Critical Role of Zinc in a New Murine Model of Enterotoxigenic Escherichia coli Diarrhea

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveler's diarrhea as well as of endemic diarrhea and stunting in children in developing areas. However, a small-mammal model has been badly needed to better understand and assess mechanisms, vaccines, and interventions. We report a murine model of ETEC diarrhea, weight loss, and enteropathy and investigate the role of zinc in the outcomes. ETEC strains producing heat-labile toxins (LT) and heat-stable toxins (ST) that were given to weaned C57BL/6 mice after antibiotic disruption of normal microbiota caused growth impairment, watery diarrhea, heavy stool shedding, and mild to moderate intestinal inflammation, the latter being worse with zinc deficiency. Zinc treatment promoted growth in zinc-deficient infected mice, and subinhibitory levels of zinc reduced expression of ETEC virulence genes cfa1, cexE, sta2, and degP but not of eltA in vitro. Zinc supplementation increased shedding and the ileal burden of wild-type (WT) ETEC but decreased shedding and the tissue burden of LT knockout (LTKO) ETEC. LTKO ETEC-infected mice had delayed disease onset and also had less inflammation by fecal myeloperoxidase (MPO) assessment. These findings provide a new murine model of ETEC infection that can help elucidate mechanisms of growth, diarrhea, and inflammatory responses as well as potential vaccines and interventions.

EnteropathogenicEscherichia coli(EPEC) Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations and Increased Intestinal Permeability in a Murine Model

“…Recently, a study demonstrated that mice are susceptible to EPEC colonization in an age and microbiota disruption-dependent manner, with the infant mice being more susceptible [30]. We have previously shown that disruption of intestinal microbiota using a broad-spectrum antibiotic cocktail enabled colonization of bacterial pathogens such as ETEC [48], C. jejuni [33] and S. flexneri [34] in C57BL/6 mice with mice developing diarrhea. This current study also used the same antibiotic cocktail to enable the assessment of disease outcomes as a result of EPEC infection.…”

“…A murine EPEC infection model able to induce changes in body weight and diarrhea, which are important outcomes in children infected by EPEC, has been needed [20]. Depletion of intestinal microbiota by antibiotics has been shown to be effective in promoting colonization by bacterial pathogens [32][33][34]. We therefore, tested whether pretreatment with antibiotics could enable the study of body weight and diarrhea in mice infected with EPEC (10 10 CFU) ( Fig 1A).…”

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice

“…We performed oral inoculations of S. flexneri 2a strain 2457T or ETEC strain H01407 (refs. 9,10 ). Mice were previously exposed to a broad-spectrum antibiotic cocktail in the drinking water (metronidazole 215 mg/L, colistin 850 U/mL, gentamicin 35 mg/L, and vancomycin 45 mg/L) for 3 days.…”

“…Quantitative analysis of S. flexneri and ETEC burdens Fresh fecal pellets were obtained from individual mice, DNA extracted, and quantitative real-time PCR performed 9,10 . DNA was isolated from fecal pellets using the QIAamp DNA stool mini kit (Qiagen).…”

“…Recently, a combined Shigella-ETEC vaccine (CVD 1208S-122), consisting of an attenuated S. flexneri 2a strain expressing the ETEC colonization factor antigen-1 (CFA/1) and the heat labile-enterotoxin (LTb), has been shown to be safe and immunogenic in guinea pigs 7,8 . In order to provide stronger evidence of its pre-clinical effects, we evaluated whether CVD 1208S-122 could induce antibody responses and confer protection against diarrhea and weight loss using two murine models of orally administered S. flexneri and ETEC infections in C57BL/6 mice 9,10 . Following antibiotic treatment, these models enable the study of clinical outcomes that mimic human disease in children induced by oral infection and enable the testing of interventions.…”