TABLE 3.

Phenotypic characterization of V. vulnificus grown at different temperatures.

Condition	Biofilm (Abs540)a	Motility rateb	Cellular-associated polysaccharides (μg/108 cells)c	Proteolytic activity (PU)d
20°C	0.027 ± 0.002	0.24 ± 0.02*	87.67 ± 0.37*	12.09 ± 6.65*
25°C	0.034 ± 0.005	1.56 ± 0.08#	95.37 ± 1.32*#	6216.17 ± 250.55#
28°C	0.035 ± 0.016	1.40 ± 0.31#	103.55 ± 2.77#	6498.76 ± 210.18#
37°C	0.066 ± 0.048	2.46 ± 0.60*#	108.68 ± 5.92#	4021.47 ± 181.49*#

^aFor biofilm quantification bacteria were grown in glass tubes for 24 h. Then, planktonic bacteria were eliminated and biofilm was quantified after staining with crystal violet by measuring absorbance at 540 nm (Abs₅₄₀). ^bMotility was measured as motility rate (colony surface in mm²/log of colony bacterial number in CFU). The assay was performed on plates of CM9-agar (0.3%) as described by Pajuelo et al. (2016). ^cTotal polysaccharide concentration was determined with Total Carbohydrate Assay Kit (BioVision) as described by the manufacturers. ^dProteolytic activity in ECPs, obtained according to Biosca and Amaro (1996), was evaluated using azocasein (Sigma) as substratum (Valiente et al., 2008b). Proteolytic activity is expressed as proteolytic units (PU) produced in each condition (Miyoshi et al., 1987). In all cases results are presented as the averages ± standard deviation of three independent experiments. *: significant differences (P < 0.05) at each temperature vs. 28°C. #: significant differences (P < 0.05) at each temperature vs. 20°C.