Biology subjects

Harrington, N. E.

Publications and source records attributed to Harrington, N. E..

9 recordsLinked to original sources

bioRxiv2026

Multiple ecological and evolutionary mechanisms drive treatment-induced antibiotic resistance

The emergence of resistance within patients during antibiotic treatment is an important cause of treatment failure. However, the ecological and evolutionary mechanisms driving within-patient emergence remain poorly understood. Here, we analysed 24,478 Pseudomonas aeruginosa isolates sampled from 180 bronchiectasis patients during a clinical trial to understand how ciprofloxacin-resistant infections emerged over a one-year period of pulse-dosing. Pre-existing resistance predominated, accelerating resistance emergence relative to patients where resistance emerged by spontaneous mutation or strain immigration. Selective sweeps of costly mutations increased resistance over time in some patients, whereas in others oscillating resistance levels were driven by antibiotic treatment and resistance-growth trade-offs between genetically divergent subpopulations. Our findings show that infections under identical treatment follow diverse and sometimes complex ecological and evolutionary paths to antibiotic resistance, with implications for better predicting and managing treatment-induced antibiotic resistance.

evolutionary biology↗

bioRxiv2026

Biofilm-deficient mutants of Pseudomonas aeruginosa have wild-type levels of antibiotic tolerance in a model of cystic fibrosis lung infection

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. This result was unexpected, given previous work in our lab that suggested physical protection of cells from colistin by the biofilm matrix was important for tolerance. The biofilm defects in the mutants were confirmed using electron and light microscopy, and cryo scanning electron microscopy was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. Hence, mature biofilm structure is not essential for tolerance, as other mechanisms may be sufficient in this environment. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

microbiology↗

bioRxiv2026

Uncovering the multifaceted mechanism of action of a historical antimicrobial

Natural products have provided most of our modern pharmacopoeia, serving as active molecules or scaffolds for active molecules. Their use in drug development is often inspired by their traditional or historical medical use. For many decades, this discovery pipeline has focused on identifying a single molecule responsible for much of the biological activity of a raw natural product preparation (e.g. a whole-plant extract) and scoping this molecule for clinical potential. However, it is increasingly realised that historical/traditional remedies with significant biological activity may owe this activity to the combined action of multiple molecules. Concomitantly, microbiologists increasingly argue that effectively fighting antimicrobial-resistant infections will rely on combination therapies that combine multiple antimicrobials and/or adjuvant molecules. We previously reconstructed a complex historical remedy, Bald's eyesalve. Our reconstruction of this remedy had strong antibiofilm activity, which relied on the presence of multiple ingredients. Here, we report that Bald's eyesalve has multiple antibacterial effects on exemplar Gram-positive (Staphylococcus aureus) and Gram-negative (Acinetobacter baumannii) pathogens. Bald's eyesalve disrupts bacterial membrane integrity; inhibits expression of genes associated with bacterial adhesins, virulence factors and efflux pumps in both S. aureus and A. baumannii; inhibits quorum sensing in S. aureus; and causes downregulation of genes involved in de novo nucleotide biosynthesis in S. aureus. Lastly, we show that this multifaceted mechanism of action makes it difficult for S. aureus, A. baumannii, and Pseudomonas aeruginosa to evolve resistance against Bald's eyesalve. Bald's eyesalve could be used to identify a defined cocktail of natural products suitable for preclinical testing as a multi-target antibacterial preparation to which resistance may arise more slowly than current single-molecule antibiotics.

microbiology↗

bioRxiv2024

Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis

BackgroundPseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments. MethodsWe have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections. ResultsWe observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms. ConclusionsOur findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes. SummaryThe largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection.

genomics↗

bioRxiv2022

Pseudomonas aeruginosa transcriptome analysis in a cystic fibrosis lung model reveals metabolic changes accompanying biofilm maturation and increased antibiotic tolerance over time.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilm infections in the lungs of people with the genetic condition cystic fibrosis (CF) that can persist for decades. There are numerous P. aeruginosa lifestyle changes associated with chronic biofilm infection cued by the CF lung environment. These include a loss of virulence, metabolic changes and increased antimicrobial tolerance. We have investigated P. aeruginosa PA14 biofilm infection over 7 d in an ex vivo pig lung (EVPL) model for CF, previously shown to facilitate formation of a clinically relevant P. aeruginosa biofilm structure with expression of key genes comparable to human infection. We have compared P. aeruginosa gene expression between sequential time points: 24 h, 48 h and 7 d post infection, and investigated tolerance to polymyxins. Our results demonstrate that the EVPL model can maintain a P. aeruginosa biofilm population, which exhibits increased antibiotic tolerance, for at least 7 d. Differential expression of antimicrobial resistance-associated genes was not observed, however there was significant upregulation of sulfur metabolism and maintenance of a structured biofilm. Our findings provide further insight into the increased P. aeruginosa antibiotic tolerance during chronic infection of the CF lung, and suggest we can cue aspects of chronic infection in 7 d under the right lab conditions.

microbiology↗

bioRxiv2021

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 chronic 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 of which 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 found to be 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 and provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance. Significance StatementPseudomonas aeruginosa lung infections that affect people with cystic fibrosis are a well-researched area, yet they continue to evade antimicrobial treatments. The lack of a laboratory model that captures all key aspects of these infections hinders not only research progression but also clinical diagnostics. We used transcriptome analysis to demonstrate how a model using pig lungs can more accurately replicate key characteristics of P. aeruginosa lung infection, including mechanisms of antibiotic resistance and infection establishment. Therefore, this model may be used in the future to further understand infection dynamics to develop novel treatments and more accurate treatment plans. This could improve clinical outcomes as well as quality of life for individuals affected by these infections.

microbiology↗

bioRxiv2020

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

BackgroundBacterial 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. MethodsIn 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. ResultsThe results demonstrated minimal effect on the bacterial load with therapeutically relevant concentrations of ciprofloxacin and meropenem, with the later 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. ConclusionsWe demonstrate a realistic model for understanding antibiotic resistance and tolerance in biofilms clinically and for molecules screening in anti-biofilm drug development. P. aeruginosa showed high levels of antibiotic tolerance, with minimal effect on bacterial load and increased proteases production, which could negatively affect lung function. This may potentially contribute to exacerbations and eventual lung failure.

microbiology↗

bioRxiv2019

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 and CF lung infection.

microbiology↗

bioRxiv2019

An ex vivo cystic 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.

microbiology↗