RESUMO
Biofilms are complex microbial communities that have a critical function in many natural ecosystems, industrial settings as well as in recurrent and chronic infections. Biofilms are highly heterogeneous and dynamic assemblages that display complex responses to varying environmental factors, and those properties present substantial challenges for their study and control. In recent years, there has been a growing interest in developing improved biofilm models to offer more precise and comprehensive representations of these intricate systems. However, an objective assessment for ascertaining the ability of biofilms in model systems to recapitulate those in natural environments has been lacking. In this Perspective, we focus on medical biofilms to delve into the current state-of-the-art in biofilm modelling, emphasizing the advantages and limitations of different approaches and addressing the key challenges and opportunities for future research. We outline a framework for quantitatively assessing model accuracy. Ultimately, this Perspective aims to provide a comprehensive and critical overview of medically focused biofilm models, with the intent of inspiring future research aimed at enhancing the biological relevance of biofilm models.
RESUMO
Antimicrobial resistance poses an escalating global threat, rendering traditional drug development approaches increasingly ineffective. Thus, novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats, effectively reducing virulence in vitro and in vivo. The idea of harnessing cooperative behaviours for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study, we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats, followed by increased antibiotic effectiveness against cheat-invaded populations, thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.
Assuntos
Antibacterianos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Percepção de Quorum , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Antibacterianos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Animais , Virulência/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
In this primer on biofilms and their role in infections, we trace the historical roots of microbial understanding from Van Leeuwenhoek's observations to Bill Costerton's groundbreaking work, which solidified biofilms' significance in infections. In vivo biofilm research, investigating patient samples and utilizing diverse host models, has yielded invaluable insights into these complex microbial communities. However, it comes with several challenges, particularly regarding replicating biofilm infections accurately in the laboratory. In vivo biofilm analyses involve various techniques, revealing biofilm architecture, composition, and behaviour, while gaps in knowledge persist regarding infection initiation and source, diversity, and the Infectious Microenvironment (IME). Ultimately, the study of biofilms in infections remains a dynamic and evolving field poised to transform our approach to combat biofilm-associated diseases.
Assuntos
Biofilmes , Infecções , Humanos , Infecções/microbiologiaRESUMO
Chronic rhinosinusitis (CRS) is a debilitating condition characterized by long-lasting inflammation of the paranasal sinuses. It affects a significant portion of the population, causing a considerable burden on individuals and healthcare systems. The pathogenesis of CRS is multifactorial, with bacterial infections playing a crucial role in CRS development and persistence. In recent years, the presence of biofilms has emerged as a key contributor to the chronicity of sinusitis, further complicating treatment and exacerbating symptoms. This review aims to explore the role of biofilms in CRS, focusing on the involvement of the bacterial species Staphylococcus aureus and Pseudomonas aeruginosa, their interactions in chronic infections, and model systems for studying biofilms in CRS. These species serve as an example of how microbial interplay can influence disease progression and exemplify the need for continued investigation and innovation in CRS research.
RESUMO
Antimicrobial resistance poses an escalating global threat, rendering traditional drug development approaches increasingly ineffective. Thus, novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats, effectively reducing virulence in vitro and in vivo. The idea of harnessing cooperative behaviors for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study, we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats, followed by increased antibiotic effectiveness against cheat-invaded populations, thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.
RESUMO
The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.
