Fighting Infectious Diseases with Antimicrobial Agents using Microfluidic Platforms

Abstract

The COVID-19 pandemic has demonstrated the impact that untreatable infectious pathogens can have on society but an existing threat, if unaddressed could be even more devastating. Excessive deaths from infectious diseases were thought to be a thing of the past but antimicrobial resistance could cause the 700,000 annual deaths reported from resistant organisms to rise. New or alternative treatments to target Gram-negative pathogens are urgently required, alongside a greater understanding of the mechanisms of resistance to currently available therapeutics. Here I aim to advance the field of antimicrobial research through the exploration of the potential alternative therapy, bacteriophages, as well as investigating novel ways of cultivating previously unidentified antibiotic producing microorganisms. I demonstrate through novel microfluidic and single cell technologies (previously underutilised for phage assessments) that strategies for phage resistance in Escherichia coli are environment – structure dependent. In more complex environments, bacteria favour phenotypic over genetic resistance, which occurs through a diverse range of mechanisms such as filamentation or reduced receptor expression, and that extensive heterogeneity exists within the population. Such findings are important to enable the evolutionary and ecological dynamics of bacteria–phage interactions to be predicted and manipulated if they are to become a viable therapy in the clinic. I further show that using these microfluidic systems, the environment where phage-bacteria interactions occur can be tightly moderated and manipulated. Exposure duration, nutrient availability and even bacterial growth phase can be altered to optimise killing efficacy and observe the different single cell phenotypes of both surviving and susceptible cells that occur in response to phage. New treatments to target potential biothreat agents are also required. Here I have shown novel treatment options for Burkholderia involving a recently environmentally isolated phage in combination with existing antibiotics. Through antibiotic-antibiotic and phage – antibiotic combinations, I have been able to demonstrate improved clearance of B. thailandensis populations in vitro whilst lowering the required concentration of antibiotic. Although studies involving bacteriophage as an antimicrobial therapy are proving promising, they have not replaced the urgent need for new antibiotics. The majority of our current antibiotics are derived from environmental bacteria, but no new compounds have been licensed for decades. Here culturing platforms were designed to facilitate the growth of previously unculturable bacteria in an estuarine mud environment in situ. This method is still low-through put and additional parallel approaches are required such as microdroplet/ microfluidic systems or culture independent approaches like metagenomics. Ultimately, the future of antibiotic discovery to target the antibiotic resistance crisis lies in combined, parallel investigations utilising all available knowledge and resources in a unified approach for the treatment of infectious diseases.