Kim Rachael Hardie

Graphical Brief diagrammatic overview of tetracyclines structure, mode of action, side-effects, spectrum of action and administration. Tetracyclines are type II polyketides consisting of four fused rings, with an oxygen-rich ‘non-modifiable’ region required to maintain antimicrobial activity. Tetracyclines target the prokaryotic ribosome by binding the 30S subunit, inhibiting polypeptide elongation. These antimicrobials have a broad spectrum of activity targeting Gram-positive and Gram-negative bacteria alongside eukaryotic parasites. Tetracyclines are administered both orally and intravenously and cause a wide range of side effects, primarily driven by the ability of tetracycline to chelate essential metal ions and bind to the mitochondrial ribosomes. The graphics were created in BioRender. Fenn, S. (2026) https://BioRender.com/fdhfh5s

Pseudomonas aeruginosa is commonly isolated from chronic wounds where it forms biofilms that are recalcitrant to current treatments, greatly impacting the morbidity and mortality of infected patients. Since P. aeruginosa dictates wound severity and treatment outcomes, this bacterium is an important target in the treatment of chronic wound infections. This study aimed to unravel the functional intricacies of a key virulence factor, the arginine-specific aminopeptidase of P. aeruginosa (AaaA), which is crucial for the establishment and persistence of chronic infections. While interrogation of genomes revealed that aaaA is highly conserved, its activity varied between chronic wound clinical isolates. To explore the phenotypic impact of AaaA in chronic infections, a collagen-based synthetic chronic wound (SCW) model was optimized to enable reproducible assessment of AaaA activity. AaaA activity was increased within the SCW compared to planktonic cultures, further supporting the hypothesis that this virulence factor is integral to chronic infections caused by P. aeruginosa . It was also shown that AaaA contributed to biofilm formation as well as providing a survival advantage within the SCW model. Overall, a novel link between AaaA and biofilm formation was verified and served to guide the optimisation of a realistic chronic wound infection model.

Introduction. Bacterial infections of skin wounds can increase hospitalization duration and lead to worse patient prognoses, especially for burn wounds and diabetic foot ulcers. The two main pathogens which infect these wounds are Pseudomonas aeruginosa and Staphylococcus aureus . However, many other species can be present in wound infections, including skin commensal bacteria such as Staphylococcus epidermidis and Micrococcus luteus . Hypothesis. It was hypothesized that co-infection alters the antibiotic resistance of each species present. Aim. To investigate dual-species commensal-pathogen co-culture and assess the potential influence on the antibiotic resistance of each species. Methodology. The commensal and pathogenic species were grown either separately or in dual-species co-culture, potentially allowing biofilm formation for 24 h and were subsequently treated with antibiotics (ciprofloxacin or tobramycin). The impact of the co-culture growth was compared with single species cultures and the effect of the antimicrobial treatment on both conditions were assessed through Minimum Biofilm Eradication Concentrations (MBECs) and bacterial viable counts. Results. The viability of each bacterial species was reduced in the presence of other species, and this translated to reduced antibiotic resistance (lower MBECs) of P. aeruginosa in particular. The resistance of the other species appeared more dependent on the specific inter-species effects. Conclusion. The inclusion of a commensal species with pathogens in co-culture reduced the antibiotic resistance, and inter-species effects influenced the viability of the pathogens. More realistic antimicrobial resistance assessment protocols accounting for microbial communities could therefore lead to more effective treatments.

Introduction. Chronic wounds are notoriously difficult to treat and are associated with decreased limb function, reduced quality of life and significant morbidity. Their recurrent nature, despite aggressive antibiotic therapy, is due in part to the presence of polymicrobial biofilms. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most frequently co-isolated pathogens in these infections and are known to form complex biofilms that hinder treatment. Hypothesis. We hypothesized that co-existence and competitive dynamics between P. aeruginosa and S. aureus in chronic wound infections are influenced by strain-specific interactions and may not rely solely on well-characterized inhibitory mechanisms such as 2-alkyl-4-quinolone (AQ) production by P. aeruginosa impacting on S. aureus fitness. Aim. To establish a polymicrobial chronic wound infection model and assess the contribution of AQ signalling and strain-specific interactions on co-existence. Methodology. We used a modified chronic wound biofilm model to co-culture matched and mismatched clinical isolate pairs of P. aeruginosa and S. aureus , collected from two different chronic wound patients. Viable bacterial counts (c.f.u.) were quantified over an 8-day period. AQ production by each P. aeruginosa strain was quantified using liquid chromatography-MS. Results. A stable culture of P. aeruginosa strains was achieved, but distinct behaviours between each S. aureus strain were seen. One matched clinical isolate pair maintained stable c.f.u. levels of both species throughout the 8-day model, indicating a compatible co-existence. In contrast, mismatched pairs showed early loss of S. aureus viability and the emergence of small colony variants after 4 days, not seen in matched pair growth. Interestingly, the most competitive P. aeruginosa strain exhibited undetectable levels of all AQs tested, indicating that its dominance was not due to AQ-mediated antagonism, as has previously been described. Conclusion. Our findings demonstrate that stable dual-species biofilm formation in chronic wounds is strain-dependent and that P. aeruginosa can impact on S. aureus fitness through AQ-independent mechanisms. These results highlight the importance of using clinical isolates in biofilm research and caution against generalizing findings from laboratory strains to complex clinical infections.

The biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan-induced antibiotic tolerance and biofilm formation in Staphylococcus aureus. Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected S. aureus biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in S. aureus biofilms required an active stringent response because biofilms of a pppGpp⁰ strain were not protected from antibiotic killing. Incubation of S. aureus with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation.

Antimicrobial resistance is a threat to public health for which new treatments are urgently required. The capability of bacteria to form biofilms is of particular concern as it enables high bacterial tolerance to conventional therapies by reducing drug diffusion through the dense, exopolymeric biofilm matrix and the upregulation of antimicrobial resistance machinery. Quorum sensing (QS), a process where bacteria use diffusible chemical signals to coordinate group behaviour, has been shown to be closely interconnected with biofilm formation and bacterial virulence in many top priority pathogens including Pseudomonas aeruginosa. Inhibition of QS pathways therefore pose an attractive target for new therapeutics. We have recently reported a new series of pqs quorum sensing inhibitors (QSIs) that serve as potentiators for antibiotics in P. aeruginosa infections. The impact on biofilms of some reported QSIs was however hindered by their poor penetration through the bacterial biofilm, limiting the potential for clinical translation. In this study we developed a series of poly(β-amino ester) (PBAE) triblock copolymers and evaluated their ability to form micelles, encapsulate a QSI and enhance subsequent delivery to P. aeruginosa biofilms. We observed that the QSI could be released from polymer micelles, perturbing the pqs pathway in planktonic P. aeruginosa. In addition, one of the prepared polymer variants increased the QSIs efficacy, leading to an enhanced potentiation of ciprofloxacin (CIP) action and therefore improved reduction in biofilm viability, compared to the non-encapsulated QSI. Thus, we demonstrate QSI encapsulation in polymeric particles can enhance its efficacy through improved biofilm penetration.

The biocompatibility, biodegradability, and responsiveness of poly( β ‐amino esters) (PBAEs) has led to their widespread use as biomaterials for drug and gene delivery. Nonetheless, the step‐growth polymerization mechanism that yields PBAEs limits the scope for their structural optimization toward specific applications because of limited monomer choice and end‐group modifications. Moreover, to date the post‐synthetic functionalization of PBAEs has relied on grafting‐to approaches, challenged by the need for efficient polymer–polymer coupling and potentially difficult post‐conjugation purification. Here a novel grafting‐from approach to grow reversible addition–fragmentation chain transfer (RAFT) polymers from a PBAE scaffold is described. This is achieved through PBAE conversion into a macromolecular chain transfer agent through a multistep capping procedure, followed by RAFT polymerization with a range of monomers to produce PBAE–RAFT hybrid triblock copolymers. Following successful synthesis, the potential biological applications of these ABA triblock copolymers are illustrated through assembly into polymeric micelles and encapsulation of a model hydrophobic drug, followed by successful nanoparticle (NP) uptake in breast cancer cells. The findings demonstrate this novel synthetic methodology can expand the scope of PBAEs as biomaterials.

The biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan induced antibiotic tolerance and biofilm formation in Staphylococcus aureus . Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected S. aureus biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in planktonic and biofilm cultures required an active stringent response because a pppGpp ⁰ strain was not protected from antibiotic killing. Incubation of S. aureus with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation. Importance: The prevalent bacterium Staphylococcus aureus infects skin lesions and indwelling devices, and this can cause sepsis with 33% mortality. Intrinsic to this is the formation of co-ordinated communities (biofilms) protected by a polysaccharide coat. S. aureus is increasingly difficult to eradicate due to its antibiotic resistance. Protection against Methicillin Resistant S. aureus (MRSA) includes pre-hospital admission washing with products containing biocides. The biocide triclosan is the predominant antibacterial compound in sewage in Ontario due to its use in household and hospital settings. Levels of triclosan accumulate with exposure in humans. The significance of our research is in identifying the mechanisms triggered by exposure of S. aureus to physiological levels of triclosan that go on to raise the tolerance of S. aureus to antibiotics and promote the formation of biofilms. This understanding will inform future criteria used to determine effective antimicrobial treatments.

Rifampicin (also known as rifampin) inhibits RNA synthesis, and is used to treat tuberculosis, leprosy, staphylococcal infections and legionnaires’ disease. It can also protect at-risk populations from Haemophilus influenzae type b and Neisseria meningitidis . It is a polyketide antibiotic and is on the World Health Organization (WHO) list of essential medicines due to its critical importance to human medicine. The adverse effect of liver toxicity is controlled by testing during prolonged treatment regimes. Rifampicin’s red–orange colour can result in the colouration of sweat, tears and urine. Resistance to rifampicin arises from mutation of the target RNA polymerase or ADP ribosylation of the antibiotic or efflux. Mycobacteria may become singularly resistant to rifampicin or as part of multidrug or extensive drug resistance.