Quality control guidelines for susceptibility testing of retapamulin (SB-275833) by reference and standardized methods.

Pleuromutilin was discovered in 1951 from an edible mushroom, Pleurotus mutilus, but it was not until the early 1970s that its potential for use as an antimicrobial was more fully recognized (2). Tiamulin and valnemulin were developed as semisynthetic pleuromutilins whose antimicrobial activity was traced to inhibition of bacterial protein synthesis following interaction with the peptidyltransferase center of the 50S ribosomal subunit (2, 3, 8, 9). Tiamulin has potent activity against anaerobes, intestinal spirochetes, and Mycoplasma spp., and it has been principally used in swine production to control grampositive and -negative pathogens (3). Retapamulin (formerly SB-275833; Fig. 1) is the first topical pleuromutilin derivative developed for human use with enhanced activity against gram-positive bacteria, including staphylococcal and streptococcal isolates (2). This report describes results from a multilaboratory trial designed to establish retapamulin quality control (QC) ranges for disk diffusion and broth microdilution MIC methods (1, 6, 7) and used study design criteria as published in the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) M23-A2 document (4). An eight-laboratory study group was recruited for the development of MIC and disk diffusion zone diameter QC guidelines for retapamulin. The QC group consisted of laboratories at the University of Alberta (Edmonton, Alberta, Canada), The Cleveland Clinic Foundation (Cleveland, OH), University of Texas Medical Center (Houston, TX), University of Rochester Medical Center (Rochester, NY), Denver Health Medical Center (Denver, CO), University of Washington (Seattle, WA), TREK Diagnostics (Cleveland, OH), and JMI Laboratories (North Liberty, IA). Each laboratory followed a protocol based on CLSI document M23-A2 specifications (4), as well as the M7-A6 method for broth microdilution antimicrobial testing (7) and M2-A8 method for disk diffusion susceptibility testing (6). The MIC portion of the study utilized frozen-form, reference broth microdilution panels prepared by TREK Diagnostics (Cleveland, OH). The panels contained four lots of cationadjusted Mueller-Hinton broth (Oxoid, Hampshire, United Kingdom; BBL, Sparks, MD; Difco, Detroit, MI [two lots]) and the same four lots of Mueller-Hinton broth supplemented with 2 to 5% lysed horse blood. Ofloxacin and erythromycin were utilized as control agents. Each laboratory tested Staphylococcus aureus ATCC 29213 and Streptococcus pneumoniae ATCC 49619 and generated a total of 560 MIC results. Of the control agent results, 99.3% were within CLSI published guidelines (1). Colony counts were performed from the broth microdilution trays by subculturing in a quantitative manner onto drug-free solid media. The counts ranged from 1.1 10 to 8.5 10 CFU/ml, with an average of 3.8 10 CFU/ml. The disk diffusion portion of the study utilized three different lots of commercially prepared Mueller-Hinton agar and three lots of Mueller-Hinton agar supplemented with 5% sheep blood (Remel, Lenexa, KS; BBL, Sparks, MD). Two different lots of retapamulin disks were utilized versus each QC strain (Oxoid lot 320100 and BBL lot 3336172). Single lots of ofloxacin, erythromycin, and ceftriaxone disks (Remel lots 290536, 308402, and 311176) were applied as internal control agents. A total of 1,350 control zone diameter results were produced, and 99.7% of reported results were within the CLSI QC ranges as published in document M100-S15 (1). Proposed retapamulin QC ranges were optimized to encompass 95.0% of all reported results, as recommended by the M23-A2 guideline (4). The MIC and disk diffusion zone diameter results were tabulated and compared by intraand interlaboratory analyses to determine potentially unacceptable technical variations. Broth or agar medium and disk lots were also compared to determine variations among manufacturers. The S. pneumoniae ATCC 49619 internal QC zone diameter results from laboratory H were determined to be “out of control”; thus, all results from that participant were deleted from the final analysis. The results of retapamulin MIC distributions among the