Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model

Gianmarco Mangiaterra,Francesca Biavasco,Emiliano Laudadio,Nicholas Cedraro,Davide Bizzaro,Cristina Minnelli,Salvatore Vaiasicca,Barbara Citterio,Giovanna Mobbili,Roberta Galeazzi

doi:10.3390/antibiotics9070399

Gianmarco Mangiaterra, Francesca Biavasco + Show 8 more

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https://doi.org/10.3390/antibiotics9070399

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Journal: Antibiotics	Publication Date: Jul 10, 2020
Citations: 9	License type: CC BY 4.0

Affiliation: Marche Polytechnic University, University of Urbino

Abstract

The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

Highlights

Pseudomonas aeruginosa causes chronic infections [1], which are the main cause of death in cystic fibrosis (CF) patients
The factors involved in viable but non-culturable (VBNC) cell induction in the lungs of CF patients are still to be elucidated, their pulmonary environment is characterized by the presence of multiple stress factors that include low-level oxygen, catabolic waste, nutrient depletion, suboptimal pH as well as antibiotics, since chronic infections are repeatedly treated with drugs such as tobramycin and ciprofloxacin
Persisters were initially described as bacterial forms which are produced stochastically and whose low metabolic activity is responsible for the failure of antibiotic treatment [17]; this is critical in immunocompromised subjects such as CF patients [18]

Summary

Introduction

Pseudomonas aeruginosa causes chronic infections [1], which are the main cause of death in cystic fibrosis (CF) patients. The factors involved in VBNC cell induction in the lungs of CF patients are still to be elucidated, their pulmonary environment is characterized by the presence of multiple stress factors that include low-level oxygen, catabolic waste, nutrient depletion, suboptimal pH as well as antibiotics, since chronic infections are repeatedly treated with drugs such as tobramycin and ciprofloxacin. The antibiotic may be found in subinhibitory concentrations in the biofilm matrix due either to its poor penetration or to its decrease between two different therapeutic cycles [9]. This is a cause of concern, in strong biofilm-producing bacteria like P. aeruginosa. Total live cells were detected by the developed qPCR protocol proven to reliably detect all viable (i.e., culturable and non-culturable) P. aeruginosa cells [8]

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