, 2004). The vast majority of bacteria in most natural, industrial, and clinical environments

live in biofilms and not as free-living or ‘planktonic’ cells that are commonly studied in the laboratory. Biofilms play a key role in the pathogenesis of many bacterial infections (Parsek & Singh, 2003). Many bacteria and pathogens utilize a biofilm strategy Ion Channel Ligand Library to survive inhospitable conditions and cause disease, including Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Escherichia coli, and Enterobacter (Bower & Daeschel, 1999; Stepanovic et al., 2004; Hanning et al., 2009; Holmberg et al., 2009; Jain & Agarwal, 2009; Yamanaka et al., 2009). Current research has shown that bacteria in a biofilm have increased resistance to host defenses and antimicrobial agents, making most biofilm infections difficult or impossible to be eradicated (Donlan & Costerton, Ku-0059436 cell line 2002; Grenier et al., 2009). However, the relationship between virulence and biofilm formation ability, and whether biofilms can exhibit altered virulence factors, has not been investigated. Therefore, the objective of this study was to determine whether biofilm cells can alter adherence, virulence, gene expression, and immunogenicity compared with planktonic cells, using an SS2 virulent strain and an avirulent strain. Three SS2 strains, two virulent and one avirulent, were used in this study. ZY05719 was isolated in Ziyang,

China, in 2005 and HA9801 was isolated by our laboratory in Jiangsu, China, in 1998. T15, the SS2 avirulent strain, was donated by Dr H. Smith (DLO-Institute for Animal Science and Health, the Netherlands)

(Vecht et al., 1997). All SS2 strains were grown in Todd–Hewitt broth (THB) at 37 °C. Planktonic cells and biofilm cell populations were investigated in this study. The collection of different cell suspensions was performed according to the method described previously (Hanning et al., 2009; Wong et al., 2010), with a few modifications. Briefly, for biofilm cultures, SS was grown in THB these medium supplemented with 1% fibrinogen in 100 mm polystyrene Petri dishes at 37 °C for 24 h. The supernatant was then removed and the plates were rinsed twice with phosphate-buffered saline (PBS, pH 7.2). Biofilms were detached by scraping and sonicated for 5 min (Bransonic 220; Branson Consolidated Ultrasonic Pty Ltd, Australia). All pelleted cultures were washed twice by resuspending pellets with vortexing; biofilm cells were collected by centrifugation. SS planktonic cells were grown in the same culture medium at 37 °C in a rotary shaker (180 r.p.m.). Planktonic cells were pelleted and washed as described in the biofilm cultures above. The production of biofilms by SS was tested using the protocol described by Grenier and colleagues, with a few modifications (Bonifait et al., 2008; Grenier et al., 2009). Briefly, an overnight culture of SS was diluted to an OD600 nm of 0.2 with fresh THB medium supplemented with 1% fibrinogen.