Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

ABSTRACTA healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

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Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids

Infection and Immunity ◽

10.1128/iai.00045-20 ◽

2020 ◽

Vol 88 (6) ◽

Author(s):

Jenessa A. Winston ◽

Alissa J. Rivera ◽

Jingwei Cai ◽

Rajani Thanissery ◽

Stephanie A. Montgomery ◽

...

Keyword(s):

Immune Response ◽

Bile Acid ◽

Inflammatory Response ◽

Innate Immune Response ◽

Innate Immune ◽

Colonization Resistance ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficile in vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficile in vivo. However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro. In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

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Repurposing the Antiamoebic Drug Diiodohydroxyquinoline for Treatment of Clostridioides difficile Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02115-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 1

Author(s):

Nader S. Abutaleb ◽

Mohamed N. Seleem

Keyword(s):

Toxin Production ◽

New Drugs ◽

Recurrence Rates ◽

Content Type ◽

Clostridioides Difficile ◽

Key Species ◽

Bacterial Inoculum ◽

Antiparasitic Drugs ◽

Time Kill

ABSTRACT Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at concentrations of 0.5 μg/ml and 2 μg/ml, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3 log10 within 6 h. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficile in vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit the growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium, and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

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In vitro antivirulence activity of baicalin against Clostridioides difficile

Journal of Medical Microbiology ◽

10.1099/jmm.0.001179 ◽

2020 ◽

Vol 69 (4) ◽

pp. 631-639

Author(s):

Abraham Joseph Pellissery ◽

Poonam Gopika Vinayamohan ◽

Kumar Venkitanarayanan

Keyword(s):

Spore Germination ◽

Type Species ◽

Brain Heart Infusion ◽

Toxin Production ◽

Strictly Anaerobic ◽

Content Type ◽

Link Type ◽

Clostridioides Difficile ◽

Spore Outgrowth

Introduction. Clostridioides difficile is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize C. difficile pathogenicity and disease outcome in affected individuals. Aim. This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in C. difficile in vitro. Methodology. Hypervirulent C. difficile isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining C. difficile toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, C. difficile spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on C. difficile toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR. Results. The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, C. difficile genes critical for pathogenesis were significantly down-regulated in the presence of baicalin. Conclusion. Our results suggest that baicalin could potentially be used to control C. difficile , and warrant future studies in vivo.

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Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽

10.1128/msphere.00963-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Diarrheal Disease ◽

Toxin Production ◽

The United States ◽

Upstream Region ◽

Pcr Analysis ◽

Bacterial Cells ◽

Master Regulator ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽

10.1128/mbio.01559-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Nicolas Kint ◽

Carolina Alves Feliciano ◽

Maria C. Martins ◽

Claire Morvan ◽

Susana F. Fernandes ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Anaerobic Bacteria ◽

Growth Defect ◽

Strictly Anaerobic ◽

Low Oxygen ◽

Air Exposure ◽

Vegetative Cells ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

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RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽

10.1128/mbio.01991-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 10

Author(s):

Adrianne N. Edwards ◽

Brandon R. Anjuwon-Foster ◽

Shonna M. McBride

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Toxin Production ◽

Toxin Gene ◽

Dna Binding Domain ◽

Content Type ◽

Toxin Genes ◽

Clostridioides Difficile ◽

Species Specific ◽

Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

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Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization

mSphere ◽

10.1128/msphere.00780-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 3

Author(s):

Olivia A. Todd ◽

Mairi C. Noverr ◽

Brian M. Peters

Keyword(s):

Staphylococcus Aureus ◽

Candida Albicans ◽

Quorum Sensing ◽

Bacterial Pathogen ◽

Toxin Production ◽

Extracellular Ph ◽

Morbidity And Mortality ◽

Alpha Toxin ◽

Content Type

ABSTRACT Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro. Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans. Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

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Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa062 ◽

2020 ◽

Vol 75 (6) ◽

pp. 1458-1465

Author(s):

C H Chilton ◽

G S Crowther ◽

C Miossec ◽

J de Gunzburg ◽

A Andremont ◽

...

Keyword(s):

Limit Of Detection ◽

Toxin Production ◽

Human Gut ◽

Antibiotic Resistant ◽

Delayed Onset ◽

Clostridioides Difficile ◽

Clostridioides Difficile Infection ◽

Clinical Benefits ◽

Bacteroides Fragilis Group

Abstract Background Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. Objectives We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. Methods C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. Results DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). Conclusions DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

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Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming ActinobacteriumCorynebacterium matruchotii

Journal of Bacteriology ◽

10.1128/jb.00783-17 ◽

2018 ◽

Vol 200 (9) ◽

pp. e00783-17 ◽

Cited By ~ 5

Author(s):

Truc Thanh Luong ◽

Reyhaneh Tirgar ◽

Melissa E. Reardon-Robinson ◽

Andrzej Joachimiak ◽

Jerzy Osipiuk ◽

...

Keyword(s):

Disulfide Bond ◽

Gene Deletion ◽

Bond Formation ◽

Toxin Production ◽

Content Type ◽

Cellular Processes ◽

Disulfide Oxidoreductase ◽

New Gene ◽

Resolution Structure

ABSTRACTThe actinobacteriumCorynebacterium matruchotiihas been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of theC. matruchotiigenome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to theCorynebacterium diphtheriaethiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure ofC. matruchotiiMdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machinein vitro, we demonstrated that MdbACmcatalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported thatmdbAis essential inC. matruchotii. Remarkably, heterologous expression of MdbACmin theC. diphtheriaeΔmdbAmutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACmis a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding inC. matruchotiiby a mechanism that is conserved inActinobacteria.IMPORTANCEThe actinobacteriumCorynebacterium matruchotiihas been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of aC. matruchotiioxidoreductase that is highly homologous to theCorynebacterium diphtheriaethiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated thatC. matruchotiiMdbA catalyzes disulfide bond formationin vitro. Furthermore, a new gene deletion method revealed that deletion ofmdbAis lethal inC. matruchotii. Remarkably,C. matruchotiiMdbA can replaceC. diphtheriaeMdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis thatC. matruchotiiutilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.

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Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity

Scientific Reports ◽

10.1038/s41598-020-65253-0 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Aleksander Mahnic ◽

Jennifer M. Auchtung ◽

Nataša Poklar Ulrih ◽

Robert A. Britton ◽

Maja Rupnik

Keyword(s):

Colonization Resistance ◽

Clostridioides Difficile

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