Phenotypic and genotypic characterisation of single and dual species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus treated with a Novel antimicrobial compound

Abstract

Bacterial infections are becoming increasingly difficult to treat due to the emergence of antimicrobial resistance, (AMR), which renders current antimicrobial therapies ineffective. Further complicating matters is the ability of bacteria to form communities called biofilms, which are infamous for their tolerance to antimicrobial therapy. Biofilm mediated tolerance and AMR contribute to disease chronicity. Consequently, there is a need for novel antimicrobial interventions. The aim of this thesis was to characterise a novel antimicrobial, HT61, against biofilms of clinically relevant bacterial pathogens, particularly Pseudomonas aeruginosa and Staphylococcus aureus.

Phenotypic studies showed that HT61 was effective against biofilms formed by Gram-positive species with limited efficacy towards biofilms of Gram-negative species. Scanning electron microscopy of HT61 treated biofilms of S. aureus and P. aeruginosa suggested a mechanism of action targeting the cell envelope. Quantitative proteomic analysis of HT61 treated S. aureus cultures supported this, identifying upregulation of proteins associated with the cell wall stress stimulon and division cell wall cluster.

To investigate the effect of interspecies interactions on bacterial adaptation to HT61, a dual species biofilm model of P. aeruginosa and S. aureus was developed and characterised. Fluctuation analysis suggested that biofilm co-culture increased the mutation rate of S. aureus over 500-fold compared to planktonic culture and almost 100-fold compared to culture as a single species biofilm.

Whole genome sequencing of single and dual species biofilm derived P. aeruginosa and S. aureus isolates revealed significant genomic variation in both coding and intergenic sequences, but no change in evolutionary trajectory between isolates derived from mono- or co-culture biofilms. Following HT61 treatment, mutations in S. aureus were identified in graS and fmtC, which encode products that modulate the cell envelope and may suggest routes to HT61 adaptation.

In summary, the data presented in this thesis suggests potential mechanisms of action and adaptation to HT61, which could inform future antimicrobial development. This thesis also reinforces the need to understand the impact of interspecies interactions on bacterial evolution and shows that biofilms are important hubs of genomic diversity, which could have dangerous implications for the emergence of AMR.

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Identifiers

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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