Clostridium difficile: Infection and Immunity

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile

Antibacterial potential of extracts of various parts of Catunaregam tomentosa (Blume ex DC) Tirveng and their effects on bacterial granularity and membrane integrity

Purpose: To investigate the antibacterial activity of extracts from Catunaregam tomentosa on Bacillus subtilis and Staphylococcus aureus, and the bacterial responses to the extracts.Methods: The antibacterial activity of fruit, leaf and stem bark extracts were  evaluated against B.subtilis (ATCC6633) and S. aureus (ATCC25923). Using a disc diffusion method, extracts at concentrations ranging from 50 – 1,000 μg/disc were tested. The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of the extracts against the test bacteria were determined. Fluorescent activated cell sorting (FACS) was used to assess the responses of both types of bacteria to the extracts.Results: The fruit and leaf extracts at 1,000 μg/disc showed optimum efficacy against B. subtilis and S. aureus with MIC of 1,000 μg/mL against both B. subtilis and S. aureus, for the fruit and the leaf extracts. With increasing doses of fruit and leaf extracts at 6 h of incubation, FACS profiles revealed that cell death for B. subtilis increased. The fruit and leaf extracts of C. tomentosa also exhibited antibacterial activity against S. aureus in a dose- and time-dependent manner. The bacteria initially lost their granularity, then lost membrane integrity, and consequently died. Conclusion: The fruit and leaf extracts of C. tomentosa exhibit significant antibacterial potential against Gram-positive bacteria by damaging bacterial granularity and membrane integrity. Keywords: Catunaregam tomentosa, Flow cytometry, Programmed cell death, Response pattern, Bacterial granularity, Membrane integrit

Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A(+)B(+)). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A

Black Soldier Fly (<i>Hermetia illucens</i>) Larvae Meal: A Sustainable Alternative to Fish Meal Proven to Promote Growth and Immunity in Koi Carp (<i>Cyprinus carpio</i> var. <i>koi</i>)

Insect meal has shown promise as a potentially sustainable source of nutrients for aquafeeds, offering an alternative to expensive and ecologically undesirable ingredients, in the context of population explosion and climate change. Despite this promising outlook, its effects on fish growth and immune responses remain to be thoroughly investigated. Our scientific goal was to experimentally test responses to replacements of the fish meal with a protein source derived from black soldier fly larvae meal (BSFLM). Possible impacts on growth, immunological response, and the expression of selected immune-system related genes were evaluated in Koi carp (Cyprinus carpio var. koi) using a biofloc culture system. Three hundred fish (20.0 ± 0.2 g) were allocated into five groups: a control group receiving a basal diet containing 0 g kg−1 BSFLM and four experimental groups in which fish meal was replaced with 50, 100, 150, and 200 g kg−1 BSFLM for eight weeks. After 4 weeks of feeding, there were no statistically significant differences in specific growth rate (SGR), feed conversion ratio (FCR), and survival rate between fish fed BSFLM-enriched diets at 50, 100, 150 g kg−1 and a control (0 g kg−1 BSFLM) diet. However, fish fed 200 g kg−1 BSFLM showed significantly improved weight gain (WG) and SGR compared to the control after 4 weeks; this difference persisted through 8 weeks (p r = 0.470) and SR (r = 0.384), respectively, with the BSFLM levels, but significant and highly correlated linear relationships were observed in WG (r = 0.917) and SGR (r = 0.912). Immunological response analysis showed slight changes in lysozyme and peroxidase levels by replacing fish meal with BSFLM, but these apparent differences were not significantly related to experimental diets. Interestingly, mRNA transcripts of immune-related genes (TNF-α, TGF-β, IL1, IL10, and hsp70) were upregulated in the groups receiving higher amounts of BSFLM, with statistically significant differences observed in certain comparisons. Our findings reveal that fish meal can be effectively replaced by BSFLM, and that this not only has a positive effect on immune-related gene expression in Koi carp, but also on growth rate, pointing to the future potential role of BSFLM as an alternative fish meal protein in aquafeed formulation

In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

<i>Bacillus cereus</i>, responsible for food poisoning, and <i>Clostridium difficile</i>, the causative agent of <i>Clostridium difficile</i>-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from <i>B. cereus</i> spore coat, exosporium and 54 proteins from the <i>C. difficile</i> coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two <i>Bacilli</i> and one <i>Clostridium</i> species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the <i>B. cereus</i> group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces