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How does infection site affect the structure and biology of bacterial biofilms?

Primary Supervisor: Dr Freya Harrison, School of Life Sciences

Secondary supervisor: Dr Kim Hardie (University of Nottingham) and Dr Meera Unnikrishnan (University of Warwick).

PhD project title: How does infection site affect the structure and biology of bacterial biofilms?

University of Registration: University of Warwick.

Project outline:

The formation of multicellular biofilms is key to the progression of many bacterial infections. Biofilms cause persistent, antibiotic-resistant infections in burn wounds, in diabetic ulcers, around the endotracheal tubes used to ventilate hospital patients, and in the lungs of people with cystic fibrosis. Biofilms are also associated with gut health and dysbiosis. The environments found in these infection sites differ, but some species of bacteria are pathogens in more than one. You will explore whether biofilms formed by the key pathogens have different structures, community interactions and responses to drugs, depending on the environment they colonise.

Biofilms: a sticky situation

In biofilms, bacteria of the same and different species aggregate by producing an extracellular matrix of carbohydrates, proteins and DNA. This protects bacteria from host clearance mechanisms and antibiotics: eradication can require an antibiotic concentration 100-1,000X higher than that needed to eradicate the same bacteria growing planktonically. This is often impossible to achieve in vivo, so biofilm infections may effectively be untreatable. Wound biofilm infections cost the NHS >£1Bn each year, and can lead to severe complications including amputation or sepsis; while 90% of people with cystic fibrosis die from respiratory failure resulting from long-term airway biofilm infection.

Our understanding of in vivo biofilms is limited, because the complexity of the environment in infection sites, and the complexity of the biofilm community, are hard to mimic using standard lab models. Some key unanswered questions are:

  • To what extent do bacteria make a different mixture of matrix polymers, or express different genes, depending on where they find themselves? This is important because a drug which penetrates a biofilm and kills a pathogen in one setting may fail to enter the biofilm matrix in another (if the environment cues production of matrix polymers that differ in size/charge), be inactivated in some environments but not others (e.g. due to pH), or be degraded by enzymes present in one context but not another (e.g. environment-dependent regulation of beta-lactamase production).
  • What do mixed-species biofilms look like, on a scale relevant to bacterial interactions? Are the different species well mixed in space, and do they organise themselves in space differently in different infections? Without knowing this, we can’t say which ecological interactions (signalling, competition, cooperation) are relevant to microbial life in different biofilms.
  • How does the host commensal microbiome - which varies at different body sites - affect how pathogens invade and establish biofilm?

The project

In this PhD, you will work with selected in vivo-like polymicrobial biofilm models that we have developed in our labs (cystic fibrosis lung, chronic skin wound, gut, ventilator tube). You will make biofilms of key pathogens (e.g. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae) and use advanced microscopy techniques to characterize at a molecular level how the biofilm matrix in your chosen environments differs using specific probes for matrix polysaccharides and eDNA. Alongside this, you will assess the spatial geography of bacteria and host cells in the models. Having made these fundamental observations, you could determine how well bacterial cell-cell signalling molecules and/or antimicrobials enter the biofilm matrix, an map the distribution of these molecules in high resolution using a state-of-the art mass spectrometry technique (Cryo-OrbiSIMS). This will enable you to move on to more detailed questions of biofilm biology, such as the role of the host microbiome in infection, the spatiotemporal pattern of gene expression, the presence of microniches with different pH or oxygen concentration, or strategies for improving drug delivery.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology

    Techniques that will be undertaken during the project:

    • Fundamental microbiology skills inc. culturing Class 2 pathogenic bacteria and a range of standard assays (reporter genes, colorimetric/chemical assays) for quantifying virulence factor expression in Gram- and Gram+ bacteria.
    • Antibiotic resistance profiling of clonal and polyclonal populations of pathogenic bacteria.
    • Basic molecular biology skills e.g. inserting antibiotic resistance marker or luminescent/fluorescent reporter construct to facilitate co-culture experiments.
    • Tissue preparation for histopathological analysis of infected animal tissue.
    • Light/epifluorescence/confocal microscopy and image analysis.
    • Cryo-OrbiSIMS: a new platform for secondary ion mass spectrometry which allows 3D imaging of biofilms in their native (hydrated) state.
    • Potentially – use of fluorescent nanosensors to characterise biofilm pH/oxygen concentration.
    • A range of parametric and non-parametric statistical analyses.
    • Awareness and promotion of 3Rs in infection research.

    Contact: Dr Freya Harrison, University of Warwick