Casey M. Theriot

Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection

Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth.

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.

Hydrolysis of organophosphorus compounds by microbial enzymes

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.

Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine

Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine. ABSTRACT It is hypothesized that the depletion of microbial members responsible for converting primary bile acids into secondary bile acids reduces resistance to Clostridium difficile colonization. To date, inhibition of C. difficile growth by secondary bile acids has only been shown in vitro. Using targeted bile acid metabolomics, we sought to define the physiologically relevant concentrations of primary and secondary bile acids present in the murine small and large intestinal tracts and how these impact C. difficile dynamics. We treated mice with a variety of antibiotics to create distinct microbial and metabolic (bile acid) environments and directly tested their ability to support or inhibit C. difficile spore germination and outgrowth ex vivo. Susceptibility to C. difficile in the large intestine was observed only after specific broad-spectrum antibiotic treatment (cefoperazone, clindamycin, and vancomycin) and was accompanied by a significant loss of secondary bile acids (deoxycholate, lithocholate, ursodeoxycholate, hyodeoxycholate, and ω-muricholate). These changes were correlated to the loss of specific microbiota community members, the Lachnospiraceae and Ruminococcaceae families. Additionally, physiological concentrations of secondary bile acids present during C. difficile resistance were able to inhibit spore germination and outgrowth in vitro. Interestingly, we observed that C. difficile spore germination and outgrowth were supported constantly in murine small intestinal content regardless of antibiotic perturbation, suggesting that targeting growth of C. difficile will prove most important for future therapeutics and that antibiotic-related changes are organ specific. Understanding how the gut microbiota regulates bile acids throughout the intestine will aid the development of future therapies for C. difficile infection and other metabolically relevant disorders such as obesity and diabetes. IMPORTANCE Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine.

Interactions Between the Gastrointestinal Microbiome and Clostridium difficile

Antibiotics have significant and long-lasting effects on the intestinal microbiota and consequently reduce colonization resistance against pathogens, including Clostridium difficile. By altering the community structure of the gut microbiome, antibiotics alter the intestinal metabolome, which includes both host- and microbe-derived metabolites. The mechanisms by which antibiotics reduce colonization resistance against C. difficile are unknown yet important for development of preventative and therapeutic approaches against this pathogen. This review focuses on how antibiotics alter the structure of the gut microbiota and how this alters microbial metabolism in the intestine. Interactions between gut microbial products and C. difficile spore germination, growth, and toxin production are discussed. New bacterial therapies to restore changes in bacteria-driven intestinal metabolism following antibiotics will have important applications for treatment and prevention of C. difficile infection.

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.

Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains.

The toxin-producing bacterium C. difficile is the leading cause of antibiotic-associated colitis, with an estimated 500,000 cases C. difficile infection (CDI) each year in the US with a cost approaching 3 billion dollars. Despite the significance of CDI, the pathogenesis of this infection is still being defined. The recent development of tractable murine models of CDI will help define the determinants of C. difficile pathogenesis in vivo. To determine if cefoperazone-treated mice could be utilized to reveal differential pathogenicity of C. difficile strains, 5–8 week old C57BL/6 mice were pretreated with a 10 d course of cefoperazone administered in the drinking water. Following a 2-d recovery period without antibiotics, the animals were orally challenged with C. difficile strains chosen to represent the potential range of virulence of this organism from rapidly fatal to nonpathogenic. Animals were monitored for loss of weight and clinical signs of colitis. At the time of harvest, C. difficile strains were isolated from cecal contents and the severity of colitis was determined by histopathologic examination of the cecum and colon. Cefoperazone treated mice challenged with C. difficile strains VPI 10463 and BI1 exhibited signs of severe colitis while infection with 630 and F200 was subclinical. This increased clinical severity was correlated with more severe histopathology with significantly more edema, inflammation and epithelial damage encountered in the colons of animals infected with VPI 10463 and BI1. Disease severity also correlated with levels of C. difficile cytotoxic activity in intestinal tissues and elevated blood neutrophil counts. Cefoperazone treated mice represent a useful model of C. difficile infection that will help us better understand the pathogenesis and virulence of this re-emerging pathogen.

Alteration of the Murine Gastrointestinal Microbiota by Tigecycline Leads to Increased Susceptibility to Clostridium difficile Infection

ABSTRACT Antibiotics can play dual roles in Clostridium difficile infection (CDI); antibiotic treatment increases the risk of CDI, and antibiotics are used to treat CDI. The glycylcycline antibiotic tigecycline has broad antimicrobial activity, yet it is rarely associated with the development of CDI, presumably due to its activity against C. difficile. In this study, we investigated how tigecycline treatment affects the structure of the gut microbiota and susceptibility to CDI by treating mice with tigecycline (n = 20) or saline (n = 8) for 10 days. A sequence analysis of the bacterial 16S rRNA gene amplicons was used to monitor changes in the fecal microbiota. A subset of the mice was followed for 5 weeks after the end of treatment. The remaining mice were challenged with C. difficile strain VPI 10463 spores 2 days after the tigecycline treatment ended. Tigecycline treatment resulted in major shifts in the gut microbiota, including large decreases in Bacteroidetes levels and large increases in Proteobacteria levels. Mice with tigecycline-altered microbial communities were susceptible to challenge with C. difficile spores and developed clinical signs of severe CDI. Five weeks after the cessation of tigecycline treatment, the recovery of the bacterial community was incomplete and diversity was lower than in the untreated controls. Antibiotics with intrinsic activity against C. difficile can still alter the microbiota in a way that leads to susceptibility to CDI after discontinuation of the drug. These results indicate that microbiotic dynamics are key in the development of CDI, and a better understanding of these dynamics may lead to better strategies to prevent and treat this disease.

Metabolic Model-Based Integration of Microbiome Taxonomic and Metabolomic Profiles Elucidates Mechanistic Links between Ecological and Metabolic Variation

Studies characterizing both the taxonomic composition and metabolic profile of various microbial communities are becoming increasingly common, yet new computational methods are needed to integrate and interpret these data in terms of known biological mechanisms. Here, we introduce an analytical framework to link species composition and metabolite measurements, using a simple model to predict the effects of community ecology on metabolite concentrations and evaluating whether these predictions agree with measured metabolomic profiles. We find that a surprisingly large proportion of metabolite variation in the vaginal microbiome can be predicted based on species composition (including dramatic shifts associated with disease), identify putative mechanisms underlying these predictions, and evaluate the roles of individual bacterial species and genes. Analysis of gut microbiome data using this framework recovers similar community metabolic trends. This framework lays the foundation for model-based multi-omic integrative studies, ultimately improving our understanding of microbial community metabolism. ABSTRACT Multiple molecular assays now enable high-throughput profiling of the ecology, metabolic capacity, and activity of the human microbiome. However, to date, analyses of such multi-omic data typically focus on statistical associations, often ignoring extensive prior knowledge of the mechanisms linking these various facets of the microbiome. Here, we introduce a comprehensive framework to systematically link variation in metabolomic data with community composition by utilizing taxonomic, genomic, and metabolic information. Specifically, we integrate available and inferred genomic data, metabolic network modeling, and a method for predicting community-wide metabolite turnover to estimate the biosynthetic and degradation potential of a given community. Our framework then compares variation in predicted metabolic potential with variation in measured metabolites’ abundances to evaluate whether community composition can explain observed shifts in the community metabolome, and to identify key taxa and genes contributing to the shifts. Focusing on two independent vaginal microbiome data sets, each pairing 16S community profiling with large-scale metabolomics, we demonstrate that our framework successfully recapitulates observed variation in 37% of metabolites. Well-predicted metabolite variation tends to result from disease-associated metabolism. We further identify several disease-enriched species that contribute significantly to these predictions. Interestingly, our analysis also detects metabolites for which the predicted variation negatively correlates with the measured variation, suggesting environmental control points of community metabolism. Applying this framework to gut microbiome data sets reveals similar trends, including prediction of bile acid metabolite shifts. This framework is an important first step toward a system-level multi-omic integration and an improved mechanistic understanding of the microbiome activity and dynamics in health and disease. IMPORTANCE Studies characterizing both the taxonomic composition and metabolic profile of various microbial communities are becoming increasingly common, yet new computational methods are needed to integrate and interpret these data in terms of known biological mechanisms. Here, we introduce an analytical framework to link species composition and metabolite measurements, using a simple model to predict the effects of community ecology on metabolite concentrations and evaluating whether these predictions agree with measured metabolomic profiles. We find that a surprisingly large proportion of metabolite variation in the vaginal microbiome can be predicted based on species composition (including dramatic shifts associated with disease), identify putative mechanisms underlying these predictions, and evaluate the roles of individual bacterial species and genes. Analysis of gut microbiome data using this framework recovers similar community metabolic trends. This framework lays the foundation for model-based multi-omic integrative studies, ultimately improving our understanding of microbial community metabolism.

Microbial and metabolic interactions between the gastrointestinal tract and Clostridium difficile infection

Antibiotics disturb the gastrointestinal tract microbiota and in turn reduce colonization resistance against Clostridium difficile. The mechanism for this loss of colonization resistance is still unknown but likely reflects structural (microbial) and functional (metabolic) changes to the gastrointestinal tract. Members of the gut microbial community shape intestinal metabolism that provides nutrients and ultimately supports host immunity. This review will discuss how antibiotics alter the structure of the gut microbiota and how this impacts bacterial metabolism in the gut. It will also explore the chemical requirements for C. difficile germination, growth, toxin production and sporulation. Many of the metabolites that influence C. difficile physiology are products of gut microbial metabolism including bile acids, carbohydrates and amino acids. To restore colonization resistance against C. difficile after antibiotics a targeted approach restoring both the structure and function of the gastrointestinal tract is needed.