Developing a Universal and Efficient Method for the Rapid Selection of Stable Fluorescent Protein-Tagged Pathogenic Vibrio Species

Abstract

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 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.

Ecological Fitness of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in a Small-Scale Population Dynamics Study

“…Previously, VpR-DSMZ and VpR-NSI were grown at 28 • C in a separate sea water source, filtered through a 0.22 µM filter and then autoclaved (NSW) prior to the niche experiments. In those trial tests, we did not see any changes in V. parahaemolyticus abundance (Thorstenson and Ullrich, 2020). After the niche experiments, we tested VpR-DSMZ in the same NSW, with an incubation at 20 • C for 72 h. Again, the concentration of V. parahaemolyticus remained stable at the starting concentration at inoculation over 72 h. Based on these experiments in the NSW, it is unlikely the decline in V. parahaemolyticus abundance observed in the SSW incubations is a specific attribute of our genetically modified bacteria, but rather is attributed to the state of the SSW used for the incubation.…”

“…Bacterial strains used in this study, and their relevant characteristics, are shown in Table 1. Previously, multiple genetically modified Vibrio clones were obtained via bi-parental mating with an Escherichia coli strain carrying a suicide vector with a Tn5 transposon coding for the red fluorescent protein (RFP) gene (dsRedExpress), the gentamycin resistance gene (Gm R ), and the chloramphenicol resistance gene (Cm R ) ( Thorstenson and Ullrich, 2020). These RFP-labeled clones behave comparably to the wild-type (WT) strain when grown in MB medium and sterilized NSW.…”

“…To see if we could amplify the RFP gene found in the transposon originally used in the construction of VvR-21 (Thorstenson and Ullrich, 2020), in the new bacterial isolates from the VvR-21 niche analysis, the rfp6 primers were used. All isolate DNA was tested alongside the DNA of VvR-21 as a positive control.…”

“…To see if the non-Vibrio growth phenomenon was repeatable, the niche analysis with VvR-21 was repeated a second time with the same source of water for the NaSW and SSW, which had been stored for five days in the dark at 4 • C. At the same time, we repeated the niche analysis with a separate strain of RFPlabeled V. vulnificus, VvR-25. This strain had been constructed in the same manner as VvR-21 and carries the same transposon (Thorstenson and Ullrich, 2020). This second experiment was to confirm that the transfer of the transposon was unique to the VvR-21 strain and not to V. vulnificus as a species.…”

“…The only time non-Vibrio growth occurred on the selective (Gm-containing) medium used for enumeration, was when VvR-21 was inoculated into the sample, providing evidence that the Gm resistance found in the non-Vibrio growth is somehow related to the VvR-21 strain and not due to natural resistance in the water sample. The Gm R cassette carried by VvR-21 is flanked by the RFP gene and a Cm R cassette (Thorstenson and Ullrich, 2020). Accordingly, non-Vibrio bacterial growth were streaked for isolation on Gm-and Cm-containing media.…”