INTEG RATED
HUMAN PRACT ICES

Introduction

What is Pseudomonas aeruginosa?

Pseudomonas aeruginosa (PA) is a Gram-negative, opportunistic pathogen that typically causes illness in individuals with weakened immune systems or compromised defenses. Its ability to form resilient biofilms to environmental stresses and survive in diverse moist environments makes it capable of thriving in conditions often inhospitable to other bacteria. Additionally, the high prevalence of highly mutatable P. aeruginosa strains enables the pathogen to acquire extremely pervasive antimicrobial resistance (AMR) [1], making the management of chronic infections particularly challenging. The extreme versatility, AMR pervasiveness, and wide range of defenses make P. aeruginosa one of the most difficult organisms to treat in modern medicine [2].

Why should we care?

Pseudomonas aeruginosa is involved in a wide range of infections and systemic conditions such as sepsis, organ failure, and multiple skin conditions. PA usually infects the lungs (predominantly in patients with cystic fibrosis (CF), ears (otitis externa), eyes (keratitis), urinary tract, and bloodstream (via open wounds/burns)). [3], [4]

P. aeruginosa infections are rare in immunocompetent individuals. In most cases of infection, a physical barrier to infection (such as skin or mucous membrane) is lost or an underlying immunodeficiency is present. [5] High-risk groups include: patients with cystic fibrosis (CF), Patients with an immunodeficiency, patients with hospital-acquired infections (HAIs), and those with non-CF bronchiectasis. In this chronic lung condition, the walls of the patient’s airways widen and become thickened due to inflammation and infection [6], often characterized by severe lung deterioration and a poorer quality of life. [7] An international observational study of ICU patients revealed that Pseudomonas aeruginosa accounted for 16.2% of all infections and was responsible for 23% of ICU-acquired infections, with the respiratory system being the most common source of P. aeruginosa infection. [8]

Specific issue

Treating these infections is extremely challenging. P. aeruginosa contains many mechanisms for antibiotic resistance and a variety of virulence factors that account for the many different sites of infection it causes. [9] Conventional treatment of PA relies heavily on the use of antibiotics such as β-lactams, aminoglycosides, quinolones, and polypeptides. However, the use of such broad-spectrum antibiotics has led to increasingly challenging treatment for the resulting antimicrobial-resistant PA strains. [10] MDR PA has multiple mechanisms of antibiotic resistance, including intrinsic antibiotic resistance, efflux systems, and antibiotic-inactivating enzymes. These allow PA to restrict membrane permeability to antimicrobials, excrete antibiotics outside the cell, and directly break down therapeutic drugs.

Additionally, the ability of PA to form a biofilm can increase antibiotic resistance and mitigate host defenses. These biofilms create physical and biochemical barriers that limit antibiotic penetration and interfere with immune responses, such as impaired antibody diffusion. This makes infections difficult to eradicate, especially on indwelling devices like catheters and ventilators. [11] Active infections involve rapid bacterial growth and acute inflammation, and are generally more responsive to treatment. Chronic infections, on the other hand, are characterized by the formation of biofilms, evasion of the immune system, and the persistent presence of bacteria. Current treatment options are ineffective for long-term control of these infections, highlighting the urgent need for innovative therapeutic approaches.

Starting Questions: How can we effectively disarm Pseudomonas aeruginosa and improve treatment outcomes for high-risk patient populations?

Given the wide range of solutions that Pseudomonas aeruginosa has to current treatment options, we aimed to design a solution to enhance current treatment options and reduce the ability of PA to harm high-risk patient groups in healthcare settings.

Our initial therapeutic solution targeted the drug efflux pump responsible for the most antibiotic resistance, MexAB-OprM. Its expression is extremely high in the cell, and has a broad substrate specificity, allowing the system to pump out a variety of antibiotics from the cell. [12] We planned to disarm Pseudomonas aeruginosa by plugging the tiny outer-membrane portion of its MexAB-OprM efflux pump with a "plug" shaped peptide made by AlphaFold and Rosetta. We hoped this strategy would lower the antibiotic resistance of PA, allowing for increased drug uptake by the pathogen. However, after conversing with stakeholders, especially Adam Chazin-Gray, from the Institute of Protein Design, who has experience developing similar protein therapeutics, we determined the delivery of our therapeutic would not be feasible.

