Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

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Nutritional Effects on Host Response to Lung Infections with Mucoid Pseudomonas aeruginosa in Mice

Infection and Immunity ◽

10.1128/iai.72.3.1479-1486.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1479-1486 ◽

Cited By ~ 15

Author(s):

Anna M. van Heeckeren ◽

Mark Schluchter ◽

Lintong Xue ◽

Juan Alvarez ◽

Steven Freedman ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Docosahexaenoic Acid ◽

Knockout Mice ◽

Host Response ◽

Inflammatory Responses ◽

Liquid Diet ◽

Wild Type ◽

Agarose Beads ◽

Lung Infections

ABSTRACT In cystic fibrosis, a recessive genetic disease caused by defects in the cystic fibrosis conductance regulator (CFTR), the main cause of death is lung infection and inflammation. Nutritional deficits have been proposed to contribute to the excessive host inflammatory response in both humans and Cftr-knockout mice. Cftr-knockout mice and gut-corrected Cftr-knockout mice expressing human CFTR primarily in the gut were challenged with Pseudomonas aeruginosa-laden agarose beads; they responded similarly with respect to bronchoalveolar lavage cell counts and levels of the acute-phase cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6. Wild-type mice fed the liquid diet used to prevent intestinal obstruction in Cftr-knockout mice had inflammatory responses to P. aeruginosa-laden agarose beads similar to those of wild-type mice fed an enriched solid diet, so dietary effects are unlikely to account for differences between wild-type mice and mice with cystic fibrosis. Finally, since cystic fibrosis patients and Cftr-knockout mice have an imbalance in fatty acids (significantly lower-than-normal levels of docosahexaenoic acid), the effects of specific supplementation with docosahexaenoic acid of wild-type and Cftr-knockout mice on their inflammatory responses to P. aeruginosa-laden agarose beads were tested. There were no significant differences (P = 0.35) in cumulative survival rates between Cftr-knockout mice and wild-type mice provided with either the liquid diet Peptamen or Peptamen containing docosahexaenoic acid. In conclusion, diet and docosahexaenoic acid imbalances alone are unlikely to explain the differences in the host response to lung infections with mucoid P. aeruginosa between mice with cystic fibrosis and their wild-type counterparts.

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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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Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections

The ISME Journal ◽

10.1038/ismej.2014.223 ◽

2014 ◽

Vol 9 (6) ◽

pp. 1391-1398 ◽

Cited By ~ 42

Author(s):

Chloe E James ◽

Emily V Davies ◽

Joanne L Fothergill ◽

Martin J Walshaw ◽

Colin M Beale ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lytic Activity ◽

Lung Infections

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Psl Produced by Mucoid Pseudomonas aeruginosa Contributes to the Establishment of Biofilms and Immune Evasion

mBio ◽

10.1128/mbio.00864-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 22

Author(s):

Christopher J. Jones ◽

Daniel J. Wozniak

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Immune Evasion ◽

Biofilm Formation ◽

Primary Concern ◽

Pulmonary Infections ◽

Chronic Infections ◽

Key Factor ◽

Current Therapies ◽

Lung Infections

ABSTRACT Despite years of research and clinical advances, chronic pulmonary infections with mucoid Pseudomonas aeruginosa remain the primary concern for cystic fibrosis patients. Much of the research on these strains has focused on the contributions of the polysaccharide alginate; however, it is becoming evident that the neutral polysaccharide Psl also contributes to biofilm formation and the maintenance of chronic infections. Here, we demonstrate that Psl produced by mucoid strains has significant roles in biofilm structure and evasion of immune effectors. Though mucoid strains produce less Psl than nonmucoid strains, the Psl that is produced is functional, since it mediates adhesion to human airway cells and epithelial cell death. Additionally, Psl protects mucoid bacteria from opsonization and killing by complement components in human serum. Psl production by mucoid strains stimulates a proinflammatory response in the murine lung, leading to reduced colonization. To determine the relevance of these data to clinical infections, we tested Psl production and biofilm formation of a panel of mucoid clinical isolates. We demonstrated three classes of mucoid isolates, those that produce Psl and form robust biofilms, those that did not produce Psl and have a poor biofilm phenotype, and exopolysaccharide (EPS) redundant strains. Collectively, these experimental results demonstrate that Psl contributes to the biofilm formation and immune evasion of many mucoid strains. This is a novel role for Psl in the establishment and maintenance of chronic pulmonary infections by mucoid strains. IMPORTANCE Cystic fibrosis patients are engaged in an ongoing battle against chronic lung infections by the bacterium Pseudomonas aeruginosa. One key factor contributing to the maintenance of chronic infections is the conversion to a mucoid phenotype, where the bacteria produce copious amounts of the polysaccharide alginate. Once the bacteria become mucoid, existing treatments are poorly effective. We proposed that mucoid bacteria produce an additional polysaccharide, Psl, which is important for their establishment and maintenance of chronic infections. This work demonstrates that Psl enhances attachment of mucoid bacteria to lung surfaces and leads to inflammation and damage in the lung. Additionally, we find that 50% of mucoid bacteria isolated from patients with chronic infections rely on Psl for the structure of their biofilm communities, suggesting that treatments against Psl should be investigated to enhance the success of current therapies. IMPORTANCE Cystic fibrosis patients are engaged in an ongoing battle against chronic lung infections by the bacterium Pseudomonas aeruginosa. One key factor contributing to the maintenance of chronic infections is the conversion to a mucoid phenotype, where the bacteria produce copious amounts of the polysaccharide alginate. Once the bacteria become mucoid, existing treatments are poorly effective. We proposed that mucoid bacteria produce an additional polysaccharide, Psl, which is important for their establishment and maintenance of chronic infections. This work demonstrates that Psl enhances attachment of mucoid bacteria to lung surfaces and leads to inflammation and damage in the lung. Additionally, we find that 50% of mucoid bacteria isolated from patients with chronic infections rely on Psl for the structure of their biofilm communities, suggesting that treatments against Psl should be investigated to enhance the success of current therapies.

