Vibrio parahaemolyticus can resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that type I pili, type IV pili, and both flagellar systems contribute to V. parahaemolyticus persistence in Pacific oysters whereas type III secretion systems and phase variation do not.T he genus Vibrio consists of a group of bacteria that naturally inhabit aquatic environments worldwide. The human pathogen Vibrio parahaemolyticus is commonly found associated with shellfish, particularly oysters (1, 2). Depuration is a controlled process in which shellfish are placed into clean seawater to reduce the bacterial contaminants in their tissues. However, depuration is not very effective at reducing the numbers of V. parahaemolyticus cells in oysters (3), and little is known about the association of V. parahaemolyticus with oysters.Both type I and type IV pili are important for bacterial attachment to a variety of surfaces (4-6). V. parahaemolyticus encodes a homologue of type I pili that is most similar to the CsuA/B operon from Acinetobacter baumannii (7) and homologues of two wellstudied type IV pili from vibrios, the mannose-sensitive hemagglutinin (MSHA) and the chitin-regulated pilus (PilA) (http://img .jgi.doe.gov/) (8, 9). Vibrio vulnificus uses PilA for persistence in Crassostrea virginica (10, 11). Both type IV pili are involved in biofilm formation in V. parahaemolyticus (12). Flagella are often critical during early stages of bacterial colonization of a surface (13,14). V. parahaemolyticus possesses a single, polar flagellum used for movement in liquid (reviewed in reference 14) as well as peritrichous (lateral) flagella for surface movement (15-17 and reviewed in reference 18). Many bacterial pathogens use type III secretion systems (T3SSs) for survival in the host by injecting virulence factors directly into the host. V. parahaemolyticus encodes two T3SSs that exhibit different phenotypes during disease (19)(20)(21). V. parahaemolyticus also exhibits "phase variation" and can switch from an opaque (OP) to a translucent (TR) phenotype based on polysaccharide production (22). In this study, we examined the role of V. parahaemolyticus pili, flagella, phase variation, and T3SSs in persistence in the Pacific oyster, Crassostrea gigas.Oysters were collected from Oregon Oyster Farm (Newport, OR) and exposed to ϳ10 5 CFU/ml of V. parahaemolyticus for 16 to 18 h at room temperature (ϳ20°C) with a recirculating pump. Algae were added to the tank to facilitate uptake according to the manufacturer's recommendation (Phytoplex; Kent Marine). Depuration was conducted at 19 to 20°C for 48 to 72 h. At each time point, animals from each exposure tank were weighed, homogenized, and diluted with phosphate-buffered saline (PBS) for enumeration on tryptic soy agar (TSA) supplemented with 1.5% NaCl and either 100 g/ml streptomycin, 60 g/ml chloramphenicol, 10 g/ml phosphomycin, or 50 g/ml kanamycin, depending on the strain (see Table S1 in the supplemental material), to eliminate naturally occurring bac...
