28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Standardizing Methods For Disk Diffusion And Broth Microdilution Susceptibility Testing Of Aquatic Isolates Ron A. Miller1, Robert D. Walker1, and Renate Reimschuessel1 1Food and Drug Administration – Center for Veterinary Medicine, Office of Research, Division of Animal and Food Microbiology, 8401 Muirkirk Rd. Laurel, MD 20708 Many bacterial pathogens isolated from aquatic animals require growth conditions that are considerably different from the growth conditions required by bacterial pathogens of warm-blooded animals. To accurately assess the susceptibility of these bacterial pathogens to antimicrobial agents it is necessary to develop appropriate standardized antimicrobial susceptibility testing (AST) methods. To facilitate this, the National Committee for Clinical Laboratory Standards (NCCLS) established an Aquaculture Working Group (AWG), charged with developing standardized AST methods for bacterial pathogens of aquatic species. The AWG recently published an NCCLS report, M42-R, which includes the standardized method for disk diffusion susceptibility testing, and quality control (QC) ranges for two ATCC® reference strains for nine antimicrobial agents commonly used in aquaculture. Members of the AWG are also developing a broth microdilution (BMD) method for testing aquatic pathogens. Following NCCLS guidelines, two ATCC® reference strains, Escherichia coli 25922 and Staphylococcus aureus 29212 have been shown to be excellent QC strains for BMD testing at 22ºC and 28ºC. These strains have a wide temperature tolerance range and are susceptible to a wide range of antimicrobial agents. In order to evaluate the utility of E. coli 25922 and S. aureus 29212 as control strains, their performance was compared to that of several aquatic isolates tested at 22ºC or 28ºC against 10 antimicrobial agents used in aquaculture. These isolates included species of Aeromonas, Vibrio, Edwardsiella, Photobacterium, and Pseudomonas. Results showed the drug concentration required to inhibit growth of the two QC organisms was in the same range as the MICs for most of the aquatic isolates tested. The next phase of the study is to demonstrate intra- and inter-laboratory reproducibility in accordance with NCCLS guidelines. The results of these studies will provide a foundation for developing international standardized BMD methods for testing aquatic animal isolates. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page