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

Freya Harrison,Esther Sweeney,Niamh E Harrington,Marwa M Hassan

doi:10.3389/fmicb.2020.568510

Freya Harrison, Esther Sweeney + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2020.568510

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Abstract

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 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.ConclusionIn 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.

Highlights

Cystic fibrosis (CF) is a genetic disease in which people have decreased mucociliary clearance in the respiratory tract, due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel (Davies, 2002; Lyczak et al, 2002). This impairment leads to a reduction in mucus clearance and increased viscosity, resulting in accumulation of microbial cells, increased bacterial adherence and inflammation and the formation of bacterial biofilm (Davies, 2002; Lyczak et al, 2002; Hoiby et al, 2010)
Biofilm infections are more difficult to eradicate due to the difference in their nature compared to non-biofilm infections; they are lifelong infections in CF
The results demonstrated an increased antibiotic tolerance in the ex vivo pig lung biofilm model (EVPL) model at concentrations >25-fold the reported sputum concentrations when tested in Mueller-Hinton broth, which even further increased when tested in artificial sputum media

Summary

Introduction

Cystic fibrosis (CF) is a genetic disease in which people have decreased mucociliary clearance in the respiratory tract, due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel (Davies, 2002; Lyczak et al, 2002). Adaptive mechanisms controlling differential gene expression of multiple virulence factors, such as efflux pumps and antibiotic-degrading enzymes, lead to antibiotic tolerance (Breidenstein et al, 2011; Taylor et al, 2014). The latter is highly dependent and varies based on the environment surrounding the biofilm (Breidenstein et al, 2011). Inaccurate diagnostics affect treatment choice, preventing bacterial clearance and potentially selecting for antibiotic resistance.

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