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      Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents

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          Abstract

          Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.

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          Most cited references32

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          Bacterial persistence as a phenotypic switch.

          A fraction of a genetically homogeneous microbial population may survive exposure to stress such as antibiotic treatment. Unlike resistant mutants, cells regrown from such persistent bacteria remain sensitive to the antibiotic. We investigated the persistence of single cells of Escherichia coli with the use of microfluidic devices. Persistence was linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates. Quantitative measurements led to a simple mathematical description of the persistence switch. Inherent heterogeneity of bacterial populations may be important in adaptation to fluctuating environments and in the persistence of bacterial infections.
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            Riddle of biofilm resistance.

            K. Lewis (2001)
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              Persister cells and tolerance to antimicrobials.

              Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                04 October 2017
                2017
                : 8
                : 1917
                Affiliations
                [1] 1Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa , Pisa, Italy
                [2] 2Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health , Berlin, Germany
                [3] 3Berlin-Brandenburger Centrum für Regenerative Therapien, Charité – Universitätsmedizin Berlin , Berlin, Germany
                [4] 4Department of Biomedical Sciences, University of Cagliari , Monserrato, Italy
                Author notes

                Edited by: Shouguang Jin, University of Florida, United States

                Reviewed by: Rodolfo García-Contreras, National Autonomous University of Mexico, Mexico; Osmar Nascimento Silva, Universidade Católica Dom Bosco, Brazil; César de la Fuente, Massachusetts Institute of Technology, United States

                *Correspondence: Giovanna Batoni, giovanna.batoni@ 123456med.unipi.it

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.01917
                5632672
                29046671
                a17dd5ae-4bc9-4df9-b0de-127508ea23a1
                Copyright © 2017 Grassi, Di Luca, Maisetta, Rinaldi, Esin, Trampuz and Batoni.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 21 July 2017
                : 20 September 2017
                Page count
                Figures: 6, Tables: 3, Equations: 0, References: 45, Pages: 12, Words: 0
                Funding
                Funded by: Università di Pisa 10.13039/501100007514
                Award ID: PRA_2017_18
                Funded by: European Society of Clinical Microbiology and Infectious Diseases 10.13039/501100001704
                Award ID: Observership Grant for LG
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                persisters,induction of persistence,staphylococcus aureus,pseudomonas aeruginosa,isothermal microcalorimetry,antimicrobial peptides

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