A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria
Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1023326/full |
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| author | Niclas Nordholt Kate O'Hara Kate O'Hara Ute Resch-Genger Mark A. T. Blaskovich Bastian Rühle Frank Schreiber |
| author_facet | Niclas Nordholt Kate O'Hara Kate O'Hara Ute Resch-Genger Mark A. T. Blaskovich Bastian Rühle Frank Schreiber |
| author_sort | Niclas Nordholt |
| collection | DOAJ |
| description | Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action. |
| first_indexed | 2024-04-13T08:13:44Z |
| format | Article |
| id | doaj.art-9531fa2f38d248fc86ada89904889a83 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T08:13:44Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9531fa2f38d248fc86ada89904889a832022-12-22T02:54:52ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-11-011310.3389/fmicb.2022.10233261023326A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteriaNiclas Nordholt0Kate O'Hara1Kate O'Hara2Ute Resch-Genger3Mark A. T. Blaskovich4Bastian Rühle5Frank Schreiber6Division of Biodeterioration and Reference Organisms (4.1), Department of Materials and the Environment, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyDivision of Biodeterioration and Reference Organisms (4.1), Department of Materials and the Environment, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyDivision of Biophotonics (1.2), Department of Analytical Chemistry, Reference Materials, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyDivision of Biophotonics (1.2), Department of Analytical Chemistry, Reference Materials, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyCentre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaDivision of Biophotonics (1.2), Department of Analytical Chemistry, Reference Materials, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyDivision of Biodeterioration and Reference Organisms (4.1), Department of Materials and the Environment, Federal Institute for Materials Research and Testing (BAM), Berlin, GermanyQuaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1023326/fullquaternary ammonium compoundQACfluorescenceantimicrobial resistanceeffluxTolC |
| spellingShingle | Niclas Nordholt Kate O'Hara Kate O'Hara Ute Resch-Genger Mark A. T. Blaskovich Bastian Rühle Frank Schreiber A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria Frontiers in Microbiology quaternary ammonium compound QAC fluorescence antimicrobial resistance efflux TolC |
| title | A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria |
| title_full | A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria |
| title_fullStr | A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria |
| title_full_unstemmed | A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria |
| title_short | A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria |
| title_sort | fluorescently labelled quaternary ammonium compound nbd dda to study resistance mechanisms in bacteria |
| topic | quaternary ammonium compound QAC fluorescence antimicrobial resistance efflux TolC |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1023326/full |
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