Plasmid Curing of Vibrio Isolates

Vibrio isolates, that harbored plasmid, were treated with acridine orange (AO) (Thermo Fisher Scientific, Waltham, MA, USA) following modifications of the method by Letchumanan et al. [19 (link)]. The isolates were grown on TSB supplemented with 1.5% of NaCl, at 30 °C for 18 h under constant agitation. After incubation, 200 µL of the aliquots were added into the tubes containing Luria Bertani (LB) broth (Oxoid, Hampshire, UK), supplemented with 1.5% of NaCl (control), and into the tubes containing LB broth, supplemented with 1.5% of NaCl and AO. The isolates were incubated at 30°C for 18 h [36 (link)]. The presence of plasmid was detected using agarose gel electrophoresis (1 % w/v) after treatment with AO. In addition, the antibiotic susceptibility test was repeated, as stated earlier, to confirm changes in resistance profiles.