The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-α and Trn-β, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [ 13 C, 15 N] Trn-α and [ 13 C, 15 N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S′-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to α-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.two-component bacteriocin | posttranslational modifications | Clostridium difficile-associated disease | peptide | NMR

Detection of C. difficile is increased in IBD outpatients in remission, and strain diversity is consistent with community acquisition from a multitude of sources.

Clostridium difficile-associated diarrhoea (CDAD) is the most common hospital-acquired diarrhoea, and is a major type of gastroenteritis infection in nursing homes and facilities for the elderly. In this study the antimicrobial activity of the two-component lantibiotic, lacticin 3147, against a range of genetically distinct C. difficile isolates was studied. The bacteriocin exhibited an MIC50 of 3.6 μg ml−1 for 10 genetically distinct C. difficile strains isolated from healthy subjects, inflammatory bowel disease patients and culture collection strains. In time-kill studies, 106 c.f.u. ml−1 C. difficile ATCC 42593 and CDAD isolate DPC 6220 were killed within 120 or 20 min incubation, respectively, at a concentration of 6 μg lacticin ml−1. Interestingly, addition of lacticin 3147 to exponentially growing cells of C. difficile ATCC 43593 caused rapid lysis of the cells after an initial lag phase, as measured by the concomitant release of the intracellular enzyme, acetate kinase. The addition of a food-grade, milk-based lacticin containing powder to faecal fermentation demonstrated that lacticin is effective in completely eliminating 106 c.f.u. C. difficile ml−1 from a model faecal environment within 30 min when present at concentrations as low as 18 μg ml−1. While other culturable microflora such as total anaerobes, bacteroides, total non-spore-forming anaerobes and total Gram-negative anaerobes were unaffected, populations of lactobacilli and bifidobacteria were reduced by 3 log cycles at bacteriocin levels sufficient to eliminate over 106 C. difficile. In light of these findings, the potential of lacticin 3147 for treatment of CDAD is discussed.