Difference between revisions of "Team:Nottingham/Project"

Description

Clostridium difficile

Clostridium difficile is a Gram-positive, rod-shaped, anaerobic bacterium and is the most common causative agent of hospital-acquired diarrhoea in the Western world. The symptoms of C. difficile infection (CDI) can range from watery diarrhoea to pseudomembranous colitis, toxic megacolon and in severe cases death. Most C. difficile strains produce two major toxins, TcdA and TcdB, which are responsible for causing the characteristic symptoms of CDI.

C. difficile is estimated to be present in the natural gut microbiota of around 4% of the healthy adult population however, exposure to broad-spectrum antibiotics, such as cephalosporins, can cause disruption to the microbiota. This disruption can promote the colonisation of toxigenic strains allowing infection to persist. It is thought that non-toxigenic strains of C. difficile can act as a probiotic by outcompeting toxigenic strains in the gut and reducing the likelihood of disease. Currently, CDI is treated using two main antibiotics, metronidazole and vancomycin however, raised concerns over the emergence of antibiotic resistance has led to a desire for alternative treatments.

Phage and phage therapy

Bacteriophages (phages) are viruses which infect bacteria and can exist anywhere bacteria are located. Phages are highly specific, only infecting a single species or strain of bacteria and can be defined as either lytic or temperate depending on the life cycle they follow.

Lytic phages exclusively follow the lytic lifecycle, following infection these phages will hijack the host cell machinery to produce multiple copies of the phage proteins. These proteins are then assembled into multiple phage progeny which burst out of the host cell and go onto infect other bacterial cells. Temperate phages can follow the lytic life cycle but are also able to follow the lysogenic life cycle. These phages can integrate their genome in the host cell chromosome upon infection where they can remain dormant for long periods of time as prophages. When conditions are favourable, usually due to host cell stress, these prophages can excise from the host cell chromosome and enter the lytic life cycle where progeny phage particles are produced. To date, all phages found to infect C. difficile are temperate phages.

Ever since the first phage was isolated their use as a potential therapeutic agent has been explored, such as in the treatment of wound infections. Traditionally such therapy relies on strictly lytic phages to wipe out the problematic/problem causing bacterial populations. Phage therapy would be an ideal alternative treatment for CDI as their highly specific nature would mean they would not disrupt the natural gut microbiota, only targeting C. difficile cells.

This could reduce the incidence of relapse by allowing the gut microbiota to remain in its protective role against future colonisation. In comparison to antibiotics, the impact of resistance to phage therapy would be minimal due to phages and bacteria co-evolving. As bacteria gain resistance to overcome phage infection, the phages can evolve to evade these systems resulting in susceptible bacterial populations which can be treated. Although phage therapy would be the ideal alternative treatment for CDI the major roadblock is that no strictly lytic phages currently exist.

Project description

The main aim of this project was to create a therapeutic for CDI that would allow the natural gut microbiota to remain unchanged and reduce the reliance on antibiotics. To achieve this goal phage therapy was selected as an appropriate alternative due to its highly specific nature. It has been shown that non-toxigenic strains of C. difficile can act as probiotics to reduce the colonisation of toxigenic C. difficile in the gut therefore, by silencing the toxin gene expression in C. difficile, non-toxigenic probiotic strains are created.

Abstract

Antibiotics serve a critical role in remedying bacterial infections, however a major disadvantage to their use is the non-specificity of broad spectrum antibiotics that drastically kills off beneficial bacteria reducing the diversity of the gut flora. The use of antibiotics allows opportunistic pathogens like Clostridium difficile to take advantage of the dysbiosis caused.

A consequence of antibiotic misuse and the capability of bacteria to readily adapt to versatile conditions, has allowed antibiotic resistance in bacteria to become a major dilemma. Each year in the United States alone 2 million people are subject to infection from antibiotic resistant bacteria. Phage therapy is an alternative to antibiotics. The goal of our project was to engineer a bacteriophage which will infect C. difficile and express genetic constructs designed to suppress toxin production. We will pursue two strategies to achieve this; asRNA and dCAS-9, both of which will target the toxin genes tcdB and tcdA. Ultimately, we aim to produce a phage therapy which will reduce toxigenicity of resident strains of C. difficile without significantly affecting the native gastrointestinal microbiota.