Assuntos
Doença Aguda , Doença Crônica , Genes Bacterianos , Oxigênio , Infecções por Pseudomonas , Pseudomonas aeruginosa , RNA Bacteriano , Animais , Humanos , Oxigênio/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fibrose Cística/microbiologia , Ferimentos e Lesões/microbiologia , Ubiquinona/biossíntese , Anaerobiose , Genes Bacterianos/genética , Sepse/complicações , Sepse/microbiologiaRESUMO
Calprotectin is a transition metal chelating protein of the innate immune response known to exert nutritional immunity upon microbial infection. It is abundantly released during inflammation and is therefore found at sites occupied by pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The metal limitation induced by this protein has previously been shown to mediate P. aeruginosa and S. aureus co-culture. In addition to the transition metal sequestration role of calprotectin, it has also been shown to have metal-independent antimicrobial activity via direct cell contact. Therefore, we sought to assess the impact of this protein on the biofilm architecture of P. aeruginosa and S. aureus in monomicrobial and polymicrobial culture. The experiments described in this report reveal novel aspects of calprotectin's interaction with biofilm communities of P. aeruginosa and S. aureus discovered using scanning electron microscopy and confocal laser scanning microscopy. Our results indicate that calprotectin can interact with microbial cells by stimulating encapsulation in mesh-like structures. This physical interaction leads to compositional changes in the biofilm extracellular polymeric substance (EPS) in both P. aeruginosa and S. aureus.
Assuntos
Biofilmes , Imunidade Inata , Complexo Antígeno L1 Leucocitário , Pseudomonas aeruginosa , Staphylococcus aureus , Antibacterianos/imunologia , Antibacterianos/farmacologia , Matriz Extracelular de Substâncias Poliméricas/genética , Matriz Extracelular de Substâncias Poliméricas/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/imunologia , Fagocitose , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologiaRESUMO
A new technique was used to measure the viscoelasticity of in vivo Pseudomonas aeruginosa biofilms. This was done through ex vivo microrheology measurements of in vivo biofilms excised from mouse wound beds. To our knowledge, this is the first time that the mechanics of in vivo biofilms have been measured. In vivo results are then compared to typical in vitro measurements. Biofilms grown in vivo are more relatively elastic than those grown in a wound-like medium in vitro but exhibited similar compliance. Using various genetically mutated P. aeruginosa strains, it is observed that the contributions of the exopolysaccharides Pel, Psl, and alginate to biofilm viscoelasticity were different for the biofilms grown in vitro and in vivo. In vitro experiments with collagen containing medium suggest this likely arises from the incorporation of host material, most notably collagen, into the matrix of the biofilm when it is grown in vivo. Taken together with earlier studies that examined the in vitro effects of collagen on mechanical properties, we conclude that collagen may, in some cases, be the dominant contributor to biofilm viscoelasticity in vivo.
Assuntos
Biofilmes , Pseudomonas aeruginosa , Animais , Colágeno/metabolismo , Colágeno/farmacologia , Camundongos , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/fisiologia , Substâncias Viscoelásticas , Ferimentos e Lesões/microbiologiaRESUMO
Biofilms are the cause of most chronic bacterial infections. Living within the biofilm matrix, which is made of extracellular substances, including polysaccharides, proteins, eDNA, lipids and other molecules, provides microorganisms protection from antimicrobials and the host immune response. Exopolysaccharides are major structural components of bacterial biofilms and are thought to be vital to numerous aspects of biofilm formation and persistence, including adherence to surfaces, coherence with other biofilm-associated cells, mechanical stability, protection against desiccation, binding of enzymes, and nutrient acquisition and storage, as well as protection against antimicrobials, host immune cells and molecules, and environmental stressors. However, the contribution of specific exopolysaccharide types to the pathogenesis of biofilm infection is not well understood. In this study we examined whether the absence of the two main exopolysaccharides produced by the biofilm former Pseudomonas aeruginosa would affect wound infection in a mouse model. Using P. aeruginosa mutants that do not produce the exopolysaccharides Pel and/or Psl we observed that the severity of wound infections was not grossly affected; both the bacterial load in the wounds and the wound closure rates were unchanged. However, the size and spatial distribution of biofilm aggregates in the wound tissue were significantly different when Pel and Psl were not produced, and the ability of the mutants to survive antibiotic treatment was also impaired. Taken together, our data suggest that while the production of Pel and Psl do not appear to affect P. aeruginosa pathogenesis in mouse wound infections, they may have an important implication for bacterial persistence in vivo.
Assuntos
Infecções por Pseudomonas , Infecção dos Ferimentos , Animais , Proteínas de Bactérias/genética , Biofilmes , Camundongos , Polissacarídeos Bacterianos/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genéticaRESUMO
The ever-growing threat of new and existing infectious diseases in combination with antimicrobial resistance requires the need for innovative and effective forms of drug delivery. Optimal drug delivery systems for existing and newly developed antimicrobials can enhance drug bioavailability, enable site-specific drug targeting, and overcome current limitations of drug formulations such as short elimination half-lives, poor drug solubility, and undesirable side effects. Nanoemulsions (NE) consist of nanometer-sized droplets stabilized by emulsifiers and are typically more stable and permeable due to their smaller particle sizes and higher surface area compared to conventional emulsions. NE have been identified as a promising means of antimicrobial delivery due to their intrinsic antimicrobial properties, ability to increase drug solubility, stability, bioavailability, organ and cellular targeting potentials, capability of targeting biofilms, and potential to overcome antimicrobial resistance. Herein, we discuss non-drug loaded essential oil-based NE that can confer antimicrobial actions through predominantly physical or biochemical mechanisms without drug payloads. We also describe drug-loaded NE for enhanced antimicrobial efficacy by augmenting the potency of existing antimicrobials. We highlight the versatility of NE to be administered through multiple different routes (oral, parenteral, dermal, transdermal, pulmonary, nasal, ocular, and rectal). We summarize recent advances in the clinical translation of antimicrobial NE and shed light on future development of effective antimicrobial therapy to combat infectious diseases.
Assuntos
Anti-Infecciosos , Nanopartículas , Óleos Voláteis , Anti-Infecciosos/farmacologia , Sistemas de Liberação de Medicamentos , Emulsões , Tamanho da Partícula , SolubilidadeRESUMO
Microbes interact in natural communities in a spatially structured manner, particularly in biofilms and polymicrobial infections. While next generation sequencing approaches provide powerful insights into diversity, metabolic capacity, and mutational profiles of these communities, they generally fail to recover in situ spatial proximity between distinct genotypes in the interactome. Hi-C is a promising method that has assisted in analysing complex microbiomes, by creating chromatin cross-links in cells, that aid in identifying adjacent DNA, to improve de novo assembly. This study explored a modified Hi-C approach involving an initial lysis phase prior to DNA cross-linking, to test whether adjacent cell chromatin can be cross-linked, anticipating that this could provide a new avenue for study of spatial-mutational dynamics in structured microbial communities. An artificial polymicrobial mixture of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli was lysed for 1-18 h, then prepared for Hi-C. A murine biofilm infection model was treated with sonication, mechanical lysis, or chemical lysis before Hi-C. Bioinformatic analyses of resulting Hi-C interspecies chromatin links showed that while microbial species differed from one another, generally lysis significantly increased links between species and increased the distance of Hi-C links within species, while also increasing novel plasmid-chromosome links. The success of this modified lysis-Hi-C protocol in creating extracellular DNA links is a promising first step toward a new lysis-Hi-C based method to recover genotypic microgeography in polymicrobial communities, with potential future applications in diseases with localized resistance, such as cystic fibrosis lung infections and chronic diabetic ulcers.
Assuntos
Fibrose Cística , Pseudomonas aeruginosa , Animais , Biofilmes , Camundongos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureusRESUMO
Standard doses of antibiotics do not efficiently treat chronic infections of the soft tissue and bone. In this Personal View, we advocate for improving treatment of these infections by taking the infectious microenvironment into account. The infectious microenvironment can cause sensitive bacteria to lose their susceptibility to antibiotics that are effective in standard laboratory susceptibility testing. We propose that bacteria behave substantially different in standard laboratory conditions than they do in actual infections. The infectious microenvironment could impose changes in growth and metabolic activity that result in increased protection against antibiotics. Therefore, we advocate that improved antibiotic treatment of chronic infection is achievable when antibiotics are recommended on the basis of susceptibility testing in relevant in vitro conditions that resemble actual infectious microenvironments. We recommend establishing knowledge of the relevant conditions of the chemical and physical composition of the infectious microenvironment. Recent advances in RNA sequencing, metabolomics, and microscopy have made it possible for the characterisation of the microenvironment of infections and to validate the clinical relevance of in vitro conditions to actual infections.
