DAO 64:211-222 (2005)  -  doi:10.3354/dao064211

Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria

R. A. Miller1,*, R. D. Walker1, J. Carson2, M. Coles3, R. Coyne4, I. Dalsgaard5, C. Gieseker1, H. M. Hsu6, J. J. Mathers7, M. Papapetropoulou8, B. Petty9, C. Teitzel10, R. Reimschuessel1

1Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, Maryland 20708, USA
2Fish Health Unit, Department of Primary Industries Water & Environment, 165 Westbury Road, Prospect Launceston, Tasmania 7250, Australia
3Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada
4Institute of Marine Research, Fish Health Division, Department of Aquaculture, Nordnesgaten 50, 5817 Bergen, Norway
5Fish Disease Laboratory, Danish Institute for Fisheries Research, Stigbøjlen 4, 1870 Frederiksberg C, Denmark
6Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin, 6101 Mineral Point Road, Madison, Wisconsin 53705, USA
7Alpharma, Animal Health Division, 400 State Street, Chicago Heights, Illinois 60411, USA
8Laboratory of Public Health, Medical School, University of Patras, 26500 Rio Patras, Greece
9Florida Department of Agriculture and Consumer Services, Division of Animal Industry, 605 East Main Street, Bartow, Florida 33830, USA
10Washington State University, Washington Animal Disease Diagnostic Laboratory, Pullman, Washington 99164, USA

ABSTRACT: A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. Quality control ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35°C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96 well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35°C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.


KEY WORDS: Minimum inhibitory concentration · Broth microdilution · Antimicrobial susceptibility testing · Quality control · Antibiotic


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