How will our project positively impact the world?

13th of August 2018

Clostridium difficile Significance, Symptoms and Cause

Clostridium difficile infections are the dominant cause of hospital-acquired diarrhea in the Western world. Outbreaks of hyper-virulent strains are also increasing, due to the high rates at which bacterial pathogens are developing antibiotic resistance [1]. More than 250,000 people require hospitalisation every year in the United States for treatment of C. difficile infections [2]. Additionally, in the US, a meta-analysis estimated that roughly 6.3 billion U.S dollars are spent annually on C. difficile attributable costs [3]. Consequently, our project focuses on a novel approach to curtail mortality and costs of treating C. difficile in the future.

C. difficile strains form spores which are released into the environment. These spores are highly resistant to environmental stresses such as ethanol, hydrogen peroxide, chloroform and heat [4], making contamination much harder to control in healthcare settings. This means outbreaks in institutions with high numbers of immunocompromised patients, whom are already at higher risk of suffering from infection, are now more likely to be exposed to the bacterium. As such, finding an alternative treatment for C. difficile infections is critical to properly managing a potential pandemic in the future.

Symptoms of a C. difficile infection can range from dehydration, fever, colonic inflammation, toxic megacolon, and in more severe cases sepsis and death [5]. Immunocompromised patients who have undergone broad-spectrum antibiotic treatment are particularly susceptible to this opportunistic pathogen. This is because their gut microbiota is more likely to exist in a dysbiotic (unbalanced) state meaning fewer commensal bacteria are available to fend off pathogens or take up space in the gastrointestinal tract, increasing the likelihood of a C. difficile infection.

Current Treatments

Generally the first step to treating a C. difficile infection is to stop taking the antibiotic that may have disrupted the gut microbiota initially, allowing C. difficile to colonise [6]. Another set of antibiotics more specific to C. difficile infections are then administered dependent on the severity of infection. The most common antibiotics used are: vancomycin, fidaxomicin, and metronidazole. A major repercussion of using antibiotics to treat C. difficile infections is the risk of further damaging other commensal bacteria in the gastrointestinal tract provoking even more dysbiosis. This chain reaction can allow C. difficile to gain a stronger footing in the gut. Another undesirable outcome is the likelihood of antibiotic resistance being developed. For example, the antibiotic vancomycin is frequently prescribed to treat C. difficile. This antibiotic is a last resort antibiotic against many multidrug resistant pathogens, meaning its use increases the likelihood of bacteria developing antibiotic resistance to it, endangering our ability to treat other bacterial infections with this antibiotic [7]. Antibiotics are also not very suitable long term treatments against C. difficile infections as 1 in 5 patients experience recurrence of infection [8]. This could further promote the likelihood of C. difficile developing antibiotic resistance. The costs of treating C. difficile are also a burden to the economy, and it is estimated a total of 34,157 US dollars are spent on attributable C. difficile costs per case [3]. As such antibiotic treatment of C. difficile is not a viable long term solution.

Alternative Treatments

Fortunately, alternative treatments already exist, some of which are being employed and are showing efficacious results. Potential remedies can range from Faecal Microbial Transplants (FMT), to pro and prebiotics. In fact, it has been shown that FMT has at least 80% success rate for treating C. difficile infections. [9] However, there are drawbacks to these treatments. For example, due to the complexity of a person’s gut microbiota, exchanging faecal matter between hosts can have unwanted long term impacts on metabolism and other aspects of a patient’s physiology that are not yet well understood [10].

Probiotic and prebiotic treatments as a preventative measure against opportunistic pathogens like C. difficile, are also promising avenues. In 2017 a cost effectiveness study was carried out by Shen et al. to determine whether hospital patients receiving antibiotic treatment should take probiotics as a preventative measure against C. difficile infections. This study provided evidence of probiotics being able to serve a corrective role in decreasing the antibiotic induced dysbiosis in the gut [11]. The only impediment of probiotic treatments, is that they work over a longer time period, and as more of a preventative measure that is not always effective. Given the challenges of existing treatments, our goal has been to find an additional and efficacious treatment for C. difficile infection.

Bacteriophage (phage) Therapy

Phage therapy is a relatively unexplored route of treatment in Western medicine compared to Eastern Europe. Primarily because in the past antibiotics have proven themselves to be a relatively fast, and broad form of treatment for bacterial infections. Resulting in less experimental and therapeutic information being needed on alternative treatments such as phage therapy for the past century[7, 12]. However, as antibiotic resistance and other detrimental factors of antibiotic intake, such as the removal of commensal bacteria, are becoming better understood, an alternative treatment that is able to avoid these consequences is important. One such treatment is the use of phages.

Generally, a phage particle consists of a protein shell enveloping genetic material, and a tail that allows attachment and insertion of the genetic material into the host bacterium. They generally have two modes of action for propagation. The lytic cycle which is the process by which a phage inserts its genome into the bacterial host, hijacks the hosts protein machinery and generates up to hundreds of copies of itself until the bacteria lyses and the phages are released to infect more bacteria [13].

Whereas, phages that follow the lysogenic cycle will integrate their genome into the bacterial host chromosome and remain dormant within the bacterium. Thereby dividing with the cell to propagate rather than killing off the host immediately as a lytic phage would. Certain environmental stresses such as antibiotics and ultraviolet light can trigger a temperate phage (a phage that has integrated its genome into a host bacterium) to switch into a lytic state thereby killing the host bacterium. It is important to understand that most phages are able to switch between lytic and lysogenic cycles depending on their inherent nature and environment, rather than strictly persisting in one type of cycle [13].

Phage therapy has remarkable features that makes it a very useful treatment. Due to phages being living organisms they have evolved alongside bacteria, meaning they are more predisposed to counter certain bacterial defense systems. Plus, unlike antibiotics some phages have evolved mechanisms to penetrate biofilms formed by bacterial aggregates, thus making them easier to disband with phage therapy compared to antibiotic treatment. Phages are also very specific in nature meaning commensal gut bacteria would not be infected by a potential phage therapy. Only a small dose of phages would be needed as they continuously replicate as they interact with their host bacterium [14, 15].

As with any solution there are likely to be drawbacks, as is also the case with phage therapy. For one, specificity acts as a double edged sword. On the one hand only the desired bacterium is likely to be infected, however finding the right phage can be time consuming and potentially lead to the patient’s condition worsening. It should also be recognized that it is an unfamiliar treatment, as such, negative long term effects to these remedies could be a potential complication in the future [14].

Our project

Today phage therapy focuses on utilizing lytic phages as a treatment however, our project centers on synthetically engineering a temperate phage to suppress the toxin production in Clostridium difficile. Our engineered phage would integrate genetic constructs designed to reduce toxin production at a transcriptional and translational level in the bacterium, thereby allowing Clostridium difficile to remain in the gastrointestinal tract as a commensal bacterium.

The rationale for this is that by keeping the C. difficile present, they can take up space in the gut and potentially outcompete other more harmful opportunistic pathogens that might try and invade the gut during a state of dysbiosis [16]. Additionally, by not rapidly lysing the C. difficile cells the release of relatively high concentrations of intracellular components containing potentially harmful enzymes and other substrates cannot interfere with the gastrointestinal tract and cause harm. This could have been a potential outcome if our project focused on using a lytic phage. A potential flaw with our lysogenic phage therapy, is the potential for unwanted horizontal transfer of certain genes. However, this is unlikely because the phage used is specific for C. difficile.

We have integrated two different DNA constructs into our lysogenic phage delivery system that both aim to suppress the toxins C. difficile synthesizes during pathogenesis. The first construct uses an antisense RNA based mechanism, which is able to hybridize to the toxin mRNA transcripts, inhibiting translation and eventually be subject to degradation by enzymes. The second construct is a dead Cas9 protein, that binds to the promoter regions of these toxins, impeding the rate at which the toxins are transcribed.

Impact

Our goal focuses on exploiting a novel combination of synthetic biology and phages to treat a bacterial disease that currently causes sizable patient mortality and economic burdens worldwide. Unfortunately, antibiotic resistance is a malignant problem that requires alternative treatments to be overcome and it is our hope that with this project we can extend knowledge of innovative ways to utilize phages in a medicinal context. Today phage therapy is largely focused on utilising lytic phages however, there are unique ways to manipulate phages at the genetic level so that they can control bacterial populations in a less absolute way which may serve more useful in certain circumstances, such as in the gut. Although phage therapy is relatively unknown to the public, we hope to draw attention to the remarkable outcomes that can be achieved from understanding and manipulating bacteriophages.

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