DAO 66:197-204 (2005)  -  doi:10.3354/dao066197

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

Geert Huys1,*, Margo Cnockaert1, Kerry Bartie2, Dang Thi Hoang Oanh3, NguyenThanh Phuong3, Temdoung Somsiri4, Supranee Chinabut4, Fatimah Md Yussoff5, Mohamed Shariff5, Mauro Giacomini6, Stefania Bertone7, Jean Swings1,8, Alan Teale2

1Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium
2Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
3Laboratory of Fish Diseases, College of Aquaculture and Fisheries, Can Tho University, Can Tho City, Vietnam
4Aquatic Animal Health Research Institute, Kasetsart University Campus, Jatujak, Bangkok 10900, Thailand
5Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
6DIST, University of Genova, Via Opera Pia 13, 16145 Genova, Italy
7RILAB stl, Via Guerrazzi 24/12B, 16146 Genova, Italy
8BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

ABSTRACT: In the course of an international research project on hazard analysis of antimicrobial resistance 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 aquaculture environments was sent by the central laboratory to the other participating laboratories. In each laboratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were performed 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 subcultures 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 distributed in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global averaging 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 indicated that the use of either ISA or MH medium in DD testing has a limited impact on the method’s precision among different laboratories. In conclusion, the current study has provided a validated SOP to promote 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 breakpoints should be specifically designed and validated for aquaculture drugs and organisms.


KEY WORDS: Antimicrobial agent susceptibility testing · Disk diffusion · Aquaculture monitoring · Intra- and interlaboratory harmonization · Standard operating procedure


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