Scope and efficacy of the broad-spectrum topical antiseptic choline geranate

Autoři: Joshua R. Greene aff001;  Kahla L. Merrett aff001;  Alexanndra J. Heyert aff001;  Lucas F. Simmons aff001;  Camille M. Migliori aff002;  Kristen C. Vogt aff003;  Rebeca S. Castro aff003;  Paul D. Phillips aff001;  Joseph L. Baker aff003;  Gerrick E. Lindberg aff001;  David T. Fox aff005;  Rico E. Del Sesto aff002;  Andrew T. Koppisch aff001
Působiště autorů: Department of Chemistry, Northern Arizona University, Flagstaff, AZ, United States of America aff001;  Department of Chemistry, Dixie State University, St. George, UT, United States of America aff002;  Department of Chemistry, The College of New Jersey, Ewing, NJ, United States of America aff003;  Center for Materials Interfaces in Research and Application, Northern Arizona University, Flagstaff, AZ, United States of America aff004;  Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM, United States of America aff005
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222211


Choline geranate (also described as Choline And GEranic acid, or CAGE) has been developed as a novel biocompatible antiseptic material capable of penetrating skin and aiding the transdermal delivery of co-administered antibiotics. The antibacterial properties of CAGE were analyzed against 24 and 72 hour old biofilms of 11 clinically isolated ESKAPE pathogens (defined as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp, respectively), including multidrug resistant (MDR) isolates. CAGE was observed to eradicate in vitro biofilms at concentrations as low as 3.56 mM (0.156% v:v) in as little as 2 hours, which represents both an improved potency and rate of biofilm eradication relative to that reported for most common standard-of-care topical antiseptics in current use. In vitro time-kill studies on 24 hour old Staphylococcus aureus biofilms indicate that CAGE exerts its antibacterial effect upon contact and a 0.1% v:v solution reduced biofilm viability by over three orders of magnitude (a 3log10 reduction) in 15 minutes. Furthermore, disruption of the protective layer of exopolymeric substances in mature biofilms of Staphylococcus aureus by CAGE (0.1% v:v) was observed in 120 minutes. Insight into the mechanism of action of CAGE was provided with molecular modeling studies alongside in vitro antibiofilm assays. The geranate ion and geranic acid components of CAGE are predicted to act in concert to integrate into bacterial membranes, affect membrane thinning and perturb membrane homeostasis. Taken together, our results show that CAGE demonstrates all properties required of an effective topical antiseptic and the data also provides insight into how its observed antibiofilm properties may manifest.

Klíčová slova:

Biology and life sciences – Microbiology – Biofilms – Bacteriology – Bacterial biofilms – Microbial control – Antimicrobials – Medical microbiology – Microbial pathogens – Bacterial pathogens – Organisms – Bacteria – Staphylococcus – Staphylococcus aureus – Biochemistry – Biochemical simulations – Lipids – Computational biology – Medicine and health sciences – Pharmacology – Drugs – Antibacterials – Pathology and laboratory medicine – Pathogens – Research and analysis methods – Biological cultures – Cell culturing techniques – Biofilm culture – Physical sciences – Chemistry – Chemical compounds – Organic compounds – Alcohols – Ethanol – Organic chemistry


1. Food, Drug Administration HHS. Safety and Effectiveness of Health Care Antiseptics; Topical Antimicrobial Drug Products for Over-the-Counter Human Use. Final rule. Fed Regist. 2017;82(242):60474–503. 29260839.

2. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999;284(5418):1318–22. doi: 10.1126/science.284.5418.1318 PubMed PMID: WOS:000080430600043. 10334980

3. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15(2):167–93. doi: 10.1128/CMR.15.2.167-193.2002 11932229; PubMed Central PMCID: PMCPMC118068.

4. Joo HS, Otto M. Molecular basis of in vivo biofilm formation by bacterial pathogens. Chem Biol. 2012;19(12):1503–13. doi: 10.1016/j.chembiol.2012.10.022 23261595; PubMed Central PMCID: PMCPMC3530155.

5. Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45(4):999–1007. doi: 10.1128/AAC.45.4.999-1007.2001 11257008; PubMed Central PMCID: PMCPMC90417.

6. Lebeaux D, Chauhan A, Rendueles O, Beloin C. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections. Pathogens. 2013;2(2):288–356. doi: 10.3390/pathogens2020288 25437038; PubMed Central PMCID: PMCPMC4235718.

