Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface

Abstract

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.

Figure 1. Effect of iron on growth of enteric bacteria.

Effect of various concentrations of ferric citrate on in vitro growth of (A) S. typhimurium, (B) C. freundii, (C) E. coli, (D) E. faecalis and (E) L. plantarum.

Figure 2. Effect of iron on bacterial adhesion to an epithelial monolayer.

Adhesion (mean+SD) of enteric bacteria to a monolayer of Caco-2 cells is given as percentage of the inoculum. A: S. typhimurium, n = 8. B: C. freundii, n = 4. C: E. coli, n = 6. D: E. faecalis, n = 6. E: L. plantarum, n = 5. Means without a common letter differ, P<0.05. Notably, adhesion data of S. typhimurium were derived from 4 separate experiments performed at 13, 15, 18 and 21 days post-seeding of Caco-2 cells. The fact that each experiment revealed the same trend is indicative for similar physiochemical properties of the monolayer at these time points.

Figure 3. Effect of iron on invasion of S. typhimurium into epithelial cells.

Invasion (mean+SD) of S. typhimurium into Caco-2 cells, n = 2. Invasion after 3.5 h is given as percentage of the inoculum. The inoculum was removed after 2 hours of adhesion time. Means of 0–10 µmol/L ferric citrate were compared by one-way ANOVA.

Figure 4. Effect of iron on the ability of S. typhimurium to cross and deteriorate an epithelial monolayer.

Effect of iron on the ability of S. typhimurium to cross an epithelial monolayer of Caco-2 cells and the integrity of this monolayer. A: The translocation is given as percentage of the inoculum (mean+SD), n = 2. Means without a common letter differ, P<0.07. B: The integrity of the Caco-2 monolayer during S. typhimurium (St) and L. plantarum (Lp) translocation, monitored by TEER measurements.

Figure 5. Effect of iron on the ability of enteric bacteria to induce cell damage.

LDH-release (mean+SD) as a measure of cell damage of Caco-2 cells upon co-incubation with S. typhimurium (St, n = 5), C. freundii (Cf, n = 4), E. coli (Ec, n = 4), E. faecalis (Ef, n = 4), and L. plantarum (Lp, n = 2) pre-incubated with or without ferric citrate. The percentage LDH release compared to the control (no bacteria) was corrected for the number of bacteria in the medium (average between t = 0 and t = 2 h). Means within a group and without a common letter differ significantly, P<0.05.