A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

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Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽

10.1128/msphere.00423-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 11

Author(s):

Robert W. McKee ◽

Carissa K. Harvest ◽

Rita Tamayo

Keyword(s):

Gene Expression ◽

Clostridium Difficile ◽

Biofilm Formation ◽

Toxin Production ◽

Intestinal Colonization ◽

Cyclic Diguanylate ◽

Signaling Molecule ◽

Content Type ◽

Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

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Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽

10.1128/mbio.00183-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 106

Author(s):

Judith H. Merritt ◽

Dae-Gon Ha ◽

Kimberly N. Cowles ◽

Wenyun Lu ◽

Diana K. Morales ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Extracellular Polysaccharide ◽

Diguanylate Cyclase ◽

Flagellar Motility ◽

Critical Question ◽

Cyclic Diguanylate ◽

Critical Signal ◽

Content Type ◽

Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

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c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

mSphere ◽

10.1128/msphere.00919-21 ◽

2021 ◽

Author(s):

Adrianne N. Edwards ◽

Caitlin L. Willams ◽

Nivedita Pareek ◽

Shonna M. McBride ◽

Rita Tamayo

Keyword(s):

Life Cycle ◽

Biofilm Formation ◽

Toxin Production ◽

Cyclic Diguanylate ◽

Content Type ◽

Critical Step ◽

Clostridioides Difficile ◽

Physiological Processes ◽

Gastrointestinal Pathogen ◽

The Impact

Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile . However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen’s life cycle, is unknown.

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Interspecies Interactions between Clostridium difficile and Candida albicans

mSphere ◽

10.1128/msphere.00187-16 ◽

2016 ◽

Vol 1 (6) ◽

Cited By ~ 39

Author(s):

Pim T. van Leeuwen ◽

Jasper M. van der Peet ◽

Floris J. Bikker ◽

Michel A. Hoogenkamp ◽

Ana M. Oliveira Paiva ◽

...

Keyword(s):

Candida Albicans ◽

Clostridium Difficile ◽

Biofilm Formation ◽

Bacterial Disease ◽

Opportunistic Pathogens ◽

Human Gut ◽

Aerobic Conditions ◽

Content Type ◽

Hypha Formation ◽

Wide Range

ABSTRACT Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C. difficile can survive under ambient aerobic conditions, which would otherwise be toxic. We also found that C. difficile affects the hypha formation of C. albicans, most likely through the excretion of p-cresol. This ultimately leads to an inability of C. albicans to form a biofilm. Our study provides new insights into interactions between C. albicans and C. difficile and bears relevance to both fungal and bacterial disease. The facultative anaerobic polymorphic fungus Candida albicans and the strictly anaerobic Gram-positive bacterium Clostridium difficile are two opportunistic pathogens residing in the human gut. While a few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, the nature of the interactions between these two microbes has not been studied thus far. In the current study, both chemical and physical interactions between C. albicans and C. difficile were investigated. In the presence of C. albicans, C. difficile was able to grow under aerobic, normally toxic, conditions. This phenomenon was neither linked to adherence of bacteria to hyphae nor to biofilm formation by C. albicans. Conditioned medium of C. difficile inhibited hyphal growth of C. albicans, which is an important virulence factor of the fungus. In addition, it induced hypha-to-yeast conversion. p-Cresol, a fermentation product of tyrosine produced by C. difficile, also induced morphological effects and was identified as an active component of the conditioned medium. This study shows that in the presence of C. albicans, C. difficile can persist and grow under aerobic conditions. Furthermore, p-cresol, produced by C. difficile, is involved in inhibiting hypha formation of C. albicans, directly affecting the biofilm formation and virulence of C. albicans. This study is the first detailed characterization of the interactions between these two gut pathogens. IMPORTANCE Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C. difficile can survive under ambient aerobic conditions, which would otherwise be toxic. We also found that C. difficile affects the hypha formation of C. albicans, most likely through the excretion of p-cresol. This ultimately leads to an inability of C. albicans to form a biofilm. Our study provides new insights into interactions between C. albicans and C. difficile and bears relevance to both fungal and bacterial disease.