Assuntos
Antibacterianos , Bactérias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , HumanosRESUMO
Novel anti-biofilm and dispersal agents are currently being investigated in an attempt to combat biofilm-associated wound infections. Glycoside hydrolases (GHs) are enzymes that hydrolyze the glycosidic bonds between sugars, such as those found within the exopolysaccharides of the biofilm matrix. Previous studies have shown that GHs can weaken the matrix, inducing bacterial dispersal, and improving antibiotic clearance. Yet, the number of GH enzymes that have been examined for potential therapeutic effects is limited. In this study, we screened sixteen GHs for their ability to disperse mono-microbial and polymicrobial biofilms grown in different environments. Six GHs, α-amylase (source: A. oryzae), alginate lyase (source: various algae), pectinase (source: Rhizopus sp.), amyloglucosidase (source: A. niger), inulinase (source: A. niger), and xylanase (source: A. oryzae), exhibited the highest dispersal efficacy in vitro. Two GHs, α-amylase (source: Bacillus sp.) and cellulase (source: A. niger), used in conjunction with meropenem demonstrated infection clearing ability in a mouse wound model. GHs were also effective in improving antibiotic clearance in diabetic mice. To examine their safety, we screened the GHs for toxicity in cell culture. Overall, there was an inverse relationship between enzyme exposure time and cellular toxicity, with twelve out of sixteen GHs demonstrating some level of toxicity in cell culture. However, only one GH exhibited harmful effects in mice. These results further support the ability of GHs to improve antibiotic clearance of biofilm-associated infections and help lay a foundation for establishing GHs as therapeutic agents for chronic wound infections.
RESUMO
Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.
Assuntos
Matriz Extracelular de Substâncias Poliméricas , Infecção dos Ferimentos , Animais , Antibacterianos/uso terapêutico , Biofilmes , Camundongos , Pseudomonas aeruginosaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that causes thousands of deaths every year in part due to its ability to form biofilms composed of bacteria embedded in a matrix of self-secreted extracellular polysaccharides (EPS), e-DNA, and proteins. In chronic wounds, biofilms are exposed to the host extracellular matrix, of which collagen is a major component. How bacterial EPS interacts with host collagen and whether this interaction affects biofilm viscoelasticity is not well understood. Since physical disruption of biofilms is often used in their removal, knowledge of collagen's effects on biofilm viscoelasticity may enable new treatment strategies that are better tuned to biofilms growing in host environments. In this work, biofilms are grown in the presence of different concentrations of collagen that mimic in vivo conditions. In order to explore collagen's interaction with EPS, nine strains of P. aeruginosa with different patterns of EPS production were used to grow biofilms. Particle tracking microrheology was used to characterize the mechanical development of biofilms over two days. Collagen is found to decrease biofilm compliance and increase relative elasticity regardless of the EPS present in the system. However, this effect is minimized when biofilms overproduce EPS. Collagen appears to become a de facto component of the EPS, through binding to bacteria or physical entanglement.
Assuntos
Biofilmes , Pseudomonas aeruginosa , Colágeno , Polissacarídeos Bacterianos , ViscosidadeRESUMO
Wound biofilms must be identified to target disruption and bacterial eradication but are challenging to detect with standard clinical assessment. This study tested whether bacterial fluorescence imaging could detect porphyrin-producing bacteria within a biofilm using well-established in vivo models. Mouse wounds were inoculated on Day 0 with planktonic bacteria (n = 39, porphyrin-producing and non-porphyrin-producing species, 107 colony forming units (CFU)/wound) or with polymicrobial biofilms (n = 16, 3 biofilms per mouse, each with 1:1:1 parts Staphylococcus aureus/Escherichia coli/Enterobacter cloacae, 107 CFU/biofilm) that were grown in vitro. Mouse wounds inoculated with biofilm underwent fluorescence imaging up to Day 4 or 5. Wounds were then excised and sent for microbiological analysis. Bacteria-matrix interaction was assessed with scanning electron microscopy (SEM) and histopathology. A total of 48 hours after inoculation with planktonic bacteria or biofilm, red fluorescence was readily detected in wounds; red fluorescence intensified up to Day 4. Red fluorescence from biofilms persisted in excised wound tissue post-wash. SEM and histopathology confirmed bacteria-matrix interaction. This pre-clinical study is the first to demonstrate the fluorescence detection of bacterial biofilm in vivo using a point-of-care wound imaging device. These findings have implications for clinicians targeting biofilm and may facilitate improved visualisation and removal of biofilms.
Assuntos
Infecção dos Ferimentos , Animais , Bactérias , Biofilmes , Camundongos , Imagem Óptica , Sistemas Automatizados de Assistência Junto ao Leito , Infecção dos Ferimentos/diagnósticoRESUMO
Proteases play an essential role in the four sequential but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. In chronic wounds, excessive protease secretion damages the newly formed extracellular matrix, thereby delaying or preventing the normal healing process. Peptide-based fluorogenic sensors provide a visual platform to sense and analyze protease activity through changes in the fluorescence intensity. Here, we have developed an integrated microfluidic chip coated with multilayered fluorogenic nanofilms that can directly monitor protease activity. Fluorogenic protease sensors were chemically conjugated to polymer films coated on the surface of parallel microfluidic channels. Capillary flow layer-by-layer (CF-LbL) was used for film assembly and combined with subsequent sensor modification to establish a novel platform sensing technology. The benefits of our platform include facile fabrication and processing, controllable film nanostructure, small sample volume, and high sensitivity. We observed increased fluorescence of the LbL nanofilms when they were exposed to model recombinant proteases, confirming their responsiveness to protease activity. Increases in the nanofilms' fluorescence intensity were also observed during incubation with liquid extracted from murine infected wounds, demonstrating the potential of these films to provide real-time, in situ information about protease activity levels.
Assuntos
Nanoestruturas , Animais , Matriz Extracelular , Camundongos , Peptídeo Hidrolases , Polímeros , CicatrizaçãoRESUMO
Staphylococcus aureus is a prominent etiological agent of suppurative abscesses. In principle, abscess formation and purulent exudate are classical physiological features of healing and tissue repair. However, S. aureus deploys two coagulases that can usurp this classical host response and form distinct abscess lesions. Here, we establish that during coinfection with coagulase producers and non-producers, coagulases are shared public goods that contribute to staphylococcal persistence, abscess formation, and disease progression. Coagulase-negative mutants that do not produce the public goods themselves are able to exploit those cooperatively secreted by producers and thereby thrive during coinfection at the expense of others. This study shows the importance of social interactions among pathogens concerning clinical outcomes.
Assuntos
Abscesso/microbiologia , Coagulase/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coagulase/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Humanos , Camundongos , Interações Microbianas , Mutação , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Opportunistic pathogens are associated with a number of chronic human infections, yet the evolution of virulence in these organisms during chronic infection remains poorly understood. Here, we tested the evolution of virulence in the human opportunistic pathogen Pseudomonas aeruginosa in a murine chronic wound model using a two-part serial passage and sepsis experiment, and found that virulence evolved in different directions in each line of evolution. We also assessed P. aeruginosa adaptation to a chronic wound after 42 days of evolution and found that morphological diversity in our evolved populations was limited compared with that previously described in cystic fibrosis (CF) infections. Using whole-genome sequencing, we found that genes previously implicated in P. aeruginosa pathogenesis (lasR, pilR, fleQ, rpoN and pvcA) contained mutations during the course of evolution in wounds, with selection occurring in parallel across all lines of evolution. Our findings highlight that: (i) P. aeruginosa heterogeneity may be less extensive in chronic wounds than in CF lungs; (ii) genes involved in P. aeruginosa pathogenesis acquire mutations during chronic wound infection; (iii) similar genetic adaptations are employed by P. aeruginosa across multiple infection environments; and (iv) current models of virulence may not adequately explain the diverging evolutionary trajectories observed in an opportunistic pathogen during chronic wound infection.