Although typhoid fever has been intensively studied, chronic typhoid carriage 
still represents a problem for the transmission and persistence of the disease 
in areas of endemicity. This chronic state is highly associated with the 
presence of gallstones in the gallbladder of infected carriers upon which 
Salmonella can form robust biofilms. However, we hypothesize that in addition to 
gallstones, the gallbladder epithelium aids in the establishment/maintenance of 
chronic carriage. In this work, we present evidence of the role of the 
gallbladder epithelium in chronic carriage by a mechanism involving invasion, 
intracellular persistence, and biofilm formation. Salmonella was able to adhere 
to and invade polarized gallbladder epithelial cells apically in the absence and 
presence of bile in a Salmonella pathogenicity island 1 (SPI-1)-dependent 
manner. Intracellular replication of Salmonella was also evident at 12 and 24 h 
postinvasion. A flowthrough system revealed that Salmonella is able to adhere to 
and form extensive bacterial foci on gallbladder epithelial cells as early as 12 
h postinoculation. In vivo experiments using a chronic mouse model of typhoid 
carriage showed invasion and damage of the gallbladder epithelium and lamina 
propria up to 2 months after Salmonella infection, with an abundant presence of 
macrophages, a relative absence of neutrophils, and extrusion of infected 
epithelial cells. Additionally, microcolonies of Salmonella cells were evident 
on the surface of the mouse gallbladder epithelia up to 21 days postinfection. 
These data reveal a second potential mechanism, intracellular persistence and/or 
bacterial aggregation in/on the gallbladder epithelium with luminal cell 
extrusion, for Salmonella maintenance in the gallbladder.