This dissertation introduces assembly of spherical particles as a novel topography-based anti-biofouling coating. It also provides new insights on the effects of surface topography, especially local curvature, on cell–surface and cell–cell interactions during the evolution of biofilms. I investigated the adhesion, colonization, and biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa on a solid coated in close-packed spheres of polystyrene, using flat polystyrene sheets as a control. The results show that, whereas flat sheets are covered in large clusters after one day, a close-packed layer of 630–1550 nm monodisperse spheres prevents cluster formation. Moreover, the film of spheres reduces the density of P. aeruginosa adhered to the solid by 80%. Our data show that when P. aeruginosa adheres to the spheres, the distribution is not random. For 630 nm and larger particles, P. aeruginosa tends to position its body in the confined spaces between particles. After two days, 3D biofilm structures cover much of the flat polystyrene, whereas 3D biofilms rarely occur on a solid with a colloidal crystal coating of 1550 nm spheres. On 450 nm colloidal crystals, the bacterial growth was intermediate between the flat and 1550 nm spheres. The initial preference for P. aeruginosa to adhere to confined spaces is maintained on the second day, even when the cells form clusters: the cells remain in the confined spaces to form non-touching clusters. When the cells do touch, the contact is usually the pole, not the sides of the bacteria. The observations are rationalized based on the potential gains and costs associated with cell-sphere and cell-cell contacts. I concluded that the anti-biofilm property of the colloidal crystals is correlated with the ability to arrange the individual cells. I showed that a colloidal crystal coating delays P. aeruginosa cluster formation on a medical-grade stainless-steel needle. This suggests that a colloidal crystal approach to biofilm inhibition might be applicable to other materials and geometries. The results presented in appendix 1 suggest that colloidal crystals can also delay adhesion of Methicillin resistant staphylococcus aureus (MRSA) while it supports selective adhesion of this bacterium to the confined spaces. / Ph. D.

It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts are responsible for responding to this tissue damage by breaking down the damaged extracellular matrix (ECM) and secreting a new ECM which aids in wound repair and supports the migration of immune cells. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound repairing fibroblasts. To address this lack of understanding, this thesis focuses on quantifying changes in fibroblast morphology, migratory behavior, and force exertion to investigate this host cell's response to representative cytotoxic (PAO1) and invasive (PA14) strains of P. aeruginosa. These assays study host cell-pathogen interactions on highly aligned nanofibers of varied spacing and diameter, which mimic the fibroblast deposited ECM and dictate fibroblast morphology. We discovered that the cytotoxic strain of P. aeruginosa induced significantly shorter fibroblast death times. Furthermore, two modes of death, sharp and gradual, were identified and found to be dependent on both fiber configuration and strain of P. aeruginosa. In addition, fibroblasts exposed to PAO1 migrating on the parallel formation were found to be significantly slower and less persistent than those exposed to PA14, however, fibroblasts exposed to both strains of bacteria were shown to exert similar forces. Lastly, exposure to PA14 led to the greatest change in actin, evident by increased actin punctae and less prominent actin stress fiber formation. / Master of Science / It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts respond to burn wounds by breaking down the damaged tissue fibers, termed extracellular matrix (ECM), and secreting a new ECM. Unfortunately, severe thermal injuries place hospitalized burn victims at high risk of infection. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound healing fibroblasts. To address this knowledge gap, this thesis focuses on quantifying changes in fibroblast shape, migratory behavior, and force exertion to investigate this host cell's response to two strains of P. aeruginosa, which employ different mechanisms of invasion. These interactions are studied on a platform of suspended nanofibers with controlled spacing and diameter, to dictate fibroblast shape and mimic the fibroblast deposited ECM. We discovered that the two strain of P. aeruginosa induced significantly different fibroblast death times. During death, it was observed that fibroblasts either balled up quickly, termed sharp death, or remained spread out, termed gradual death, dependent upon fibroblast shape and strain of P. aeruginosa introduced. In addition, significant differences in migration speed and persistence were found between fibroblasts exposed to the two strains of bacteria, however, both groups were shown to exert similar forces. Lastly, the fibrous proteins which make up the cytoskeleton of the cell, actin stress fibers, were found to vary among the control and bacteria treated cells.

Part I: Signaling Role of the Sensor Kinase RetS in Biofilm formation Regulation of Pseudomonas aeruginosa-<br />The opportunistic human pathogen Pseudomonas aeruginosa causes both acute and chronic infections in predisposed individuals. Acute infections require a functional Type Three Secretion System (TTSS), which mediates the translocation of select cytotoxins into host cells. Chronic infections, the leading cause of death among cystic fibrosis patients, are characterized by drug-resistant biofilms formation. To regulate gene expression, Pseudomonas aeruginosa utilizes two-component regulatory systems (TCS). Specifically, we focus on the TCS signaling kinase RetS, which is a critical repressor of biofilm formation. The signaling mechanism of RetS is unusual. According to recent findings and one hypothesis, RetS employs a novel signaling mechanism involving direct binding to the signaling kinase GacS, thereby repressing the GacS-induced biofilm formation. RetS is believed to be regulated by the interaction of its periplasmic sensory domain (RetSperi) with an unknown ligand. As such, RetSperi is a potential drug target. We hypothesized that ligand-binding shifts the equilibrium between the formation of a RetS homo-dimer and the RetS-GacS complex by tuning the homo-dimerization of the RetSperi. While the molecular signal that regulates RetS is unknown, our structural studies of the sensory domain suggest that this ligand is a carbohydrate-based moiety. Unchanged biofilm-EPS production phenotype of RetSperi ligand binding site mutants indicates that the natural ligand is not from Pseudomonas aeruginosa.<br />Additional experiments unambiguously determined that the sensory domain forms a stable homodimer. Adding to the complexity of the system, we have identified<br />two possible dimer interfaces in our in vitro assays. However, inconsistent with the current model, elimination of RetSperi results in a slightly increased biofilm EPS production phenotype. Therefore, with the previous demonstration that RetS is able to dephosphorylate GacS, we propose an alternative hypothesis: the RetS kinase domain serves as a phosphatase for phosphorylated GacS; this phosphatase activity is tuned by signaling sensing on RetSperi. Finally, to provide an important piece of information for understanding the molecular basis of RetS-GacS signaling, we have developed a crystallization-based structure determination strategy in order to reveal the precise RetS-GacS interaction pattern.<br /><br />PartII: The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology-<br />germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-"?lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft. / Ph. D.

The Type III secretion system (T3SS) of Pseudomonas aeruginosa uses a needle-like protein apparatus to detect eukaryotic host cells and translocate effectors directly into the host cell. The effectors are also known as cytotoxins, which cause disruption of a series of signaling events in the host cell, facilitating the infection by P. aeruginosa. As the T3SS is antigenic and the expression of T3SS is energy-consuming, it is highly regulated where several regulatory proteins interact with each other and control the expression of T3SS genes. Among these proteins, ExsA, the master regulator of T3SS in P. aeruginosa, is of great importance as it is a transcriptional activator that activates the expression of all T3SS genes. Also, as ExsA belongs to the AraC protein family which only exists in bacteria and fungi, it makes an excellent potential target for drugs against P. aeruginosa related infections. With a combination of molecular biology tools and structural biology methods, we solved the N-terminal domain structure of the ExsA protein in P. aeruginosa. The model of the ExsA N-terminal domain has enriched our knowledge about ExsA dimerization and can serve as the base for mapping the interaction interfaces on ExsA and ExsD. Further, we have found two homologues of ExsA by structural alignment, which share a lot of similarities and have conserved amino acid residues that are important for ligand binding. The fact that both of these two proteins are regulated by small ligands rather than proteins also raises the possibility that ExsA may have a second regulatory mechanism under which ExsA is regulated by a small ligand, which so far has not been observed or reported by researchers. In order to map the binding site of ExsA on its anti-activator ExsD, we removed the coiled-coil region (amino acid residue 138-202, the potential binding site) of ExsD, based on the  structure of ExsD. We surprisingly found that the ExsD variant without the coiled-coil region readily inhibits ExsA-dependent in vitro transcription. This result rules out other possibilities and makes us focus on the N-terminus and adjacent regions of ExsD for the interface with ExsA. Moreover, in order to gain a comprehensive understanding of the dynamics of the regulation of T3SS in P. aeruginosa, we have begun to build a mathematical model of the T3SS regulatory pathways. We are measuring the cellular concentrations of T3SS regulatory proteins with quantitative molecular biology methods such as quantitative western blot, quantitative PCR and quantitative mass spectrometry. We have determined the cellular level of ExsA and ExsD proteins under different physiological conditions, and found that some factors such as temperature have a significant impact on the levels of ExsA and ExsD. This study has thus unveiled some unknown features of the T3SS of P. aeruginosa and its related infections. / Master of Science

This study demonstrates that the surface motility of the bacterium Pseudomonas aeruginosa is dependent on the surface chemistry of the underlying substrate. In particular, cells on hydrophobic polydimethylsiloxane (PDMS) have a speed that is on average 38% greater than on hydrophilic PDMS. These results were obtained using time-lapse microscopy of bacteria exposed to continuously flowing tryptic soy broth growth medium at 37 ⁰C. Not only are the mean speeds different, the distributions of speeds are also different: on the hydrophobic substrate, a smaller proportion of bacteria move by less than about one body-length (~3 µm) in 60 minutes. In addition, the surface chemistry affects the orientation of the cells: there is a greater fraction of "walking" bacteria on the hydrophobic surface. Sensitivity to the substrate surface chemistry occurs despite the presence of a complex mix of substances in the growth medium and offers hope that surface chemistry can be used to tune motility and the progression to biofilm formation. Additionally, the effect of reducing the near-surface Young's modulus of the PDMS from 7000 to 70 kPA is investigated. For the lower modulus material, there is an increase in the likelihood of a bacterium executing sudden, high angle turns. This is evident in images with a framerate of one frame per 0.22s. However, the impact of these turns is averaged out over longer times such that the mean speed over periods of more than about one minute is the same for bacteria on both the high and the low modulus materials. Consequently, except over very short time intervals, Young's modulus in the surface region is not effective as a means of modulating motile behavior. / Master of Science / This study demonstrates that the ability of the bacterium Pseudomonas aeruginosa to move on a solid surface is dependent on the surface chemistry of the underlying substrate. In particular, cells on hydrophobic polydimethylsiloxane (PDMS) have a speed that is on average 38% greater than on hydrophilic PDMS. These results were obtained using time-lapse microscopy of bacteria exposed to continuously flowing growth medium at 37 ⁰C. Not only are the mean speeds different, the distributions of speeds are also different: on the hydrophobic substrate, a smaller proportion of bacteria move by less than about one body-length (~3 µm) in 60 minutes. In addition, the surface chemistry affects the orientation of the cells: there is a greater fraction of vertically-oriented bacteria on the hydrophobic surface. Additionally, the effect of reducing the stiffness of the PDMS from 7000 to 70 kPA is investigated. For the less stiff material, there is an increase in the likelihood of a bacterium executing sudden, high angle turns. This is evident in images with a framerate of one frame per 0.22s. However, the impact of these turns is averaged out over longer times such that the mean speed over periods of more than about one minute is the same for bacteria on both the high and the low stiffness materials. Consequently, except over very short time intervals, stiffness in the surface region is not effective as a means of changing patterns of surface-bound P. aeruginosa movement.

The core of the dissertation lies in developing two novel systems bioengineering approaches, a synthetic Escherichia coli killer-prey microecology, and a combined infection-inflammation NET-array system, to investigate the role of the mechanochemical complexity of the microenvironment in driving the microbe-microbe and host-microbe interactions, respectively. Herein, the first part of the dissertation includes designing and engineering a synthetic E. coli killer-prey microecological system where we quantified the quorum-sensing mediated interactions between the engineered killer and prey E. coli bacterial strains plated on nutrient-rich media. In this work, we developed the plate assay followed by plasmid sequencing and computational modeling that emphasizes the concept of the constant evolution of species or acquired resistance in the prey E. coli, in the vicinity of the killer strain. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. Through time-lapse imaging on petri plates automated using a scanner, we recorded the "kill wave" that originates outside the killer colony and travels outward as the prey dies. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. However, some of the prey colonies evolve to be resistant to the effects of AHL secreted by the killer. These prey colonies resistant to the killer were then selected and confirmed by plasmid sequencing. Using this empirical data, we developed the first ecological model emphasizing the concept of the constant evolution of species, where the survival of the prey species is dependent on the location (distance from the killer) or the evolution of resistance. The importance of this work lies in the context of the evolution of antibiotic-resistant bacterial strains and in understanding the communication between the microbial consortia, such as in the gut microbiome. Further, the second part of the dissertation includes quantifying the interactions between immune cells (primary healthy human neutrophils) and motile Pseudomonas aeruginosa bacteria in an inflammation-rich microenvironment. Neutrophils, being the first responding immune cells to infection, defend by deploying various defense mechanisms either by phagocytosing and killing the pathogen intracellularly or through a suicidal mechanism of releasing their DNA to the extracellular space in the form of Neutrophil Extracellular Traps (NETs) to trap the invading pathogens. Although the release of NETs is originally considered a protective mechanism, it is shown to increase the inflammation levels in the host if unchecked, ultimately resulting in end-organ damage (especially lung and kidney damage), as with the severe cases of sepsis and COVID-19. In our work, we developed a combined infection-inflammation NET-array system integrated with a live imaging assay to quantify the spatiotemporal dynamics of NET release in response to P. aeruginosa infection in an inflammatory milieu at a single-cell resolution. Importantly, we found increased NET release to P. aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. Our device platform is unique in that the nanoliter well-assisted individual neutrophil trapping enables us to quantify NET release with single-cell precision. Besides, incorporating confined side loops in the device helped us study the role of mechanical confinement on NET release, showing reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our microsystem. In summary, our work emphasizes the importance of studying the heterogeneity of NET release in host defense and inflammation. In the future, our system can be used for screening novel neutrophil-based immunotherapies and serve as a valuable research tool in precision medicine. / Doctor of Philosophy / The microenvironment plays a vital role in shaping the interactions within microbes and between the host and the microbes. Microbes use quorum-sensing-based chemical signaling to adapt to the environmental stresses in a microecology (be it a soil microecology or the gut microbiome). They communicate with each other with the help of these chemicals to regulate their population density (to mutual benefit in the case of a biofilm formation or to compete for resources in the case of a predator-prey model). In the first part of the dissertation, we utilize this quorum-sensing approach to study the spatiotemporal dynamics of the interactions between two engineered killer and prey Escherichia coli bacterial strains on a nutrient-rich agar plate in real-time. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. Further, through sequencing, we found that some of the prey colonies acquired resistance to the effects of AHL secreted by the killer. We then developed a computational model that recapitulates our experimental results, emphasizing the concept of the constant evolution of species or acquired resistance. The importance of this work lies in using experimental and computational approaches to better understand the evolution of multidrug-resistant (MDR) bacterial strains. Next, we investigated the interactions between primary human neutrophils (first responding immune cell type to infection) and motile Pseudomonas aeruginosa bacteria in the second part of the dissertation, explicitly focusing on quantifying neutrophil extracellular traps (NETs) release. With increasing concerns regarding the role of the dysregulated NET release in exaggerated inflammatory responses in the host, it is imperative to quantify NET release precisely at a single-cell level in a controlled microenvironment. To this end, we engineered a combined infection-inflammation NET-array device with 1024 nanoliter wells per device and achieved single-cell level trapping of neutrophils in these wells. Our device platform is unique in that the individual wells of the device have constricted side loops, which helps us better understand the role of mechanical confinement on NET release from an engineering standpoint. We then used the NET-array system to quantify the spatiotemporal dynamics of NET release to P. aeruginosa in an inflammatory mediator-rich microenvironment. Importantly, we found heightened NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. We also demonstrated reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our combined infection-inflammation microsystem. Especially with the increasing complexity of the intercellular cues at the site of infection, by integrating our microfluidic method with the conventional reductionist approaches, we can better solve the intricate puzzles of the immune cell decision-making processes at a single-cell level. Our study highlights the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.

Little is known concerning the production and significance of bacterial pigments. There is seemingly an open field for studying the effects produced by varying the nutritive content of culture media upon which organisms are grown. This has led to an especial interest in, and the purpose of this investigation.

Lactate is an important metabolic intermediate in mammals, and increased lactate production has been shown to occur under inflammatory conditions. Invading bacteria can utilize this lactate as a carbon source for growth and persistence in infection contexts, and a deeper understanding of bacterial lactate metabolism is therefore essential for treating such infections. One bacterial species that can utilize lactate for growth and that is often found in environments where lactate accumulates is the opportunistic pathogen Pseudomonas aeruginosa.  P. aeruginosa is most known for its colonization of the lungs of people with cystic fibrosis and of chronic wounds, environments where lactate concentrations can range from 10-40 mM. This thesis uncovers the details of lactate metabolism in P. aeruginosa, including the regulation of its lactate utilization genes, and elucidates several aspects of cell metabolism found to influence lactate consumption.  Chapter 1 provides a background into the prevalence of lactate as a major metabolic intermediate in mammals and the rationale for why it has become such a well-studied compound. This chapter also touches on the diversity of lactate utilization enzymes and their regulation across various bacterial species, and takes a closer look at P. aeruginosa’s ability to cause infection. Since much of P. aerugionsa’s success as a pathogen is linked to aspects of its metabolism, a comprehensive picture of the core pathways that support its growth and survival has the potential to reveal drug targets or inform therapeutic strategies. Chapter 2 dives deeper into the regulatory mechanisms underpinning lactate utilization in P. aeruginosa and explores the reasoning behind P. aeruginosa’s possession of two, seemingly redundant L-lactate dehydrogenase genes: lldD and lldA. The chapter discusses how the two unique regulators of these genes - LldR and LldS, respectively - confer distinct conditional sensitivities on the expression of lldD and lldA, especially with respect to iron and glycolate concentrations. These diverse inputs allow P. aeruginosa to adapt its lactate utilization to specific environments. Chapter 3 takes a closer look at glycolate metabolism in P. aeruginosa, since glycolate is structurally similar to lactate and, as described in Chapter 2, has been identified as a potent inhibitor of LldD-dependent lactate metabolism. Although evidence suggests glycolate is also present in infection sites, little is known about its metabolism, especially in pathogenic bacteria. Within this chapter, my co-authors and I demonstrate that expression of the P. aeruginosa glcDEFG operon is responsive to glycolate, and that the operon is expressed in the absence of added glycolate, suggesting that this metabolite is produced endogenously. We speculate that the main source of this glycolate is glyoxal/methylglyoxal detoxification, a process whereby toxic metabolic byproducts are converted into either glycolate or lactate. The fact that glyoxal/methylglyoxal detoxification produces both glycolate and lactate underscores the high degree of cross-talk between the bioactivities of these two metabolites. Finally, Chapter 4 goes into more detail about a core theme introduced in the other chapters - how P. aeruginosa adapts its metabolism in response to changing oxygen and nutrient conditions. P. aeruginosa possesses two rubredoxin genes, which encode small soluble electron carriers believed to help it cope with oxidative stress. This chapter demonstrates that induction of the rubredoxin genes in liquid culture may occur at key time points associated with oxidative stress and metabolic shifts, and may be linked to changes in lactate and glycolate metabolism. This thesis lays the groundwork for understanding aspects of P. aeruginosa physiology that have yet to be fully explored, including the regulatory relationships between detoxification mechanisms and central metabolism, the condition-dependent control of metabolic pathways that affects physiological differentiation in multicellular structures and in infection sites, and the potential for neighboring species in polymicrobial infections to influence P. aeruginosa physiology and virulence. This work will hopefully bring to light the need to study these metabolic and regulatory pathways, not just in Pseudomonas spp., but in other organisms as well, as many of these core biochemical processes are evolutionarily conserved.

Pseudomonas aeruginosa, a notorious opportunistic pathogen, is a leading cause of hospital-acquired infections. The newest generation of β-lactam antibiotics, the carbapenems, are often used to treat multi-drug resistant (MDR) P. aeruginosa infections. Treatment of P. aeruginosa has become increasingly difficult due to its remarkable ability to resist antibiotics through various intrinsic and acquired mechanisms. Physicians rely on a limited group of antibiotics to treat these infections, but many P. aeruginosa isolates are evolving to become resistant to all available antibiotics, including carbapenems. The multifaceted mechanisms underlying P. aeruginosa antibiotic resistance include β -lactamases, efflux pumps, altered membrane porins, and antibiotic binding site mutations of the penicillin binding proteins. There is an urgent need for continued research to better understand the resistance mechanisms used by P. aeruginosa, in order to develop novel therapeutic strategies. The purpose of this project was to investigate the effect of β-lactam antibiotics used in combination with the known efflux pump inhibitor mefloquine, on the growth of MDR P. aeruginosa. The effect of the combination of mefloquine andβ-lactams was investigated in vitro using the checkerboard method. In vitro assays showed that mefloquine when combined with certain β-lactam antibiotics produced no significant additional inhibition than the β-lactams antibiotics alone on MDR P. aeruginosa. Mefloquine, in combination with various β-lactams, did not restore clinically relevant sensitivity, even in those isolates where resistance is thought to be mediated by efflux pumps.

Pseudomonas aeruginosa (PA) is a gram-negative bacillus well known for colonizing human respiratory airways and causing opportunistic infections. Children with neuromuscular disease (NMD) including cerebral palsy (CP) and severe upper airway obstruction who get infected with PA, their chances of experiencing a severe illness, being admitted to a pediatric intensive care unit, and extended or repeated hospital stays increase dramatically. These patients often need a surgical procedure called tracheostomy which act as a channel for microbes to enter lower respiratory tract and increase infections, despite its well documented impact as an opportunistic pathogen comprehensive investigation into the diversity of PA in such vulnerable populations is limited. To fill this gap in knowledge we perform whole genome sequencing (WGS) and phenotypic analysis of 40 PA isolates from the respiratory tract of this susceptible population with and without tracheotomies. Pangenome analysis showed highly variable genome content with 16,212 total genes of which 2326 are core genes. MLST revealed diverse sequence types (STs) among the studied population with 21 known and 10 new STs. Genotypic analysis revealed moderate variations in the antimicrobial resistance determinants and virulence factors among all isolates. In total 8 serogroups were identified, with serogroups O6 and O11 accounting for 70% of all the isolates. Genotypic diversity was observed in overall population however comparative analysis among tracheostomized and non-tracheostomized patient groups showed significant similarity which aligns with the phenotypic analysis revealing significant similarity with minor differences in biofilm formation, motility, hemolysis production, and pigment production. Last, we explored putative healthcare transmission and identified three potential transmission events. These findings provide insight into how WGS along with phenotypic analysis can help us better understand population dynamics, epidemiology, virulence profile and antibiotic resistance profile of PA contributing to respiratory infections which has valuable therapeutic implications for epidemiology and disease management.