Clostridioides difficile colonization is not mediated by bile salts and requires Stickland fermentation of proline in an in vitro model of infection.

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile.

IMPORTANCE: Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile.

Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is anaerobic bacterium which colonizes the mammalian GI tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucus layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well-understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed that defects in adhesion by mutants deficient in flagella, but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus.

Identification of Simplified Microbial Communities That Inhibit Clostridioides difficile Infection through Dilution/Extinction.

The gastrointestinal microbiome plays an important role in limiting susceptibility to infection with Clostridioides difficile To better understand the ecology of bacteria important for C. difficile colonization resistance, we developed an experimental platform to simplify complex communities of fecal bacteria through dilution and rapidly screen for their ability to resist C. difficile colonization after challenge, as measured by >100-fold reduction in levels of C. difficile in challenged communities. We screened 76 simplified communities diluted from cultures of six fecal donors and identified 24 simplified communities that inhibited C. difficile colonization in vitro Sequencing revealed that simplified communities were composed of 19 to 67 operational taxonomic units (OTUs) and could be partitioned into four distinct community types. One simplified community could be further simplified from 56 to 28 OTUs through dilution and retain the ability to inhibit C. difficile We tested the efficacy of seven simplified communities in a humanized microbiota mouse model. We found that four communities were able to significantly reduce the severity of the initial C. difficile infection and limit susceptibility to disease relapse. Analysis of fecal microbiomes from treated mice demonstrated that simplified communities accelerated recovery of indigenous bacteria and led to stable engraftment of 19 to 22 OTUs from simplified communities. Overall, the insights gained through the identification and characterization of these simplified communities increase our understanding of the microbial dynamics of C. difficile infection and recovery.IMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrhea and a significant health care burden. Fecal microbiota transplantation is highly effective at treating recurrent C. difficile disease; however, uncertainties about the undefined composition of fecal material and potential long-term unintended health consequences remain. These concerns have motivated studies to identify new communities of microbes with a simpler composition that will be effective at treating disease. This work describes a platform for rapidly identifying and screening new simplified communities for efficacy in treating C. difficile infection. Four new simplified communities of microbes with potential for development of new therapies to treat C. difficile disease are identified. While this platform was developed and validated to model infection with C. difficile, the underlying principles described in the paper could be easily modified to develop therapeutics to treat other gastrointestinal diseases.

Amyloid Beta peptides differentially affect hippocampal theta rhythms in vitro.

Soluble amyloid beta peptide (A ß ) is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble A ß alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different A ß peptides, we also compared Aß 25-35 and Aß 1-42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 µ M). We found that Aß 25-35 reduces, with less potency than Aß 1-42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aß 25-35 but was reduced by Aß 1-42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.