An international multilaboratory collaborative study was conducted to develop standard media and consensus methods for the performance and quality control of antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A reference strain from the American Type Culture Collection was designated for each species, which was to be used for quality control purposes. Repeat testing of replicate samples of each reference strain by participating laboratories utilizing both methods and different lots of media enabled a 3-to 4-dilution MIC range to be established for drugs in several different classes, including tetracyclines, macrolides, ketolides, lincosamides, and fluoroquinolones. This represents the first multilaboratory collaboration to standardize susceptibility testing methods and to designate quality control parameters to ensure accurate and reliable assay results for mycoplasmas and ureaplasmas that infect humans.
Methods for in vitro antimicrobial susceptibility testing of mycoplasmas were first described in the 1960s (6). Despite numerous publications during the ensuing years that have reported the activities of antimicrobial agents against these organisms, there have been no universally accepted or standardized broth dilution-or agar-based methods designating the optimum testing conditions, pH, media, length of incubation, quality control (QC) MIC reference ranges, or reference strains. The lack of a consensus method for MIC determination, coupled with complex cultivation requirements, has resulted in considerable confusion regarding the antimicrobial activities of various drugs against these fastidious organisms.To address the need for a standard method for performing and validating in vitro susceptibility tests for human mycoplasmas and ureaplasmas, the Clinical and Laboratory Standards (CLSI) Subcommittee on Antimicrobial Susceptibility Testing of Human Mycoplasmas devised a series of studies involving a total of 10 laboratories from 3 different countries representing academia, industry, and government. Sequential evaluations of both brothand agar-based methods were done with Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma species. The methods included commercial and individual laboratory-produced media and the testing of multiple reference strains for evaluation as QC strains. The QC strains and their respective MIC reference ranges were designated for several drug classes, including macrolides, ketolides, lincosamides, tetracyclines, and fluoroquinolones for each organism.
MATERIALS AND METHODSThe CLSI mandates specific protocols and numbers of participating laboratories for determining MIC reference ranges for QC purposes and for the actual measurement of MICs. These requirements have evolved over the years and have now become quite stringent (2). However, the fastidious nature, complex media and incubation requirements, and relatively slow growth for some mycoplasmal species necessitated s...