Building a better biofilm - formation of in vivo-like biofilm structures by Pseudomonas aeruginosa in a porcine model of cystic fibrosis lung infection

Pseudomonas aeruginosa biofilm infections in the cystic fibrosis (CF) lung are highly resistant to current antimicrobial treatments and are associated with increased mortality rates. The existing models for such infections are not able to reliably mimic the clinical biofilms observed. We aimed to further optimise an ex vivo pig lung (EVPL) model for P. aeruginosa CF lung infection that can be used to increase understanding of chronic CF biofilm infection. The EVPL model will facilitate discovery of novel infection prevention methods and treatments, and enhanced exploration of biofilm architecture. We investigated purine metabolism and biofilm formation in the model using transposon insertion mutants in P. aeruginosa PA14 for key genes: purD, gacA and pelA. Our results demonstrate that EVPL recapitulates a key aspect of in vivo P. aeruginosa infection metabolism, and that the pathogen forms a biofilm with a clinically realistic structure not seen in other in vitro studies. Two pathways known to be required for in vivo biofilm infection - the Gac regulatory pathway and production of the Pel exopolysaccharide - are essential to the formation of this mature, structured biofilm on EVPL tissue. We propose the high-throughput EVPL model as a validated biofilm platform to bridge the gap between in vitro work and CF lung infection

Predicting antibiotic-associated virulence of pseudomonas aeruginosa using an ex vivo lung biofilm model

Background: Bacterial biofilms are known to have high antibiotic tolerance which directly affects clearance of bacterial infections in people with cystic fibrosis (CF). Current antibiotic susceptibility testing methods are either based on planktonic cells or do not reflect the complexity of biofilms in vivo. Consequently, inaccurate diagnostics affect treatment choice, preventing bacterial clearance and potentially selecting for antibiotic resistance. This leads to prolonged, ineffective treatment. Methods: In this study, we use an ex vivo lung biofilm model to study antibiotic tolerance and virulence of Pseudomonas aeruginosa. Sections of pig bronchiole were dissected, prepared and infected with clinical isolates of P. aeruginosa and incubated in artificial sputum media to form biofilms, as previously described. Then, lung-associated biofilms were challenged with antibiotics, at therapeutically relevant concentrations, before their bacterial load and virulence were quantified and detected, respectively. Results: The results demonstrated minimal effect on the bacterial load with therapeutically relevant concentrations of ciprofloxacin and meropenem, with the latter causing an increased production of proteases and pyocyanin. A combination of meropenem and tobramycin did not show any additional decrease in bacterial load but demonstrated a slight decrease in total proteases and pyocyanin production. Conclusion: In this initial study of six clinical isolates of P. aeruginosa showed high levels of antibiotic tolerance, with minimal effect on bacterial load and increased proteases production, which could negatively affect lung function. Thus, the ex vivo lung model has the potential to be effectively used in larger studies of antibiotic tolerance in in vivo-like biofilms, and show how sub optimal antibiotic treatment of biofilms may potentially contribute to exacerbations and eventual lung failure. We demonstrate a realistic model for understanding antibiotic resistance and tolerance in biofilms clinically and for molecules screening in anti-biofilm drug development

Transcriptome analysis of Pseudomonas aeruginosa biofilm infection in an ex vivo pig model of the cystic fibrosis lung

Pseudomonas aeruginosa is the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a P. aeruginosa biofilm infection, highlighted by previous RNA-sequencing studies. We compared the P. aeruginosa PA14 transcriptome in an ex vivo pig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM). P. aeruginosa was grown in the EVPL model for 1, 2 and 7 days, and in vitro in SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each cued a transcriptome distinct from that seen in SCFM in vitro. The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative to in vitro SCFM was similar to CF sputum versus in vitro conditions. Hence, the EVPL model can replicate key aspects of in vivo biofilm infection that are missing from other current models. It provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance that quickly drives P. aeruginosa into a chronic-like infection phenotype

An ex vivocystic fibrosis model recapitulates key clinical aspects of chronic Staphylococcus aureus infection

Staphylococcus aureus is one of the most prevalent organisms isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. Clinical studies are limited in scope by age and health of participants and in vitro studies are not always able to accurately recapitulate chronic disease characteristics such as the development of small colony variants. Further, animal models also do not fully represent features of clinical disease: in particular, mice are not readily colonized by S. aureus and when infection is established it leads to the formation of abscesses, a phenomenon almost never observed in the human CF lung. Here, we present details of the development of an existing ex vivo pig lung model of CF infection to investigate the growth of S. aureus. We show that S. aureus is able to establish infection and demonstrates clinically significant characteristics including small colony variant phenotype, increased antibiotic tolerance and preferential localisation in mucus. Tissue invasion and the formation of abscesses were not observed, in line with clinical data

Antibiotic efficacy testing in an ex vivo model of Pseudomonas aeruginosa and Staphylococcus aureus biofilms in the Cystic Fibrosis lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo pig lung, EVPL)

