Alison D. O’Brien

Shiga Toxin–Producing Escherichia coli in Montana: Bacterial Genotypes and Clinical Profiles

The diseases and virulence genes associated with Shiga toxin-producing Escherichia coli (STEC) are characterized incompletely. We analyzed, by polymerase chain reaction, 82 STEC isolates collected prospectively in Montana and profiled associated illnesses by patient chart review. All E. coli O157:H7 contained stx2-group genes, as well as eae, iha, espA, and ehxA; 84% contained stx1. Non-O157:H7 STEC less frequently contained stx1 (P=.046), stx2 (P<.001), iha (P<.001), eae, and espA (P=.039 for both), were isolated less often from patients treated in emergency departments (P=.022), and tended to be associated less frequently with bloody diarrhea (P=.061). There were no significant associations between stx genotype and bloody diarrhea, but isolates containing stx2c or stx(2d-activatable) were recovered more often from patients who underwent diagnostic or therapeutic procedures (P=.033). Non-O157:H7 STEC are more heterogeneous and cause bloody diarrhea less frequently than do E. coli O157:H7. Bloody diarrhea cannot be attributed simply to the stx genotype of the infecting organism.

Pathogenesis of Shiga-Toxin Producing Escherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) are food-borne pathogens that cause hemorrhagic colitis and a serious sequela, the hemolytic uremic syndrome (HUS). The largest outbreaks of STEC are due to a single E. coli serotype, O157:H7, although non-O157 serotypes also cause the same diseases. Two immunologically distinct Stxs are found in E. coli, Stx1 and Stx2. The Stxs are AB₅ toxins that halt protein synthesis in the host cell, a process that may lead to an apoptotic cell death. Stx-mediated damage to renal glomerular endothelial cells is hypothesized as the precipitating event for HUS. A subset of STEC referred to as the enterohemorrhagic E. coli has the capacity to intimately attach to and efface intestinal epithelial cells, a pathology called the A/E lesion. The A/E lesion is mediated by the adhesin intimin, its bacterially encoded receptor, Tir, and effectors secreted through a type III secretion system. The proteins needed for the A/E lesion are encoded within a large pathogenicity island called the locus of enterocyte effacement or LEE. There are several animal models for STEC infection, but no one model fully represents the spectrum of STEC illness. Currently there is no cure for STEC infection, and therapies are based mainly on alleviating symptoms. However, chimeric or humanized monoclonal antibodies have been developed that neutralize the Stxs, and those therapies may be able to prevent the development of HUS in an STEC-infected patient.

Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease

Significance We demonstrated that dietary fiber content affects susceptibility to Shiga toxin (Stx)-producing Escherichia coli (STEC) infection in mice. We showed that high fiber diet (HFD)-fed mice had elevated levels of butyrate, a beneficial gut metabolite that paradoxically enhances the cell-killing capacity of Stx. We also found that the amount of gut bacteria in HFD-fed mice increased whereas the percent of commensal Escherichia species (spp) decreased compared with animals fed a low fiber diet (LFD). These changes led to higher E. coli O157:H7 colonization levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals. The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.

Influence of Host Genes on Resistance of Inbred Mice to Lethal Infection with Salmonella typhimurium

Salmonella typhi, the causative agent of typhoid fever, is avirulent for mice. The parenteral 50% lethal dose (LD50) of S. typhi for mice of all inbred strains examined to date is ≥108 bacteria (Gerichter 1960; Carter and Collins 1974a; O’Brien 1982) unless the animals are pretreated with iron and/or an iron chelater (Powell et al. 1980; O’Brien 1982) or the mice are challenged intraperitoneally with S. typhi suspended in hog gastric mucin (Nungester et al. 1936; Spaun 1964). By contrast, Salmonella typhimurium primarily evokes gastroenteritis in man, but causes a typhoid fever-like disease in mice (called murine typhoid). Both human typhoid and murine typhoid are systemic illnesses. In mice infected orally with S. typhimurium or in humans who ingest food or water contaminated with S. typhi, the bacteria either multiply in the small bowel or directly penetrate the intestinal mucosa without apparent enteric colonization (Gerichter 1960; Hornick etal. 1970; Hohman 1978). Studies in mice have established that the foci from which salmonellae disseminate are the Peyer’s patches of the small intestine (Carter and Collins 1974b; Hohman et al. 1978). The bacteria apparently gain access to the circulation via the lymphatics, seed the reticuloendothelial cell system (RES), and replicate within splenic and hepatic tissues.

