Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores.

Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

Susceptibility of hamsters to clostridium difficile isolates of differing toxinotype

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ~21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression

Association between proton pump inhibitor therapy and clostridium difficile infection: a contemporary systematic review and meta-analysis.

Abstract Introduction Emerging epidemiological evidence suggests that proton pump inhibitor (PPI) acid-suppression therapy is associated with an increased risk of Clostridium difficile infection (CDI). Methods Ovid MEDLINE, EMBASE, ISI Web of Science, and Scopus were searched from 1990 to January 2012 for analytical studies that reported an adjusted effect estimate of the association between PPI use and CDI. We performed random-effect meta-analyses. We used the GRADE framework to interpret the findings. Results We identified 47 eligible citations (37 case-control and 14 cohort studies) with corresponding 51 effect estimates. The pooled OR was 1.65, 95% CI (1.47, 1.85), I2 = 89.9%, with evidence of publication bias suggested by a contour funnel plot. A novel regression based method was used to adjust for publication bias and resulted in an adjusted pooled OR of 1.51 (95% CI, 1.26–1.83). In a speculative analysis that assumes that this association is based on causality, and based on published baseline CDI incidence, the risk of CDI would be very low in the general population taking PPIs with an estimated NNH of 3925 at 1 year. Conclusions In this rigorously conducted systemic review and meta-analysis, we found very low quality evidence (GRADE class) for an association between PPI use and CDI that does not support a cause-effect relationship

The Ecology of Antibiotic Use in the ICU: Homogeneous Prescribing of Cefepime but Not Tazocin Selects for Antibiotic Resistant Infection

Background: Antibiotic homogeneity is thought to drive resistance but in vivo data are lacking. In this study, we determined the impact of antibiotic homogeneity per se, and of cefepime versus antipseudomonal penicillin/beta-lactamase inhibitor combinations (APP-beta), on the likelihood of infection or colonisation with antibiotic resistant bacteria and/or two commonly resistant nosocomial pathogens (methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa). A secondary question was whether antibiotic cycling was associated with adverse outcomes including mortality, length of stay, and antibiotic resistance

Evaluation of a hand-held far-ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens

<p>Abstract</p> <p>Background</p> <p>Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against <it>Clostridium difficile</it> spores, but also safe and rapid. The Sterilray™ Disinfection Wand device is a hand-held room decontamination technology that utilizes far-ultraviolet radiation (185-230 nm) to kill pathogens.</p> <p>Methods</p> <p>We examined the efficacy of disinfection using the Sterilray device in the laboratory, in rooms of hospitalized patients, and on surfaces outside of patient rooms (i.e. keyboards and portable medical equipment). Cultures for <it>C. difficile</it>, methicillin-resistant <it>Staphylococcus aureus</it> (MRSA), and vancomycin-resistant <it>Enterococcus</it> (VRE) were collected from commonly-touched surfaces before and after use of the Sterilray device.</p> <p>Results</p> <p>On inoculated surfaces in the laboratory, application of the Sterilray device at a radiant dose of 100 mJ/cm² for ~ 5 seconds consistently reduced recovery of <it>C. difficile</it> spores by 4.4 CFU log₁₀, MRSA by 5.4 log₁₀CFU and of VRE by 6.9 log₁₀CFU. A >3 log₁₀ reduction of MRSA and VRE was achieved in ~2 seconds at a lower radiant dose, but killing of <it>C. difficile</it> spores was significantly reduced. On keyboards and portable medical equipment that were inoculated with <it>C. difficile</it> spores, application of the Sterilray device at a radiant dose of 100���mJ/cm² for ~ 5 seconds reduced contamination by 3.2 log₁₀CFU. However, the presence of organic material reduced the lethal effect of the far-UV radiation. In hospital rooms that were not pre-cleaned, disinfection with the Sterilray device significantly reduced the frequency of positive <it>C. difficile</it> and MRSA cultures (<it>P</it> =0.007).</p> <p>Conclusions</p> <p>The Sterilray™ Disinfection Wand is a novel environmental disinfection technology that rapidly kills <it>C. difficile</it> spores and other healthcare-associated pathogens on surfaces. However, the presence of organic matter reduces the efficacy of far-UV radiation, possibly explaining the more modest results observed on surfaces in hospital rooms that were not pre-cleaned.</p