scholarly journals Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum

2015 ◽  
Vol 113 (2) ◽  
pp. E110-E116 ◽  
Author(s):  
Sebastian H. Kopf ◽  
Alex L. Sessions ◽  
Elise S. Cowley ◽  
Carmen Reyes ◽  
Lindsey Van Sambeek ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Heavy Water ◽  
Growth Rates ◽  
Physiological State ◽  
Full Range ◽  
New Drugs ◽  
Chronic Infections ◽  
Preclinical Development ◽  
Laboratory Models

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogenStaphylococcus aureusdirectly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results revealS. aureusgeneration times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth ofS. aureuson average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

scholarly journals Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

2014 ◽  
Vol 82 (11) ◽  
pp. 4477-4486 ◽  
Author(s):  
Kasper N. Kragh ◽  
Morten Alhede ◽  
Peter Ø. Jensen ◽  
Claus Moser ◽  
Thomas Scheike ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Growth Rates ◽  
Polymorphonuclear Leukocytes ◽  
Growth Patterns ◽  
Tissue Samples ◽  
Content Type ◽  
Growth Physiology

ABSTRACTCystic fibrosis (CF) patients have increased susceptibility to chronic lung infections byPseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate thein vivogrowth physiology ofP. aeruginosawithin lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescencein situhybridization (PNA-FISH)-based method was used to estimate thein vivogrowth rates ofP. aeruginosadirectly in lung tissue samples from CF patients and the growth rates ofP. aeruginosain infected lungs in a mouse model. The growth rate ofP. aeruginosawithin CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect onP. aeruginosaby PMNs was also observedin vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding thatP. aeruginosagrowth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2consumption, which slows the growth ofP. aeruginosain infected CF lungs. In support of this, the growth ofP. aeruginosawas significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronicP. aeruginosainfection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Interference ofPseudomonas aeruginosasignalling and biofilm formation for infection control

2010 ◽  
Vol 12 ◽  
Author(s):  
Thomas Bjarnsholt ◽  
Tim Tolker-Nielsen ◽  
Niels Høiby ◽  
Michael Givskov
Keyword(s):  
Cystic Fibrosis ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Antibiotic Tolerance ◽  
Chronic Infections ◽  
Major Task ◽  
Vitro Data ◽  
In Vitro Data

Pseudomonas aeruginosais the best described bacterium with regards to quorum sensing (QS), in vitro biofilm formation and the development of antibiotic tolerance. Biofilms composed ofP. aeruginosaare thought to be the underlying cause of many chronic infections, including those in wounds and in the lungs of patients with cystic fibrosis. In this review, we provide an overview of the molecular mechanisms involved in QS, QS-enabled virulence, biofilm formation and biofilm-enabled antibiotic tolerance. We now have substantial knowledge of the multicellular behaviour ofP. aeruginosain vitro. A major task for the future is to investigate how such in vitro data correlate with the in vivo behaviour ofP. aeruginosa, and how to treat chronic infections of this bacterium in patients.

scholarly journals Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Majken Sønderholm ◽  
Kasper Nørskov Kragh ◽  
Klaus Koren ◽  
Tim Holm Jakobsen ◽  
Sophie E. Darch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Electron Acceptor ◽  
Chronic Wounds ◽  
Alginate Bead ◽  
Alginate Beads ◽  
Model System ◽  
Chronic Infections ◽  
Content Type

ABSTRACT Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3 − as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3 − as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

scholarly journals An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum

2021 ◽  
Vol 11 ◽  
Author(s):  
Alexander C. Graf ◽  
Johanna Striesow ◽  
Jan Pané-Farré ◽  
Thomas Sura ◽  
Martina Wurster ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Extraction ◽  
Arginine Deiminase ◽  
Microbial Pathogens ◽  
Chronic Infections ◽  
16S Sequencing ◽  
Bacterial Proteins ◽  
Human Proteins ◽  
Important Basis

