Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Systematic analyses identify modes of action of ten clinically relevant biocides and antibiotic antagonism in Acinetobacter baumannii

Nature Microbiology volume 8, pages 1995–2005 (2023)Cite this article

Subjects

Abstract

Concerns exist that widespread use of antiseptic or disinfectant biocides could contribute to the emergence and spread of multidrug-resistant bacteria. To investigate this, we performed transposon-directed insertion-site sequencing (TraDIS) on the multidrug-resistant pathogen, Acinetobacter baumannii, exposed to a panel of ten structurally diverse and clinically relevant biocides. Multiple gene targets encoding cell envelope or cytoplasmic proteins involved in processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, were identified to have effects on bacterial fitness upon biocide exposure, suggesting that these compounds may have intracellular targets in addition to their known effects on the cell envelope. As cell respiration genes are required for A. baumannii fitness in biocides, we confirmed that sub-inhibitory concentrations of the biocides that dissipate membrane potential can promote A. baumannii tolerance to antibiotics that act intracellularly. Our results support the concern that residual biocides might promote antibiotic resistance in pathogenic bacteria.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Biocide TraDIS data.
Fig. 2: Genes with TraDIS insertion changes during biocide exposure.
Fig. 3: Effect of biocides on membrane potential.
Fig. 4: Biocides’ impact on acriflavine intracellular accumulation and cell membrane permeability.
Fig. 5: Biocides and antibiotics interactions in A. baumannii.

Similar content being viewed by others

Data availability

TraDIS sequencing data were deposited in the European Nucleotide Database under project number PRJEB8707. Source data are provided with this paper.

References

  1. O’Neil, J. Tackling a Crisis for the Health and Wealth of Nations (World Health Organization, 2014).

  2. Darby, E. M. et al. Molecular mechanisms of antibiotic resistance revisited. Nat. Rev. Microbiol. 21, 280–295 (2023).

    Article  CAS  PubMed  Google Scholar 

  3. Peleg, A. Y., Seifert, H. & Paterson, D. L. Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev. 21, 538–582 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  4. Wong, D. et al. Clinical and pathophysiological overview of Acinetobacter infections: a century of challenges. Clin. Microbiol. Rev. 30, 409–447 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. McDonnell, G. & Russell, A. D. Antiseptics and disinfectants: activity, action, and resistance. Clin. Microbiol. Rev. 12, 147–179 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Webber, M. A. et al. Parallel evolutionary pathways to antibiotic resistance selected by biocide exposure. J. Antimicrob. Chemother. 70, 2241–2248 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fernández-Cuenca, F. et al. Reduced susceptibility to biocides in Acinetobacter baumannii: association with resistance to antimicrobials, epidemiological behaviour, biological cost and effect on the expression of genes encoding porins and efflux. J. Antimicrob. Chemother. 70, 3222–3229 (2015).

    Article  PubMed  Google Scholar 

  8. Alekshun, M. N. & Levy, S. B. The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol. 7, 410–413 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Rajamohan, G., Srinivasan, V. B. & Gebreyes, W. A. Novel role of Acinetobacter baumannii RND efflux transporters in mediating decreased susceptibility to biocides. J. Antimicrob. Chemother. 65, 228–232 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Liau, S. Y., Read, D. C., Pugh, W. J., Furr, J. R. & Russell, A. D. Interaction of silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions. Lett. Appl. Microbiol. 25, 279–283 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Morones-Ramirez, J. R., Winkler, J. A., Spina, C. S. & Collins, J. J. Silver enhances antibiotic activity against Gram-negative bacteria. Sci. Transl. Med. 5, 190ra181 (2013).

    Article  Google Scholar 

  12. Dibrov, P., Dzioba, J., Gosink, K. K. & Häse, C. C. Chemiosmotic mechanism of antimicrobial activity of Ag(+) in Vibrio cholerae. Antimicrob. Agents Chemother. 46, 2668–2670 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gupta, A., Matsui, K., Lo, J. F. & Silver, S. Molecular basis for resistance to silver cations in Salmonella. Nat. Med. 5, 183–188 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Franke, S., Grass, G. & Nies, D. H. The product of the ybdE gene of the Escherichia chromosome is involved in detoxification of silver ions. Microbiology 147, 965–972 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. McMurry, L. M., Oethinger, M. & Levy, S. B. Triclosan targets lipid synthesis. Nature 394, 531–532 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Chen, Y., Pi, B., Zhou, H., Yu, Y. & Li, L. Triclosan resistance in clinical isolates of Acinetobacter baumannii. J. Med. Microbiol. 58, 1086–1091 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Hassan, K. A., Liu, Q., Henderson, P. J. F. & Paulsen, I. T. Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems. mBio 6, e01982-14 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Langridge, G. C. et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res. 19, 2308–2316 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  19. Cain, A. K. et al. A decade of advances in transposon-insertion sequencing. Nat. Rev. Genet. 21, 526–540 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hassan, K. A. et al. Fluorescence-based flow sorting in parallel with transposon insertion site sequencing identifies multidrug efflux systems in Acinetobacter baumannii. mBio 7, e01200-16 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Barquist, L. et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 32, 1109–1111 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hood, M. I., Becker, K. W., Roux, C. M., Dunman, P. M. & Skaar, E. P. Genetic determinants of intrinsic colistin tolerance in Acinetobacter baumannii. Infect. Immun. 81, 542–551 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Turnbough, C. L. & Switzer, R. L. Regulation of pyrimidine biosynthetic gene expression in bacteria: repression without repressors. Microbiol. Mol. Biol. Rev. 72, 266–300 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kenyon, J. J., Nigro, S. J. & Hall, R. M. Variation in the OC locus Acinetobacter baumannii genomes predicts extensive structural diversity in the lipoologosaccharide. PLoS ONE 9, e107833 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Arroyo, L. A. et al. The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17978 and clinical isolates through phosphoethanolamine modification of lipid A. Antimicrob. Agents Chemother. 55, 3743–3751 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Boll, J. M. et al. Reinforcing lipid A acylation on the cell surface of Acinetobacter baumannii promotes cationic antimicrobial peptide resistance and desiccation survival. mBio 6, e00478-15 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kenyon, J. J. & Hall, R. M. Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes. PLoS ONE 8, e62160 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lees-Miller, R. G. et al. A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii. Mol. Microbiol. 89, 816–830 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Geisinger, E. & Isberg, R. R. Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii. PLoS Pathog. 11, e1004691 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Luke, N. R. et al. Identification and characterization of a glycosyltransferase involved in Acinetobacter baumannii lipopolysaccharide core biosynthesis. Infect. Immun. 78, 2017–2023 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kenyon, J. J., Holt, K. E., Pickard, D., Dougan, G. & Hall, R. M. Insertions in the OCL1 locus of Acinetobacter baumannii lead to shortened lipooligosaccharides. Res. Microbiol. 165, 472–475 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Pakharukova, N. et al. Structural insight into archaic and alternative chaperone-usher pathways reveals a novel mechanism of pilus biogenesis. PLoS Pathog. 11, e1005269 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Coyne, S., Courvalin, P. & Périchon, B. Efflux-mediated antibiotc resistance in Acinetobacter spp. Antimicrob. Agents Chemother. 55, 947–953 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Magnet, S., Courvalin, P. & Lambert, T. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob. Agents Chemother. 45, 3375–3380 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yoon, E. J., Courvalin, P. & Grillot-Courvalin, C. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and AdeRS mutations. Antimicrob. Agents Chemother. 57, 2989–2995 (2013).

    Article  CAS  PubMed Central  Google Scholar 

  36. Hassan, K. A. et al. Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins. Proc. Natl Acad. Sci. USA 110, 20254–20259 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sugawara, E. & Nikaido, H. Properties of AdeABC and AdeIJK efflux systems of Acinetobacter baumannii compared with those of the AcrAB-TolC system of Escherichia coli. Antimicrob. Agents Chemother. 58, 7250–7257 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Leus, I. V. et al. Substrate specificities and efflux efficiencies of RND efflux pumps of Acinetobacter baumannii. J. Bacteriol. https://doi.org/10.1128/JB.00049-18 (2018).

  39. Rosenfeld, N., Bouchier, C., Courvalin, P. & Périchon, B. Expression of the resistance-nodulation-cell division pump AdeIJK in Acinetobacter baumannii is regulated by AdeN, a TetR-type regulator. Antimicrob. Agents Chemother. 56, 2504–2510 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rajamohan, G., Srinivasan, V. B. & Gebreyes, W. A. Molecular and functional characterization of a novel efflux pump, AmvA, mediating antimicrobial and disinfectant resistance in Acinetobacter baumannii. J. Antimicrob. Chemother. 65, 1919–1925 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Hassan, K. A. et al. Roles of DHA2 family transporters in drug resistance and iron homeostasis in Acinetobacter spp. J. Mol. Microbiol. Biotechnol. 20, 116–124 (2011).

    CAS  PubMed  Google Scholar 

  42. Orth, P., Schnappinger, D., Hillen, W., Saenger, W. & Hinrichs, W. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat. Struct. Biol. 7, 215–219 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Li, L., Hassan, K. A., Brown, M. H. & Paulsen, I. T. Rapid multiplexed phenotypic screening identifies drug resistance functions for three novel efflux pumps in Acinetobacter baumannii. J. Antimicrob. Chemother. 71, 1223–1232 (2016).

    Article  PubMed  Google Scholar 

  44. Jung, W. K. et al. Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Appl. Environ. Microbiol. 74, 2171–2178 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Taber, H. W., Mueller, J. P., Miller, P. F. & Arrow, A. S. Bacterial uptake of aminoglycoside antibiotics. Microbiol. Rev. 51, 439–457 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Holtje, J. V. Induction of streptomycin uptake in resistant strains of Escherichia coli. Antimicrob. Agents Chemother. 15, 177–181 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nikaido, H. & Thanassi, D. G. Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples. Antimicrob. Agents Chemother. 37, 1393–1399 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Piddock, L. J. Mechanism of quinolone uptake into bacterial cells. J. Antimicrob. Chemother. 27, 399–403 (1991).

    Article  CAS  PubMed  Google Scholar 

  49. Morente, E. O. et al. Biocide tolerance in bacteria. Int. J. Food Microbiol. 162, 13–25 (2013).

    Article  CAS  Google Scholar 

  50. Zhao, Y. et al. Evidence for co-selection of antibiotic resistance genes and mobile genetic elements in metal polluted urban soils. Sci. Total Environ. 656, 512–520 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Short, F. L.et al. Benzalkonium chloride antagonises aminoglycoside antibiotics and promotes evolution of resistance. Ebiomedicine https://doi.org/10365310.1016/j.ebiom.2021.103653 (2021).

  52. Nordholt, N., Kanaris, O., Schmidt, S. B. I. & Schreiber, F. Persistence against benzalkonium chloride promotes rapid evolution of tolerance during periodic disinfection. Nat. Commun. 12, 6792 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Papkou, A., Hedge, J., Kapel, N., Young, B. & MacLean, R. C. Efflux pump activity potentiates the evolution of antibiotic resistance across S. aureus isolates. Nat. Commun. 11, 3970 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. El Meouche, I. & Dunlop, M. J. Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation. Science 362, 686–690 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Fridman, O., Goldberg, A., Ronin, I., Shoresh, N. & Balaban, N. Q. Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature 513, 418–421 (2014).

    Article  CAS  PubMed  Google Scholar 

  56. Van den Bergh, B. et al. Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence. Nat. Microbiol. 1, 16020 (2016).

    Article  PubMed  Google Scholar 

  57. Baym, M. et al. Spatiotemporal microbial evolution on antibiotic landscapes. Science 353, 1147–1151 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Levin-Reisman, I. et al. Antibiotic tolerance facilitates the evolution of resistance. Science 355, 826–830 (2017).

    Article  CAS  PubMed  Google Scholar 

  59. Gallagher, L. A. et al. Resources for genetic and genomic analysis of emerging pathogen Acinetobacter baumannii. J. Bacteriol. 197, 2027–2035 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Odermatt, A., Krapf, R. & Solioz, M. Induction of the putative copper ATPase, CopA and CopB, of Enterococcus hirae by Ag+ and Cu2+ extrusion by CopB. Biochem. Biophys. Res. Commun. 202, 44–48 (1994).

    Article  CAS  PubMed  Google Scholar 

  61. Maillard, J. Y. & Hartemann, P. Silver as an antimicrobial: facts and gaps in knowledge. Crit. Rev. Microbiol. 39, 473–483 (2013).

    Article  Google Scholar 

  62. Srinivasan, V. B., Rajamohan, G. & Gebreyes, W. A. Role of AbeS, a novel efflux pump of the SMR family of transporters, in resistance to antimicrobial agents in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53, 5312–5316 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ogase, H., Nagal, I., Kameda, K., Kume, S. & Ono, S. Identification and quantitative analysis of degradation products of chlorhexidine with chlorhexidine-resistant bacteria with three-dimensional high performance liquid chromatography. J. Appl. Bacteriol. 73, 71–78 (1992).

