ETEC biofilms are regulated by magnesium and lactate bioavailability.

The prevailing dogma is that enterotoxigenic Escherichia coli (ETEC) use plasmid-borne colonization factors (CFs) to adhere to the intestinal epithelium, where the organisms proliferate and produce their diarrhea-causing virulence factors, the heat-stable (ST) and/or heat-labile (LT) enterotoxins. However, vaccines that target major CF antigens fail to induce complete protective immunity, indicating that ETEC may also use other antigens to colonize the small intestines. We previously demonstrated that ST intoxication limits magnesium bioavailability in the intestinal lumen, but the role of magnesium in ETEC pathogenesis has not been rigorously evaluated. Here, we demonstrate that addition of magnesium at concentrations found in the intestinal mucosa promotes biofilm formation in ETEC H10407 and other clinical isolates, especially in the presence of lactate. ETEC H10407 biofilms fail to express high levels of colonization factor antigen I(CFA/I) fimbriae, but ETEC H10407 biofilms remain significantly better than planktonic counterparts at adhering to intestinal epithelial cells. Furthermore, ETEC H10407 biofilms are more acid-resistant than their planktonic counterparts, indicating that biofilms may promote survival through gastric acidity. Finally, using intragastric infection of neonatal mice, ETEC H10407 biofilms are significantly more virulent than their planktonic counterparts. Scanning electron micrographs of biofilm-infected mice show ETEC H10407 adheres to small intestinal villi. Thus, ETEC may respond to changes in environmental conditions to alter adherence mechanisms. Therefore, identification of biofilm antigens should be prioritized in ETEC vaccine development.