7. Lebeaux D, Ghigo JM, Beloin C. Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics. Microbiol Mol Biol R. 2014;78(3):510–43. doi: 10.1128/Mmbr.00013-14 PubMed PMID: WOS:000341639400007. 25184564

8. Davies D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov. 2003;2(2):114–22. doi: 10.1038/nrd1008 12563302.

9. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358(9276):135–8. doi: 10.1016/s0140-6736(01)05321-1 PubMed PMID: WOS:000169837700032. 11463434

10. Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Ch. 2000;44(7):1818–24. doi: 10.1128/Aac.44.7.1818–1824.2000 PubMed PMID: WOS:000087961400008.

11. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J, et al. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ Microbiol. 2013;15(10):2865–78. doi: 10.1111/1462-2920.12155 23751003; PubMed Central PMCID: PMCPMC4045617.

12. Antibiotic Resistance Threats in the United States [Internet]. 2013

13. Solomon SL, Oliver KB. Antibiotic resistance threats in the United States: stepping back from the brink. Am Fam Physician. 2014;89(12):938–41. 25162160.

14. Tam VH, Rogers CA, Chang KT, Weston JS, Caeiro JP, Garey KW. Impact of Multidrug-Resistant Pseudomonas aeruginosa Bacteremia on Patient Outcomes. Antimicrob Agents Ch. 2010;54(9):3717–22. doi: 10.1128/Aac.00207-10 PubMed PMID: WOS:000281005900028. 20585122

15. Zakrewsky M, Banerjee A, Apte S, Kern TL, Jones MR, Sesto RE, et al. Choline and Geranate Deep Eutectic Solvent as a Broad-Spectrum Antiseptic Agent for Preventive and Therapeutic Applications. Adv Healthc Mater. 2016;5(11):1282–9. doi: 10.1002/adhm.201600086 26959835.

16. Zakrewsky M, Lovejoy KS, Kern TL, Miller TE, Le V, Nagy A, et al. Ionic liquids as a class of materials for transdermal delivery and pathogen neutralization. Proc Natl Acad Sci U S A. 2014;111(37):13313–8. doi: 10.1073/pnas.1403995111 25157174; PubMed Central PMCID: PMCPMC4169946.

17. Busetti A, Crawford DE, Earle MJ, Gilea MA, Gilmore BF, Gorman SP, et al. Antimicrobial and antibiofilm activities of 1-alkylquinolinium bromide ionic liquids. Green Chem. 2010;12(3):420–5. doi: 10.1039/b919872e PubMed PMID: WOS:000275379600012.

18. Carson L, Chau PKW, Earle MJ, Gilea MA, Gilmore BF, Gorman SP, et al. Antibiofilm activities of 1-alkyl-3-methylimidazolium chloride ionic liquids. Green Chem. 2009;11(4):492–7. doi: 10.1039/b821842k PubMed PMID: WOS:000264978500010.

19. Lovejoy KS, Corley C. A., Cope E. K., Valentine M. C, Leid J. G., Purdy G. M., et al. Utilization of Metal Halide Species Ambiguity to Develop Amorphous, Stabilized Pharmaceutical Agents As Ionic Liquids. Cryst Growth Des. 2012;12:5357–64.

20. Pendleton JN, Gilmore BF. The antimicrobial potential of ionic liquids: A source of chemical diversity for infection and biofilm control. Int J Antimicrob Ag. 2015;46(2):131–9. doi: 10.1016/j.ijantimicag.2015.02.016 PubMed PMID: WOS:000360705500001. 25907139

21. Lovejoy KS, Lou AJ, Davis LE, Sanchez TC, Iyer S, Corley CA, et al. Single-pot extraction-analysis of dyed wool fibers with ionic liquids. Anal Chem. 2012;84(21):9169–75. doi: 10.1021/ac301873s 23066794.

22. Lovejoy KS, Purdy GM, Iyer S, Sanchez TC, Robertson A, Koppisch AT, et al. Tetraalkylphosphonium-based ionic liquids for a single-step dye extraction/MALDI MS analysis platform. Anal Chem. 2011;83(8):2921–30. doi: 10.1021/ac102944w 21410201.

