Intra- and interlaboratory performance of antibiotic disk-diffusion-susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments

In the course of an international research project on hazard analysis of antimicrobial resis- tance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquacul- ture environments was sent by the central laboratory to the other participating laboratories. In each lab- oratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were per- formed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of sub- cultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distrib- uted in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global aver- aging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indi- cated that the use of either ISA or MH medium in DD testing has a limited impact on the method's preci- sion among different laboratories. In conclusion, the current study has provided a validated SOP to pro- mote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive break- points should be specifically designed and validated for aquaculture drugs and organisms.