Pseudomonas aeruginosaAlginate Overproduction Promotes Coexistence withStaphylococcus aureusin a Model of Cystic Fibrosis Respiratory Infection

ABSTRACTWhile complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example,Pseudomonas aeruginosaandStaphylococcus aureusare often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis thatP. aeruginosaand/orS. aureusadopts phenotypes that allow coexistence during infection. We compared competitive interactions ofP. aeruginosaandS. aureusisolates from mono- or coinfected CF patients employingin vitrococulture models.P. aeruginosaisolates from monoinfected patients were more competitive towardS. aureusthanP. aeruginosaisolates from coinfected patients. We also observed that the least competitiveP. aeruginosaisolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to killS. aureus. Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureusactivity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids—each required for efficient killing ofS. aureus. These studies demonstrate alginate overproduction may be an important factor drivingP. aeruginosacoinfection withS. aureus.IMPORTANCENumerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains ofP. aeruginosais sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompeteS. aureus, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoidP. aeruginosacan coexist withS. aureus. Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.

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Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms

Journal of Bacteriology ◽

10.1128/jb.00159-20 ◽

2020 ◽

Vol 202 (18) ◽

Cited By ~ 1

Author(s):

Giulia Orazi ◽

Fabrice Jean-Pierre ◽

George A. O’Toole

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Respiratory Infections ◽

Multiple Infection ◽

Bacterial Biofilms ◽

Interspecies Interactions ◽

Chronic Infections ◽

Content Type

ABSTRACT The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

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Rapid diversification of Pseudomonas aeruginosa in cystic fibrosis lung-like conditions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721270115 ◽

2018 ◽

Vol 115 (42) ◽

pp. 10714-10719 ◽

Cited By ~ 20

Author(s):

Alana Schick ◽

Rees Kassen

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Chronic Infection ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Evolutionary Diversification ◽

Nutritional Conditions ◽

Patient Health ◽

Rapid Diversification

Chronic infection of the cystic fibrosis (CF) airway by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality for adult CF patients. Prolonged infections are accompanied by adaptation of P. aeruginosa to the unique conditions of the CF lung environment, as well as marked diversification of the pathogen into phenotypically and genetically distinct strains that can coexist for years within a patient. Little is known, however, about the causes of this diversification and its impact on patient health. Here, we show experimentally that, consistent with ecological theory of diversification, the nutritional conditions of the CF airway can cause rapid and extensive diversification of P. aeruginosa. Mucin, the substance responsible for the increased viscosity associated with the thick mucus layer in the CF airway, had little impact on within-population diversification but did promote divergence among populations. Furthermore, in vitro evolution recapitulated traits thought to be hallmarks of chronic infection, including reduced motility and increased biofilm formation, and the range of phenotypes observed in a collection of clinical isolates. Our results suggest that nutritional complexity and reduced dispersal can drive evolutionary diversification of P. aeruginosa independent of other features of the CF lung such as an active immune system or the presence of competing microbial species. We suggest that diversification, by generating extensive phenotypic and genetic variation on which selection can act, may be a key first step in the development of chronic infections.

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Poly (acetyl arginyl) glucosamine attenuates Pseudomonas aeruginosa in a rat lung infection model

10.1101/2021.09.15.460521 ◽

2021 ◽

Author(s):

Bryan Garcia ◽

Melissa S. McDaniel ◽

Allister J. Loughran ◽

J. Dixon Johns ◽

Vidya Narayanaswamy ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Respiratory Infections ◽

Bacterial Species ◽

Membrane Integrity ◽

Opportunistic Pathogen ◽

Infection Model ◽

Total Biomass ◽

Chronic Infections

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer which has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity which can normalize rheologic properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, which may warrant further evaluation as a therapeutic.

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Pseudomonas aeruginosa Volatilome Characteristics and Adaptations in Chronic Cystic Fibrosis Lung Infections

mSphere ◽

10.1128/msphere.00843-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Trenton J. Davis ◽

Ava V. Karanjia ◽

Charity N. Bhebhe ◽

Sarah B. West ◽

Matthew Richardson ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Chemical Diversity ◽

Chronic Infections ◽

Lung Function Decline ◽

Content Type ◽

Flight Mass Spectrometry ◽

Patient Health ◽

Lung Infections

ABSTRACT Pseudomonas aeruginosa chronic lung infections in individuals with cystic fibrosis (CF) significantly reduce quality of life and increase morbidity and mortality. Tracking these infections is critical for monitoring patient health and informing treatments. We are working toward the development of novel breath-based biomarkers to track chronic P. aeruginosa lung infections in situ. Using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC–TOF-MS), we characterized the in vitro volatile metabolomes (“volatilomes”) of 81 P. aeruginosa isolates collected from 17 CF patients over at least a 5-year period of their chronic lung infections. We detected 539 volatiles produced by the P. aeruginosa isolates, 69 of which were core volatiles that were highly conserved. We found that each early infection isolate has a unique volatilome, and as infection progresses, the volatilomes of isolates from the same patient become increasingly dissimilar, to the point that these intrapatient isolates are no more similar to one another than to isolates from other patients. We observed that the size and chemical diversity of P. aeruginosa volatilomes do not change over the course of chronic infections; however, the relative abundances of core hydrocarbons, alcohols, and aldehydes do change and are correlated with changes in phenotypes associated with chronic infections. This study indicates that it may be feasible to track P. aeruginosa chronic lung infections by measuring changes to the infection volatilome and lays the groundwork for exploring the translatability of this approach to direct measurement using patient breath. IMPORTANCE Pseudomonas aeruginosa is a leading cause of chronic lung infections in cystic fibrosis (CF), which are correlated with lung function decline. Significant clinical efforts are therefore aimed at detecting infections and tracking them for phenotypic changes, such as mucoidy and antibiotic resistance. Both the detection and tracking of lung infections rely on sputum cultures, but due to improvements in CF therapies, sputum production is declining, although risks for lung infections persist. Therefore, we are working toward the development of breath-based diagnostics for CF lung infections. In this study, we characterized of the volatile metabolomes of 81 P. aeruginosa clinical isolates collected from 17 CF patients over a duration of at least 5 years of a chronic lung infection. We found that the volatilome of P. aeruginosa adapts over time and is correlated with infection phenotype changes, suggesting that it may be possible to track chronic CF lung infections with a breath test.

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Rapid diversification of Pseudomonas aeruginosa in cystic fibrosis lung-like conditions

10.1101/231050 ◽

2017 ◽

Author(s):

Alana Schick ◽

Rees Kassen

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Chronic Infection ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Chronic Infections ◽

Evolutionary Diversification ◽

Nutritional Conditions ◽

Patient Health

AbstractChronic infection of the cystic fibrosis (CF) airway by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality for adult CF patients. Prolonged infections are accompanied by adaptation of P. aeruginosa to the unique conditions of the CF lung environment as well as marked diversification of the pathogen into phenotypically and genetically distinct strains that can coexist for years within a patient. Little is known, however, about the causes of this diversification and its impact on patient health. Here, we show experimentally that, consistent with ecological theory of diversification, the nutritional conditions of the CF airway can cause rapid and extensive diversification of P. aeruginosa. The increased viscosity associated with the thick mucous layer in the CF airway had little impact on within-population diversification but did promote divergence among populations. Notably, in vitro evolution recapitulated patho-adaptive traits thought to be hallmarks of chronic infection, including reduced motility and increased biofilm formation, and the range of phenotypes observed in a collection of clinical isolates. Our results suggest that nutritional complexity and reduced dispersal can drive evolutionary diversification of P. aeruginosa independent of other features of the CF lung such as an active immune system or the presence of competing microbial species. They also underscore the need to obtain diverse samples of P. aeruginosa when developing treatment plans. We suggest that diversification, by generating extensive phenotypic and genetic variation on which selection can act, may be a key first step in the transition from transient to chronic infection.Significance StatementChronic infection with the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of lung transplant or death in cystic fibrosis patients. P. aeruginosa diversifies in the CF lung, although why this happens remains a mystery. We allowed P. aeruginosa to evolve in the laboratory under a range of conditions approximating the CF lung. The diversity of evolved populations was highest, and most closely resembled the range of phenotypes among clinical isolates, in environments resembling the spectrum of nutritional resources available in the CF lung. Our results point to the nutritional complexity of the CF lung as a major driver of diversification and they suggest that diversity could be important in the development of chronic infections.

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PSEUDOMONAS AERUGINOSA VOLATILOME CHARACTERISTICS AND ADAPTATIONS IN CHRONIC CYSTIC FIBROSIS LUNG INFECTIONS

10.1101/2020.06.13.126698 ◽

2020 ◽

Author(s):

Trenton J. Davis ◽

Ava V. Karanjia ◽

Charity N. Bhebhe ◽

Sarah B. West ◽

Matthew Richardson ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Chemical Diversity ◽

Chronic Infections ◽

Flight Mass Spectrometry ◽

Detection And Tracking ◽

Sputum Production ◽

Patient Health ◽

Lung Infections

ABSTRACTPseudomonas aeruginosa chronic lung infections in individuals with cystic fibrosis (CF) significantly reduce quality of life and increase morbidity and mortality. Tracking these infections is critical for monitoring patient health and informing treatments. We are working toward the development of novel breath-based biomarkers to track chronic P. aeruginosa lung infections in situ. Using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS), we characterized the in vitro volatile metabolomes (or volatilomes) of 81 P. aeruginosa isolates collected from 17 CF patients over at least a five-year period of their chronic lung infections. We detected 539 volatiles produced by the P. aeruginosa isolates, 69 of which were core volatiles that were highly conserved. We found that each early infection isolate has a unique volatilome, and as infection progresses, the volatilomes of isolates from the same patient become increasingly dissimilar, to the point that these intra-patient isolates are no more similar to one another than to isolates from other patients. We observed that the size and chemical diversity of P. aeruginosa volatilomes do not change over the course of chronic infections; however, the relative abundances of core hydrocarbons, alcohols, and aldehydes do change, and are correlated to changes in phenotypes associated with chronic infections. This study indicates that it may be feasible to track P. aeruginosa chronic lung infections by measuring changes to the infection volatilome, and lays the groundwork for exploring the translatability of this approach to direct measurement using patient breath.IMPORTANCEPseudomonas aeruginosa is a leading cause of chronic lung infections in cystic fibrosis (CF), and are correlated with lung function declines. Significant clinical efforts are, therefore, aimed at detecting infections and tracking them for phenotypic changes, such as mucoidy and antibiotic resistance. Both the detection and tracking of lung infections relies on sputum cultures, but due to improvements in CF therapies, sputum production is declining though risks for lung infections persist. Therefore, we are working toward the development of breath-based diagnostics for CF lung infections. In this study we characterized of the volatile metabolomes of 81 P. aeruginosa clinical isolates collected from 17 CF patients over a duration of at least five years of a chronic lung infection. We found that the volatilome of P. aeruginosa adapts over time, and correlates to infection phenotype changes, suggesting it may be possible to track chronic CF lung infections with a breath test.

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Inactivation of the Mismatch Repair System in Pseudomonas aeruginosa Attenuates Virulence but Favors Persistence of Oropharyngeal Colonization in Cystic Fibrosis Mice

Journal of Bacteriology ◽

10.1128/jb.00120-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3665-3668 ◽

Cited By ~ 37

Author(s):

Ana Mena ◽

María D. Maciá ◽

Nuria Borrell ◽

Bartolomé Moya ◽

Teresa de Francisco ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Mismatch Repair ◽

Respiratory Infections ◽

Repair System ◽

Chronic Infections ◽

Mismatch Repair System ◽

Colonization Potential

ABSTRACT The inactivation of the mismatch repair (MMR) system of Pseudomonas aeruginosa modestly reduced in vitro fitness, attenuated virulence in murine models of acute systemic and respiratory infections, and decreased the initial oropharyngeal colonization potential. In contrast, the inactivation of the MMR system favored long-term persistence of oropharyngeal colonization in cystic fibrosis mice. These results may help in understanding the reasons for the low and high prevalences, respectively, of hypermutable P. aeruginosa strains in acute and chronic infections.

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Pseudomonas aeruginosa enhances the efficacy of norfloxacin against Staphylococcus aureus biofilms

10.1101/2020.03.26.010959 ◽

2020 ◽

Author(s):

Giulia Orazi ◽

Fabrice Jean-Pierre ◽

George A. O’Toole

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Respiratory Infections ◽

Multiple Infection ◽

Bacterial Biofilms ◽

Interspecies Interactions ◽

Chronic Infections ◽

Susceptibility Profiles

AbstractThe thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both have been associated with worse lung function. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including HQNO, siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa to shift S. aureus norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates S. aureus membrane potential, and furthermore, depletion of the S. aureus proton-motive force recapitulates the effect of P. aeruginosa supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus. From these results, we hypothesize that exposure to P. aeruginosa exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus to efflux norfloxacin. Our results illustrate that microbially-derived products can greatly alter the ability of antimicrobial agents to kill bacterial biofilms.ImportancePseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and non-healing diabetic foot ulcers. Co-infection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with one organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

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In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis

Microorganisms ◽

10.3390/microorganisms9030478 ◽

2021 ◽

Vol 9 (3) ◽

pp. 478

Author(s):

Ersilia Vita Fiscarelli ◽

Martina Rossitto ◽

Paola Rosati ◽

Nour Essa ◽

Valentina Crocetta ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Multidrug Resistant ◽

In Vitro Test ◽

Chronic Infections ◽

Hospital Environments ◽

Vitro Test ◽

General Reliability ◽

Phage Activity

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

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Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

mBio ◽

10.1128/mbio.00966-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 88

Author(s):

Rasmus Lykke Marvig ◽

Søren Damkiær ◽

S. M. Hossein Khademi ◽

Trine M. Markussen ◽

Søren Molin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Iron Acquisition ◽

Chronic Infections ◽

Content Type ◽

Mortality And Morbidity ◽

Airway Infections ◽

Host Evolution ◽

Promoter Mutations

ABSTRACTPseudomonas aeruginosaairway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist,P. aeruginosadepends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and thePseudomonasheme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation ofP. aeruginosato the host environment. Here we investigated the within-host evolution of the transmissibleP. aeruginosaDK2 lineage. We found positive selection for promoter mutations leading to increased expression of thephusystem. By mimicking conditions of the CF airwaysin vitro, we experimentally demonstrate that increased expression ofphuRconfers a growth advantage in the presence of hemoglobin, thus suggesting thatP. aeruginosaevolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additionalP. aeruginosalineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages,phuRpromoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment ofP. aeruginosainfections in CF patients.IMPORTANCEMost bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogenPseudomonas aeruginosato cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens’ ability to acquire iron. Here, we investigated the within-host evolution ofP. aeruginosa, and we found evidence thatP. aeruginosaduring long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

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