Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage

Abstract

Salmonella enterica serovar Typhi can colonize the gallbladder and persist in an asymptomatic carrier state that is frequently associated with the presence of gallstones. We have shown that salmonellae form bile-mediated biofilms on human gallstones and cholesterol-coated surfaces in vitro. Here, we test the hypothesis that biofilms on cholesterol gallbladder stones facilitate typhoid carriage in mice and men. Naturally resistant (Nramp1(+/+)) mice fed a lithogenic diet developed cholesterol gallstones that supported biofilm formation during persistent serovar Typhimurium infection and, as a result, demonstrated enhanced fecal shedding and enhanced colonization of gallbladder tissue and bile. In typhoid endemic Mexico City, 5% of enrolled cholelithiasis patients carried serovar Typhi, and bacterial biofilms could be visualized on gallstones from these carriers whereas significant biofilms were not detected on gallstones from Escherichia coli infected gallbladders. These findings offer direct evidence that gallstone biofilms occur in humans and mice, which facilitate gallbladder colonization and shedding.

Fig. 1.

Effects of an 8-week lithogenic diet (mouse chow supplemented with 1% cholesterol and 0.5% cholic acid) in mice naturally resistant to serovar Typhimurium infections (n = 30, two independent experiments). (A) Cholesterol diet-induced gallstones in the gallbladder of Nramp1^+/+ 129 × 1/SvJ mice. (B) Gallbladder stones were removed and photographed with a 25-gauge needle for scale.

Fig. 2.

CFU enumerations (CFU/mL) from organs, bile, and gallstones of 129 × 1/SvJ mice fed normal chow or lithogenic diet at days 3, 7, and 21 post i.p.infections with 1.4 × 10⁴ serovar Typhimurium 14028s (n = 42, 21 mice for each diet group, 7 per time point; two individual experiments; means + SD are given). (A) Intake of excessive dietary cholesterol did not enhance general infection or spleen burden at any time point. (B) The presence of gallstones significantly increased the amount of salmonellae recovered from gallbladder tissues at late time points compared with normal mouse chow controls during persistent murine infections. (C) Bacterial numbers from gallbladder bile increased significantly with time postinfection in mice fed a lithogenic diet for 8 weeks compared to infected mice fed normal chow. (D) Salmonellae were recovered from homogenized gallstones, and CFU values increased over time postinfection. No bacteria were recovered from the gallstones of uninfected mice. *, statistical significance (P < 0.01) based on a two-tailed Student t test.

Fig. 3.

Serovar Typhimurium is shed in fecal matter from infected 129 × 1/SvJ mice. Mice harboring cholesterol gallstones shed three logs more serovar Typhimurium than those on a normal chow diet. Means + SD are given for two independent experiments using seven infected animals per diet group. *, statistical significance (P < 0.01) based on a two-tailed Student t test.

Fig. 4.

Serovar Typhimurium biofilm formation on cholesterol gallstones at late time points during chronic murine infection. (A) SEM micrograph showing the surface of a cholesterol gallstone taken from an uninfected 129 × 1/SvJ control mouse fed a lithogenic diet. B and C show bacterial biofilms on gallstones taken from two different mice infected with serovar Typhimurium for 21 days.

Fig. 5.

Gallbladder stones from an asymptomatic typhoid carrier in Mexico City support biofilm formation. SEM micrographs show serovar Typhi bacilli embedded in biofilms on the surfaces of gallstones at magnifications of 1,500× (A), 2,000× (B), 2,400× (C), and 16,000× (D).

Fig. 6.

Biofilm formation on human gallstones was observed only in patients seeking cholecystectomy that were positive for typhoid carriage. SEM micrographs show bacterial biofilms on gallstones of two asymptomatic typhoid carriers in A and B. (C) Biofilm formation was not detected on the gallstone of one of the serovar Typhi carriers. The unique surface texture and pigment were suggestive of black, calcium bilirubinate gallstones. (D) Absence of biofilm formation on gallbladder stones from a patient positive for E. coli and S. flexneri.