In vitro efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent Clostridium difficile

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.

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Effect of Antibiotic Treatment on Growth of and Toxin Production by Clostridium difficile in the Cecal Contents of Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.8.3529-3532.2005 ◽

2005 ◽

Vol 49 (8) ◽

pp. 3529-3532 ◽

Cited By ~ 50

Author(s):

Nicole J. Pultz ◽

Curtis J. Donskey

Keyword(s):

Clostridium Difficile ◽

Antibiotic Treatment ◽

Subcutaneous Administration ◽

Toxin Production ◽

In Vitro Growth ◽

Anaerobic Microflora

ABSTRACT In mice, subcutaneous administration of antibiotics that disrupt the anaerobic microflora (i.e., clindamycin, piperacillin-tazobactam, and ceftriaxone) facilitated in vitro growth of and toxin production by Clostridium difficile in cecal contents, whereas antibiotics that cause minimal disruption of the anaerobic microflora (i.e., levofloxacin, cefepime, and aztreonam) did not.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.45682-0 ◽

2004 ◽

Vol 53 (11) ◽

pp. 1129-1136 ◽

Cited By ~ 76

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.

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Mutation of the Ferric Uptake Regulator (FUR) Severely Impairs Toxin Production in a Human in vitro Gut Model of Clostridium Difficile Infection

Gastroenterology ◽

10.1016/s0016-5085(17)30522-x ◽

2017 ◽

Vol 152 (5) ◽

pp. S48

Author(s):

Tanya Monaghan ◽

Caroline Chilton ◽

Magdalena Fit ◽

Anthony Buckley ◽

Sarah Kuehne ◽

...

Keyword(s):

Clostridium Difficile ◽

Clostridium Difficile Infection ◽

Toxin Production ◽

Ferric Uptake Regulator

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Genome Characterization of a Novel Binary Toxin-Positive Strain of Clostridium difficile and Comparison with the Epidemic 027 and 078 Strains

10.20944/preprints201705.0206.v1 ◽

2017 ◽

Author(s):

Zhong Peng ◽

Sidi Liu ◽

Xiujuan Meng ◽

Wan Liang ◽

Biao Tang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Clostridium Difficile ◽

Genome Size ◽

Genome Sequence ◽

Gc Content ◽

Antibiotic Resistance Genes ◽

Toxin Production ◽

Genome Comparison ◽

Binary Toxin ◽

Severe Diarrhea

A novel binary toxin-positive non-027, non-078 Clostridium difficile strain designated LC693 whose sequence type was ST201 was isolated from the fecal sample of a patient with severe diarrhea in China. To understand the pathogenesis basis of C. difficile ST201, this recently recovered isolate LC693 was then chosen for whole genome sequencing. The project finally generated an estimated genome size of approximately 4.07 Mbp. The genome sequence was then analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. Genome size of the three ST201 strains ranged from 4.07 Mb~4.16 Mb, with an average GC content between 28.5%~28.9%. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance, suggesting that they may have a strong antibiotic resistance. The ST201 strains contained a typical clade 3 specific PaLoc with a Tn6218 element inserted, and those genes harbored on their PaLoc that participated in the toxin expression and regulation were highly homologues to the epidemic 027 and 078 strains, with the exception of tcdC. A truncated TcdC was found in the ST201 strains, which is suggestive to have a contribution to the toxin production of the ST201 strains. In addition, the ST201 strains contained intact binary toxin coding and regulation genes, which is also proposed to contribute to the virulence. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for the C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The identification of those genes also contributes to the pathogenesis of the ST201 strain. To our knowledge, this is the first study that the genome sequence of C. difficile ST201 was discussed in detail, and the present study would have a contribution to understanding the pathogenesis basis of C. difficile ST201.

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Effects of Exposure of Clostridium difficile PCR Ribotypes 027 and 001 to Fluoroquinolones in a Human Gut Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00306-08 ◽

2008 ◽

Vol 53 (2) ◽

pp. 412-420 ◽

Cited By ~ 35

Author(s):

Katie Saxton ◽

Simon D. Baines ◽

Jane Freeman ◽

Rachael O'Connor ◽

Mark H. Wilcox

Keyword(s):

Clostridium Difficile ◽

Gut Microbiota ◽

Spore Germination ◽

Toxin Production ◽

Human Gut ◽

Ribotype 027 ◽

Resistant Mutants ◽

High Level ◽

Pcr Ribotype 027

ABSTRACT The incidence of Clostridium difficile infection is increasing, with reports implicating fluoroquinolone use. A three-stage chemostat gut model was used to study the effects of three fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) on the gut microbiota and two epidemic C. difficile strains, strains of PCR ribotypes 027 and 001, in separate experiments. C. difficile total viable counts, spore counts, and cytotoxin titers were determined. The emergence of C. difficile isolates with reduced antibiotic susceptibility was monitored with fluoroquinolone-containing medium, and molecular analysis of the quinolone resistance-determining region was performed. C. difficile spores were quiescent in the absence of fluoroquinolones. Instillation of each fluoroquinolone led to C. difficile spore germination and high-level cytotoxin production. High-level toxin production occurred after detectable spore germination in all experiments except those with C. difficile PCR ribotype 027 and moxifloxacin, in which marked cytotoxin production preceded detectable germination, which coincided with isolate recovery on fluoroquinolone-containing medium. Three C. difficile PCR ribotype 027 isolates and one C. difficile PCR ribotype 001 isolate from fluoroquinolone-containing medium exhibited elevated MICs (80 to ≥180 mg/liter) and possessed mutations in gyrA or gyrB. These in vitro results suggest that all fluoroquinolones have the propensity to induce C. difficile infection, regardless of their antianaerobe activities. Resistant mutants were seen only following moxifloxacin exposure.

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