23. Rogers RD, Gurau G. Is "choline and geranate" an ionic liquid or deep eutectic solvent system? Proc Natl Acad Sci U S A. 2018;115(47):E10999. doi: 10.1073/pnas.1814976115 30401741

24. Banerjee A, Ibsen K, Brown T, Chen R, Agatemor C, Mitragotri S. Reply to Rogers and Gurau: Definitions of ionic liquids and deep eutectic solvents. Proc Natl Acad Sci U S A. 2018;115(47):E11000–E1. doi: 10.1073/pnas.1815526115 30401740

25. Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther. 2013;11(3):297–308. doi: 10.1586/eri.13.12 23458769.

26. Leid JG, Ditto AJ, Knapp A, Shah PN, Wright BD, Blust R, et al. In vitro antimicrobial studies of silver carbene complexes: activity of free and nanoparticle carbene formulations against clinical isolates of pathogenic bacteria. J Antimicrob Chemother. 2012;67(1):138–48. doi: 10.1093/jac/dkr408 21972270; PubMed Central PMCID: PMCPMC3236053.

27. Frederix M, Hutter K, Leu J, Batth TS, Turner WJ, Ruegg TL, et al. Development of a native Escherichia coli induction system for ionic liquid tolerance. PLoS One. 2014;9(7):e101115. doi: 10.1371/journal.pone.0101115 24983352; PubMed Central PMCID: PMCPMC4077768.

28. Ruegg TL, Kim EM, Simmons BA, Keasling JD, Singer SW, Lee TS, et al. An auto-inducible mechanism for ionic liquid resistance in microbial biofuel production. Nat Commun. 2014;5:3490. doi: 10.1038/ncomms4490 24667370.

29. Pasenkiewicz-Gierula M, Rog T, Murzyn K. Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane: a molecular modeling study. Biophysical Journal. 2007:643a-a. PubMed PMID: WOS:000243972404483.

30. Yoo B, Zhu Y, Maginn EJ. Molecular Mechanism of Ionic-Liquid-Induced Membrane Disruption: Morphological Changes to Bilayers, Multilayers, and Vesicles. Langmuir. 2016;32(21):5403–11. doi: 10.1021/acs.langmuir.6b00768 27159842.

31. Gilbert P, Moore LE. Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol. 2005;99(4):703–15. doi: 10.1111/j.1365-2672.2005.02664.x 16162221.

32. Shai Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta. 1999;1462(1–2):55–70. doi: 10.1016/s0005-2736(99)00200-x 10590302.

33. Wimley WC. Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol. 2010;5(10):905–17. doi: 10.1021/cb1001558 20698568; PubMed Central PMCID: PMCPMC2955829.

34. Bertelsen K, Dorosz J, Hansen SK, Nielsen NC, Vosegaard T. Mechanisms of peptide-induced pore formation in lipid bilayers investigated by oriented 31P solid-state NMR spectroscopy. PLoS One. 2012;7(10):e47745. doi: 10.1371/journal.pone.0047745 23094079; PubMed Central PMCID: PMCPMC3475706.

35. Grage SL, Afonin S, Kara S, Buth G, Ulrich AS. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides. Front Cell Dev Biol. 2016;4:65. doi: 10.3389/fcell.2016.00065 27595096; PubMed Central PMCID: PMCPMC4999517.

36. Huang HW. Molecular mechanism of antimicrobial peptides: the origin of cooperativity. Biochim Biophys Acta. 2006;1758(9):1292–302. doi: 10.1016/j.bbamem.2006.02.001 16542637.

37. Khandelia H, Ipsen JH, Mouritsen OG. The impact of peptides on lipid membranes. Biochim Biophys Acta. 2008;1778(7–8):1528–36. doi: 10.1016/j.bbamem.2008.02.009 18358231.

38. Ludtke S, He K, Huang H. Membrane thinning caused by magainin 2. Biochemistry. 1995;34(51):16764–9. doi: 10.1021/bi00051a026 8527451.

39. Toke O. Antimicrobial peptides: new candidates in the fight against bacterial infections. Biopolymers. 2005;80(6):717–35. doi: 10.1002/bip.20286 15880793.

40. Institute CaLS. Performance Standards for Antimicrobial Susceptibility Testing, 25th informational supplement. Clinical and Laboratory Standards Institute; 2015.

41. Tanner EEL, Ibsen KN, Mitragotri S. Transdermal insulin delivery using choline-based ionic liquids (CAGE). J Control Release. 2018;286:137–44. doi: 10.1016/j.jconrel.2018.07.029 30026081.