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Potential of preventing Pseudomonas aeruginosa lung infections in cystic fibrosis patients: Experimental studies in animals

Apmis ◽

10.1111/j.1600-0463.1996.tb05581.x ◽

1996 ◽

Vol 104 (S63) ◽

pp. 5-42 ◽

Cited By ~ 13

Author(s):

HELLE KROGH JOHANSEN

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Experimental Studies ◽

Lung Infections

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Mechanism of Pseudomonas aeruginosa persistence during treatment with broad-spectrum cephalosporins of lung infections in patients with cystic fibrosis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.31.9.1438 ◽

1987 ◽

Vol 31 (9) ◽

pp. 1438-1439 ◽

Cited By ~ 4

Author(s):

A B Schryvers ◽

J Ogunariwo ◽

S Chamberland ◽

A J Godfrey ◽

H R Rabin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Broad Spectrum ◽

Lung Infections

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Genotypic and phenotypic diversity of Staphylococcus aureus from cystic fibrosis lung infections and their interactions with Pseudomonas aeruginosa

10.1101/814152 ◽

2019 ◽

Author(s):

Eryn E. Bernardy ◽

Robert A. Petit ◽

Vishnu Raghuram ◽

Ashley M. Alexander ◽

Timothy D. Read ◽

...

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Phenotypic Diversity ◽

Laboratory Strain ◽

Toxin Production ◽

Reference Isolate ◽

Patient Health ◽

Lung Infections

AbstractPseudomonas aeruginosa and Staphylococcus aureus are the most common bacteria that infect the respiratory tract of individuals with the genetic disease cystic fibrosis (CF); in fact, S. aureus has recently overtaken P. aeruginosa to become the most common. Substantial research has been performed on the epidemiology of S. aureus in CF; however, there appears to be a gap in knowledge in regard to the pathogenesis of S. aureus in the context of CF lung infections. Most studies have focused on a few S. aureus isolates, often exclusively laboratory adapted strains, and how they are killed by P. aeruginosa. Because of this, little is known about the diversity of S. aureus CF lung isolates in both virulence and interaction with P. aeruginosa. To begin to address this gap in knowledge, we recently sequenced 65 clinical S. aureus isolates from the Emory CF Biospecimen Registry and Boston Children’s Hospital, including the reference isolate JE2, a USA300 strain. Here, we analyzed antibiotic resistance genotypes, sequence type, clonal complex, spa type, and agr type of these isolates. We hypothesized that major virulence phenotypes of S. aureus that may be associated with CF lung infections, namely toxin production and mucoid phenotype, would be retained in these isolates. To test our hypothesis, we plated on specific agars and found that most isolates can hemolyze both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), consistent with virulence retention in CF lung isolates. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus CF isolates, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.Author SummaryStaphylococcus aureus is now the most frequently detected pathogen in the lungs of individuals who have cystic fibrosis (CF), followed closely by Pseudomonas aeruginosa. When these two pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus. Here we present an in-depth study of 64 S. aureus CF clinical isolates where we investigated genetic diversity utilizing whole genome sequencing, virulence phenotypes, and interactions with P. aeruginosa. We have found that S. aureus isolated from the CF lung are phylogenetically diverse, most retain known virulence factors, and they vary in interactions with P. aeruginosa from highly sensitive to completely tolerant. Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

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Scnn1b-Transgenic BALB/c Mice as a Model of Pseudomonas aeruginosa Infections of the Cystic Fibrosis Lung

Infection and Immunity ◽

10.1128/iai.00237-20 ◽

2020 ◽

Vol 88 (9) ◽

Author(s):

Kristen J. Brao ◽

Brendan P. Wille ◽

Joshua Lieberman ◽

Robert K. Ernst ◽

Mark E. Shirtliff ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Immune Cell ◽

Opportunistic Pathogen ◽

Sodium Absorption ◽

Lung Pathology ◽

Wild Type ◽

Content Type ◽

Lung Infections ◽

Content Modeling

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1β (IL-1β), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.

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Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

Journal of Bacteriology ◽

10.1128/jb.00300-20 ◽

2020 ◽

Vol 203 (2) ◽

pp. e00300-20

Author(s):

Katie V. Farrant ◽

Livia Spiga ◽

Jane C. Davies ◽

Huw D. Williams

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Immune System ◽

Hypochlorous Acid ◽

Secretion System ◽

Content Type ◽

Type 3 Secretion System ◽

Lung Infections ◽

Type 3

ABSTRACTPseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

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