As our primary goal remained to reduce the impact of P. aeruginosa on immunocompromised patients and alleviate the burden of AMR strains on hospital settings, we decided to gravitate towards PA’s mechanisms for attacking host cells, one of which is the release of extracellular toxins. Virulence factors, including extracellular toxins, allow PA to adhere to tissue surfaces, spread damaged tissues within the host, and improve the bacteria’s chances of survival by securing nutrients. [13] Exotoxin A (ExoA) is the most toxic virulence factor of Pseudomonas aeruginosa [14] and causes disease by inhibition of protein synthesis, direct cytopathic effects, and interference with cellular immune functions of the host. [15]

Understanding ExoA

Protein visualisation of Exotoxin A from PyMOL

ExoA is composed of three functional domains. The N-terminal receptor-binding domain (domain I - pink) facilitates attachment to host cells by binding to receptors like CD91, which are found on the exterior membrane of a wide variety of cells including epithelial cells, immune cells, and endothelial cells. [16] Once bound, ExoA then undergoes endocytosis, whereby the translocation domain (domain II - yellow) enables the toxin to cross cellular membranes via endosomal pathways, reaching the host cell cytoplasm. Once inside, the C-terminal domain (domain III - cyan) possesses ADP-ribosyltransferase activity, which enzymatically modifies and inactivates the host cell’s eukaryotic elongation factor 2 (eEF-2), halting protein synthesis and inducing apoptosis (cell death) in human cells. [17]

Protein visualisation of our inhibitor (green) bound to ExoA (cyan)

Exotoxin A Inhibition

Our goal is to lower the virulence of Pseudomonas aeruginosa by neutralizing ExoA, thereby protecting host tissues, reducing infection severity, and improving health outcomes for vulnerable patients with bronchiectasis.

Antivirulence FAQs

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Goals and Visions

Impact

We aim to understand the impact of our therapy on high-risk patient populations, such as those with bronchiectasis, who are particularly vulnerable to Pseudomonas infections. Our goal is to maximize the clinical benefits of our therapeutic across hospital, outpatient, and resource-limited settings, ensuring reduced inflammation, slower disease progression, and slower resistance development. By focusing on these vulnerable groups, our approach has the potential to improve patient outcomes while simultaneously alleviating current healthcare burdens. Finally, to implement this treatment effectively, we aim to optimize its delivery and scale it in a manner that supports both the target patient populations and the practitioners responsible for administering the therapy.

Implementation

We considered a variety of implementation options, including a prophylactic coating on catheters and other medical equipment, IV, a gel, and dry powder. An IV implementation would most commonly be used for a large variety of situations and would be relatively efficient to integrate provided the protein was water soluble. A gel would be used to help stabilize our peptide and allow more variety in application. A dry powder would be stable, and could be nebulized for lung infection. All of these options require a lot of thinking about the impact of our project, the ethics, and further work required for FDA approval.

Market and Policy

Our goal is to clearly communicate the process of bringing a hydrogel drug and inhalant for PA to patients, including preclinical development, clinical trials, and regulatory approval. We want to ensure healthcare providers, patients, and stakeholders understand each step of the pathway and the safety measures involved. By emphasizing transparency in this journey, we will aim to build trust and awareness around the treatment’s potential impact.

Society and Ethics

Our solution raises important questions about ethics, safety, and equitable access since it is a non-traditional therapeutic approach. We recognize that developing a non-traditional approach requires balancing innovation with the concerns of patients, clinicians, and policymakers. By engaging with stakeholders and reviewing literature, we were able to explore potential off-target effects, regulations surrounding therapies that directly address toxins, and how to ensure our therapy is not only safe and responsible, but also accessible across different healthcare settings and environments in practice.

Stakeholder Timeline

Impact

P. aeruginosa has a prevalence of 7.1% - 7.3% amongst all healthcare-associated infections, manifesting as pneumonia, surgical site infections, urinary tract infections, etc. [37] Particularly in the ICU, Pseudomonas is responsible for 26% of all ventilator-associated pneumonia infections (VAP) [32] and 10% of all catheter-associated UTIs (CAUTI). [33] Based on valuable insights from clinicians and infectious disease experts, Dr. Erin McCreary, Dr. Julie Ann Justo, and Dr. Emily Heil, our primary goal inhibiting ExoA should be to alter Pseudomonas replication or fitness. If successful, this technology could be implemented through coating catheters or other biomedical devices with antivirulence agents to prevent biofilm formation and infection. They recommended we investigate whether our therapeutic modulation of ExoA, as a virulence factor, would result in changes to local tissues and possibly improve clinical cure rates and mortality rates.

