Viability during storage and stability of plasmids during storage and subculturing in strains of Vibrio anguillarum

The stability of plasmids, 3.3 kb -similar to 200 kb, in 8 strains of Vibrio anguillarum displaying different plasmid profiles and reactions with O-antisera was investigated over an 18 months period. All plasmid profiles proved to be resistant to storage at different temperatures but strains stored at 37 degrees C were only viable for a short period of maximum 2 months. Strains stored at 5 degrees C and 20 degrees C were viable for a longer period. Viable strains maintained their plasmid profile throughout the experiment, except in 2 cases where a 67 kb and a 200 kb plasmid were lost. Strains stored at -80 degrees C all remained viable and maintained their plasmid profile throughout the study. By subcultivating daily for up to 100 successive days, most strains maintained their plasmid profiles. Only 2 strains lost their plasmid. When picking 100 single colonies from agar plates, none of the colonies showed plasmid profiles deviating from the expected. The results suggest that plasmid profiles among V. anguillarium are very stable during subculturing, storage and laboratory handling using standard laboratory procedures, and thus, reliable for epidemiological investigations. In a second experiment, 2 pairs of 2 strains were grown together in mixed cultures. They were identical in all traits, except that one strain in each pair harboured the 67 kb pJM1-like virulence plasmid, whereas the other had lost this plasmid. The result showed that the growth rate was the same for strains with and without the plasmid, indicating that under laboratory conditions, this plasmid is neither a benefit nor the opposite for bacterial growth.