TY - JOUR
T1 - Unlocking the Gates
T2 - A Novel Diagnostic Molecule for Quantifying Efflux Levels in Gram-Positive Bacteria
AU - Patil, Mrunal
AU - Munteanu, Tatiana
AU - Brasseur, Gaël
AU - Ferreira, Carolina
AU - Costa, Sofia Santos
AU - Couto, Isabel
AU - Athar, Mohd
AU - Asunis, Elisa
AU - Vargiu, Attilio Vittorio
AU - Viveiros, Miguel
AU - DiGiorgio, Carole
AU - Brunel, Frédéric
AU - Raimundo, Jean-Manuel
AU - Camplo, Michel
AU - Siri, Olivier
AU - Bolla, Jean Michel
N1 - Funding Information:
All authors contributed equally to this work. This project was partially funded by the A*Midex Foundation Interdisciplinarity Grant \u201CElisa.\u201D M.P. was supported by the CNRS\u2010Innovation \u201CPhenux\u201D Grant. M.A. and A.V.V. gratefully acknowledge the \u201COne Health Basic and Translational Research Actions addressing Unmet Needs on Emerging Infectious Diseases (INF\u2010ACT)\u201D, PNRR, mission 4, component 2, investment 1.3, project number foundation by the Italian Ministry of University and Research PE00000007 (University of Cagliari). M.A. and A.V.V. received financial support from the NIAID/NIH grant no. R01AI136799. Additional support came from Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia (FCT, Portugal) through funds to GHTM \u2013 UID/04413/2020, LA\u2010REAL \u2013 LA/P/0117/2020, to project BIOSAFE (c, PTDC/CAL\u2010EST/30713/2017), and to the Project DREBI, Ref. 2022.07931.PTDC ( http://doi.org/10.54499/2022.07931.PTDC ). CF was supported by grant UI/BD/154472/2022 from FCT, Portugal. SSC was supported by FCT through CEECINST/00042/2021/CP1773/CT0009 ( https://doi.org/10.54499/CEECINST/00042/2021/CP1773/CT0009 ). The authors are grateful to Michael J. Rybak, Wayne State University, USA, and Dr. Lorena Tuchscherr, Jena University Hospital, Germany, for providing strains of mepA and norA mutants, respectively. Special thanks to Jean\u2010Michel Jault and C\u00E9dric Orelle from Universit\u00E9 Lyon, France, for supplying PatA/PatB and BmrA efflux mutants. The authors also appreciate the encouragement of SATT\u2010SUD and Idylle Labs, Paris, for their assistance in transforming this concept. The authors thank Dr. V\u00E9ronique Sinou (MCT, AMU, Marseille) for providing ESKAPE reference bacteria and SAK1758 bacteria. The authors thank Paolo Ruggerone (University of Cagliari) for valuable discussions. Most importantly, the authors acknowledge the tireless work of the IHTM team in Lisbon. The authors also extend our gratitude to Serhii Krykun (L'Institut national des sciences appliqu\u00E9es de Rouen) and Simon Pascal (University of Nantes) for their technical assistance. S. aureus
Publisher Copyright:
© 2025 The Author(s). Advanced Healthcare Materials published by Wiley-VCH GmbH.
PY - 2025/3/11
Y1 - 2025/3/11
N2 - Efflux-mediated antibiotic resistance poses a significant global threat, affecting diverse bacterial species. Clinicians recognize the danger of efflux mechanisms during antibiotic treatment, yet precise diagnostic tools remain unavailable. The antibiogram currently infers abnormal efflux pump activity in clinical isolates, which is subsequently confirmed through transcriptomic or genomic analysis. This study harnesses the colorimetric, fluorescence, and solubility properties of phenazinium derivatives to develop a rapid protocol for detecting bacterial efflux. Among several synthesized phenazinium compounds, the compound demonstrating differential MIC in Staphylococcus efflux mutants and exhibiting appropriate physicochemical properties is selected. A diagnostic protocol is developed using the selected compound to assess efflux levels, categorized as no, weak, or strong, through colorimetry and spectroscopy techniques. Testing on Gram-positive efflux mutants and clinical Staphylococcus isolates further validates the approach. In-silico docking analysis confirms the interaction between the chosen compound and the NorA efflux pump in S. aureus. Flow cytometry is employed to re-evaluate the detection assays. The developed molecule and protocol represent the first known method to evaluate efflux levels in any Gram-positive species through a streamlined and user-friendly process. This pioneering test significantly advances the epidemiological analysis of efflux mechanisms and enables more informed therapeutic decision-making, leading to more effective treatment.
AB - Efflux-mediated antibiotic resistance poses a significant global threat, affecting diverse bacterial species. Clinicians recognize the danger of efflux mechanisms during antibiotic treatment, yet precise diagnostic tools remain unavailable. The antibiogram currently infers abnormal efflux pump activity in clinical isolates, which is subsequently confirmed through transcriptomic or genomic analysis. This study harnesses the colorimetric, fluorescence, and solubility properties of phenazinium derivatives to develop a rapid protocol for detecting bacterial efflux. Among several synthesized phenazinium compounds, the compound demonstrating differential MIC in Staphylococcus efflux mutants and exhibiting appropriate physicochemical properties is selected. A diagnostic protocol is developed using the selected compound to assess efflux levels, categorized as no, weak, or strong, through colorimetry and spectroscopy techniques. Testing on Gram-positive efflux mutants and clinical Staphylococcus isolates further validates the approach. In-silico docking analysis confirms the interaction between the chosen compound and the NorA efflux pump in S. aureus. Flow cytometry is employed to re-evaluate the detection assays. The developed molecule and protocol represent the first known method to evaluate efflux levels in any Gram-positive species through a streamlined and user-friendly process. This pioneering test significantly advances the epidemiological analysis of efflux mechanisms and enables more informed therapeutic decision-making, leading to more effective treatment.
KW - chemical synthesis
KW - clinical strains
KW - diagnostic
KW - efflux pump
KW - fluorescence
UR - http://www.scopus.com/inward/record.url?scp=105000425120&partnerID=8YFLogxK
U2 - 10.1002/adhm.202404145
DO - 10.1002/adhm.202404145
M3 - Article
C2 - 40066601
AN - SCOPUS:105000425120
SN - 2192-2640
JO - Advanced Healthcare Materials
JF - Advanced Healthcare Materials
M1 - 2404145
ER -