Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens

Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmentedP. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies ofP. piscicida on lawns of the naturally occurring marine pathogensVibrio vulnificus ,Vibrio parahaemolyticus ,Vibrio cholerae ,Photobacterium damselae , andShewanella algae . Inhibition also occurred on lawns ofStaphylococcus aureus but not onEscherichia coli O157:H7 orSalmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all threeP. piscicida strains. Direct overlay of thePseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface ofP. piscicida and a novel mechanism of direct transfer of these vesicles toV. parahaemolyticus . Vesicles digested holes inV. parahaemolyticus cells, while theP. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by whichP. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCE Pseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains ofP. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by whichP. piscicida killsV. parahaemolyticus . It involves the direct transfer of apparently lytic vesicles from the surface of thePseudoalteromonas strains to the surface ofVibrio cells, with subsequent digestion of holes in theVibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls.Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use ofP. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.