An overview on deficit and requirements of the Irish national soil phosphorus balance

peer-reviewedPhosphorus (P) is an essential life-supporting nutrient for which there is no substitute. Modern farming practice and food production are supported by the application of mineral P fertiliser derived from finite mined phosphate rock. The European Union does not have indigenous mineral phosphate reserves, which poses a significant issue to food security. This research paper assesses the potential of indigenous recycled P sources to replace imported P fertiliser within the Republic of Ireland. The research is undertaken at NUTS 3 (Nomenclature of Territorial Units) regional level, the nutrient soil P requirement is established, and the extent to which the regional production of indigenous recycled P sources can offset this requirement is determined. The soil P requirement was derived from analyzing the regional soil P indexes, stocking rate and land-use. It was established that to optimise Irish agricultural production, approximately 95,500 t of P fertiliser is required by Irish agriculture per annum. Indigenous P sources were reviewed to determine their contribution to the Irish P balance; the sources included sewage sludge, dairy processing waste, and animal manures. Regional indigenous P quantities vary greatly with the South-West Region producing the largest quantity of indigenous recycled P at 42.4% of required P than the Mid-West Region only producing 22.0% of its P requirement indigenously. Sources of indigenous P also vary greatly from region to region depending on population and industry, with the highest quantity of sewage sludge being produced in the Dublin plus Mid-East Region while the greatest contributor of dairy waste is the South-West Region. In total, over 28,500 t of P is recovered from indigenous sources per annum. This indicates that approximately 30% of the national P requirement could be met by indigenous P recycling

Results from Ireland\u27s 2014 report card on physical activity in children and youth

Background: Physical activity (PA) levels are a key performance indicator for policy documents in Ireland. The first Ireland Report Card on Physical Activity in Children and Youth aims to set a robust baseline for future surveillance of indicators related to PA in children and youth. Methods: Data collected between 2003-2010 on more than 35,000 7- to 18-year-old children and youth were used and graded using a standardized grading system for 10 indicators. Results: Grades assigned for the indicators were as follows: overall physical activity levels, D-; sedentary behavior (TV viewing), C-; organized sport participation, C-: physical education, D-; active play, inconclusive (INC); active transportation, D; school, C-, community and the built environment, B; family, INC; and government, INC. Conclusions: PA recommendations exist in Ireland but this Report Card has shown that participation is still low. A number of promising policies, programs and services are in place but these require thorough evaluation and adequate resourcing. Agreement and implementation of a common framework for the systematic surveillance of indictors related to PA of children and youth is necessary to monitor change over time and ensure the impact of promising work is captured

Combining SNAPs with antibiotics shows enhanced synergistic efficacy against S. <i>aureus</i> and P. <i>aeruginosa</i> biofilms (vol 9, 36, 2023)

Correction to: npj Biofilms and Microbiomes, published online 08 June 2023 In this article the funding from MRC Doctoral Training Partnership [grant number MR/N014294/1] was omitted. The original article has been corrected

Results from Ireland north and south\u27s 2016 report card on physical activity for children and youth

Background: Physical activity (PA) is a key performance indicator for policy documents in both the Republic of Ireland and Northern Ireland. Building on baseline grades set in 2014, Ireland\u27s second Report Card on Physical Activity for Children and Youth allows for continued surveillance of indicators related to PA in children and youth. Methods: Data and information were extracted and collated for 10 indicators and graded using an international standardized grading system. Results: Overall, 7 grades stayed the same, 2 increased, and 1 decreased. Grades were assigned as follows: Overall PA, D (an increase); Sedentary Behavior (TV), C-; Physical Education, D-; Active Play, Incomplete/Inconclusive (INC); Active Transportation, D; School, D (a decrease); Home (Family), INC; Community and the Built Environment, B+ (an increase); and Government, INC. Unlike 2014\u27s report card, different grades for the Republic (C-) and Northern Ireland (C+) were assigned for Organized Sport Participation. Conclusions: Although the grade for Overall PA levels increased to a D, this may reflect the increased quality and quantity of data available. The double burden of low PA and high sedentary levels are concerning and underscore the need for advocacy toward, and surveillance of, progress in achieving targets set by the new National Physical Activity Plan in the Republic and obesity and sport plans in the North

Results from Ireland's 2014 Report Card on Physical Activity in Children and Youth

peer-reviewedBackground: Physical activity (PA) levels are a key performance indicator for policy documents in Ireland. The first Ireland Report Card on Physical Activity in Children and Youth aims to set a robust baseline for future surveillance of indicators related to PA in children and youth. Methods: Data collected between 2003-2010 on more than 35,000 7- to 18-year-old children and youth were used and graded using a standardized grading system for 10 indicators. Results: Grades assigned for the indicators were as follows: overall physical activity levels, D-; sedentary behavior (TV viewing), C-; organized sport participation, C-: physical education, D-; active play, inconclusive (INC); active transportation, D; school, C-, community and the built environment, B; family, INC; and government, INC. Conclusions: PA recommendations exist in Ireland but this Report Card has shown that participation is still low. A number of promising policies, programs and services are in place but these require thorough evaluation and adequate resourcing. Agreement and implementation of a common framework for the systematic surveillance of indictors related to PA of children and youth is necessary to monitor change over time and ensure the impact of promising work is captured.peer-reviewe