxicity, oral or parenteral dosing, and good potency enhanced by very favorable pharmakinetics. Fluoroquinolone therapy is often prescribed empirically or directed by susceptibility test results for multitudes of infections including nosocomial and community-acquired respiratory tract therapy (sinusitis, pneumonia and chronic bronchitis). BMS-284756 is a novel des-F(6)-quinolone (formerly T-3811ME) with a broad spectrum of activity. While it may be no more active than ciprofloxacin or levofloxacin against many Gram-negative bacteria, it is consistently more active against the Gram-positive bacteria. Animal model studies show BMS-284756 has greater bioavailability than ciprofloxacin and is less toxic than levofloxacin. The study is designed to evaluate the in vitro susceptibility testing criteria of BMS-284756 by comparing over 30 species of Gram-positive and -negative bacteria by reference broth microdilution (BMD), agar dilution, and disk diffusion methods. Comparisons between reference and Etest MICs were also performed to access accuracy of the agar stable gradient methodology. The proposed breakpoint of  4 g/ml was used for some MIC comparisons. 1. Biedenbach DJ, Croco MAT, Barrett TJ, Jones RN. Comparative in vitro activity of gatifloxacin against Stenotrophomonas maltophilia and Burkholderia species isolates including evaluation of disk diffusion and Etest methods. Eur J Clin Microbiol Infect Dis 1999; 18:428-431. 2. Fung-Tomc JC, Minassian B, Kolek B, Huczko E, Aleksunes L, Stickle T, Tasho T, Gradelski E, Valera L, Bonner DP. (2000a). Antibacterial spectrum of a novel des-fluoro(6)quinolone, BMS-284756. Antimicrob Agents Chemother 44:3351-3356. 3. Hoellman DB, Kelly LM, Jacobs MR, Appelbaum PC. (2001). Comparative antianaerobic activity of BMS-284756. Antimicrobial Agents Chemother 45, 589-592. 4. National Committee for Clinical Laboratory Standards (NCCLS). (2000). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Document M7-A5. Wayne, PA:NCCLS. 5. National Committee for Clinical Laboratory Standards (NCCLS). (2000). Performance standards for antimicrobial disk susceptibility tests, seventh edition. Approved standard, M2-A7. Wayne, PA:NCCLS. 6. National Committee for Clinical Laboratory Standards (NCCLS). (2001). Development of in vitro susceptibility testing criteria and quality-control parameters. Approved guideline M23-A2. Wayne, PA:NCCLS. 7. 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: In vitro susceptibility of 269 non-fermentative Gram-negative blood stream infection isolates from the SENTRY Program (2000). Comparison of BMS-284756 Etest versus BMD MICs FIGURE 7: Scattergram comparing the reference agar dilution BMS-284756 MICs and zone diameters around 5-g disks (137 strains of N. gonorrhoeae; r=0.78) FIGURE 3: Scattergrams comparing the BMS-284756 MIC with the zone of inhibition around a 5-g disk when testing P. aeruginosa, (124 strains). Vertical broken lines indicate preliminary zone diameter breakpoint criteria correlating to the proposed  4 g/ml MIC indicating susceptibility. Acinetobacter (43) P. aeruginosa (129) Stenotrophomonas/Burkholderia spp. (97) TOTAL (269) 0 0 0 0 5 6 4 15 4 38 31 73 0 33 29 62 0 1 12 13 0 0 5 5 MATERIALS AND METHODS Isolates (3,328 strains) were selected from various clinical organism collections at the University of Iowa College of Medicine (Iowa City, IA), JMI Laboratories, and the SENTRY Antimicrobial Surveillance Program (1997-2001). These isolates belong to over 30 different genus groups. Most isolates were stored at -70C or below in serum or lysed blood, or at room temperature in distilled water (Pseudomonas spp.). aOnly on-scale results for both tests were used to assess essential agreement (168 organism tests). bEssential agreement  one log2 dilution was 89.3% (97.0%  two log2 dilutions steps). A156-23