From these clinician interviews, we learned that although ExoA exposure led to human cell death, the need for our therapeutic approach for clinicians wasn’t in preventing tissue necrosis. Instead, it could serve as an adjunctive therapy to reduce Pseudomonas virulence and buy time for existing treatments to take effect, which has the potential to benefit a broader patient group. Following discussions with Dr. Ajai Dandekar, we were encouraged to explore the potential of our therapy in managing persistent infections, as existing treatments were often insufficient in rapidly controlling inflammation and bacterial load—especially in chronic airway conditions like cystic fibrosis, where ongoing infection and inflammation are major challenges.

P. aeruginosa (PA) is the main driver for morbidity and mortality of cystic fibrosis (CF) patients [34]. In a healthy lung, inhaled bacteria are cleared primarily by the mucociliary escalator, where mucus traps particles and cilia sweep them up and out of the airways to be swallowed or coughed out. [35] Patients with CF have an impaired mucociliary escalator because a defective CFTR protein leads to a lack of salt and water on the airway surface, causing the mucus to be thick and sticky. This trapped, thickened mucus allows PA to flourish, and cannot be properly cleared by the cilia, leading to airway inflammation and chronic infections. [36]

Similarly, PA is one of the most frequently isolated organisms in patients [37] with non-CF bronchiectasis. In these chronic infections, where PA persists in patients despite immune responses, antibiotic resistance is accelerated by the presence of “hyper-mutators”. These PA organisms have an increased mutation rate (up to 1000 times that of wild-type PA) that allows them to acquire resistance even more rapidly. [38] Over time, in patients with CF and bronchiectasis, each subsequent generation of PA becomes more resistant to antibiotics, making infections significantly more difficult to treat over time. [39] The current management of P. aeruginosa in non-CF bronchiectasis typically involves using continuous antimicrobial dosing to treat exacerbations and prevent decline in lung function, highlighting the necessity for alternative therapeutic options.

Our goal is to slow disease progression and maximize clinical benefits for patients with cystic fibrosis and bronchiectasis by developing an antivirulence therapy targeting ExoA inhibition. Based on discussions with stakeholders, our drug would be best suited as an adjunctive therapy. An adjunctive therapy is a therapy used in addition to a primary treatment (in this case, antibiotics), improving its effectiveness or helping to prevent a condition’s return. [40] For example, one stakeholder suggested utilizing our inhibitor alongside Tobramycin, an antibiotic effective against Gram-negative bacteria, such as PA. Our therapy would decrease the fitness of the bacteria, slowing resistance development and buying time for the antibiotic and immune system to clear it.

Another potential use case of our therapeutic is to reduce the persistence of chronic Pseudomonas aeruginosa lung infections in patients with CF and Bronchiectasis by reducing biofilm formation in biofilm-growing mucoid strains, a type of PA strain that forms complex biofilms by producing a mucoid exopolysaccharide (EPS) layer. These biofilms cause chronic infections since they are resistant to both antibiotics and immune system defenses. As a result, the body creates a strong antibody response, leading to chronic inflammation, a major cause of lung tissue damage in CF. [41] Based on expert advice from Dr. Chris Goss and Dr. Siddhartha Kapnadak, our adjunctive therapies primarily aim to reduce inflammatory damage rather than directly decrease bacterial load, acting as a biofilm disruptor and minimizing inflammation damage via virulence reduction. This strategy would in turn slow the growth of P. aeruginosa and lead to better patient outcomes with reduced lung tissue damage.

Currently, a significant challenge to these types of treatments is overcoming the mucus barrier lining the lungs. If our therapy was used adjunctively to Trikafta (a prescription medicine primarily used in CF patients to aid in mucus circulation) [42], we could bypass the mucus layer and directly treat the lungs.

Patients with cystic fibrosis and bronchiectasis often have complex treatment plans. To support them, we aim to provide ExAway for long-term management in outpatient clinics and for use during acute exacerbations or initial diagnosis in hospitals. By reducing PA’s virulence, slowing resistance development, and improving patient outcomes, ExAway aims to decrease hospital admissions, emergency visits, and long-term healthcare costs associated with unmanaged disease progression. Ultimately, our goal is to develop adaptable treatment plans for different healthcare system capabilities, reducing the impact of P. aeruginosa infections on patients most affected.

Implementation

We considered many different methods of delivery for our therapy, each with their own strengths and weaknesses, including but not limited to: an IV bag, a dry powder, and a prophylactic coating on catheters.

IV

Dr. Brian Werth believed an IV could be a great method of delivery as it can be used for a number of different infections, and as long as our peptide was water soluble, and stable. Among chronically infected patients, vein space is limited, so adding another IV may be additionally taxing on patients, and require more time for providers to find available veins.

Dry Powder

A dry powder appeared to have the most promise as it would be stable, and could be inhaled, or mixed into a solution. One of the most promising applications was an inhalable nebulization. This would see a lot more use for patients with Cystic Fibrosis, as PA infections in the lungs are much more common for those with CF. Additionally, Dr. Dandekar mentioned that it would be important for our dry powder to be pre-mixed with antibiotics. This would take the step of mixing the two drugs off of healthcare providers.

Prophylactic coating

Adding our therapeutic as a coating to catheters and other indwelling medical devices was initially promising as it could decrease the probability of hospital related infection. However, when we asked Dr. Dandekar about this idea, he stated that it would prove to be ineffective as the largest concern in early hospital related infection is the formation of a biofilm, which our therapy would not impact enough to make a sizable difference.

Possible limitations

Despite the possibilities of our project, we heard a number of concerns from researchers we talked to. One of these is among chronically infected patients, exotoxins are often downregulated in PA. Dr. Dandekar mentioned at the point where PA is identified, this downregulation rarely occurs. This would mean our therapeutic could be prescribed before its efficacy would be impacted by downregulation. Dr. Werth mentioned PA expresses several redundant exotoxins, so targeting ExoA would limit the impact of our therapeutic.

Cotherapy benefits

We believe when implementing our project into a clinical setting, it is important to provide our therapy as a pre-mixed solution with current antibiotics. This would take a step off healthcare workers’ hands and would make it easier to get FDA approval. Dr. Werth told us it is very difficult to get an adjunctive therapy through FDA approval if it is not already mixed together. This is because our therapy by itself does very little to fight infection, but when mixed with antibiotics would prove more effective than antibiotics alone. In terms of what antibiotic we should use, Dr. Werth said that we should use the standard of care, meaning we would most likely use ceftolozane-tazobactam or ceftazidime-avibactam. [43]

Market and Policy

ExAway is a unique antivirulence therapy in the drug market. Unlike existing treatments that focus on killing P. aeruginosa (PA), our inhibitor specifically targets and neutralizes the exotoxin A (ExoA).

Currently, the antimicrobial therapeutic market value for PA was estimated to be around 4.9 billion USD in 2022, exhibiting a Compound Annual Growth Rate (CAGR) of approximately 8.10% [44]. This market expansion is due to the rise of hospital-related PA infections, as well as the increase in multidrug-resistant PA.[45] This rise has led to an excessive burden on the healthcare system and expensive treatment costs. A 2015 study found that hospital-acquired pneumonia in patients in the ICU with PA infections incurred an additional $180,000 in treatment costs compared to patients without. [46] Especially in patients with CF, initial P. aeruginosa infection increased annual per-patient costs by an average of over $18,500. [47]

Due to the rising demand for alternative solutions, there is a significant opportunity to develop solutions that help reduce the burden of PA infection. The most common treatment regimen is a two-drug combination of antibiotics, typically a β-lactam with an aminoglycoside. [48] As PA develops resistance, we continuously develop new antibiotics to combat the pathogen’s resistance. A 2019 study found that those with multidrug-resistant (MDR) PA incurred an average adjusted excess cost of $22,370 per case compared to those with non-MDR infections. [49] Because our therapeutic targets a virulence factor, we do not add any evolutionary pressure for PA to develop resistance. By targeting the bacteria's most potent virulence factor, ExoA, we protect host cells and tissues from its devastating effects, reducing infection severity and improving health outcomes for vulnerable patients with bronchiectasis. This unique focus on virulence neutralization, rather than bacterial eradication, essentially creates an evolutionary dead end where the bacteria cannot easily adapt to overcome the treatment. For the market, this translates to a longer-lasting product with sustained efficacy. Our therapy would additionally help minimize excess healthcare costs associated with prolonged treatments, hospitalizations, and managing resistant infections with antibiotics.

