World-wide increases in Vibrio-associated diseases have been reported in aquaculture and humans in co-occurrence with increased sea surface temperatures. Twelve species of Vibrio are known to cause disease in humans, but three species dominate the number of human infections world-wide: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Fluorescent protein (FP)-labelled bacteria have been used to make great progress through in situ studies of bacterial behavior in mixed cultures or

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... within host tissues. Currently, FP-labelling methods specific for Vibrio species are still limited by time-consuming counterselection measures that require the use of modified media and temperatures below the optimal growth temperature of many Vibrio species. Within this study, we used a previously reported R6K-based suicide delivery vector and two newly constructed transposon variants to develop a tailored protocol for FP-labelling V. cholerae, V. parahaemolyticus, and V. vulnificus environmental isolates within two days of counterselection against the donor Escherichiacoli. This herein presented protocol worked universally across all tested strains (30) with a conjugation efficiency of at least two transconjugants per 10,000 recipients.