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.

Ricin Crosses Polarized Human Intestinal Cells and Intestines of Ricin-Gavaged Mice without Evident Damage and Then Disseminates to Mouse Kidneys

Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

When a healthy diet turns deadly

The health benefits of a high fiber diet (HFD) result in part from the action of metabolic end products made by gut commensals on the host epithelium. Butyrate is one such beneficial metabolite; however, butyrate paradoxically enhances the capacity of Escherichia coli-produced Shiga toxin type 2 (Stx2) to kill tissue culture cells. We recently showed that mice fed an HFD exhibited increased butyrate in gut contents and had an altered intestinal microbiota with reduced numbers of Escherichia species. Furthermore, mice fed an HFD and infected with Stx-producing E. coli (STEC) were colonized to a higher degree, lost more weight and succumbed to infection at greater rates compared with STEC-infected low fiber diet animals. The HFD animals showed higher levels of the Stx receptor globotriaocylceramide (Gb3) in both the gut and kidneys. We speculate that an HFD that leads to increased intestinal butyrate and Gb3 in the intestines and kidneys may explain the higher rate of the hemolytic uremic syndrome in females over males.

Expression and contribution to virulence of each polysaccharide capsule of Bacillus cereus strain G9241

Bacillus cereus strain G9241 was isolated from a patient with pneumonia who had an anthrax-like illness. Like Bacillus anthracis, the virulence of G9241 is dependent on two large plasmids. In G9241 those plasmids are pBCXO1 and pBC210. There is a multi-gene capsule locus on each of these virulence plasmids, and both capsules are produced by G9241 in vitro and in mice. The hasACB operon on pBCXO1 is responsible for production of a hyaluronic acid (HA) capsule. The locus on pBC210 encodes a putative tetrasaccharide (TS) capsule that assembles in a Wzy-dependent manner. We found that the pBC210 capsule locus is transcribed as two operons and identified the promoter regions responsible for transcription. We constructed isogenic mutants to assess the role of genes in the two TS capsule operons in production of the capsule. Spores of strains deficient in production of either the HA or TS capsule were inoculated subcutaneously or intranasally into A/J and C57BL/6 mice to determine the lethal dose 50% of each bacterial mutant by each route of infection. The loss of the HA capsule attenuated G9241 more than the loss of the TS capsule for both infection routes in both mouse strains. Overall, our data further characterize the unique TS capsule on pBC210 and demonstrate that the two capsules do not have the same impact on virulence of G9241.

Genetic Control of Murine Resistance to Salmonella Typhimurium Infection

Mice infected with Salmonella typhimurium develop a disease which is similar in its pathogenesis to typhoid fever (42). This facultative intracellular organism multiplies in the phagocytic cells of the murine reticuloendothelial cell system, and unrestricted bacterial growth results in death of the host. However, mice of various inbred strains differ in response to S. typhimurium infection. Some strains of mice invariably succumb to infection with < 10 organisms, whereas other strains survive challenge doses of ≥ 104 bacteria (21, 24). Webster, Schott and Gowen (9, 34, 39–41) were the first to recognize that this differential susceptibility was genetically regulated, and they subsequently developed salmonella-susceptible and resistant mouse strains (39). Although their pioneering work was performed in the 1930’s, the delineation of the genes involved in resistance to murine typhoid occurred only recently.