scholarly journals Human Hypervirulent Clostridium difficile Strains Exhibit Increased Sporulation as Well as Robust Toxin Production

2010 ◽  
Vol 192 (19) ◽  
pp. 4904-4911 ◽  
Author(s):  
Michelle Merrigan ◽  
Anilrudh Venugopal ◽  
Michael Mallozzi ◽  
Bryan Roxas ◽  
V. K. Viswanathan ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Severe Disease ◽  
Toxin Production ◽  
Negative Regulator ◽  
Comparative Genomic ◽  
Toxin Gene ◽  
Antibiotic Associated Diarrhea ◽  
Quantitative Examination ◽  
Mechanistic Basis

ABSTRACT Toxigenic Clostridium difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth and are the leading cause of antibiotic-associated diarrhea. C. difficile isolates of the molecular type NAP1/027/BI have been associated with severe disease and hospital outbreaks worldwide. It has been suggested that these “hypervirulent” strains produce larger amounts of toxin and that a mutation in a putative negative regulator (TcdC) allows toxin production at all growth phases. To rigorously explore this possibility, we conducted a quantitative examination of the toxin production of multiple hypervirulent and nonhypervirulent C. difficile strains. Toxin gene (tcdA and tcdB) and toxin gene regulator (tcdR and tcdC) expression was also monitored. To obtain additional correlates for the hypervirulence phenotype, sporulation kinetics and efficiency were measured. In the exponential phase, low basal levels of tcdA, tcdB, and tcdR expression were evident in both hypervirulent and nonhypervirulent strains, but contrary to previous assumptions, toxin levels were below the detectable thresholds. While hypervirulent strains displayed robust toxin production during the stationary phase of growth, the amounts were not significantly different from those of the nonhypervirulent strains tested; further, total toxin amounts were directly proportional to tcdA, tcdB, and tcdR gene expression. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Comparative genomic analyses of the closely related nonhypervirulent strains VPI 10463 (the highest toxin producer) and 630 (the lowest toxin producer) revealed polymorphisms in the tcdR ribosome binding site and the tcdR-tcdB intergenic region, suggesting that a mechanistic basis for increased toxin production in VPI 10463 could be increased TcdR translation and read-through transcription of the tcdA and tcdB genes. Hypervirulent isolates produced significantly more spores, and did so earlier, than all other isolates. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.

Toxin A and B genes expression of Clostridium difficile in the sub-minimum inhibitory concentration of clindamycin, vancomycin and in combination with ceftazidime

2020 ◽  
Author(s):  
Mohammad Moradi ◽  
Shahla Mansouri ◽  
Nouzar Nakhaee ◽  
Farhad Sarafzadeh ◽  
Ebrahim Rezazadeh Zarandi
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Inhibitory Concentration ◽  
Toxin Production ◽  
Toxin Gene ◽  
Transcription Level ◽  
The Third ◽  
Genes Expression ◽  
Antibiotic Associated Diarrhea ◽  
Toxin Gene Expression

Background and Objectives: Antibiotics prescribed for infections have diverse effects on microbiota and the pathogen Clostridium difficile (C. difficile) as the most important antibiotic-associated diarrhea. This study aims to determine the gene expression of toxins A and B at the transcription level in the sub-MIC of vancomycin (VAN), clindamycin (CLI), and cef- tazidime (CAZ) alone and in combination. Materials and Methods: The MIC and fractional inhibitory concentration (FIC) of two C. difficile samples (a clinical isolate and ATCC 9689) were determined by microdilution and checkerboard microdilution methods, respectively. The total RNA was extracted from the medium inoculated with ~106  CFU/mL of fresh bacteria in the pre-reduced medium containing  ½ MIC of antibiotics alone and ½ FIC of antibiotics in combination. Real-time PCR was performed by sybrGreen methods in triplicate, and the data were analyzed by the comparative ∆∆CT  method. Results: All antibiotics except CAZ (alone and in combination) decreased the gene expression of toxins A and B within 24 hours. VAN and CLI reduced toxin gene expression within 24 and 48 hours. However, CAZ alone and in combination with VAN as well as CLI increased the gene expression of toxins A and B. Conclusion: The results confirmed toxin gene transcription and toxin production are associated with the type of isolates and antibiotics, as well as the combined form of antibiotics. This could be the reason which can explain the occurrence of C. difficile infection among patients who were treated with the third generation of cephalosporins alone and in combination with another antibiotic in the form of combinational therapy.

