Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model.

Gianmarco Mangiaterra,Francesca Biavasco,Barbara Citterio,Salvatore Vaiasicca,Elisa Carotti,Nicholas Cedraro,Anna La Teana

doi:10.3390/ijms22041628

Gianmarco Mangiaterra, Francesca Biavasco + Show 5 more

Open Access

https://doi.org/10.3390/ijms22041628

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Abstract

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

Highlights

Pseudomonas aeruginosa lung infections represent the main cause of morbidity and mortality for Cystic Fibrosis (CF) patients, who suffer from recurrent pulmonary exacerbations and decreased lung function [1]
To further demonstrate the reliability of our results, we have developed in vitro biofilms of a P. aeruginosa strain producing the Green Fluorescent Protein (GFP), and exposed them to stress conditions, mimicking those found in the CF lung environment between antibiotic treatments, i.e., starvation and low drug concentrations
As the gfp(ASV) gene was cloned under the influence of the araBAD promoter, the ability of the recombinant strain to produce the GFP protein was assessed by fluorescence microscopy and flow cytometry, after the exposure for 3 h to 0.2% arabinose

Summary

Introduction

Pseudomonas aeruginosa lung infections represent the main cause of morbidity and mortality for Cystic Fibrosis (CF) patients, who suffer from recurrent pulmonary exacerbations and decreased lung function [1]. Patients are often subjected to heavy and cyclic antibiotic treatments [2], which fail to completely eradicate the pathogen though, since, after a time period of apparent infection clearance, the symptoms recrudescence is observed and the same P. aeruginosa strain is isolated again in sputum bacterial culture [3]. Such recurrence is attributed to the occurrence of persistent bacterial forms, which have been defined as cells able to tolerate high bactericidal antibiotic concentrations, without the involvement of a heritable genetic factor [4]. An interesting hypothesis is the involvement of low drug concentrations in the development and maintenance of these specific phenotypes [10,11], according to the hormesis theory, which describes the dual, concentration-dependent effect of antimicrobial compounds, i.e., bactericidal at high doses and gene expression regulators at subinhibitory concentrations [12]

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