Effect of salinity and incubation time of planktonic cells on biofilm formation, motility, exoprotease production, and quorum sensing of Aeromonas hydrophila

• Salinity controls biofilms formation by Aeromonas hydrophila.
• Planktonic age is important for controlling biofilms and quorum sensing.
• Salinity modulates quorum sensing, motility and exoprotease activity.
• Water salinity contributes to biofilms formation on crab shell by A. hydrophila.

The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by Aeromonas hydrophila on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30 °C for 24 h and 96 h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but Aeromonas-selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain Chromobacterium violaceum CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with Vibrio harveyi BB170. The biofilm structure at various salinities (0–3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of A. hydrophila grown at 0–0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1–3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0–0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1–3%) induced stress in A. hydrophila. The present study provides insight into the ecology of A. hydrophila growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater.