{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T17:38:16Z","timestamp":1790876296166,"version":"4.1.0"},"reference-count":23,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T00:00:00Z","timestamp":1689638400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Cystic Fibrosis Research Foundation","award":["FFC#13\/2017"],"award-info":[{"award-number":["FFC#13\/2017"]}]},{"name":"Italian Cystic Fibrosis Research Foundation","award":["FFC#16\/2019"],"award-info":[{"award-number":["FFC#16\/2019"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with\/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin\u2013antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.<\/jats:p>","DOI":"10.3390\/ijms241411618","type":"journal-article","created":{"date-parts":[[2023,7,19]],"date-time":"2023-07-19T00:47:38Z","timestamp":1689727658000},"page":"11618","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5035-5879","authenticated-orcid":false,"given":"Gianmarco","family":"Mangiaterra","sequence":"first","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via S. Chiara 27, 61029 Urbino, Italy"},{"name":"Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Cedraro","sequence":"additional","affiliation":[{"name":"Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0160-1022","authenticated-orcid":false,"given":"Salvatore","family":"Vaiasicca","sequence":"additional","affiliation":[{"name":"Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Via Tronto 10\/a, 60020 Ancona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3346-6379","authenticated-orcid":false,"given":"Barbara","family":"Citterio","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via S. Chiara 27, 61029 Urbino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5619-7912","authenticated-orcid":false,"given":"Emanuela","family":"Frangipani","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via S. Chiara 27, 61029 Urbino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Biavasco","sequence":"additional","affiliation":[{"name":"Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0993-5967","authenticated-orcid":false,"given":"Carla","family":"Vignaroli","sequence":"additional","affiliation":[{"name":"Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e00138-18","DOI":"10.1128\/CMR.00138-18","article-title":"Cystic Fibrosis and Pseudomonas aeruginosa: The Host-Microbe Interface","volume":"32","author":"Malhotra","year":"2019","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1038\/s41579-019-0196-3","article-title":"Definitions and guidelines for research on antibiotic persistence","volume":"17","author":"Balaban","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e00249-18","DOI":"10.1128\/JB.00249-18","article-title":"Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells","volume":"200","author":"Ayrapetyan","year":"2018","journal-title":"J. Bacteriol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1111\/1751-7915.13039","article-title":"Molecular viability testing of viable but non-culturable bacteria induced by antibiotic exposure","volume":"11","author":"Lee","year":"2018","journal-title":"Microb. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Deschaght, P., Schelstraete, P., Van Simaey, L., Vanderkercken, M., Raman, A., Mahieu, L., Van Daele, S., De Baets, F., and Vaneechoutte, M. (2013). Is the improvement of CF patients, hospitalized for pulmonary exacerbation, correlated to a decrease in bacterial load?. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0079010"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mangiaterra, G., Cedraro, N., Vaiasicca, S., Citterio, B., Galeazzi, R., Laudadio, E., Mobbili, G., Minnelli, C., Bizzaro, D., and Biavasco, F. (2020). Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model. Antibiotics, 9.","DOI":"10.3390\/antibiotics9070399"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1007\/5584_2020_570","article-title":"Diffusion and Characterization of Pseudomonas aeruginosa Aminoglycoside Resistance in an Italian Regional Cystic Fibrosis Centre","volume":"1323","author":"Mangiaterra","year":"2021","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Mangiaterra, G., Amiri, M., Di Cesare, A., Pasquaroli, S., Manso, E., Cirilli, N., Citterio, B., Vignaroli, C., and Biavasco, F. (2018). Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: A validation study. BMC Infect. Dis., 18.","DOI":"10.1186\/s12879-018-3612-9"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e00019-18","DOI":"10.1128\/CMR.00019-18","article-title":"Epidemiology, Biology, and Impact of Clonal Pseudomonas aeruginosa Infections in Cystic Fibrosis","volume":"31","author":"Parkins","year":"2018","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1038\/nrmicro3270","article-title":"Microbiological effects of sublethal levels of antibiotics","volume":"12","author":"Andersson","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"258","DOI":"10.3389\/fmicb.2014.00258","article-title":"The importance of the viable but non-culturable state in human bacterial pathogens","volume":"5","author":"Li","year":"2014","journal-title":"Front. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"626715","DOI":"10.3389\/fmicb.2021.626715","article-title":"Gene Expression Profiling of Pseudomonas aeruginosa Upon Exposure to Colistin and Tobramycin","volume":"12","author":"Amman","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1021\/acs.jnatprod.0c01151","article-title":"The Natural Alkaloid Berberine Can Reduce the Number of Pseudomonas aeruginosa Tolerant Cells","volume":"84","author":"Mangiaterra","year":"2021","journal-title":"J. Nat. Prod."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1111\/j.1365-2958.2008.06152.x","article-title":"Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes","volume":"68","author":"Pamp","year":"2008","journal-title":"Mol. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e01846-20","DOI":"10.1128\/AAC.01846-20","article-title":"Pseudomonas aeruginosa Polynucleotide Phosphorylase Controls Tolerance to Aminoglycoside Antibiotics by Regulating the MexXY Multidrug Efflux Pump","volume":"65","author":"Fan","year":"2021","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Dufour, D., Zhao, H., Gong, S.G., and L\u00e9vesque, C.M. (2022). A DNA-Damage Inducible Gene Promotes the Formation of Antibiotic Persisters in Response to the Quorum Sensing Signaling Peptide in Streptococcus mutans. Genes, 13.","DOI":"10.3390\/genes13081434"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5216","DOI":"10.1038\/s41467-019-13021-8","article-title":"Quorum sensing modulates the formation of virulent Legionella persisters within infected cells","volume":"10","author":"Personnic","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_18","unstructured":"Clinical and Laboratory Standards Institute (2017). Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Seventh Informational Supplement M100-S27, CLSI."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1404","DOI":"10.1128\/AAC.44.5.1404-1406.2000","article-title":"Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus","volume":"44","author":"Kaatz","year":"2000","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1093\/jac\/dkv496","article-title":"New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection","volume":"71","author":"Revest","year":"2016","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Aparna, V., Dineshkumar, K., Mohanalakshmi, N., Velmurugan, D., and Hopper, W. (2014). Identification of natural compound inhibitorsbfor multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0101840"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1089\/107662903322762743","article-title":"Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains","volume":"9","author":"Oh","year":"2003","journal-title":"Microb. Drug Resist."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Coelho, P., Oliveira, J., Fernandes, I., Ara\u00fajo, P., Pereira, A.R., Gameiro, P., and Bessa, L.J. (2021). Pyranoanthocyanins Interfering with the Quorum Sensing of Pseudomonas aeruginosa and Staphylococcus aureus. Int. J. Mol. Sci., 2021 22.","DOI":"10.3390\/ijms22168559"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/14\/11618\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:14:21Z","timestamp":1760127261000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/14\/11618"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,7,18]]},"references-count":23,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["ijms241411618"],"URL":"https:\/\/doi.org\/10.3390\/ijms241411618","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,7,18]]}}}