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Effects of Surotomycin on Clostridium difficile Viability and Toxin ProductionIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00275-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4199-4205 ◽

Cited By ~ 20

Author(s):

Laurent Bouillaut ◽

Shonna McBride ◽

Joseph A. Sorg ◽

Diane J. Schmidt ◽

José M. Suarez ◽

...

Keyword(s):

Clostridium Difficile ◽

Stationary Phase ◽

Virulence Factors ◽

Toxin Production ◽

Exponential Phase ◽

The Other ◽

Stages Of Development ◽

Content Type ◽

Other Hand

ABSTRACTThe increasing incidence and severity of infection byClostridium difficilehave stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treatingC. difficileinfection and of a third antimicrobial, surotomycin, to killC. difficilecells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis.

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Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere ◽

10.1128/msphere.00572-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 3

Author(s):

Kelvin G. K. Goh ◽

Danilo G. Moriel ◽

Steven J. Hancock ◽

Minh-Duy Phan ◽

Mark A. Schembri

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Biofilm Formation ◽

Secretion System ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Type V Secretion ◽

K 12 ◽

Type V

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

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Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00265-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2682-2691 ◽

Cited By ~ 15

Author(s):

Yi Wang ◽

Sok Ho Kim ◽

Ramya Natarajan ◽

Jason E. Heindl ◽

Eric L. Bruger ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Direct Synthesis ◽

Wild Type ◽

Cyclic Diguanylate ◽

Content Type ◽

Exogenous Addition ◽

In The Wild ◽

Growth Requirement ◽

Transposon Insertions

ABSTRACTIn bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene inAgrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants forodcgrew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite inA. tumefaciensand is synthesized from putrescine inA. tumefaciensvia the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to theodcmutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for theodc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. Theodcmutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue.IMPORTANCEPolyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. InAgrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential forA. tumefaciensgrowth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

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Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release inClostridium difficile

mBio ◽

10.1128/mbio.00648-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 18

Author(s):

Sandra Wydau-Dematteis ◽

Imane El Meouche ◽

Pascal Courtin ◽

Audrey Hamiot ◽

René Lai-Kuen ◽

...

Keyword(s):

Health Care ◽

Clostridium Difficile ◽

Stationary Phase ◽

Catalytic Domain ◽

Brain Heart Infusion ◽

Surface Protein ◽

Wild Type ◽

Content Type ◽

Lytic Transglycosylase ◽

Cell Autolysis

ABSTRACTClostridium difficileis the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis ofC. difficileis mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that aC. difficilecwp19mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributionsin vitrodepending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells ofC. difficilethat mediates the release of the toxins from the cell cytosol in response to specific environment conditions.IMPORTANCEClostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains ofC. difficilegenerally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase inC. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes toC. difficilecell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

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The Mannitol Utilization System of the Marine Bacterium Zobellia galactanivorans

Applied and Environmental Microbiology ◽

10.1128/aem.02808-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1799-1812 ◽

Cited By ~ 19

Author(s):

Agnès Groisillier ◽

Aurore Labourel ◽

Gurvan Michel ◽

Thierry Tonon

Keyword(s):

Abc Transporter ◽

Biochemical Characterization ◽

Heterotrophic Bacteria ◽

Dry Weight ◽

Sole Source ◽

Content Type ◽

Wide Range ◽

Marine Heterotrophic Bacteria ◽

Operon Gene ◽

Living Organisms

ABSTRACTMannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria.Zobellia galactanivorans(Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH ofZ. galactanivoranswas active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K+. Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture ofZ. galactanivoransin the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes ofFlavobacteriaceaeamong the 76 publicly available at the time of the analysis. It is not conserved in allBacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes.

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Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02760-16 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 24

Author(s):

Seema U. Nayak ◽

J. McLeod Griffiss ◽

Jeffrey Blumer ◽

Mary Ann O'Riordan ◽

Wesley Gray ◽

...

Keyword(s):

Clostridium Difficile ◽

Adverse Events ◽

Intestinal Flora ◽

Systemic Exposure ◽

Trna Synthetase ◽

Health Concern ◽

Pharmacokinetic Analysis ◽

Content Type ◽

Wide Range ◽

Methionyl Trna Synthetase

ABSTRACT Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.)

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