1222. Ceftolozane-Tazobactam Heteroresistance in Cystic Fibrosis Related Pseudomonas aeruginosa Infections

Abstract Background Cystic fibrosis (CF) patients are often colonized with Pseudomonas aeruginosa (PSA). During treatment, PSA can develop subpopulations exhibiting variable in vitro antimicrobial susceptibility patterns. Heteroresistance may underlie the reported discordant in vitro results and clinical responses to various antimicrobials. Here, we sought to examine the presence and nature of PSA heteroresistance to ceftolozane-tazobactam (C-T) in isolates originating from CF pulmonary exacerbations. Methods Respiratory cultures from 26 adult CF patients were collected. From each sample, 5-10 PSA colonies were selected. Susceptibility testing was conducted via E-test for C-T, ceftazidime-avibactam (CZA), and imipenem-relebactam (I-R). Polyclonal-heteroresistance (PHR) was defined as the presence of different susceptibility profiles among the colonies that originated from a single patient specimen. Population analysis profile (PAPs) were performed to assess the presence of monoclonal-heteroresistance (MHR), defined as ≥ 4 fold change in the C-T MIC from a single colony over 24-48 hours. Results 246 PSA isolates from 26 adult CF patients were included. The C-T MIC50 and MIC90 were 1/4 and ≥ 256/4 µg/mL, respectively (Figure 1). Sixteen of the 26 patients (62%) demonstrated ≥ 2 fold change in C-T MIC between isolates from the same culture. Of these 16 isolates, the fold change in C-T MIC was >2 fold for 7 isolates (27%) and resulted in a susceptibility interpretation change in 6 of the isolates (23%). Of the 32 isolates that underwent PAP testing, 7 grew on MH plates at 2-fold the C-T MIC concentration. One isolate, PSA 1311, demonstrated growth on PAPs up to 4 fold the MIC (16/4 µg/mL) (Figure 2). Figure 1. Ceftolozane-tazobactam, ceftazidime-avibactam, and imipenem-relebactam MIC distributions against PSA isolates from 26 adult CF patients Figure 2. Monoclonal heteroresistance to C-T (PSA 1311 [C-T MIC 1/4 µg/mL] PAPs at 2, 4, 6, and 8-fold the C-T MIC). Conclusion Susceptibilities to C-T and CZA were similar across our CF PSA isolates. Comparatively, I-R retained better in vitro potency. C-T PHR exists among PSA isolates in the majority of our CF patients. Approximately 25% of these PHR isolates resulted in susceptibility interpretation changes supporting concerns surrounding the utility of traditional susceptibility testing methodology for CF isolates. These data suggest MHR also exists, albeit rare in this small subset. Additional data are needed to better understand these results in clinical context. Disclosures David E. Greenberg, MD, Shionogi (Grant/Research Support)Solenic Medical (Shareholder)

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Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis

mSphere ◽

10.1128/msphere.00343-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 5

Author(s):

Jeffrey M. Flynn ◽

Lydia C. Cameron ◽

Talia D. Wiggen ◽

Jordan M. Dunitz ◽

William R. Harcombe ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Susceptibility Testing ◽

Anaerobic Bacteria ◽

Content Type ◽

Growth Environment ◽

Polymicrobial Infections

ABSTRACT A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro. However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a “weakest-link” approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them. IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa. The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

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1594. Ceftolozane–Tazobactam Demonstrates Higher In Vitro Susceptibility than Ceftazidime–Avibactam Against Pseudomonas aeruginosa Isolated from Respiratory Tract of Adult Cystic Fibrosis Patients

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1458 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S581-S582

Author(s):

Patrick James Nolan ◽

Tiffeny Smith ◽

James D Finklea ◽

Leah Cohen ◽

Raksha Jain

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Susceptibility Testing ◽

Clinical Laboratory ◽

Multidrug Resistant ◽

Vitro Activity ◽

Drug Resistant ◽

In Vitro Activity ◽

In Vitro Susceptibility

Abstract Background Pseudomonas aeruginosa is a commonly isolated pathogen in adults with cystic fibrosis (CF). Antimicrobial resistance is an escalating problem due to chronic colonization and frequent antimicrobial exposure. Ceftolozane–tazobactam (C/T) and ceftazidime–avibactam (CZA) exhibit promising activity against antimicrobial-resistant organisms, including P. aeruginosa. In this study, we compared in vitro activity of C/T and CZA against P. aeruginosa isolated from respiratory cultures obtained from adult patients with CF. Methods This is a retrospective study of respiratory cultures positive for P. aeruginosa collected from adult CF patients between January 1, 2015 to November 30, 2018. The first isolate per patient per year that underwent susceptibility testing for C/T, CZA, and colistin were included in the study. All isolates underwent in-house susceptibility testing for 9 anti-pseudomonal agents according to the methodology established by the Clinical Laboratory Standards Institute (CLSI). Susceptibility testing of C/T, CZA, and colistin were performed by a reference lab. Isolates were classified into 3 drug-resistant categories using the following definition: multidrug-resistant (MDR) non-susceptible (NS) to ≥1 agent in ≥3 different antimicrobial classes, extensive drug-resistant (XDR) NS to 4 or 5 different classes, and pan drug-resistant (PDR) NS to all 6 classes except colistin. Results Forty-two P. aeruginosa respiratory isolates from 32 patients with CF were included. The overall susceptibility to C/T and CZA was 59.5% and 42.9%, respectively. Thirty-eight (90%) isolates were considered MDR with susceptibility of 55.3% to C/T and 44.7% to CZA. Among the 11 XDR isolates, susceptibility to C/T was 81.8% vs. CZA 72.7%. Susceptibility to C/T vs. CZA was also higher (37.5% vs. 25%) among the 24 PDR isolates. Conclusion Among P. aeruginosa isolated from CF respiratory cultures, C/T appears to have better in vitro activity compared with CZA, and remained true among isolates considered XDR and PDR. These results suggest using C/T while awaiting susceptibilities when standard anti-pseudomonal agents cannot be used. Future studies evaluating clinical outcomes for the treatment of pulmonary CF exacerbations are needed to assess the applicability of in vitro susceptibility data. Disclosures All authors: No reported disclosures.

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In vitro biofilm formation and antibiotic susceptibility of Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis

Journal of Medical Science ◽

10.20883/jms.2016.179 ◽

2016 ◽

Vol 85 (4) ◽

pp. 245-253

Author(s):

Jolanta Długaszewska ◽

Marta Antczak ◽

Izabella Kaczmarek ◽

Renata Jankowiak ◽

Malgorzata Buszkiewicz ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Susceptibility Testing ◽

Testing Method ◽

Airway Infections ◽

Microdilution Broth

Background: Pseudomonas aeruginosa is the predominant cause of airway infections in patients with cystic fibrosis (CF) as a result of its ability to form biofilm. Resistance to antimicrobial agents is the most important feature of biofilm infection. The aim of this study was to evaluate biofilm formation and to compare antibiotic susceptibility of P. aeruginosa living in two modes of growth: planktonic and biofilm, isolated from respiratory tract of CF patients. Methods: Biofilm formation and biofilm susceptibility to antibiotics were determined using modified microtitere plate method. For susceptibility testing of planktonic culture to antibiotics serial microdilution broth method were used.Results: More than 95% of isolates were capable to form biofilm. Isolates grown as biofilms were more resistant to tested antibiotics compared to those grown planktonically. Ciprofloxacin showed the highest activity against P. aeruginosa biofilm. In contrast, no bacteriostatic activity was obtain for the highest concentration of piperacillin tested against most of P. aeruginosa strains growing in a biofilm (BIC > 4096 mg/L).Conclusions: Our study indicates the need to develop a standardized susceptibility testing method for biofilm mode of growth of pathogens. It appears that it is appropriate to introduce a biofilm susceptibility testing to routinely performed tests, as the effect of antibiotics on biofilm eradication may be variable and unpredictable.

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In vitro biofilm formation and antibiotic susceptibility of Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis

Journal of Medical Science ◽

10.20883/179 ◽

2016 ◽

Vol 85 (4) ◽

pp. 245 ◽

Cited By ~ 2

Author(s):

Jolanta Długaszewska ◽

Marta Antczak ◽

Izabella Kaczmarek ◽

Renata Jankowiak ◽

Malgorzata Buszkiewicz ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Susceptibility Testing ◽

Testing Method ◽

Airway Infections ◽

Microdilution Broth

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Multicentre study of the in vitro activity of ceftolozane/tazobactam and other commonly used antibiotics against Pseudomonas aeruginosa isolates from patients in the UK

JAC-Antimicrobial Resistance ◽

10.1093/jacamr/dlaa024 ◽

2020 ◽

Vol 2 (2) ◽

Cited By ~ 1

Author(s):

Adela Alvarez-Buylla ◽

Mike Allen ◽

Dan Betts ◽

Sean Bennett ◽

Irene Monahan ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibacterial Agents ◽

Susceptibility Testing ◽

Vitro Activity ◽

In Vitro Activity ◽

Routine Testing ◽

Informed Treatment ◽

The Uk

Abstract Objectives To evaluate the in vitro activity of ceftolozane/tazobactam and other commonly used antipseudomonal antibiotics against geographically spread Pseudomonas aeruginosa isolates in the UK using disc susceptibility testing. Methods The in vitro activity of ceftolozane/tazobactam and nine other commonly used antipseudomonal antibiotics was evaluated. Isolates were collected between January 2015 and April 2018. Susceptibility results were interpreted using EUCAST 2018 criteria. Results Overall, 1326 clinical isolates from 14 centres in the UK were tested. The majority of the isolates were collected from non-cystic fibrosis (non-CF) patients (n = 1123, 85.0%). In addition, 199 cystic fibrosis (CF) isolates were collected from 10 centres. Overall susceptibility to ceftolozane/tazobactam was 89.3% (n = 1181), which included 128 CF and 1053 non-CF isolates. The other antibacterial agents with the highest susceptibility were tobramycin (92.4%, n = 1221) and piperacillin/tazobactam (90.7%, n = 1199). Susceptibility to all antibacterial agents was lower for CF isolates. Piperacillin/tazobactam was the most active of the antibacterial agents tested, followed by ceftolozane/tazobactam (70.4% and 64.3%, respectively), and <60% of CF isolates were susceptible to ceftazidime and the carbapenems. The reason for the higher rates of susceptibility to piperacillin/tazobactam and lower susceptibility to ceftazidime compared with other studies is unclear. Conclusions The data presented here support the need to investigate the place of ceftolozane/tazobactam as a treatment option in the management of pseudomonal infections, particularly in patients with CF. The results highlight the importance of routine testing of new antibacterial agents and of making the data available to clinicians to make appropriate and informed treatment choices.

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Contemporary analysis of ETEST for antibiotic susceptibility and minimum inhibitory concentration agreement against Pseudomonas aeruginosa from patients with cystic fibrosis

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00415-0 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Maxwell J. Lasko ◽

Holly K. Huse ◽

David P. Nicolau ◽

Joseph L. Kuti

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Susceptibility Testing ◽

Inhibitory Concentration ◽

Package Insert ◽

Broth Microdilution ◽

In Vitro Susceptibility ◽

Resistant Strains ◽

Drug Resistant Strains

Abstract Objectives Cystic fibrosis (CF) acute pulmonary exacerbations are often caused by Pseudomonas aeruginosa, including multi-drug resistant strains. Optimal antibiotic therapy is required to return lung function and should be guided by in vitro susceptibility results. There are sparse data describing ETEST performance for CF isolates using contemporary isolates, methods and interpretation, as well as novel antibiotics, such as ceftazidime–avibactam and ceftolozane–tazobactam. Methods Pseudomonas aeruginosa (n = 105) isolated during pulmonary exacerbation from patients with CF were acquired from 3 US hospitals. Minimum inhibitory concentrations (MICs) were assessed by reference broth microdilution (BMD) and ETEST for aztreonam, cefepime, ceftazidime, ceftazidime–avibactam, ceftolozane–tazobactam, ciprofloxacin, levofloxacin, meropenem, piperacillin–tazobactam, and tobramycin. Broth microdilution was conducted in concordance with the Clinical and Laboratory Standards Institute M100. ETEST methodology reflected package insert recommendations. Performance of ETEST strips was evaluated using the Food and Drug Administration (FDA) and Susceptibility Testing Manufacturers Association (STMA) guidance. Results Of the 105 P. aeruginosa included, 46% had a mucoid phenotype. ETEST MICs typically read 0–1 dilution higher than BMD for all drugs. Categorical agreement and essential agreement ranged from 64 to 93% and 63 to 86%, respectively. The majority of observed errors were minor. A single very major error occurred with ceftazidime (4.2%). For ceftazidime–vibactam, 2 very major errors were observed and both were within essential agreement. Major errors occurred for aztreonam (3.3%), cefepime (9.4%), ceftazidime–avibactam (5.3%, adjusted 2.1%), ceftolozane–tazobactam (1%), meropenem (3.3%), piperacillin–tazobactam (2.9%), and tobramycin (1.5%). Conclusions ETEST methods performed conservatively for most antibiotics against this challenging collection of P. aeruginosa from patients with CF.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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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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Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa529 ◽

2020 ◽

Author(s):

María Díez-Aguilar ◽

Marta Hernández-García ◽

María-Isabel Morosini ◽

Ad Fluit ◽

Michael M Tunney ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Spontaneous Mutation ◽

Lung Surfactant ◽

Vitro Activity ◽

In Vitro Activity ◽

Broth Microdilution ◽

Agar Dilution

Abstract Background Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. Objectives To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. Methods MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time–kill assays. Resistant mutants were studied by WGS. Results The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1–5 h. Murepavadin MICs were 2–9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). Conclusions Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

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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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