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TheShigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects ofShigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence inShigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of theEnterobacteriaceae . Characterization ofS. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile inS. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate thatS. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activateS. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulatesShigella survival and virulence during host transit and subsequent colonic infection.

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts