Ingrid L. Bergin

The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection

Clostridium difficile infection (CDI) arises in the setting of antibiotic administration where disruption of the normal indigenous gut microbiota leads to susceptibility to C. difficile colonization and colitis. Using a murine model of CDI, we demonstrate that changes in the community structure of the indigenous gut microbiota are associated with the loss of colonization resistance against C. difficile. Several antibiotic regimens were tested in combination for the ability to overcome colonization resistance, including a five antibiotic cocktail consisting of kanamycin, gentamicin, colistin, metronidazole, and vancomycin administered in drinking water for three days, a single intraperitoneal dose of clindamycin or 10 days of cefoperazone in drinking water. Following antibiotic treatment animals were challenged with 105 colony forming units of C. difficile strain VPI 10463 via oral gavage. Animals that received the antibiotic cocktail and clindamycin prior to C. difficile challenge followed one of two clinical courses, either becoming clinically ill and moribund within 2-4 days post challenge, or remaining clinically well. Animals that became clinically ill developed histologically severe colitis. These histopathologic findings were significantly less severe in animals that remained clinically well. Analysis of 16S rRNA gene sequences retrieved from gut tissue at necropsy demonstrated that Proteobacteria dominated the gut microbiota in clinically ill animals. In contrast, the gut microbial community of clinically well animals more closely resembled untreated animals, which were dominated by members of the Firmicutes. All animals that received cefoperazone treatment prior to C. difficile challenge were clinically ill and moribund by 2-5 days post challenge in a dose dependent manner. The gut communities in these animals were dominated by C.difficile suggesting that cefoperazone treatment resulted in a greater loss in colonization resistance. Thus, the severity of colitis that arises in this system reflects the interplay between the expansion of C. difficile in the gut community and the ecologic dynamics of the indigenous microbial community as it recovers from antibiotic perturbation. We demonstrate that altering the balance of these two opposing processes alters clinical outcome and thus may lead to novel preventative and therapeutic approaches for CDI.

Suppression of Clostridium difficile in the Gastrointestinal Tracts of Germfree Mice Inoculated with a Murine Isolate from the Family Lachnospiraceae

ABSTRACT The indigenous microbial community of the gastrointestinal (GI) tract determines susceptibility to Clostridium difficile colonization and disease. Previous studies have demonstrated that antibiotic-treated mice challenged with C. difficile either developed rapidly lethal C. difficile infection or were stably colonized with mild disease. The GI microbial community of animals with mild disease was dominated by members of the bacterial family Lachnospiraceae, while the gut community in moribund animals had a predominance of Escherichia coli. We investigated the roles of murine Lachnospiraceae and E. coli strains in colonization resistance against C. difficile in germfree mice. Murine Lachnospiraceae and E. coli isolates were cultured from wild-type mice. The ability of each of these isolates to interfere with C. difficile colonization was tested by precolonizing germfree mice with these bacteria 4 days prior to experimental C. difficile challenge. Mice precolonized with a murine Lachnospiraceae isolate, but not those colonized with E. coli, had significantly decreased C. difficile colonization, lower intestinal cytotoxin levels and exhibited less severe clinical signs and colonic histopathology. Infection of germfree mice or mice precolonized with E. coli with C. difficile strain VPI 10463 was uniformly fatal by 48 h, but only 20% mortality was seen at 2 days in mice precolonized with the Lachnospiraceae isolate prior to challenge with VPI 10463. These findings confirm that a single component of the GI microbiota, a murine Lachnospiraceae isolate, could partially restore colonization resistance against C. difficile. Further study of the members within the Lachnospiraceae family could lead to a better understanding of mechanisms of colonization resistance against C. difficile and novel therapeutic approaches for the treatment and prevention of C. difficile infection.

Helicobacter pylori Infection and High Dietary Salt Independently Induce Atrophic Gastritis and Intestinal Metaplasia in Commercially Available Outbred Mongolian Gerbils

Risk factors for development of gastric adenocarcinoma include high dietary salt and Helicobacter pylori infection. Few animal models exist for the laboratory investigation of these factors. We examined gastric pathology resulting from H. pylori infection and high dietary salt as independent variables in commercially available, outbred Mongolian gerbils. Gastric adenocarcinoma and its precursor lesion, intestinal metaplasia, have been previously reported in inbred Mongolian gerbils (MGS/Sea) infected either with clinical isolates of H. pylori or with the strain ATCC 43504. In contrast, we utilized outbred gerbils [Crl:(MON)] infected with the Sydney strain of H. pylori. After 37 weeks, five of five infected animals had atrophic gastritis and intestinal metaplasia. These lesions were similar in description and time of appearance to the lesions reported in inbred gerbils. Atrophic gastritis and intestinal metaplasia also developed in six of six uninfected, outbred gerbils fed a 2.5% salt diet for 56 weeks. In contrast to the H. pylori-infected animals, these lesions were present without concurrent gastric inflammation. The outbred Mongolian gerbil therefore provides an excellent animal model for the study of several gastric cancer risk factors.

Dynamics and Establishment of Clostridium difficile Infection in the Murine Gastrointestinal Tract

ABSTRACT Clostridium difficile infection (CDI) following antibiotic therapy is a major public health threat. While antibiotic disruption of the indigenous microbiota underlies the majority of cases of CDI, the early dynamics of infection in the disturbed intestinal ecosystem are poorly characterized. This study defines the dynamics of infection with C. difficile strain VPI 10463 throughout the gastrointestinal (GI) tract using a murine model of infection. After inducing susceptibility to C. difficile colonization via antibiotic administration, we followed the dynamics of spore germination, colonization, sporulation, toxin activity, and disease progression throughout the GI tract. C. difficile spores were able to germinate within 6 h postchallenge, resulting in the establishment of vegetative bacteria in the distal GI tract. Spores and cytotoxin activity were detected by 24 h postchallenge, and histopathologic colitis developed by 30 h. Within 36 h, all infected mice succumbed to infection. We correlated the establishment of infection with changes in the microbiota and bile acid profile of the small and large intestines. Antibiotic administration resulted in significant changes to the microbiota in the small and large intestines, as well as a significant shift in the abundance of primary and secondary bile acids. Ex vivo analysis suggested the small intestine as the site of spore germination. This study provides an integrated understanding of the timing and location of the events surrounding C. difficile colonization and identifies potential targets for the development of new therapeutic strategies.

Dose-dependent incidence of hepatic tumors in adult mice following perinatal exposure to bisphenol A.

Background: Bisphenol A (BPA) is a high production volume chemical with hormone-like properties that has been implicated as a potential carcinogen. Early-life exposure has been linked to increased risk for precancerous lesions in mammary and prostate glands and the uterus, but no prior study has shown a significant association between BPA exposure and cancer development. Objective: We explored the effects of BPA exposure during gestation and lactation on adult incidence of hepatic tumors in mice. Methods: Isogenic mice were perinatally exposed to BPA through maternal diets containing one of four environmentally relevant doses of BPA (0, 50 ng, 50 μg, or 50 mg per kilogram of diet), and we followed approximately one male and one female per litter until they were 10 months of age. Animals were tested for known risk factors for hepatocellular carcinoma, including bacterial and viral infections. Results: We found dose-dependent incidence of hepatic tumors in 10-month-old BPA-exposed mice. Of the offspring examined, 23% presented with hepatic tumors or preneoplastic lesions. We observed a statistically significant dose–response relationship, with an odds ratio for neoplastic and preneoplastic lesions of 7.23 (95% CI: 3.23, 16.17) for mice exposed to 50 mg BPA/kg diet compared with unexposed controls. Observed early disease onset, absence of bacterial or viral infection, and lack of characteristic sexual dimorphism in tumor incidence support a nonclassical etiology. Conclusions: To our knowledge, this is the first report of a statistically significant association between BPA exposure and frank tumors in any organ. Our results link early-life exposure to BPA with the development of hepatic tumors in rodents, and have potential implications for human health and disease. Citation: Weinhouse C, Anderson OS, Bergin IL, Vandenbergh DJ, Gyekis JP, Dingman MA, Yang J, Dolinoy DC. 2014. Dose-dependent incidence of hepatic tumors in adult mice following perinatal exposure to bisphenol A. Environ Health Perspect 122:485–491; http://dx.doi.org/10.1289/ehp.1307449

Novel calicivirus identified in rabbits, Michigan, USA.

This virus is distinct from rabbit hemorrhagic disease virus.