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

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
David S. Courson ◽  
Elizabeth M. Garrett ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Biochemical Characterization ◽  
Toxin Production ◽  
Physiological Adaptation ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Wide Range

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.

scholarly journals The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 187
Author(s):  
Miad Elahi ◽  
Haruyuki Nakayama-Imaohji ◽  
Masahito Hashimoto ◽  
Ayano Tada ◽  
Hisashi Yamasaki ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Toxin Production ◽  
Inhibitory Effect ◽  
Proteinase K ◽  
Toxin Gene ◽  
Bacteroides Thetaiotaomicron ◽  
Human Gut ◽  
Polysaccharide Fraction ◽  
Pathway Gene ◽  
Cell Autolysis

Disruption of the human gut microbiota by antibiotics can lead to Clostridium difficile (CD)-associated diarrhea. CD overgrowth and elevated CD toxins result in gut inflammation. Herein, we report that a gut symbiont, Bacteroides thetaiotaomicron (BT), suppressed CD toxin production. The suppressive components are present in BT culture supernatant and are both heat- and proteinase K-resistant. Transposon-based mutagenesis indicated that the polysaccharide metabolism of BT is involved in the inhibitory effect. Among the genes identified, we focus on the methylerythritol 4-phosphate pathway gene gcpE, which supplies the isoprenoid backbone to produce the undecaprenyl phosphate lipid carrier that transports oligosaccharides across the membrane. Polysaccharide fractions prepared from the BT culture suppressed CD toxin production in vitro; the inhibitory effect of polysaccharide fractions was reduced in the gcpE mutant (ΔgcpE). The inhibitory effect of BT-derived polysaccharide fraction was abrogated by lysozyme treatment, indicating that cellwall-associated glycans are attributable to the inhibitory effect. BT-derived polysaccharide fraction did not affect CD toxin gene expression or intracellular toxin levels. An autolysis assay showed that CD cell autolysis was suppressed by BT-derived polysaccharide fraction, but the effect was reduced with that of ΔgcpE. These results indicate that cell wall-associated glycans of BT suppress CD toxin release by inhibiting cell autolysis.

scholarly journals The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

2016 ◽  
Vol 84 (12) ◽  
pp. 3434-3444 ◽  
Author(s):  
Kevin O. Childress ◽  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Clostridium Difficile ◽  
Molecular Mechanisms ◽  
Toxin Production ◽  
Negative Regulator ◽  
Hamster Model ◽  
Content Type ◽  
Sporulation Initiation

The formation of spores is critical for the survival ofClostridium difficileoutside the host gastrointestinal tract. Persistence ofC. difficilespores greatly contributes to the spread ofC. difficileinfection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation toC. difficilepathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved inC. difficileand few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion ofCD1492resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in theCD1492mutant. Deletion of CD1492 also resulted in decreased toxin productionin vitroand in decreased virulence in the hamster model of CDI. Further, theCD1492mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lowersigDandrstAtranscription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence thatC. difficilesporulation is regulated differently from that of other endospore-forming species.

Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe

The Lancet ◽  
2005 ◽  
Vol 366 (9491) ◽  
pp. 1079-1084 ◽  
Author(s):  
Michel Warny ◽  
Jacques Pepin ◽  
Aiqi Fang ◽  
George Killgore ◽  
Angela Thompson ◽  
...  
Keyword(s):  
North America ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Toxin Production

Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods

2004 ◽  
Vol 53 (11) ◽  
pp. 1129-1136 ◽  
Author(s):  
Patrizia Spigaglia ◽  
Paola Mastrantonio
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Negative Regulator ◽  
Resistance Pattern ◽  
Binary Toxin ◽  
Genetic Lineages ◽  
Time Periods ◽  
Subinhibitory Concentrations

Recent studies have shown that Clostridium difficile strains with variant toxins and those with resistance to macrolide–lincosamide–streptogramin B (MLSB) are increasingly causing severe disease and outbreaks in hospital settings. Here, the pathogenicity locus (PaLoc), the acquisition of binary toxin, and the genotypic and phenotypic characteristics of antibiotic resistance of 74 C. difficile clinical strains isolated from symptomatic patients in Italy during different time periods were studied. These strains were found to belong to two different lineages, and those isolated before 1991 were genetically unrelated to the more recent strains. The majority of recent C. difficile strains showed variations in toxin genes and in the toxin negative regulator (tcdC) and had the binary toxin. In 62 % of them, variations in tcdC and the presence of the binary toxin were associated. Five classes of susceptibility/resistance pattern (EC-a to -e) for erythromycin and clindamycin were identified in all strains studied. Most of the recent isolates belonged to EC-d and EC-e and, although erythromycin-resistant in vitro, did not harbour the commonly associated ermB determinant. Interestingly, two strains of the EC-d class were resistant to clindamycin only after induction with subinhibitory concentrations of the antibiotic. A decrease in tetracycline and chloramphenicol MIC values was also observed in the recently isolated strains, associated with less frequent detection of the catD and tetM genes. Two tetM-positive strains were resistant in vitro only after induction with subinhibitory concentrations of the antibiotic. The acquisition of the binary toxin, the possible increase in toxin production due to a mutated negative regulator and a decrease in the fitness cost as a result of lower levels of antibiotic resistance or other mechanisms may have led to the successful establishment of these new phenotypes, with potentially serious clinical implications.

scholarly journals Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Brintha P. Girinathan ◽  
Marc Monot ◽  
Daniel Boyle ◽  
Kathleen N. McAllister ◽  
Joseph A. Sorg ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Toxin Production ◽  
Hospitalized Patients ◽  
Host Tissue ◽  
Hospital Environment ◽  
Pivotal Role ◽  
Toxin Gene ◽  
Bacterial Cells ◽  
Significant Morbidity ◽  
Content Type

ABSTRACT C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291. Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291.

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