    Article  CAS  Google Scholar 

  64. Su, X. Z., Chen, J., Mizushima, T., Kuroda, T. & Tsuchiya, T. AbeM, an H+-coupled Acinetobacter baumannii multidrug efflux pump belonging to the MATE family of transporters. Antimicrob. Agents Chemother. 49, 4362–4364 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhu, L., Lin, J., Ma, J., Cronan, J. E. & Wang, H. Triclosan resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase. Antimicrob. Agents Chemother. 54, 689–698 (2010).

    Article  CAS  PubMed  Google Scholar 

  66. Chesney, J. A., Eaton, J. W. & Mahoney, J. R. Bacterial glutathione: a sacrificial defense against chlorine compounds. J. Bacteriol. 178, 2131–2135 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Fried, V. A. & Novick, A. Organic solvents as probes for the structure and function of the bacterial membrane: effects of ethanol on the wild type and an ethanol-resistant mutant of Escherichia coli K-12. J. Bacteriol. 114, 239–248 (1973).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by NHMRC (National Health and Medical Research Council) project grants APP1127615, APP1060895 and APP1165135 to I.T.P. and K.A.H. This work was also supported by ARC (Australian Research Council) Centre of Excellence in Synthetic Biology grant CE200100029 to I.T.P. The sequencing was supported by Wellcome Trust grant WT098051. Construction of the transposon mutant library was supported by Wellcome Trust grant WT100087/Z/12/Z. I.T.P. was supported by ARC Laureate Fellowship FL140100021. A.K.C. was supported by ARC DECRA (Discovery Early Career Research Award) Fellowship DE180100929. F.L.S. was supported by ARC DECRA Fellowship DE200101524. K.A.H. was supported by ARC Future Fellowship FT180100123.

Author information

Author notes

  1. Francesca L. Short

    Present address: Infection Program and Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

  2. Karl A. Hassan & Varsha Naidu

    Present address: School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia

  3. Alaska Pokhrel

    Present address: Australian Institute for Microbiology and Infection (AIMI), University of Technology, Sydney, New South Wales, Australia

  4. Julian Parkhill

    Present address: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

Authors and Affiliations

  1. ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, New South Wales, Australia

    Liping Li, Francesca L. Short, Karl A. Hassan, Varsha Naidu, Alaska Pokhrel, Farzana T. Prity, Bhumika S. Shah, Amy K. Cain & Ian T. Paulsen

  2. School of Natural Sciences, Macquarie University, Sydney, New South Wales, Australia

    Liping Li, Karl A. Hassan, Varsha Naidu, Stephanie S. Nagy, Farzana T. Prity, Bhumika S. Shah, Nusrat Afrin, Amy K. Cain & Ian T. Paulsen

  3. Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), University of Cambridge, Cambridge, UK

    Stephen Baker

  4. The Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK

    Julian Parkhill

Authors
  1. Liping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca L. Short
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karl A. Hassan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Varsha Naidu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alaska Pokhrel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephanie S. Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Farzana T. Prity
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bhumika S. Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nusrat Afrin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stephen Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Julian Parkhill
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Amy K. Cain
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ian T. Paulsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.T.P., K.A.H. and L.L. conceptualized this project. S.B. constructed the A. baumannii transposon mutant library. L.L. performed TraDIS assays. A.K.C. and J.P. developed and performed TraDIS sequencing, transposon insertion read mapping and statistical analysis. L.L. and A.K.C. analysed TraDIS data. L.L. formed hypotheses including that biocides dissipate membrane potential. I.T.P., F.L.S. and L.L. formed the hypothesis of the antagonism between biocides and antibiotics. L.L., F.L.S., V.N., A.P., S.S.N., F.T.P., B.S.S. and N.A. performed the follow-up experiments. L.L., I.T.P. and A.K.C. wrote the manuscript. K.A.H., F.L.S. and J.P. reviewed and improved the manuscript.

Corresponding authors

Correspondence to Amy K. Cain or Ian T. Paulsen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Nicholas Jakubovics, Jessica Blair and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 TraDIS data validation.

The growth curves of an individual transposon mutant were compared to the parental strain A. baumannii AB5075, with or without biocide treatment. The data are presented as mean values +/- standard deviation, from three independent biological replicates. The data was processed and plotted by Graph Pad Prism version 10.0.0 (131).

Extended Data Fig. 2 Comparisons of biocide tolerance phenotype among different transposon mutants of the same gene.