Rapid Kinetics of Size and pH-Dependent Dissolution and Aggregation of Silver Nanoparticles in Simulated Gastric Fluid

As silver nanoparticles (AgNPs) are used in a wide array of commercial products and can enter the human body through oral exposure, it is important to understand the fundamental physical and chemical processes leading to changes in nanoparticle size under the conditions of the gastrointestinal (GI) tract. Rapid AgNP growth was observed using nanoparticle tracking analysis with 30 s resolution over a period of 17 min in simulated gastric fluid (SGF) to explore rapid kinetics as a function of pH (SGF at pH 2, 3.5, 4.5 and 5), size (20 and 110 nm AgNPs), and nanoparticle coating (citrate and PVP). Growth was observed for 20 nm AgNP at each pH, decreasing in rate with increasing pH, with the kinetics shifting from second-order to first-order. The 110 nm AgNP showed growth at ≤3.5 pH, with no growth observed at higher pH. This behavior can be explained by the generation of Ag+ in acidic environments, which precipitates with Cl-, leading to particle growth and facilitating particle aggregation by decreasing their electrostatic repulsion in solution. These results highlight the need to further understand the importance of initial size, physicochemical properties, and kinetics of AgNPs after ingestion to assess potential toxicity.

TcsL Is an Essential Virulence Factor in Clostridium sordellii ATCC 9714

ABSTRACT Clostridium sordellii is an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis of C. sordellii infections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids into C. sordellii using RP4-mediated conjugation from Escherichia coli and have successfully used these techniques to insertionally inactivate the tcsL gene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections by C. sordellii ATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor in C. sordellii and opens the way for in-depth studies of this important human pathogen at the molecular level.

Repeated dose (28-day) administration of silver nanoparticles of varied size and coating does not significantly alter the indigenous murine gut microbiome

Abstract Silver nanoparticles (AgNPs) have been used as antimicrobials in a number of applications, including topical wound dressings and coatings for consumer products and biomedical devices. Ingestion is a relevant route of exposure for AgNPs, whether occurring unintentionally via Ag dissolution from consumer products, or intentionally from dietary supplements. AgNP have also been proposed as substitutes for antibiotics in animal feeds. While oral antibiotics are known to have significant effects on gut bacteria, the antimicrobial effects of ingested AgNPs on the indigenous microbiome or on gut pathogens are unknown. In addition, AgNP size and coating have been postulated as significantly influential towards their biochemical properties and the influence of these properties on antimicrobial efficacy is unknown. We evaluated murine gut microbial communities using culture-independent sequencing of 16S rRNA gene fragments following 28 days of repeated oral dosing of well-characterized AgNPs of two different sizes (20 and 110 nm) and coatings (PVP and Citrate). Irrespective of size or coating, oral administration of AgNPs at 10 mg/kg body weight/day did not alter the membership, structure or diversity of the murine gut microbiome. Thus, in contrast to effects of broad-spectrum antibiotics, repeat dosing of AgNP, at doses equivalent to 2000 times the oral reference dose and 100–400 times the effective in vitro anti-microbial concentration, does not affect the indigenous murine gut microbiome.

Effects of particle size and coating on toxicologic parameters, fecal elimination kinetics and tissue distribution of acutely ingested silver nanoparticles in a mouse model

Abstract Consumer exposure to silver nanoparticles (AgNP) via ingestion can occur due to incorporation of AgNP into products such as food containers and dietary supplements. AgNP variations in size and coating may affect toxicity, elimination kinetics or tissue distribution. Here, we directly compared acute administration of AgNP of two differing coatings and sizes to mice, using doses of 0.1, 1 and 10 mg/kg body weight/day administered by oral gavage for 3 days. The maximal dose is equivalent to 2000× the EPA oral reference dose. Silver acetate at the same doses was used as ionic silver control. We found no toxicity and no significant tissue accumulation. Additionally, no toxicity was seen when AgNP were dosed concurrently with a broad-spectrum antibiotic. Between 70.5% and 98.6% of the administered silver dose was recovered in feces and particle size and coating differences did not significantly influence fecal silver. Peak fecal silver was detected between 6- and 9-h post-administration and <0.5% of the administered dose was cumulatively detected in liver, spleen, intestines or urine at 48 h. Although particle size and coating did not affect tissue accumulation, silver was detected in liver, spleen and kidney of mice administered ionic silver at marginally higher levels than those administered AgNP, suggesting that silver ion may be more bioavailable. Our results suggest that, irrespective of particle size and coating, acute oral exposure to AgNP at doses relevant to potential human exposure is associated with predominantly fecal elimination and is not associated with accumulation in tissue or toxicity.