A tripartite strategy for controlling Clostridioides difficile

Gut bacteria can exchange DNA in many ways to resist antibiotics and cause untreatable infections. Our research on understanding how and when antibiotic resistance genes (ARGs) are exchanged with other bacteria, and finding new treatment agents should ultimately reduce these occurrences and make infections more easily treated.

This research is focused on Clostridioides difficile, an important human pathogen that causes infection with high illness and death rates. C. difficile found in animals and the environment are capable of causing human disease. Antibiotic therapy that disrupts the balance of microbes in the gut is a major risk factor for C. difficile infection (CDI), hence antibiotic treatment of CDI often fails. Also C. difficile is constantly evolving to be antibiotic-resistant because of gene exchange events that involve contact with other cells, bacterial viruses (known as phages), and external DNA such as transposons. Some of these events occur more frequently than others probably because of environmental factors, and understanding these is important for controlling ARG exchange events. For this, we examine C. difficile cells in contact with other bacteria, phages, and external DNA under different environmental conditions that mimic gut conditions such as presence of antibiotics and changing pH, and measure differences in ARG being exchanged between cells.

We are investigating new agents that kill or enhance antibiotic-killing of C. difficile as possible treatments for infection. Two such agents we investigate are phages and cationic peptides. Phages are natural enemies of bacteria, and many phages found so far can be genetically altered to efficiently kill C. difficile. We do this by removing phage genes that prevent efficient killing of bacteria, forcing the phage to replicate and break apart its bacterial host cell after infection. Cationic peptides are short pieces of positively-charged proteins that disrupt the cell wall or DNA of bacteria. In this research we will be testing a synthetic cationic peptide for its ability to enhance the activity of antibiotics against C. difficile by mixing them together and adding them to actively growing cells.

We also look for agents that protect patients from recurrent CDI, which is a serious problem in about 20% of CDI patients. Probiotics are harmless bacteria that help our gut resist colonisation by pathogens. We are testing the ability of different types of probiotics to stop C. difficile from colonising a human gut model that mimics the natural microbial environment in humans suffering from CDI. This is done by growing bacteria from faecal samples of healthy humans in three flasks of pH, nutrient, and oxygen-free conditions similar to a human large intestine. Antibiotics and C. difficile are then added to the flasks to establish "infection", more antibiotics are added to remove C. difficile and simulate a recurring infection. Probiotics are then added to the system and checked to see if the probiotic prevents C. difficile. We also investigate the ability of harmless C. difficile (which lack the ability to produce toxins) to compete with toxin-producing C. difficile strains and prevent infection. So far we have found a harmless strain that prevents growth and toxin production by a superbug strain of C. difficile under a wide range of experimental conditions and this has not been shown before.

This three-pronged approach to control C. difficile in acquiring ARG, growth, and re-colonisation will open new avenues for developing treatments for CDI.

Clostridioides difficile is an anaerobic gut pathogen and recurring infections are difficult to treat. C. difficile infection (CDI) is facilitated by gut microflora dysbiosis, commonly caused by antibiotic therapy. C. difficile genomes are diverse with a core-/pan-genomic split of 29/71%, and mobile genetic elements can be transmitted by conjugation, transduction, and transformation, but it is unknown which mechanism is optimal. Our research aims to control C. difficile by targeting three areas - antimicrobial resistance acquisition, cell viability, and gut colonisation through three objectives: i) examine environmental factors of resistance gene dissemination, ii) develop phage and cationic peptides active against C. difficile, iii) develop probiotics as CDI prophylactic/treatment intervention agents. To examine gene transmission, newly isolated C. difficile in UK farms and public spaces will be conditionally induced for phage (e.g. exposure to various antimicrobials and pH) and tested for transduction of resistance genes in batch cultures and a CDI gut model. Conjugation and transformation will also be assayed in batch culture. C. difficile membrane vesicles (MV) are relatively unstudied; we will examine MV for gene transfer to bacterial cells, with and without phage. To develop C. difficile-specific phage and cationic peptides, CRISPR-based deletion of lysogeny genes will convert known temperate phages to virulent, while spontaneous lytic phage mutants will be selected by chelating agents. Phage host range, lysogeny, and resistance will be examined in batch culture and a gut model. A cationic polymer will be tested for potentiation of antibiotics and antisense PNA in vitro. To develop probiotics against colonising C. difficile, various species will be tested for preventing primary or secondary CDI and microbial interactions will be mapped. Research outcomes will lead to greater understanding of resistance transmission and intervention agents.