Influence of incubation time on antimicrobial susceptibility testing of pathogenic Vibrio anguillarum and Vibrio vulnificus isolated from fish

Sandrine Baron, Daniela Ceccarelli, Inger Dalsgaard, Sophie A. Granier, Olga Haenen, Eva Jansson, Lone Madsen, Eric Jouy, Isabelle Kempf, Emeline Larvor, Thierry Morin, Joop Testerink, Kees Veldman, Kári Karbech Mouritsen, Betty Van Gelderen, Michal Voorbergen-Laarman, Eva Säker, Eva Blomkvist, Peter Smith*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

8 Citations (Scopus)


A multi-laboratory study was performed to investigate the most suitable incubation time for susceptibility tests of fish pathogens Vibrio anguillarum and Vibrio vulnificus performed at 28 °C. An isolate set consisting of 30 V. anguillarum and 26 V. vulnificus was used by four participating laboratories in Denmark, France, Sweden, and the Netherlands. Inhibition zone diameters were determined by agar disc diffusion for eight agents and Minimum Inhibitory Concentration (MIC) values were determined for seven agents using the standard CLSI testing protocols for non-fastidious organisms that specify 24-28 h incubation. In this work an additional set of readings was made after 48 h incubation. In total, 1120 paired zone sizes and 399 paired MIC observations were made at the two incubation times. Examination of the data demonstrated that incubation time had a small but statistically significant effect on the numerical values of susceptibility measures. However, the effects of incubation time on the precision of the data sets and the categorisation of isolates based on the application of epidemiological cut-off values were slight and statistically non significant. These analyses suggest that the susceptibility of these Vibrio species could be established using protocols that specify either 24-28 h or 44-48 h incubation. This study does not provide evidence that prolonged incubation to 48 h improves the quality of data generated by the tests. Therefore, it is recommended that the existing standard CLSI protocols with 24-28 h at 28 °C should be adopted for susceptibility testing of V. anguillarum and V. vulnificus.

Original languageEnglish
Article number735258
Publication statusPublished - 15 Jul 2020


  • CLSI susceptibility testing
  • Disc diffusion
  • Incubation time
  • MIC
  • Vibrio anguillarum
  • Vibrio vulnificus


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