One of the greatest challenges in the aquaculture world is health maintenance and the control of disease outbreaks. Fish farmers throughout history have tried a range of compounds to treat fish ailments, including salt, asphalt, and brandy.

The last century saw major progress in the isolation and identification of the microorganisms that cause disease in aquatic animals. Concurrent to the advances to diagnose diseases in aquatic species, scientists were developing more effective means of fighting them with the production of antimicrobial substances. While there are currently over 70 known aquatic bacterial pathogens, there are numerous instances where isolated organisms from the aquatic environment remain either partially or completely unidentified.

Susceptibility testing of aquatic bacteria is used when there is a concern that the organism may possess resistance mechanisms against commonly used antimicrobial agents. To adequately assess the effect of antimicrobial use on the aquatic environmental bacteria, it is imperative that standardized methods are developed and that protocols are updated as more data become available.^1,2

In June 2006, the Clinical and Laboratory Standards Institute (CLSI) released the latest approved-level guidelines for performing susceptibility testing on bacteria isolated from aquatic animals: Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline (M42-A) and Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline (M49-A). 

“These documents satisfy a long-time need for standardized susceptibility testing methods in aquaculture. They provide a central core set of testing methods for the aquatic animal medicine field,” says Ron Miller, MS, U.S. Food and Drug Administration (FDA) Center for Veterinary Medicine. 

Two commonly used drug susceptibility testing methods exist. In the first type, bacteria are isolated, added to an agar plate, a paper disk saturated with the drug is added to the agar surface, and the inoculated plate is incubated. The distance to which the bacteria are inhibited from growth around the disk corresponds to their level of sensitivity to the drug.

The second type of drug susceptibility testing, the dilution technique, involves bacteria being exposed to varying concentrations of a drug. In broth microdilution tests, the drug is diluted in wells in a microtiter plate. The bacteria are then added to the wells, incubated, and allowed to grow. The lowest drug concentration for which there is no bacterial growth in the wells corresponds to that isolate’s minimal inhibitory concentration (MIC). An MIC can be used to predict the effectiveness of therapy, but should not be used without also considering additional information, including the host-drug interaction and the bacterial pathogen.

The disk diffusion method is the most commonly used laboratory technique for susceptibility testing. It is simple, cost-effective, and easy to perform. However, dilution testing is becoming increasingly popular, since an MIC provides additional information to the clinician regarding the concentration of antimicrobial agents needed to inhibit or eliminate the infectious organism.

CLSI document M42-A provides veterinary microbiologists with the most up-to-date techniques for disk diffusion susceptibility testing of aquatic species isolates. M49-A provides methods for determining MICs of aquatic bacteria by broth micro- and macrodilution.

Both M42-A and M49-A include quality control and quality assurance parameters for both fastidious and nonfastidious aquatic bacterial pathogens; tables outlining antimicrobial agents used in global aquaculture; methods for preparing stock solutions and dilutions of antimicrobial agents; protocols for data comparison between laboratories; and nonfastidious aquatic bacterial pathogen testing. ^1,2

The development process for the guidelines began in 1998 during the Workshop on MIC Methodologies in Aquaculture, held in Weymouth, England. Since the workshop, two separate and coordinated multilaboratory studies were initiated to develop the standard methods for susceptibility testing in aquatic animals, taking into account such criteria as incubation conditions and identifying quality control (QC) limits for potential QC strains. As noted on the Department of Microbiology, National University of Ireland, Galway website, “There is a real and very exciting possibility that the final outcome will be methods of truly international acceptability.”

Peter Smith, PhD, from the National University of Ireland, Galway, calls the new CLSI documents “a first essential step in an international effort to develop programs enabling scientists the world over to monitor the spread of antibiotic resistance among aquatic bacteria.”

Smith, who participated in the development of the guidelines, says the extensive collaboration has resulted in “a standard, universal language in which we can investigate the epidemiology of resistance and discuss therapies for the aquatic environment.”

Renate Reimschuessel, PhD, VMD, U.S. Food and Drug Administration (FDA) Center for Veterinary Medicine, explains “The (CLSI) standards themselves provide methods that can be applied globally and have been awaited by the fish medicine community for decades.” These documents include the best thinking of global scientists in the field and their recommendations for conducting susceptibility testing in organisms that prefer or require conditions such as lower temperatures, semisolid media, or supplemented media. Ultimately, these new guidelines allow aquatic animal disease diagnostic laboratories around the world to provide consistent and reliable testing methods, greater accuracy in interpreting results, and the ability to compare quality test data and results among laboratories.

References

Clinical and Laboratory Standards Institute (CLSI). Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline. CLSI document M42-A (ISBN 1-56238-611-5). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006.

Clinical and Laboratory Standards Institute (CLSI). Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline. CLSI document M49-A (ISBN 1-56238-612-3). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006.
[ return to top ]