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

P6342Subcutaneous delivery of NPA7, first-in-class novel bispecific designer peptide: enhances cardiorenal function and suppresses renin and aldosterone in vivo and in vitro

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Sugihara ◽  
T Ichiki ◽  
Y Chen ◽  
G J Harty ◽  
D M Heublen ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Ex Vivo ◽  
Plasma Renin ◽  
New Drugs ◽  
Preclinical Development ◽  
Effective Delivery ◽  
Canine Serum ◽  
Canine Plasma

Abstract Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P<0.05 vs. BL. Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.

scholarly journals Parallel Evolution in Pseudomonas aeruginosa over 39,000 Generations In Vivo

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Holly K. Huse ◽  
Taejoon Kwon ◽  
James E. A. Zlosnik ◽  
David P. Speert ◽  
Edward M. Marcotte ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Chronic Infection ◽  
Parallel Evolution ◽  
Chronic Infections ◽  
Content Type ◽  
Airway Infections ◽  
Chronic Colonization

ABSTRACTThe Gram-negative bacteriumPseudomonas aeruginosais a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung,P. aeruginosabecomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits ofP. aeruginosaduring chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000in vivogenerations. We identified 24 genes that were commonly regulated by all 3P. aeruginosalineages, including several genes encoding traits previously shown to be important forin vivogrowth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial forP. aeruginosachronic colonization of the CF lung.IMPORTANCEDeadly diseases like AIDS, malaria, and tuberculosis are the result of long-term chronic infections. Pathogens that cause chronic infections adapt to the host environment, avoiding the immune response and resisting antimicrobial agents. Studies of pathogen adaptation are therefore important for understanding how the efficacy of current therapeutics may change upon prolonged infection. One notorious chronic pathogen isPseudomonas aeruginosa, a bacterium that causes long-term infections in individuals with the heritable disease cystic fibrosis (CF). We used gene expression profiles to identify 24 genes that commonly changed expression over time in 3P. aeruginosalineages, indicating that these changes occur in parallel in the lungs of individuals with CF. Several of these genes have previously been shown to encode traits critical forin vivo-relevant processes, suggesting that they are likely beneficial adaptations important for chronic colonization of the CF lung.

scholarly journals In Situ Growth Rates and Biofilm Development of Pseudomonas aeruginosa Populations in Chronic Lung Infections

2007 ◽  
Vol 190 (8) ◽  
pp. 2767-2776 ◽  
Author(s):  
Lei Yang ◽  
Janus A. J. Haagensen ◽  
Lars Jelsbak ◽  
Helle Krogh Johansen ◽  
Claus Sternberg ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Growth Rates ◽  
Growth Dynamics ◽  
Chronic Infections ◽  
Lung Infections ◽  
New Perspective ◽  
Biofilm Development

ABSTRACT The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

Natural Anti-inflammatory compounds as Drug candidates in Alzheimer’s disease

2020 ◽  
Vol 27 ◽  
Author(s):  
Reyaz Hassan Mir ◽  
Abdul Jalil Shah ◽  
Roohi Mohi-ud-din ◽  
Faheem Hyder Potoo ◽  
Mohd. Akbar Dar ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Natural Products ◽  
Protective Action ◽  
New Drugs ◽  
Huperzine A ◽  
Brain Disorder ◽  
Anti Inflammatory

: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder characterized by memory impairment, dementia, oxidative stress in elderly people. Currently, only a few drugs are available in the market with various adverse effects. So to develop new drugs with protective action against the disease, research is turning to the identification of plant products as a remedy. Natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Phytochemicals including Curcumin, Resveratrol, Quercetin, Huperzine-A, Rosmarinic acid, genistein, obovatol, and Oxyresvertarol were reported molecules for the treatment of AD. Several alkaloids such as galantamine, oridonin, glaucocalyxin B, tetrandrine, berberine, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in-vivo. In conclusion, natural products from plants represent interesting candidates for the treatment of AD. This review highlights the potential of specific compounds from natural products along with their synthetic derivatives to counteract AD in the CNS.

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