scholarly journals Emergence of a non-sporulating secondary phenotype in Clostridium (Clostridioides) difficile ribotype 078 isolated from humans and animals

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
M. C. Connor ◽  
J. W. McGrath ◽  
G. McMullan ◽  
N. Marks ◽  
M. Guelbenzu ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Toxin Production ◽  
Key Factors ◽  
Phenotypic Characteristics ◽  
Clostridioides Difficile ◽  
Ribotype 078 ◽  
Developed World ◽  
Harsh Conditions ◽  
Secondary Phenotype ◽  
Antibiotic Associated Diarrhoea

Abstract Clostridium (Clostridioides) difficile is a Gram positive, spore forming anaerobic bacterium that is a leading cause of antibiotic associated diarrhoea in the developed world. C. difficile is a genetically diverse species that can be divided into 8 phylogenetically distinct clades with clade 5 found to be genetically distant from all others. Isolates with the PCR ribotype 078 belong to clade 5, and are often associated with C. difficile infection in both humans and animals. Colonisation of animals and humans by ribotype 078 raises questions about possible zoonotic transmission, and also the diversity of reservoirs for ribotype 078 strains within the environment. One of the key factors which enables C. difficile to be a successful, highly transmissible pathogen is its ability to produce oxygen resistant spores capable of surviving harsh conditions. Here we describe the existence of a non-sporulating variant of C. difficile ribotype 078 harbouring mutations leading to premature stop codons within the master regulator, Spo0A. As sporulation is imperative to the successful transmission of C. difficile this study was undertaken to investigate phenotypic characteristics of this asporogenous phenotype with regards to growth rate, antibiotic susceptibility, toxin production and biofilm formation.

scholarly journals c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

mSphere ◽  
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.

scholarly journals Ebselen Exhibits Antimicrobial Activity Against Clostridioides difficile By Disrupting Redox Associated Metabolism

2020 ◽  
Author(s):  
Ravi K.R. Marreddy ◽  
Abiola O. Olaitan ◽  
Jordan N. May ◽  
Min Dong ◽  
Julian G. Hurdle
Keyword(s):  
Toxin Production ◽  
Covalent Modification ◽  
Intestinal Bleeding ◽  
Recurrence Rates ◽  
Antibiotic Resistant ◽  
Clostridioides Difficile ◽  
Growth Inhibitory ◽  
Protein Thiols ◽  
Ribotype 078 ◽  
Almost All

ABSTRACTHigh recurrence rates and spread of antibiotic-resistant strains necessitate the need for alternative therapeutics for Clostridioides difficile infections (CDIs). Ebselen, a reactive organoselenium compound inhibits C. difficile virulence toxins TcdA and TcdB, by covalently binding to their cysteine protease domains. Ebselen is thought to lack antibacterial activity against C. difficile cells and its anti-toxin action is suggested to be solely responsible for its efficacy. However, C. difficile has several essential cysteine-containing enzymes that could be potential sites for covalent modification by ebselen; hence, we re-evaluated its anti-C. difficile properties. In BHI agar, ebselen inhibited almost all C. difficile strains (MICs of 2-8 µg/ml), with ribotype 078 being intrinsically resistant (MIC>64 µg/ml). Wilkins-Chalgren and Brucella agars are recommended media for anaerobic susceptibility testing. Ebselen was either slightly attenuated by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in Brucella agars. Transcriptome analysis showed ebselen altered redox-associated processes, cysteine metabolism and significantly enhanced the expression of Stickland proline metabolism to likely regenerate NAD+ from NADH. Intracellularly cells increased the uptake of cysteines, depleted non-protein thiols and disrupted NAD+/NADH redox ratio. Growth inhibitory concentrations of ebselen also reduced toxin and spore production. Taken together, ebselen has bactericidal activity against C. difficile, with multiple mechanisms of action that negatively impacts toxin production and sporulation. To harness the polypharmacological properties of ebselen, chemical optimization is warranted, especially to obtain derivatives that could be effective in severe CDI, where intestinal bleeding could occur.

scholarly journals Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bryan Angelo P. Roxas ◽  
Jennifer Lising Roxas ◽  
Rachel Claus-Walker ◽  
Anusha Harishankar ◽  
Asad Mansoor ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Diarrheal Disease ◽  
The United States ◽  
Genomic Islands ◽  
Rapid Expansion ◽  
Phylogenomic Analysis ◽  
Community Settings ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

scholarly journals Biofilm Formation in Xanthomonas arboricola pv. pruni: Structure and Development

Agronomy ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 546
Author(s):  
Pilar Sabuquillo ◽  
Jaime Cubero
Keyword(s):  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Environmental Changes ◽  
Nutrient Content ◽  
Infection Process ◽  
Bacterial Attachment ◽  
Key Factors ◽  
Cell Structures ◽  
Microscopy Techniques

Xanthomonasarboricola pv. pruni (Xap) causes bacterial spot of stone fruit and almond, an important plant disease with a high economic impact. Biofilm formation is one of the mechanisms that microbial communities use to adapt to environmental changes and to survive and colonize plants. Herein, biofilm formation by Xap was analyzed on abiotic and biotic surfaces using different microscopy techniques which allowed characterization of the different biofilm stages compared to the planktonic condition. All Xap strains assayed were able to form real biofilms creating organized structures comprised by viable cells. Xap in biofilms differentiated from free-living bacteria forming complex matrix-encased multicellular structures which become surrounded by a network of extracellular polymeric substances (EPS). Moreover, nutrient content of the environment and bacterial growth have been shown as key factors for biofilm formation and its development. Besides, this is the first work where different cell structures involved in bacterial attachment and aggregation have been identified during Xap biofilm progression. Our findings provide insights regarding different aspects of the biofilm formation of Xap which improve our understanding of the bacterial infection process occurred in Prunus spp and that may help in future disease control approaches.

scholarly journals CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diogo Martins ◽  
Michael A. DiCandia ◽  
Aristides L. Mendes ◽  
Daniela Wetzel ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Life Cycle ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Well Being ◽  
Human Intestine ◽  
Healthy Humans ◽  
Clostridioides Difficile ◽  
Nutritional Conditions ◽  
Sporulation Initiation

AbstractBacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

scholarly journals Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽  
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.

scholarly journals Temporal Variations in Patterns of Clostridioides difficile Strain Diversity and Antibiotic Resistance in Thailand

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 714
Author(s):  
Supapit Wongkuna ◽  
Tavan Janvilisri ◽  
Matthew Phanchana ◽  
Phurt Harnvoravongchai ◽  
Amornrat Aroonnual ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Susceptibility ◽  
Reference Strain ◽  
Toxin Production ◽  
Temporal Variations ◽  
Toxin Gene ◽  
Data Sets ◽  
Clostridioides Difficile ◽  
Life Threatening ◽  
Susceptibility Profiles

Clostridioides difficile has been recognized as a life-threatening pathogen that causes enteric diseases, including antibiotic-associated diarrhea and pseudomembranous colitis. The severity of C. difficile infection (CDI) correlates with toxin production and antibiotic resistance of C. difficile. In Thailand, the data addressing ribotypes, toxigenic, and antimicrobial susceptibility profiles of this pathogen are scarce and some of these data sets are limited. In this study, two groups of C. difficile isolates in Thailand, including 50 isolates collected from 2006 to 2009 (THA group) and 26 isolates collected from 2010 to 2012 (THB group), were compared for toxin genes and ribotyping profiles. The production of toxins A and B were determined on the basis of toxin gene profiles. In addition, minimum inhibitory concentration of eight antibiotics were examined for all 76 C. difficile isolates. The isolates of the THA group were categorized into 27 A−B+CDT− (54%) and 23 A-B-CDT- (46%), while the THB isolates were classified into five toxigenic profiles, including six A+B+CDT+ (23%), two A+B+CDT− (8%), five A−B+CDT+ (19%), seven A−B+CDT− (27%), and six A−B−CDT− (23%). By visually comparing them to the references, only five ribotypes were identified among THA isolates, while 15 ribotypes were identified within THB isolates. Ribotype 017 was the most common in both groups. Interestingly, 18 unknown ribotyping patterns were identified. Among eight tcdA-positive isolates, three isolates showed significantly greater levels of toxin A than the reference strain. The levels of toxin B in 3 of 47 tcdB-positive isolates were significantly higher than that of the reference strain. Based on the antimicrobial susceptibility test, metronidazole showed potent efficiency against most isolates in both groups. However, high MIC values of cefoxitin (MICs 256 μg/mL) and chloramphenicol (MICs ≥ 64 μg/mL) were observed with most of the isolates. The other five antibiotics exhibited diverse MIC values among two groups of isolates. This work provides evidence of temporal changes in both C. difficile strains and patterns of antimicrobial resistance in Thailand.

scholarly journals Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile

2021 ◽  
Author(s):  
Yannick D. N. Tremblay ◽  
Benjamin A. R. Durand ◽  
Audrey Hamiot ◽  
Isabelle Martin-Verstraete ◽  
Marine Oberkampf ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Clostridioides Difficile

scholarly journals Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
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.

scholarly journals Make and break the alarmone: regulation of (p)ppGpp synthetase/hydrolase enzymes in bacteria

2019 ◽  
Vol 43 (4) ◽  
pp. 389-400 ◽  
Author(s):  
Séverin Ronneau ◽  
Régis Hallez
Keyword(s):  
Cell Cycle ◽  
Stress Response ◽  
Stress Responses ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Second Messengers ◽  
Molecular Devices ◽  
Environmental Cues ◽  
Fluctuating Environments ◽  
Harsh Conditions

ABSTRACTBacteria use dedicated mechanisms to respond adequately to fluctuating environments and to optimize their chances of survival in harsh conditions. One of the major stress responses used by virtually all bacteria relies on the sharp accumulation of an alarmone, the guanosine penta- or tetra-phosphate commonly referred to as (p)ppGpp. Under stressful conditions, essentially nutrient starvation, these second messengers completely reshape the metabolism and physiology by coordinately modulating growth, transcription, translation and cell cycle. As a central regulator of bacterial stress response, the alarmone is also involved in biofilm formation, virulence, antibiotics tolerance and resistance in many pathogenic bacteria. Intracellular concentrations of (p)ppGpp are determined by a highly conserved and widely distributed family of proteins called RelA-SpoT Homologs (RSH). Recently, several studies uncovering mechanisms that regulate RSH activities have renewed a strong interest in this field. In this review, we outline the diversity of the RSH protein family as well as the molecular devices used by bacteria to integrate and transform environmental cues into intracellular (p)ppGpp levels.

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