The transposon mutants of five genes, that showed changes in transposon insertion reads in the libraries treated by biocides, were chosen to be tested through growth curve assays. Two unique Tn5 transposon mutants of each of the five genes were tested. The data are presented as mean values +/- standard deviation, from three independent biological replicates. The TraDIS results and transposon insertion sites of the mutants of choice are presented in Extended Data Table 3. The data was processed and plotted by Graph Pad Prism version 10.0.0 (131).

Extended Data Fig. 3 Validation of drug efflux pump’s role in biocide resistance.

Growth curves of an individual transposon mutant of an efflux gene or the transcriptional regulator of an efflux gene were compared to the parental strain A. baumannii AB5075, with or without biocide treatment. The data are presented as mean values +/- standard deviation, from three independent biological replicates. The data was processed and plotted by Graph Pad Prism 10.0.0 (131).

Extended Data Fig. 4 TCA cycle, electron transfer and cell division.

The heatmap represents transposon insertion read fold change of each gene, which is colour coded with darker blue indicating higher decrease in Tn5 insertion read coverage and darker red higher increase. The genes are grouped by cellular pathways, with colour scheme underneath.

Extended Data Fig. 5 The transposon mutant of ΔamvA derived from A. baumannii AB5075 accumulates more acriflavine than the parental strain.

The difference of acriflavine accumulation between ΔamvA and the parental strain were measured through flow cytometry (BD InfluxTM Cell Sorter). Each curve shows the fluorescence intensity for 50,000 cells. The cell populations show fluorescence profiles based on the concentration of acriflavine in the cell cytoplasm.

Extended Data Fig. 6 Flow cytometry gating and control in membrane potential (DiOC2(3)) assays.

For each culture, a tight single population was detected and gated, as shown in the three dot plots in the top panel. Because we were measuring the membrane potential of the whole cell population, we gated the single tight population (>85% of the total event) and recorded the total population for follow-up analysis. The dot plots from the top left to the top right are the cells with no treatment, the cells with DiOC2(3) treatment only, and the cells with DiOC2(3) and CCCP treatments, respectively. CCCP is a proton ionophore and a positive control for inducing dissipation of membrane potential in this study. DiOC2(3) is excitable by blue laser (488 nm) and emit green (530/40) and red (610/40) light. The ratio of DiOC2(3) red/green fluorescence is positively correlated to cell membrane potential. As shown in the bottom left panel, the cells without DiOC2(3) treatment had a lot lower fluorescence signal than the cells that was treated by DiOC2(3). This served as a background control that the fluorescence signals from the membrane potential assays (main text Fig. 3) are from intracellular DiOC2(3), rather than from cell auto fluorescence. As shown in the bottom right panel, CCCP induced a larger drop in red fluorescence than in green fluorescence, which resulted to a decrease in the ratio of red/green fluorescence (please also refer to Fig. 3 in the main text), indicating a drop in cell membrane potential. This indicated that DiOC2(3) is suitable for measuring membrane potential in A. baumannii via BD InfluxTM Cell Sorter. The dot plots and histograms were generated by BD InfluxTM Cell Sorter Sortware.

Extended Data Fig. 7 Flow cytometry gating and control in SYTOX Green and acriflavine assays.

The gating strategy and rationale in these assays are similar to the membrane potential assays. We were measuring and comparing the intracellular fluorescent dye concentration of the whole cell population, so we gated the tight single population, as shown in the three dot plots in the top panel. Both SYTOX Green and acriflavine can be excited by blue laser (488 nm) and emit green light (530/40 nm). The bottom left panel shows that the cells treated by SYTOX Green emitted stronger green light than the cells without treatment, and the same for acriflavine in the bottom right panel. This guaranteed that the fluorescent events obtained in Fig. 4 in the main text were from cells containing either SYTOX Green or acriflavine and the fluorescence shifts were positively correlative to the intracellular concentration difference of either of the fluorescent dyes. The dot plots and histograms were generated by BD InfluxTM Cell Sorter Sortware.

Extended Data Table 1 Current knowledge of antibacterial actions and resistance mechanisms of the biocides tested in this study and a summary of TraDIS assay outcomes
Full size table
Extended Data Table 2 Biocide MIC values and their concentrations used in this study
Full size table
Extended Data Table 3 Transposon mutants of A. baumannii AB5075 selected for growth curve assays in this study
Full size table

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Table 1.

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 4

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, L., Short, F.L., Hassan, K.A. et al. Systematic analyses identify modes of action of ten clinically relevant biocides and antibiotic antagonism in Acinetobacter baumannii. Nat Microbiol 8, 1995–2005 (2023). https://doi.org/10.1038/s41564-023-01474-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-023-01474-z

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology