The problem now

Clostridioides difficile infection (CDI) is the leading cause of hospital- and nursing home- acquired infection in the developed world. Today, nearly half of all hospitalised patients are prescribed antibiotics. These antibiotics drastically alter the gut flora, killing both beneficial and pathogenic bacteria alike. Such a shift in the dynamics of the gut microbiome allows opportunistic bacteria like C. difficile to propagate. The resulting toxins produced by C. difficile can lead to diarrhoea, bowel perforation and severe dehydration with significant fatalities. Presently, patients over the age of 65 account for more than 80% of the deaths from CDI. As such, our ever-aging populations coupled with the inevitable rise of antibiotic resistance makes C. difficile a major threat.

So how bad is C. difficile?

There are approximately 500,000 infections per year in the US alone. Of those 500,000 infections 30,000 result in death1. Moreover, there are also severe economic repercussions of CDI. In fact, extra costs associated with treatment amount to around $4.8 billion USD per year in the US. CDI places a severe burden on healthcare systems around the world. Additionally, CDI has a 20% to 30% rate of recurrence2. This makes it significantly more troublesome to treat, especially with regards to the proliferation of resistant strains.

These repeated infections can be partly attributed to C. difficile spore formation. These spores have resistance to heat, stomach acid, antibiotics and even bleach, allowing the C. difficile to persist in both the gut and the external environment. In fact, such spores are “plentiful in healthcare facilities” as reported by Leffler et al. (2015)3, thus contributing to the patient-to-patient spread and recurrence rate. Combined, these factors illustrate the need for an alternative and effective treatment.

Our Solution

We hope to change these alarming statistics with a novel treatment against C. difficile infections. Our project, ProQuorum, combines the power of probiotics and quorum sensing to cure CDI.

Specifically, we are engineering Lactobacillus reuteri, a natural resident of the human gut, to detect and kill virulent C. difficile bacteria. These super-probiotic bacteria, upon detection of the C. difficile, will release a C. difficile.-specific endolysin. This cleaves C. difficile’s peptidoglycan cell wall and causes lysis.

What was the inspiration for our project?

When we first began brainstorming for our project, our team had a wide variety of ideas which coupled synthetic biology to solving current societal issues. Several environmental projects were researched, including the production of biofuels using cyanobacteria to combat climate change and the optimisation of ethanol production using E. coli.

However our initial survey data suggested that the public was most interested in therapeutic applications of genetic engineering, see our public engagement page. So we shifted our focus onto therapeutic projects, which also drew our attention because of their potential to directly address a harmful disease. Phage therapy, bacteria that could treat coeliac disease and asthma treatments were all thoroughly researched. All these projects provided solutions to important cases, but we wanted our project to have a maximal impact and felt that our final chosen project achieved just that.

Many of us were concerned about the ever-looming threat of antibiotic resistance, which was really brought home at the Biohackathon, hosted by the Oxford University Biotech Society, where we discovered the scope of C. difficile infection (CDI). The Centres for Disease Control (CDC) cites C. difficile as one of the 3 most “urgent threats” with regard to antimicrobial resistance. Of the three, it has the highest incidence of infections per year (CDC, 2015). We decided to take it upon ourselves to respond to the CDC’s call to arms and provide an alternative treatment for C. difficile infection, rather than the standard antibiotic treatment. We hoped to create a system which combatted C. difficile to provide a more specific alternative to antibiotic treatment.

Our vision became much more tangible when we discovered that an effective endolysin against C. difficile called CD27L already existed. Although the core of our original idea has not changed, our project has greatly evolved since its inception. Both our integrated human practices and modelling have proved crucial in deciding the direction that we have taken for ProQuorum.

At first, we were unsure on the choice of the bacteria that we would use as our chassis. It was Dr. David Eyre, a C. difficile expert, who first suggested that we explore Lactobacillus strains, as these bacteria are already well adapted to grow in the human gut. We then decided to use L. reuteri as our chassis due to its intrinsic resistance to metrodinazole, vanocymin and fidoamicin 4 and its ability to secrete reuterin, a broad spectrum antibiotic when in the presence of glycerol. Together, these tools make wild-type L. reuteri an effective C. difficile suppressant.

