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

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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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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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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Impact of estradiol on biofilm formation of Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients

10.5194/biofilms9-107 ◽

2020 ◽

Author(s):

Faria Afzal ◽

Tony Le Gall ◽

Tristan Montier ◽

Mareike Müller

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Gender Gap ◽

Pathogenic Bacteria ◽

Total Biomass ◽

Biofilm Growth ◽

Underlying Mechanisms ◽

Biofilm Structure ◽

The University

Women with Cystic Fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization of pathogenic bacteria like Pseudomonas aeruginosa (PA). Reasons for this &#8220;CF gender gap&#8221; until now have not yet been fully clarified and are assumed to be multifactorial. This study aims to shed light on the contribution of sex hormones to the CF-Gender gap considering microbial endocrinology. Therefor the study investigates whether the sex hormone estradiol, whose blood serum concentrations are significantly fluctuating within the female cycle and during pregnancy, has a regulatory influence on the development of PA biofilms in the context of CF. For that purpose, biofilms of PA isolates from CF-patients are treated in vitro with various estradiol concentrations and are examined in a comparative study quantitatively regarding the total biomass, e.g. via crystal violet staining, and qualitatively, e.g. via scanning electron microscopy, to characterize the ultrastructure of the biofilm. We observed that estradiol induces biofilm-forming capacity of CF-PA-isolates with respect to the total biomass and modulates the biofilm structure especially concerning the distribution and clustering of bacteria. The observed in vitro correlation between estradiol concentration and extent of biofilm growth provides a possible microbiological explanation for gender differences in CF disease progression. These insights and further research on possible underlying mechanisms might be relevant in the long-term for new approaches in personalized treatment for female CF patients. Acknowledgement This work is supported by a financial grant from Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V., the Christiane Herzog foundation. We further thank the Equal Opportunities Office of the University Siegen as well as the DAAD PPP Frankreich (Project-ID 55976814) for financial support. &#160;

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Polymicrobial Interactions in the Cystic Fibrosis Airway Microbiome Impact the Antimicrobial Susceptibility of Pseudomonas aeruginosa

Antibiotics ◽

10.3390/antibiotics10070827 ◽

2021 ◽

Vol 10 (7) ◽

pp. 827

Author(s):

Emma Reece ◽

Pedro H. de Almeida Bettio ◽

Julie Renwick

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Airway Disease ◽

Chronic Infections ◽

Sensitivity Testing ◽

Antimicrobial Sensitivity ◽

Cystic Fibrosis Airway ◽

Airway Microbiome ◽

Diverse Community

Pseudomonas aeruginosa is one of the most dominant pathogens in cystic fibrosis (CF) airway disease and contributes to significant inflammation, airway damage, and poorer disease outcomes. The CF airway is now known to be host to a complex community of microorganisms, and polymicrobial interactions have been shown to play an important role in shaping P. aeruginosa pathogenicity and resistance. P. aeruginosa can cause chronic infections that once established are almost impossible to eradicate with antibiotics. CF patients that develop chronic P. aeruginosa infection have poorer lung function, higher morbidity, and a reduced life expectancy. P. aeruginosa adapts to the CF airway and quickly develops resistance to several antibiotics. A perplexing phenomenon is the disparity between in vitro antimicrobial sensitivity testing and clinical response. Considering the CF airway is host to a diverse community of microorganisms or ‘microbiome’ and that these microorganisms are known to interact, the antimicrobial resistance and progression of P. aeruginosa infection is likely influenced by these microbial relationships. This review combines the literature to date on interactions between P. aeruginosa and other airway microorganisms and the influence of these interactions on P. aeruginosa tolerance to antimicrobials.

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Comparison between the in-vitro activity of new agents on Pseudomonas aeruginosa isolates from cystic fibrosis patients and other chronic infections

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/14.1.25 ◽

1984 ◽

Vol 14 (1) ◽

pp. 25-29 ◽

Cited By ~ 5

Author(s):

B. Gordts ◽

C. Vandenborre ◽

Ph. VanderAuwera ◽

J. P. Butzler

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Vitro Activity ◽

In Vitro Activity ◽

Chronic Infections ◽

New Agents

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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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Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response

Cell Death and Disease ◽

10.1038/s41419-021-03526-w ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Melissa S. Phuong ◽

Rafael E. Hernandez ◽

Daniel J. Wolter ◽

Lucas R. Hoffman ◽

Subash Sad

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Cell Death ◽

Inflammatory Cytokines ◽

Immune Cell ◽

Host Cells ◽

Respiratory Pathogen ◽

Cell Death Pathways ◽

The Relationship

AbstractPseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.

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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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