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OC-043 Mutation of the Ferric Uptake Regulator (FUR) Severely Impairs Toxin Production in a Human in vitro Gut Model of Clostridium Difficile Infection
  1. TM Monaghan1,
  2. CH Chilton2,
  3. M Fit3,
  4. AM Buckley2,
  5. SA Kuehne3,
  6. MH Wilcox2,
  7. A Cockayne3
  1. 1NIHR Biomedical Research Unit in Gastrointestinal and Liver Diseases, University of Nottingham, Nottingham
  2. 2Leeds Institute for Molecular Medicine, University of Leeds, Leeds
  3. 3Clostridia Research Group, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

Abstract

Introduction Regulation of iron uptake and utilisation is critical for bacterial growth and for prevention of iron toxicity. To date, little research has been carried out on iron uptake mechanisms and their regulation in Clostridium difficile. However, analysis of available C. difficile genome sequences reveals the presence of multiple iron-uptake systems and regulators underlining the importance of iron acquisition for clostridial growth. In this study, we investigated the phenotypic effects of the ferrous iron uptake system FeoB1 and the ferric uptake regulator and iron-dependent global gene regulator Fur in C. difficile.

Methods ClosTron mutagenesis was used to generate knockout mutants in a single feoB1 and fur homologue in C. difficile 630Δerm, which were then inoculated into an in vitro human gut model to investigate relative propensity to induce C. difficile infection (CDI). Three parallel triple-stage chemostat gut models were primed with human faecal emulsions and spiked with C. difficile spores (~107 spores) from each mutant in addition to the 630Δerm wild-type parental strain. Bacterial populations were allowed to equilibrate before simulated CDI was induced by instillation of clindamycin (33.9 mg/L, four times daily for 7 days). Serial samples were collected for enumeration of microflora populations, C. difficile vegetative cells, spores and measurement of cytotoxin titres.

Results Cytotoxicity assays revealed that the fur mutant strain produced considerably lower toxin levels (~1000 fold lower) than the feoB1 and wild type strain. Following clindamycin exposure, all three C. difficile strains germinated and exhibited sustained vegetative proliferation (~5.5/6 log10 cfu/mL). The feoB1 mutant strain germinated slightly earlier than the other strains, which may have been influenced by the slightly lower clindamycin levels in the feoB1 model. Indeed, compared to wild type, higher minimum inhibitory concentrations were observed for both mutant strains, indicating reduced susceptibility to clindamycin. In all three models, the introduction of clindamycin caused a decline in Bifidobacteria (3.5 log10 cfu/mL), Clostridia (~3 log10 cfu/mL) and Lactobacilli (~2 log10 cfu/mL) with increases in Enterococci and Enterobacteriaceae (2–4 log10 cfu/mL). However, no specific microflora changes correlated with the strain of C. difficile used in each of the models.

Conclusion These findings reveal the important role of the Fur system in regulating the expression of C. difficile toxins. Modulation of iron homeostasis may represent a potential novel therapeutic or preventative strategy against CDI.

Disclosure of Interest None Declared

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