flexneri phage SfV, E. coli prophage e14 and lambda. The characterization of serotype-converting phage SfI enhances our understanding of serotype conversion of S. flexneri. Methods Bacterial strains, media and culture S. flexneri serotype 1a Small molecule library manufacturer strain 019 [16] was used as the source for induction of phage SfI. S. flexneri strain 036 (serotype Y) was used as the host for phage infection and large volume propagation of SfI [16]. One hundred and thirty two S. flexneri strains of 12 serotypes (17 serotype 1a, 5

serotype 1b, 10 serotype 2a, 10 serotype 2b, 10 serotype 3a, 2 serotype 3b, 5 serotype 4a, 5 serotype 4b, 4 serotype 5a, 10 serotype find more Y, 24 serotype X and 30 serotype Xv) were used for phage host range

detection. All S. flexneri strains learn more used in this study were isolated from diarrheal patients in China, or purchased from National Collection of Type Cultures (NCTC), UK. S. flexneri strains were serologically identified using Shigella antisera Kits (Denka Seiken, Japan) and monoclonal antibody reagents (Reagensia AB, Sweden). S. flexneri strains were routinely cultured on LB agar or in LB broth with shaking at 37°C. Induction of phage SfI Induction of phage SfI was performed as methods described by Mavris et al.[8]. Briefly, a freshly grown colony of strain 019 was incubated in 10 ml LB broth overnight with vigorous shaking. After being induced for 30 min at 56°C with aeration, the cultures were centrifuged, and the supernatants were filtered through a 0.22 mm membrane filter (Promega) to remove bacterial cells. The filtrates were either used directly for phage infection assay or stored Silibinin at 4°C with addition of 10%

(v/v) chloroform. Phage infection and lysogenization S. flexneri strain 036 cells were prepared using the methods for phage lambda [29]. Phage infection and lysogenization were performed using the methods described previously [16]. The serotypes of isolated colonies were identified by slide agglutination assay. Large volume phage purification was performed on S. flexneri strain 036, according to the methods for phage SfII [8]. Electron microscopy The purified phages were absorbed on carbon-coated copper grids (300 mesh) and negatively stained with 2% (w/v) sodium phosphotungstate (pH 7.0). Samples were visualized with a Hitachi 600 electron microscope at 80 kV. Host range detection To determine the host range of phage SfI, one hundred and thirty two S. flexneri strains of 12 serotypes were infected with SfI. The preparation of component cells, phage infection and lysogen isolation were performed as methods for strain 036 above. The SfI host range was determined by observing the presence of plaques and serologically identification of the lysogens.