Currently, the antivirulence therapeutic market is an emerging segment within the broader anti-infectives market. While precise market data are limited due to the novelty of this approach, the market is expected to grow, with a greater focus on this approach alongside traditional antibiotics. Currently, the most popular anti-virulence therapies on the market are antibody-based agents, which similarly target and neutralize bacterial toxins. These include Bezlotoxumab and Raxibacumab, which prevent the recurrence of C. difficile infection in high-risk adult patients by neutralizing toxin B and treat/prevent inhalational anthrax, respectively. For instance, the global market for Raxibacumab was valued at approximately $101.5 million in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 14.2% from 2024 to 2031. [50], [51] While it is important to note that antivirulence therapeutics are still quite new and many healthcare providers are hesitant to prescribe these therapeutics, there is a clear growing market demand for such innovative solutions.

Here, we briefly examine the demand and existing market for our two possible implementation strategies:

Demand for Prophylactic Gel Coating

Based on the feedback from the clinicians we interviewed, a prophylactic gel coating on various medical devices to reduce bacterial growth has long been desired, despite challenges to implementation and safety concerns. The non-antimicrobial mechanism offers significant benefits, particularly in minimizing the development of resistance. However, studying this population presents unique challenges, requiring carefully designed trials with comprehensive safety monitoring, clear inclusion and exclusion criteria, and patient-centric approaches to ensure participation while minimizing burden.

Demand for Nebulized Inhalant

Based on feedback from providers of CF and bronchiectasis patients, our inhibitor could have another potential use as adjunctive therapy. This implementation would develop a product dissolved in sterile saline for nebulization and create a temperature-resistant dry powder. This solution would be more feasible than a prophylactic coating and more predictable in terms of market strategy. The global nebulizer market overall was valued at $1.22 billion in 2024 and is expected to grow at a CAGR of over 6% through 2030, with CFTR modulators dominating the market and accounting for the largest revenue share of 44.0% in 2024. [51] This indicates that there is a substantial and growing market opportunity for our inhibitor-based nebulized therapy, driven by increasing demand for respiratory treatments in CF and bronchiectasis patients.

Society and Ethics

Developing an antivirulence therapy for P. aeruginosa raises important societal and ethical questions that differ from those arising with traditional antibiotics. One major issue we considered was equitable access. Dr. Leo Morales, Co-Director for the Latino Center of Health at the University of Washington as well as Professor and Assistant Dean for Healthcare Equity and Quality in the UW School of Medicine, explained how access to life-saving technologies and treatments is often determined not by patient need, but rather by structural barriers such as limited insurance coverage and tight state budgets for public insurance. He also noted inequities still persist even when a therapy has already been proven effective, since economic discrimination is built into the healthcare system. While large hospitals or tertiary care facilities may have more safeguards against economic discrimination for critical cases, patients frequenting community hospitals or those without strong insurance can still struggle with access, leaving the most vulnerable populations at risk of being left behind. Because of this, it is crucial to design inclusive clinical trials and keep up continuous community involvement to increase affordability so that our therapy doesn’t remain accessible only to niche or wealthier populations.

In addition to equitable access, safety is another important consideration. As mentioned in our implementation section, one concern about our solution is the effectiveness of targeting only Exotoxin A. P. aeruginosa produces multiple toxins; while targeting ExoA may reduce its virulence, other toxins can still contribute to infection. This makes it especially important to conduct careful testing, helping us determine whether or not targeting ExoA has a meaningful effect on patient outcomes. It is also essential to ensure our solution doesn’t create new risks or cause any unintended effects to human cells, such as immune reactions. Research has shown immune responses to P. aeruginosa infections are often complex and constantly evolving [52] .

Dr. Morales also emphasized the importance of ongoing community presence and involvement throughout the development of our project, especially in engaging with immunocompromised patients and other groups vulnerable to P. aeruginosa infections. This could also include partnerships with local organizations and patient advocacy groups, ultimately helping us connect with the community our therapy is designed for and get a better understanding of their needs, ensuring equitable distribution. The success of our project is not only defined by its efficiency in a clinical setting, but also by whether or not those in need can access our therapy across different healthcare settings.

Citations