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

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

Antibiofilm efficacy of antibiotic-loaded synthetic calcium sulphate beads in a P. aeruginosa/S. aureus co-culture model for prosthetic infections

Bacterial biofilms play a key role in prosthetic infection (PI) pathogenesis. Establishment of the biofilm phenotype confers the bacteria with significant tolerance to systemic antibiotics and the host immune system meaning thorough debridement and prosthesis removal often remain the only possible course of treatment. Protection of the prosthesis and dead-space management may be achieved through the use of antibiotic loaded cements and beads to release high concentrations of antibiotics at the surgical site. The antibacterial and antibiofilm efficacy of these materials is poorly understood in the context of mixed species models, such as are often encountered clinically

Dataset: Proteomic response of S. aureus to HT61

Dataset supports: Frapwell, C.J. et al (2020). Antimicrobial activity of the quinoline derivative HT61 against Staphylococcus aureus biofilms. Antimicrobial Agents and Chemotherapy. Data set containing mass spectrometry results for S. aureus UAMS-1 planktonic and biofilm cultures, before and after treatment with HT61.</span

Prevention of <i>Propionibacterium acnes</i> biofilm formation in prosthetic infections <i>in vitro</i>

Background: The role of Propionibacterium acnes in shoulder arthroplasty and broadly in orthopaedic prosthetic infections (PIs) has historically been underestimated, with biofilm formation identified as a key virulence factor attributed to invasive isolates. With an often indolent clinical course, P. acnes infection can be difficult to detect and treat. The present study investigates absorbable cements loaded with a broad-spectrum antibiotic combination as an effective preventative strategy to combat P. acnes biofilms.Methods: P. acnes biofilm formation on an unloaded synthetic calcium sulfate (CaSO4) bone void filler cement bead was evaluated by scanning electron microscopy (SEM) over 14 days. Beads loaded with tobramycin alone, vancomycin alone (as comparative controls) and a vancomycin &amp; tobramycin dual treatment were assessed for their ability to eradicate planktonic P. acnes, prevent biofilm formation and eradicate preformed biofilms was also evaluated using viable cell counts, confocal microscopy and SEM.Results: P. acnes surface colonisation and biofilm formation on unloaded CaSO4 beads was slow. Beads loaded with antibiotics were able to kill planktonic cultures of 106 CFU/ml, prevent bacterial colonisation and significantly reduce biofilm formation over periods of weeks. Complete eradication of established biofilms was achieved with a contact time of one week.Conclusions: This study demonstrates that antibiotic-loaded CaSO4 beads may represent an effective antibacterial and antibiofilm strategy to combat PIs in which P. acnes is involved.Level of Evidence: Basic Science Study<br/

Increased rates of genomic mutation in a biofilm co-culture model of Pseudomonas aeruginosa and Staphylococcus aureus

Biofilms are major contributors to disease chronicity and are typically multi-species in nature. Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of morbidity and mortality in a variety of chronic diseases but current in vitro dual-species biofilms models involving these pathogens are limited by short co-culture times (24 to 48 hours). Here, we describe the establishment of a stable (240 hour) co-culture biofilm model of P. aeruginosa and S. aureus that is reproducible and more representative of chronic disease. The ability of two P. aeruginosa strains, (PAO1 and a cystic fibrosis isolate, PA21), to form co-culture biofilms with S. aureus was investigated. Co-culture was stable for longer periods using P. aeruginosa PA21 and S. aureus viability within the model improved in the presence of exogenous hemin. Biofilm co-culture was associated with increased tolerance of P. aeruginosa to tobramycin and increased susceptibility of S. aureus to tobramycin and a novel antimicrobial, HT61, previously shown to be more effective against non-dividing cultures of Staphylococcal spp. Biofilm growth was also associated with increased short-term mutation rates; 10-fold for P. aeruginosa and 500-fold for S. aureus. By describing a reproducible 240 hour co-culture biofilm model of P. aeruginosa and S. aureus, we have shown that interspecies interactions between these organisms may influence short-term mutation rates and evolution, which could be of importance in understanding the adaptive processes that lead to the development of antimicrobial resistance