Cefoperazone-treated Mouse Model of Clinically-relevant <em>Clostridium difficile</em> Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20–30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile

Novel therapies and preventative strategies for primary and recurrent Clostridium difficile infections

Clostridium difficile is the leading infectious cause of antibiotic‐associated diarrhea and colitis. C. difficile infection (CDI) places a heavy burden on the healthcare system, with nearly half a million infections yearly and an approximate 20% recurrence risk after successful initial therapy. The high incidence has driven new research on improved prevention such as the emerging use of probiotics, intestinal microbiome manipulation during antibiotic therapies, vaccinations, and newer antibiotics that reduce the disruption of the intestinal microbiome. While the treatment of acute C. difficile is effective in most patients, it can be further optimized by adjuvant therapies that improve the initial treatment success and decrease the risk of subsequent recurrence. Finally, the high risk of recurrence has led to multiple emerging therapies that target toxin activity, recovery of the intestinal microbial community, and elimination of latent C. difficile in the intestine. In summary, CDIs illustrate the complex interaction among host physiology, microbial community, and pathogen that requires specific therapies to address each of the factors leading to primary infection and recurrence.Clostridium difficile is the leading infectious cause of antibiotic‐associated diarrhea and colitis. This has driven new research on improving the prevention, primary treatment, and reduction of recurrence of C. difficile infection. This review summarizes current therapy recommendations for C. difficile infection, indicates areas for improvement, and describes the new emerging drugs and treatments that we hope will address these areas.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147228/1/nyas13958.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147228/2/nyas13958_am.pd

Probiotic colonization dynamics after oral consumption of VSL#3® by antibiotic-treated mice

Background: The ability of probiotic strains to provide health benefits to the host partially hinges on the survival of gastrointestinal passage and temporary colonization of the digestive tract. This study aims to investigate the colonization profile of individual probiotic strains comprising the commercial product VSL#3® and determine their impact on the host intestinal microbiota.Methods: Using a cefoperazone-treated mouse model of antibiotic treatment, we investigated the impact of oral gavage with ~108 CFU commercial VSL#3® product on the intestinal microbiota using 16S-based amplicon sequencing over 7 days. Results: Results showed that probiotic strains in the formulation were detected in treated murine fecal samples, with early colonization by Streptococcus thermophilus and Lactiplantibacillus plantarum subsp. plantarum, and late colonization by Lacticaseibacillus paracasei subsp. paracasei, Bifidobacterium breve and Bifidobacterium animalis subsp. lactis. Overall, VSL#3® consumption is associated with increased alpha diversity in the cecal microbial community, which is important in the context of antibiotic consumption. Probiotic supplementation resulted in an expansion of Proteobacteria, Bacteroidetes, and Actinobacteria, especially Bifidobacteriaceae and Lachnospiraceae, which are associated with Clostridioides difficile resistance in the murine gut.Conclusion: This study illustrates the need for determining the ability of probiotics to colonize the host and impact the gut microbiota, and suggests that multiple doses may be warranted for extended transient colonization. In addition, follow-up studies should determine whether VSL#3® can provide resistance against C. difficile colonization and disease in a mouse model

A Small Molecule-Screening Pipeline to Evaluate the Therapeutic Potential of 2-Aminoimidazole Molecules Against Clostridium difficile

Antibiotics are considered to be the first line of treatment for mild to moderately severe Clostridium difficile infection (CDI) in humans. However, antibiotics are also risk factors for CDI as they decrease colonization resistance against C. difficile by altering the gut microbiota and metabolome. Finding compounds that selectively inhibit different stages of the C. difficile life cycle, while sparing the indigenous gut microbiota is important for the development of alternatives to standard antibiotic treatment. 2-aminoimidazole (2-AI) molecules are known to disrupt bacterial protection mechanisms in antibiotic resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, but are yet to be evaluated against C. difficile. A comprehensive small molecule-screening pipeline was developed to investigate how novel small molecules affect different stages of the C. difficile life cycle (growth, toxin, and sporulation) in vitro, and a library of commensal bacteria that are associated with colonization resistance against C. difficile. The initial screening tested the efficacy of eleven 2-AI molecules (compound 1 through 11) against C. difficile R20291 compared to a vancomycin (2 μg/ml) control. Molecules were selected for their ability to inhibit C. difficile growth, toxin activity, and sporulation. Further testing included growth inhibition of other C. difficile strains (CD196, M68, CF5, 630, BI9, M120) belonging to distinct PCR ribotypes, and a commensal panel (Bacteroides fragilis, B. thetaiotaomicron, C. scindens, C. hylemonae, Lactobacillus acidophilus, L. gasseri, Escherichia coli, B. longum subsp. infantis). Three molecules compound 1 and 2, and 3 were microbicidal, whereas compounds 4, 7, 9, and 11 inhibited toxin activity without affecting the growth of C. difficile strains and the commensal microbiota. The antimicrobial and anti-toxin effects of 2-AI molecules need to be further characterized for mode of action and validated in a mouse model of CDI

Cefoperazone-treated Mouse Model of Clinically-relevant <em>Clostridium difficile</em> Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20–30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile

Image_1_A Small Molecule-Screening Pipeline to Evaluate the Therapeutic Potential of 2-Aminoimidazole Molecules Against Clostridium difficile.TIFF

<p>Antibiotics are considered to be the first line of treatment for mild to moderately severe Clostridium difficile infection (CDI) in humans. However, antibiotics are also risk factors for CDI as they decrease colonization resistance against C. difficile by altering the gut microbiota and metabolome. Finding compounds that selectively inhibit different stages of the C. difficile life cycle, while sparing the indigenous gut microbiota is important for the development of alternatives to standard antibiotic treatment. 2-aminoimidazole (2-AI) molecules are known to disrupt bacterial protection mechanisms in antibiotic resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, but are yet to be evaluated against C. difficile. A comprehensive small molecule-screening pipeline was developed to investigate how novel small molecules affect different stages of the C. difficile life cycle (growth, toxin, and sporulation) in vitro, and a library of commensal bacteria that are associated with colonization resistance against C. difficile. The initial screening tested the efficacy of eleven 2-AI molecules (compound 1 through 11) against C. difficile R20291 compared to a vancomycin (2 μg/ml) control. Molecules were selected for their ability to inhibit C. difficile growth, toxin activity, and sporulation. Further testing included growth inhibition of other C. difficile strains (CD196, M68, CF5, 630, BI9, M120) belonging to distinct PCR ribotypes, and a commensal panel (Bacteroides fragilis, B. thetaiotaomicron, C. scindens, C. hylemonae, Lactobacillus acidophilus, L. gasseri, Escherichia coli, B. longum subsp. infantis). Three molecules compound 1 and 2, and 3 were microbicidal, whereas compounds 4, 7, 9, and 11 inhibited toxin activity without affecting the growth of C. difficile strains and the commensal microbiota. The antimicrobial and anti-toxin effects of 2-AI molecules need to be further characterized for mode of action and validated in a mouse model of CDI.</p

The Stickland Reaction Precursor trans-4-Hydroxy-l-Proline Differentially Impacts the Metabolism of Clostridioides difficile and Commensal Clostridia

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. IMPORTANCE Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal Clostridia strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline in vitro. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile