Inspection report Bicton College

Dates of inspection: 4–8 October 200

Emerging Applications of Bacterial Spores in Nanobiotechnology

Bacterial spores are robust and dormant life forms with formidable resistance properties, in part, attributable to the multiple layers of protein that encase the spore in a protective and flexible shield. The coat has a number of features pertinent to the emerging field of nanobiotechnology including self-assembling protomers and the capacity for engineering and delivery of foreign molecules. This review gives an account of recent progress describing the use of the spore, and specifically, the spore coat as a vehicle for heterologous antigen presentation and protective immunization (vaccination). As interest in the spore coat increases it seems likely that they will be exploited further for drug and enzyme delivery as well as a source of novel self-assembling proteins

M-World, S-World: Achieving wisdom in online groups

I live in M-World. You live in M-World too. In my M-World I don’t exist. In your M-World I exist but you don’t. In your M-World you don’t exist. I don’t meet me in my M-World and you don’t meet you in your M-World. But I meet you all the time and you regularly bump into me too. We are strangers to ourselves in our M-Worlds. That is just the way of it. When you and I are at our best with each other we leave our M-Worlds and come together for a while, we meet in another place. Let’s call it S-World. S-World is very, very similar to M-World. It is really, really close, closer than the hundredth of the width of a butterfly’s wing. You could not put a piece of paper between M-World and S-World. But they are so very, very different and they occupy very different places, they are an infinity apart. In M-World I am isolated, my perspective is stranded to itself, my thoughts are my own and my journey is the journey of the solitary. But, when we meet in S-World I am part of a community, my perspective is confronted and completed by yours and others, my thoughts are shared, and my journey is not so lonely. The problem seems to be that S-World is hard to find. But is it? Oddly people who need each other find each other effortlessly in S-World. All M-World separation disappears, the needy are united in an instant. When the need is great, in an instant they are all moved to and share in S-World. But people, even really, really clever and talented people who do not want to need each other can never find S-World, not even if they are all together in the same room, sitting right next to each other for hours and hours and hours, looking right at each other. They remain in their M-Worlds where they do not even meet themselves

Biological Containment of Genetically Modified<i> Bacillus subtilis</i>

ABSTRACT Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis , we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB , using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes. IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment. </jats:p

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.Peer reviewedFinal Published versio

Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

<p>Abstract</p> <p>Background</p> <p>Spore-forming <it>Bacilli </it>are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI)-tract of animals. Isolates of various <it>Bacillus </it>species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms.</p> <p>Results</p> <p>We report the annotation of carbohydrate active enzymes (CAZymes) of two pigmented <it>Bacilli </it>isolated from the human GI-tract and belonging to the <it>Bacillus indicus </it>and <it>B. firmus </it>species. A high number of glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both <it>Bacilli</it>. Experimental results on biofilm formation confirmed genomic data on the potentials of <it>B. indicus </it>HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both <it>Bacilli </it>to degrade mammalian glycans.</p> <p>Conclusions</p> <p>CAZy analyses of the genomes of the two pigmented <it>Bacilli</it>, compared to other <it>Bacillus </it>species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented <it>Bacilli </it>are adapted to the intestinal environment and are suited to grow in and colonize the human gut.</p

Mucosal antibodies to the C terminus of toxin A prevent colonization of Clostridium difficile

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile. These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission