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

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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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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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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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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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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Heterogenous response to R-pyocin killing in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

10.1101/2020.08.05.238956 ◽

2020 ◽

Author(s):

Madeline Mei ◽

Jacob Thomas ◽

Stephen P. Diggle

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Genotypic Diversity ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Bacterial Populations ◽

Heterogeneous Populations ◽

Lung Infections ◽

The Impact ◽

Lps Binding

AbstractBacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources and (ii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.ImportanceR-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection, however little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics, as well as evolve phenotypic and genotypic diversity over time; thus it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is likely heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

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413. Differences in Inflammatory Mechanisms in Pseudomonas aeruginosa and Staphyloccoccus auereus Infections in Cystic Fibrosis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.486 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S209-S209

Author(s):

Melissa S Phuong ◽

Rafael E Hernandez ◽

Subash Sad

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Cell Death ◽

Cytokine Production ◽

Neutral Red ◽

Chronic Infections ◽

Neutral Red Assay ◽

The One ◽

The University

Abstract Background Chronic bacterial lung infections are the primary cause of morbidity and mortality in cystic fibrosis (CF). The most common CF pathogens, Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus), are common commensal or environmental organisms that adapt to the CF lung. We sought to investigate whether adaptation from early lung colonizer to chronic pathogen alters the bacterial effects on host inflammation. Methods P. aeruginosa (n = 25) and S. aureus (n = 25) isolates from CF patients with early and chronic infections were acquired from Seattle Children’s CF. Environmental (n = 8) and clinical, non-CF P. aeruginosa (n = 8) isolates were obtained from the University of Ottawa. P. aeruginosa reference strain PA14 and PA14 transposon mutants for T3SS and flagellin were used to observe the relationship between cell death and cytokine production. We infected THP-1-derived macrophages (PMA differentiated) in vitro for 3 hours with various MOIs. We subsequently measured cell death of THP-1-derived macrophages using neutral red assay and cytokine production using ELISAs. Results Infections with PA14 mutants and non-CF P. aeruginosa isolates demonstrated that rapid cell death of THP-1-derived macrophages caused a reduction in cytokine production relative to strains that did not cause as much cell death. At 10 MOI, early P. aeruginosa isolates from CF patients induced more THP-1-derived macrophage cell death compared with chronic isolates (P < 0.0001). Chronic P. aeruginosa isolates induced greater production of TNF, IL-8, and IL-6 (P < 0.01, P < 0.0001, and P < 0.0001, respectively) compared with early strains. No difference in IL-1β production was observed. When controlling for cell death between the two groups by using heat-killed bacteria, the only difference maintained was in TNF production (P < 0.01). Between early and chronic S. aureus isolates, the one difference observed was greater IL-8 production among early isolates (P < 0.01). Conclusion Chronic P. aeruginosa isolates from CF patients induce less cell death but more TNF, IL-8, and IL-6 production compared with early isolates. This suggests that P. aeruginosa producing chronic infections induce inflammatory signals that may contribute to increased morbidity among CF patients. Disclosures All authors: No reported disclosures.

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Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

Infection and Immunity ◽

10.1128/iai.00575-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4462-4473 ◽

Cited By ~ 53

Author(s):

Michelle A. Laskowski ◽

Barbara I. Kazmierczak

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutational Analysis ◽

Response Regulator ◽

Acute Infection ◽

Opportunistic Pathogen ◽

Sensor Kinase ◽

Chronic Infections ◽

Reciprocal Regulation ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

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Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

10.1101/2020.02.25.964320 ◽

2020 ◽

Cited By ~ 2

Author(s):

Wai Leong ◽

Carla Lutz ◽

Jonathan Williams ◽

Yan Hong Poh ◽

Benny Yeo Ken Yee ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Late Stage ◽

Early Stage ◽

Acanthamoeba Castellanii ◽

Opportunistic Pathogen ◽

Human Neutrophils ◽

Phenotypic Traits ◽

Chronic Infections

AbstractThe opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types.Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

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