Having decided on our chassis, we wanted to maximise the efficiency of our treatment. We had originally thought that it was best for our transformed Lactobacillus--which we call ProQuorum-- to constantly produce our CD27L endolysin. In this scenario, a patient would ingest our probiotic, and our transformed L. reuteri would begin growing in the gut, similar to what may happen after ingesting Lactobacillus cultures in yogurt. The L. reuteri would then produce a steady supply of the CD27L endolysin to ultimately kill off the C. difficile and resolve the infection.

However, after modelling two different systems, where the endolysin was either produced continually or only when induced, we realized that cell stress and its effect on growth rate needed to be taken into account. We found that induction was critical to our system’s success and began the hunt for a C. difficile-sensing switch.

With the help of bacterial signalling expert Professor George Wadhams, we decided to sense C. difficileby turning its own molecular machinery against itself, by incorporating the C. difficilequorum sensing mechanism into our system. Under the control of this system, our L. reuteri would only secrete our endolysin in the presence of a quorum of C. difficile. Not only did this create a dynamic system which limits perturbations to the gut flora through exogenous endolysin secretion, but it also sparked the name for our project: ProQuorum, a fusion of “probiotic” and “quorum sensing”.

We believe we have made great strides in the eventual completion of this project. We have shown the specificity and efficacy of our CD27L and validated a method of transformation it into L. reuteri--a difficult non-traditional bacteria to work with. Although we have had some success in transforming part of the quorum sensing system into E. coli, we are not yet at the stage where our ProQuorum bacteria are able to detect C. diffficle’s auto-inducing peptide (AIP).

Why SynBio?

Synthetic biology allows for a specific and targeted approach for our C. difficile treatment. Current methods of treating C. difficile involve further antibiotic treatment (namely vancomycin) or fecal transplants. Traditional broad-spectrum antibiotics, like vancomycin, drastically alter a patient’s gut flora and have been less effective against recurrent CDI. Fecal transplants, while proving to be an effective alternative to antibiotics, often require an invasive colonoscopy for the fecal sample to be introduced to the colon. They also pose the risk of transmitting other pathogens, including viral infections. Both options are costly, at over $3,000 USD per treatment.

A Synthetic Biology approach would address both of these issues. Firstly, ProQuorum preserves patients’ unique microbiome through the high specificity of its endolysin. The ProQuorum detection system ensures that the C. difficile-specific endolysin is only secreted when C. difficile is present. By introducing the endolysin into L. reuteri bacteria, its production is confined to the gut, providing a more targeted approach than simply introducing the CD27L endolysin intravenously (IV). Moreover, high local endolysin concentrations are more likely tolerated to ensure C. difficile lysis without significant intervention of the human immune system, a genuine concern if endolysin is administered through IV. As a result, this makes Synthetic Biology necessary to achieve both specificity and potency in combating CDI.


# Reference
1 “Nearly Half a Million Americans Suffered from Clostridium Difficile Infections in a Single Year | CDC Online Newsroom | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention,
2 Balsells, Evelyn, et al. “Global Burden of Clostridium Difficile Infections: a Systematic Review and Meta-Analysis.” Journal of Global Health, vol. 9, no. 1, 2018, doi:10.7189/jogh.09.010407.
3 Leffler, Daniel A., and J. Thomas Lamont. “Clostridium Difficile Infection.” New England Journal of Medicine, vol. 372, no. 16, 2015, pp. 1539–1548., doi:10.1056/nejmra1403772.
4 Spinler, Jennifer K., et al. “Next-Generation Probiotics Targeting Clostridium Difficile through Precursor-Directed Antimicrobial Biosynthesis.” Infection and Immunity, vol. 85, no. 10, 2017, doi:10.1128/iai.00303-17.