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

Harrington, Niamh E.; Sweeney, Esther; Harrison, Freya

doi:10.1016/j.bioflm.2020.100024

Cited by 44 publications

(56 citation statements)

References 61 publications

(64 reference statements)

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“…Figure 1 represents a schematic diagram of the work flow as previously described. Supplementary Figures S3 , S4 show pieces of tissues infected with P. aeruginosa strains after 7 days of biofilm formation and the mucoid phenotype of the strains in the tissues, respectively, to represent chronic CF infection ( Harrington et al, 2020 ). Our previous work with the EVPL model confirms that P. aeruginosa forms structured biofilms in this model [Alcian blue staining confirms presence of exopolysaccharide matrix, and mutant studies showed formation of structured aggregates on tissue required the gacS/gacA pathway and pel polysaccharide ( Harrington et al, 2020 )].…”

Section: Resultsmentioning

confidence: 99%

Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model

et al. 2020

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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.

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Section: Resultsmentioning

confidence: 99%

Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model

et al. 2020

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“…50% of the original BODIPY-colistin dose being recovered, consistent with colistin binding plastic (data not shown). model, (24 )), this suggests a sensible maximum biofilm volume to use is 8.8 µl. Figure 3b shows the predicted BODIPY-colistin concentrations in the biofilms assuming this volume.…”

Section: Pa14 Sed6 Sed8mentioning

confidence: 97%

“…aeruginosa biofilms depending on strain and culture conditions. We aimed first to compare the tolerance of P. aeruginosa to colistin using planktonic microdilution in standard antibiotic susceptibility testing medium (cation-adjusted Muller-Hinton broth, caMHB) and synthetic CF sputum medium (SCFM, (23)); biofilm eradication assays using the same media in a Calgary device; and in an ex vivo model of CF biofilm which combines SCFM and pig bronchiolar tissue (24,25). We then aimed to use fluorescently-labelled colistin to measure the percentage of a dose which was able to enter the biofilm matrix in the ex vivo CF model.…”

Section: Full Textmentioning

confidence: 99%

“…We selected CF isolates SED6, SED8, SED17 and SED19 for further work with the ex vivo pig lung model, as these had a range of MIC/MBEC values and included two isolates for which MBEC=MIC in SCFM (SED6, SED8) and two for which MBEC>MIC in SCFM (SED17, SED19). The ex vivo pig lung model (EVPL) was prepared as previously described (24,25) aeruginosa has been shown to produce CF-like biofilm in this model (24).…”

Section: Standards For Antimicrobial Susceptibility Testing) Mbec Tementioning

confidence: 99%

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Effect of host-mimicking medium and biofilm growth on the ability of colistin to killPseudomonas aeruginosa

Sweeney

Sabnis

Edwards

et al. 2020

Preprint

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In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in cystic fibrosis-mimicking medium versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and R&D pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

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“…In an in vivo murine chronic wound model, oxidative stress and community composition alone do not characterize the microbial community metabolism (Dhall et al, 2014 ). Similarly, tissue models, including an ex vivo porcine lung model, investigate only the growth of a single pathogen at a time (Harrison et al, 2014 ; Dumigan et al, 2019 ; Harrington et al, 2020 ; Sweeney et al, 2019 ). To better understand chronic infections, it is important to consider the entire microbial community.…”

Section: Introductionmentioning

confidence: 99%

Thriving Under Stress: Pseudomonas aeruginosa Outcompetes the Background Polymicrobial Community Under Treatment Conditions in a Novel Chronic Wound Model

Phan

Ranjbar

Kagawa

et al. 2020

Front. Cell. Infect. Microbiol.

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In vitro infection models are important for studying the effects of antimicrobials on microbial growth and metabolism. However, many models lack important biological components that resemble the polymicrobial nature of chronic wounds or infections. In this study, we developed a perfused meat model that supports the growth of the human pathogen Pseudomonas aeruginosa in a native meat microbial background to investigate the impact of antibiotics and hydrogen peroxide on polymicrobial community growth and metabolism. P. aeruginosa plays an important role as an etiological agent involved in chronic infections and is a common opportunistic pathogen. Chemical stressors in the form of hydrogen peroxide, carbenicillin, and gentamicin were perfused through the meat with polymicrobial growth on the surface. The relative abundances of P. aeruginosa and the background microbial community were analyzed by cell viability assays, and metabolic changes of the entire community in response to different antimicrobial treatments were characterized by GC-MS analysis of volatile organic compounds. The meat background community was characterized by amplicon sequencing. Relative densities of P. aeruginosa and background microbiota were similar under control conditions. Antimicrobial stressors, even at sub-inhibitory, physiologically relevant concentrations, spurred P. aeruginosa dominance of the meat surface community. Volatile metabolite ion intensity levels showed that antibacterial treatments drive changes in microbial metabolism. The abundance of the P. aeruginosa- derived metabolite, acetophenone, remained stable with treatment, whereas the relative abundances of 2-butanone, 2-nonanone, and 2-aminoacetophenone changed in response to treatment, suggesting these could serve as biomarkers of infection. Our model recapitulates some of the physiological conditions of chronic wounds and facilitates high throughput experiments without the high cost of in vivo models. Expanded use of this perfusion model will contribute to the understanding of polymicrobial growth and metabolism in the context of chronic wounds and infections.

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

Cited by 44 publications

References 61 publications

Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model

Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model

Effect of host-mimicking medium and biofilm growth on the ability of colistin to killPseudomonas aeruginosa

Thriving Under Stress: Pseudomonas aeruginosa Outcompetes the Background Polymicrobial Community Under Treatment Conditions in a Novel Chronic Wound Model

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