A novel murine infection model for Shiga toxin-producing Escherichia coli

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates attaching and effacing (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease

Gut Microbiome Perturbations Induced by Bacterial Infection Affect Arsenic Biotransformation

Exposure to arsenic affects large human populations worldwide and has been associated with a long list of human diseases, including skin, bladder, lung, and liver cancers, diabetes, and cardiovascular disorders. In addition, there are large individual differences in susceptibility to arsenic-induced diseases, which are frequently associated with different patterns of arsenic metabolism. Several underlying mechanisms, such as genetic polymorphisms and epigenetics, have been proposed, as these factors closely impact the individuals’ capacity to metabolize arsenic. In this context, the role of the gut microbiome in directly metabolizing arsenic and triggering systemic responses in diverse organs raises the possibility that perturbations of the gut microbial communities affect the spectrum of metabolized arsenic species and subsequent toxicological effects. In this study, we used an animal model with an altered gut microbiome induced by bacterial infection, 16S rRNA gene sequencing, and inductively coupled plasma mass spectrometry-based arsenic speciation to examine the effect of gut microbiome perturbations on the biotransformation of arsenic. Metagenomics sequencing revealed that bacterial infection significantly perturbed the gut microbiome composition in C57BL/6 mice, which in turn resulted in altered spectra of arsenic metabolites in urine, with inorganic arsenic species and methylated and thiolated arsenic being perturbed. These data clearly illustrated that gut microbiome phenotypes significantly affected arsenic metabolic reactions, including reduction, methylation, and thiolation. These findings improve our understanding of how infectious diseases and environmental exposure interact and may also provide novel insight regarding the gut microbiome composition as a new risk factor of individual susceptibility to environmental chemicals.National Institute of Environmental Health Sciences (Massachusetts Institute of Technology. Center for Environmental Health Sciences Grant P30 ES002109)National Institute of Environmental Health Sciences (University of North Carolina. Center for Environmental Health and Susceptibility Grant P30 ES010126

Enteric Infection with Citrobacter rodentium Induces Coagulative Liver Necrosis and Hepatic Inflammation Prior to Peak Infection and Colonic Disease

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.United States. Army Research Office (Institute for Soldier Nanotechnology grant 6915539 (SRT))National Institutes of Health (U.S.) (Grant P01 CA026731)National Institutes of Health (U.S.) (Grant P30 ES02109)National Institutes of Health (U.S.) (Toxicology Training grant ES-070220

PLS-DA and OPLS component contributions for discrimination (R² Y) and variance (Q²) of necrosis at 3 DPI.

<p>Model results are indicated based on using serum or tissue targets alone as well as combined models using the top variables from each independent model.</p

<i>C. rodentium</i>-induced colonic effects in C57BL/6 mice.

<p>(<b>A</b>) Mock inoculated animals at 0 DPI with normal colonic architecture where epithelial integrity and goblet cells appear intact. (<b>B</b>) Colon at 3 DPI showing epithelial defects at the top of the crypt. (<b>C</b>) Colon at 14 DPI demonstrating hyperplastic crypts and depletion of goblet cells. (<b>D</b>) <i>C. rodentium</i> induced statistically significant histological changes as early as 7 DPI (inflammation, edema, epithelial defects) in colonic sections and found to be most dramatic at 14 DPI. Crypt atrophy and minimal dysplastic changes were only noticeable at 14 DPI. Changes in inflammation, edema, epithelial defects, and hyperplasia as early as 3 DPI were noted, but failed to reach statistical significance (Kruskal-Wallis non-parametric test with Dunn's multiple comparison test: <b>*</b> P<0.05, <b>**</b> P<0.01, <b>***</b> P<0.001). Symbols indicate individual animals and lines indicate group means.</p

Serum-specific OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI.

<p>OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI using serum cytokines and chemistries (X) for prediction of ALT levels (Y). (<b>A</b>) Mice segregated well in the predictive component (principal component 1) with an R²X (1) = 0.35, indicating this component captured ∼35% of the variance present in the X variables. (<b>B</b>) The predictive weight and covariation of serum targets (<b>X variables, </b><b>black triangles</b>) in relation to serum ALT (<b>Y, </b><b>red square</b>). (<b>C</b>) The variables that best separate the two pathological states in relation to the predictive component. (<b>D</b>) Variables in projection (VIPs) for the predictive component where values >1 are have positive influence in determining ALT levels, and VIP <1 have less predictive influence. (<b>E</b>) Observed vs predicted plot for ALT resulted in a R² = 0.9892 indicating a highly predictive model based on serum cytokines. (<b>F</b>) Table representing the VIPs for the predictive component for serum ALT.</p

<i>C. rodentium</i>-induced necrosis and histological liver changes in C57BL/6 mice.

<p>(<b>A</b>) Control livers appeared normal with minimal observable histology. (<b>B</b>) At 3 DPI the appearance of a focus of lobular injury and inflammation indicating a pro-inflammatory state (indicated by <b>#</b>). (<b>C</b>) Multifocal venous thrombi and associated periportal hepatocellular coagulative necrosis (indicated by <b>*</b>) was observed at 3 DPI suggestive of thrombotic ischemic injury. (<b>D</b>) Higher magnification (400×) view showing necrotic hepatocytes with eosinophilic cytoplasm, appearance of pyknotic or absence of hepatic nuclei, and loss of normal cellular architecture. (<b>E</b>) <i>C. rodentium</i> induced histological changes as early as 3 DPI in liver sections, statistically significant at 7 DPI (portal, lobular, interface inflammation, # lobes with >5 inflammatory foci, and hepatitis index score), with moderate improvement by 14 DPI. (<b>F</b>) The degree of necrosis determined by pathological assessment as well as serum ALT measurements. (<b>G</b>) The pattern of necrosis was assessed as centrilobular, midzonal, or periportal in distribution. (Kruskal-Wallis with Dunn's post test compared to controls: <b>*</b> P<0.05, <b>**</b> P<0.01, <b>***</b> P<0.001). Symbols indicate individual animals and lines indicate group means.</p

Serum-specific PLS-DA analysis of <i>C. rodentium</i> infected C57BL/6 mice at 3 DPI.

<p>(<b>A</b>) Partial least squares discriminate analysis (PLS-DA) showing separation of mice with and without necrosis using the first two principal components. (<b>B</b>) Cytokine covariation based on class discrimination using cytokine targets as independent variables (<b>X, black triangles</b>) and pathological states (presence of absence of necrotic lesions) as the dependent dummy variables (<b>Y, red squares</b>). (<b>C</b>) Variables in projection (VIPs) for principal components 1 and 2, where targets with values >1 have positive influence in discriminating between classes. Table represents the serum-specific VIPs and their respective scores that best discriminate necrotic from non-necrotic mice at 3 DPI.</p