Pathogenicity & a bedside real-time detection assay for clostridium difficile in the faeces of hospitalized patients

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates

Biocide Resistance and Transmission of Clostridium difficile Spores Spiked onto Clinical Surfaces from an American Health Care Facility

Clostridium difficile is the primary cause of antibiotic-associated diarrhea globally. In unfavourable environments the organism produces highly resistant spores which can survive microbicidal insult. Our previous research determined the ability of C. difficile spores to adhere to clinical surfaces; finding that spores had marked different hydrophobic properties and adherence ability. Investigation into the effect of the microbicide sodium dichloroisocyanurate on C. difficile spore transmission revealed that sub-lethal concentrations increased spore adherence without reducing viability. The present study examined the ability of spores to transmit across clinical surfaces and their response to an in-use disinfection concentration of 1,000-ppm of chlorine-releasing agent sodium dichloroisocyanurate. In an effort to understand if these surfaces contribute to nosocomial spore transmission, surgical isolation gowns, hospital-grade stainless steel and floor vinyl were spiked with 1 × 106 spores/ml of two types of C. difficile spore preparations: crude spores and purified spores. The hydrophobicity of each spore type versus clinical surface was examined via plate transfer assay and scanning electron microscopy. The experiment was repeated and spiked clinical surfaces were exposed to 1,000-ppm sodium dichloroisocyanurate at the recommended 10-min contact time. Results revealed that the hydrophobicity and structure of clinical surfaces can influence spore transmission and that outer spore surface structures may play a part in spore adhesion. Spores remained viable on clinical surfaces after microbicide exposure at the recommended disinfection concentration demonstrating ineffectual sporicidal action. This study showed that C. difficile spores can transmit and survive between varying clinical surfaces despite appropriate use of microbicides. IMPORTANCE Clostridium difficile is a healthcare-acquired organism and the causative agent of antibiotic-associated diarrhoea. Its spores are implicated in faecal to oral transmission from contaminated surfaces in the healthcare environment due to their adherent nature. Contaminated surfaces are cleaned using high-strength chemicals to remove and kill the spores; however, despite appropriate infection control measures, there is still high incidence of C. difficile infection in patients in the US. Our research examined the effect of a high-strength biocide on spores of C. difficile which had been spiked onto a range of clinically relevant surfaces including isolation gowns, stainless steel and floor vinyl. This study found that C. difficile spores were able to survive exposure to appropriate concentrations of biocide; highlighting the need to examine the effectiveness of infection control measures to prevent spore transmission, and consideration of the prevalence of biocide resistance when decontaminating healthcare surfaces.</jats:p

Contribution of spores to the ability of Clostridium difficile to adhere to surfaces.

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species

Extraction and sensitive detection of toxins A and B from the human pathogen Clostridium difficile in 40 seconds using microwave-accelerated metal-enhanced fluorescence.

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile

Detection of target DNA within various concentrations of gDNA by MAMEF.

<p>Various concentrations of <i>C. difficile</i> gDNA (strain CD630; ATCC, USA) were tested in the MAMEF platform. Concentrations of 10 ng and 5 ng were microwave irradiated for 8 seconds at 70% power. gDNA was subjected to disruption to increase the chances of the detection probes accessing the toxin specific sequences. The data presented is the result from a single reproduced assay. (A) The fluorescent signal intensities generated from toxin A detection within microwaved gDNA (excitation at 495 nm, emission at 519 nm). (B) The fluorescent signal intensities generated from toxin B detection within gDNA (excitation at 590 nm, emission at 617 nm). The control was PBS buffer.</p

Table of Oligonucleotides.

<p>Anchor and capture probes, and synthetic oligonucleotides target regions used in the MAMEF assay for toxins A and B are shown, along with the corresponding fluorophore used.</p><p>Table of Oligonucleotides.</p

Schematic configuration of 3 piece DNA detection assay.

<p>This configuration was used for detection of both toxin A and toxin B probes. The anchor probe (17 oligonucleotides) was anchored to the SiFs by addition of the thiol group. The fluorescent probe (22 oligonucleotides) was attached to an Alexa at the 3′ end. Upon hybridization with target DNA, the 3 piece assay is formed, and the fluorophore labeled probe is plasmon enhanced.</p

Gold lysing triangle on Starfrost slide and a typical Silver Island Film (SiFs).

<p>(A) Gold triangle with silicone isolators added to create lysing chamber. (B) The SiF here is at an OD₄₅₀ of 0.43 and has multi-well silicone isolators added. Each well can hold an individual DNA assay reaction; hence multiple repeats can be conducted.</p

Detection of DNA from microwaved <i>C. difficile</i> spore preparation in PBS using MAMEF.

<p>Spores of <i>C. difficile</i> from strain CD630 at a range of concentrations 10, 100, 1000 and 10 000 cfu were suspended in PBS and subjected to microwave irradiation and screened for the presence of toxin A and toxin B using MAMEF. (A) Various fluorescent signal intensities generated from detection of toxin A within gDNA from irradiated spores (excitation at 495 nm, emission at 519 nm). (B) The various fluorescent signal intensities generated from detection of toxin B within gDNA from irradiated spores (excitation at 590 nm, emission at 617 nm).</p