42. Ibsen KN, Ma H., Banerjee A., Tanner E.L., Nangia S., Mitragotri S. Mechanism of Antibacterial Activity of Choline-Based Ionic Liquids (CAGE). ACS Biomater Sci Eng. 2018;4:2370–9.

43. Solinski AE, Koval AB, Brzozowski RS, Morrison KR, Fraboni AJ, Carson CE, et al. Diverted Total Synthesis of Carolacton-Inspired Analogs Yields Three Distinct Phenotypes in Streptococcus mutans Biofilms. J Am Chem Soc. 2017;139(21):7188–91. doi: 10.1021/jacs.7b03879 28502178; PubMed Central PMCID: PMCPMC5891724.

44. Garrison AT, Abouelhassan Y, Kallifidas D, Tan H, Kim YS, Jin S, et al. An Efficient Buchwald-Hartwig/Reductive Cyclization for the Scaffold Diversification of Halogenated Phenazines: Potent Antibacterial Targeting, Biofilm Eradication, and Prodrug Exploration. J Med Chem. 2018;61(9):3962–83. doi: 10.1021/acs.jmedchem.7b01903 29638121.

45. Camplin AL, Maddocks SE. Manuka honey treatment of biofilms of Pseudomonas aeruginosa results in the emergence of isolates with increased honey resistance. Ann Clin Microbiol Antimicrob. 2014;13:19. doi: 10.1186/1476-0711-13-19 24884949; PubMed Central PMCID: PMCPMC4023514.

46. Capriotti K, Pelletier J., Barone S., Capriotti J. Efficacy of Dilute Povidone-Iodine against Multi-Drug Resistant Bacterial Biofilms, Fungal Biofilms and Fungal Spores. Clin Res Dermatol. 2018;5:1–5.

47. Johani K, Malone M, Jensen SO, Dickson HG, Gosbell IB, Hu H, et al. Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo. J Antimicrob Chemother. 2018;73(2):494–502. doi: 10.1093/jac/dkx391 29165561; PubMed Central PMCID: PMCPMC5890786.

48. Justo JA, Bookstaver PB. Antibiotic lock therapy: review of technique and logistical challenges. Infect Drug Resist. 2014;7:343–63. doi: 10.2147/IDR.S51388 25548523; PubMed Central PMCID: PMCPMC4271721.

49. Banerjee A, Ibsen K, Brown T, Chen R, Agatemor C, Mitragotri S. Ionic liquids for oral insulin delivery. Proc Natl Acad Sci U S A. 2018;115(28):7296–301. doi: 10.1073/pnas.1722338115 29941553; PubMed Central PMCID: PMCPMC6048483.

50. Banerjee A, Ibsen K, Iwao Y, Zakrewsky M, Mitragotri S. Transdermal Protein Delivery Using Choline and Geranate (CAGE) Deep Eutectic Solvent. Adv Healthc Mater. 2017;6(15). doi: 10.1002/adhm.201601411 28337858.

51. International A. E2799-Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay. West Conshohocken, PA, USA: ASTM International. p. 9.

52. Case DA, Betz R.M., Cerutti D.S., Cheatham T.E III., Darden T.A., Duke R.E. et al. AMBER. University of California, San Francisco; 2016.

53. Babuji Y, Brizius A., Chard K., Foster I., Katz D.S., Wilde M., et al. Introducing Parsl: A Python Parallel Scripting Library. Zenodo2017.

54. Dickson CJ, Madej BD, Skjevik ÅA, Betz RM, Teigen K, Gould IR, et al. Lipid14: The Amber Lipid Force Field. Journal of Chemical Theory and Computation. 2014;10(2):865–79. doi: 10.1021/ct4010307 24803855

55. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79(2):926–35. doi: 10.1063/1.445869

56. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. Journal of Computational Chemistry. 2004;25(9):1157–74. doi: 10.1002/jcc.20035 15116359

57. Skjevik ÅA, Madej BD, Dickson CJ, Teigen K, Walker RC, Gould IR. All-atom lipid bilayer self-assembly with the AMBER and CHARMM lipid force fields. Chemical Communications. 2015;51(21):4402–5. doi: 10.1039/c4cc09584g 25679020

58. Martínez L, Andrade R, Birgin EG, Martínez JM. PACKMOL: A package for building initial configurations for molecular dynamics simulations. Journal of Computational Chemistry. 2009;30(13):2157–64. doi: 10.1002/jcc.21224 19229944

Článek vyšel v časopise


2019 Číslo 9