Abstract
Emergence of Candida auris, a multidrug-resistant yeast, demonstrates the urgent need for novel antifungal agents. Human antimicrobial peptides (AMPs) are naturally occurring molecules with wide spectrum antimicrobial activity, particularly against a variety of fungi. Therefore, this study examined the antifungal activity of seven different human AMPs against C. auris following the CLSI guidelines. The antifungal activity was further assessed using time kill curve and cell viability assays. For combination interaction, effectiveness of these peptides with three antifungals, fluconazole, amphotericin B, and caspofungin was done following standard protocols. To elucidate the antifungal mechanism, the effects of peptides on membrane permeability were investigated using propidium iodide staining method and confocal imaging. Antifungal susceptibility results showed that all the examined peptides possessed fungicidal effect against C. auris at different levels, with human β-defensin-3 being the most potent antifungal with MIC values ranging from 3.125 to 12.5 µg/ml. Time kill curves further confirmed the killing effect of all the tested peptides. Viability assay showed a significant decrease in the percentage of viable cells exposed to different inhibitory and fungicidal concentrations of each peptide (p < 0.01). Furthermore, peptides showed mostly synergistic interaction when combined with conventional antifungal drugs, with caspofungin showing 100% synergy when combined with different AMPs. As antifungal mechanism, peptides disrupted the membrane permeability at concentrations that correlated with the inhibition of growth. Overall, the findings of this study point towards the application of the tested peptides as a monotherapy or as a combination therapy with antifungal drugs to treat multidrug-resistant C. auris infections.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All the associated data is provided with this manuscript.
References
Ahmad A, Wani MY, Khan A, Manzoor N, Molepo J (2015) Synergistic interactions of eugenol-tosylate and its congeners with fluconazole against Candida albicans. PLoS ONE 10(12):e0145053. https://doi.org/10.1371/journal.pone.0145053
Baddley JW, Pappas PG (2005) Antifungal combination therapy. Drugs 65(11):1461–1480. https://doi.org/10.2165/00003495-200565110-00002
Benfield AH, Henriques ST (2020) Mode-of-action of antimicrobial peptides: membrane disruption vs. intracellular mechanisms. Front Med Technol 2:610997. https://doi.org/10.3389/fmedt.2020.610997
Bondaryk M, Staniszewska M, Zielińska P, Urbańczyk-Lipkowska Z (2017) Natural antimicrobial peptides as inspiration for design of a new generation antifungal compounds. Journal of Fungi 3(3):46. https://doi.org/10.3390/jof3030046
Buda De Cesare G, Cristy SA, Garsin DA, Lorenz MC (2020) Antimicrobial peptides: a new frontier in antifungal therapy. mBio 11(6):e02123–20. https://doi.org/10.1128/mBio.02123-20
Candoni A, Caira M, Cesaro S, Busca A, Giacchino M, Fanci R, Delia M, Nosari A, Bonini A, Cattaneo C, Melillo L, Caramatti C, Milone G, Scime’ R, Picardi M, Fanin R, Pagano L (2014) Multicentre surveillance study on feasibility, safety and efficacy of antifungal combination therapy for proven or probable invasive fungal diseases in haematological patients: the SEIFEM real-life combo study. Mycoses 57(6):342–350. https://doi.org/10.1111/myc.12161
CDC (2019) Candida auris: information for laboratorians and health professionals. US Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, Georgia. Available online: https://www.cdc.gov/fungal/candida-auris/health-professionals.html. Accessed 29 April 2022
Choi KY, Chow LN, Mookherjee N (2012) Cationic host defence peptides: multifaceted role in immune modulation and inflammation. J Innate Immun 4(4):361–370. https://doi.org/10.1159/000336630
Chow NA, de Groot T, Badali H, Abastabar M, Chiller TM, Meis JF (2019) Potential fifth clade of Candida auris, Iran, 2018. Emerg Infect Dis 25(9):1780–1781. https://doi.org/10.3201/eid2509.190686
Ciociola T, Giovati L, Conti S, Magliani W, Santinoli C, Polonelli L (2016) Natural and synthetic peptides with antifungal activity. Future Med Chem 8(12):1413–1433. https://doi.org/10.4155/fmc-2016-0035
CLSI (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-3rd-ed. CLSI document M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA
Dahiya S, Chhillar AK, Sharma N, Choudhary P, Punia A, Balhara M, Kaushik K, Parmar VS (2020) Candida auris and nosocomial infection. Curr Drug Targets 21(4):365–373. https://doi.org/10.2174/1389450120666190924155631
Dal Mas C, Rossato L, Shimizu T, Oliveira EB, da Silva Junior PI, Meis JF, Colombo AL, Hayashi MAF (2019) Effects of the natural peptide crotamine from a South American rattlesnake on Candida auris, an emergent multidrug antifungal resistant human pathogen. Biomolecules 9(6):205. https://doi.org/10.3390/biom9060205
Ebenhan T, Gheysens O, Kruger HG, Zeevaart JR, Sathekge MM (2014) Antimicrobial peptides: their role as infection-selective tracers for molecular imaging. Biomed Res Int 2014:867381. https://doi.org/10.1155/2014/867381
Fakhim H, Chowdhary A, Prakash A, Vaezi A, Dannaoui E, Meis JF, Badali H (2017) In vitro interactions of echinocandins with triazoles against multidrug-resistant Candida auris. Antimicrob Agents Chem 61(11):e01056-e1117. https://doi.org/10.1128/AAC.01056-17
Fernández de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD (2020) Antifungal peptides as therapeutic agents. Front Cell Infect Microbiol 10:105. https://doi.org/10.3389/fcimb.2020.00105
Hancock RE, Chapple DS (1999) Peptide antibiotics. Antimicrob Agents Chemother 43(6):1317–1323. https://doi.org/10.1128/AAC.43.6.1317
Helmerhorst EJ, Reijnders IM, van ’t Hof W, Veerman EC, NieuwAmerongen AV (1999) A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Lett 449(2–3):105–110. https://doi.org/10.1016/s0014-5793(99)00411-1
Huang Q, Fei J, Yu HJ, Gou YB, Huang XK (2014) Effects of human β-defensin-3 on biofilm formation-regulating genes dltB and icaA in Staphylococcus aureus. Mol Med Rep 10(2):825–831. https://doi.org/10.3892/mmr.2014.2309
Jaggavarapu S, Burd EM, Weiss DS (2020) Micafungin and amphotericin B synergy against Candida auris. Lancet Microbe 1(8):e314–e315. https://doi.org/10.1016/S2666-5247(20)30194-4
Kang HK, Kim C, Seo CH, Park Y (2017) The therapeutic applications of antimicrobial peptides (AMPs): a patent review. J Microbiol 55(1):1–12. https://doi.org/10.1007/s12275-017-6452-1
Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA (1998) Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother 42(5):1207–1212. https://doi.org/10.1128/AAC.42.5.1207
Krishnakumari V, Rangaraj N, Nagaraj R (2008) Antifungal activity of human beta defensins HBD-1-3 and their C-terminal analogs Phd1-3. Antimicrob Agents Chem 53(1):256–260. https://doi.org/10.1128/AAC.00470-08
Kubiczek D, Raber H, Gonzalez-García M, Morales-Vicente F, Staendker L, Otero-Gonzalez AJ, Rosenau F (2020) Derivates of the antifungal peptide Cm-p5 inhibit development of Candida auris biofilms in vitro. Antibiotics 9(7):363. https://doi.org/10.3390/antibiotics9070363
Lin P, Li Y, Dong K, Li Q (2015) The antibacterial effects of an antimicrobial peptide human β-defensin 3 fused with carbohydrate-binding domain on Pseudomonas aeruginosa PA14. Curr Microbiol 71(2):170–176. https://doi.org/10.1007/s00284-015-0814-x
Lockhart SR, Berkow EL, Chow N, Welsh RM (2017) Candida auris for the clinical microbiology laboratory: not your grandfather’s Candida species. Clin Microbiol Newsl 39(13):99–103. https://doi.org/10.1016/j.clinmicnews.2017.06.003
Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, Colombo AL, Calvo B, Cuomo CA, Desjardins CA, Berkow EL, Castanheira M, Magobo RE, Jabeen K, Asghar RJ, Meis JF, Jackson B, Chiller T, Litvintseva AP (2017) Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis 64(2):134–140. https://doi.org/10.1093/cid/ciw691
Lone SA, Ahmad A (2019) Candida auris-the growing menace to global health. Mycoses 62(8):620–637. https://doi.org/10.1111/myc.12904
Lupetti A, Paulusma-Annema A, Welling MM, Dogterom-Ballering H, Brouwer CP, Senesi S, Van Dissel JT, Nibbering PH (2003) Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species. Antimicrob Agents Chemother 47(1):262–267. https://doi.org/10.1128/AAC.47.1.262-267.2003
Lyman M, Forsberg K, Reuben J, Dang T, Free R, Seagle EE, Sexton DJ, Soda E, Jones H, Hawkins D, Anderson A, Bassett J, Lockhart SR, Merengwa E, Iyengar P, Jackson BR, Chiller T (2021) Notes from the field: transmission of pan-resistant and echinocandin-resistant Candida auris in health care facilities-Texas and the District of Columbia, January–April 2021. MMWR Morb Mortal Wkly Rep 70(29):1022–1023. https://doi.org/10.15585/mmwr.mm7029a2
MacCallum DM, Desbois AP, Coote PJ (2013) Enhanced efficacy of synergistic combinations of antimicrobial peptides with caspofungin versus Candida albicans in insect and murine models of systemic infection. Eur J Clin Microbiol Infect Dis 32(8):1055–1062. https://doi.org/10.1007/s10096-013-1850-8
Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol 6:194. https://doi.org/10.3389/fcimb.2016.00194
Maiti S, Patro S, Purohit S, Jain S, Senapati S, Dey N (2014) Effective control of Salmonella infections by employing combinations of recombinant antimicrobial human β-defensins hBD-1 and hBD-2. Antimicrob Agents Chemother 58(11):6896–6903. https://doi.org/10.1128/AAC.03628-14
Malmsten M (2014) Antimicrobial peptides. Ups J Med Sci 119(2):199–204. https://doi.org/10.3109/03009734.2014.899278
Mercer DK, O’Neil DA (2020) Innate inspiration: antifungal peptides and other immunotherapeutics from the host immune response. Front Immunol 11:2177. https://doi.org/10.3389/fimmu.2020.02177
Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29(9):464–472. https://doi.org/10.1016/j.tibtech.2011.05.001
Osei Sekyere J (2018) Candida auris: a systematic review and meta-analysis of current updates on an emerging multidrug-resistant pathogen. Microbiologyopen 7(4):e00578. https://doi.org/10.1002/mbo3.578
Ostrowsky B, Greenko J, Adams E, Quinn M, O'Brien B, Chaturvedi V, Berkow E, Vallabhaneni S, Forsberg K, Chaturvedi S, Lutterloh E, Blog D (2020) Candida auris isolates resistant to three classes of antifungal medications—New York, 2019. MMWR Morb Mortal Wkly Rep 69(1):6–9. https://doi.org/10.15585/mmwr.mm6901a2
Pathirana RU, Friedman J, Norris HL, Salvatori O, McCall AD, Kay J, Edgerton M (2018) ’Fluconazole-resistant Candida auris is susceptible to salivary histatin 5 killing and to intrinsic host defenses. Antimicrob Agents Chem 62(2):e01872-e1917. https://doi.org/10.1128/AAC.01872-17
Polesello V, Segat L, Crovella S, Zupin L (2017) Candida infections and human defensins. Protein Pept Lett 24(8):747–756. https://doi.org/10.2174/0929866524666170807125245
Puri S, Edgerton M (2014) How does it kill?: understanding the candidacidal mechanism of salivary histatin 5. Eukaryot Cell 13(8):958–964. https://doi.org/10.1128/EC.00095-14
Raber HF, Sejfijaj J, Kissmann AK, Wittgens A, Gonzalez-Garcia M, Alba A, Vázquez AA, Morales Vicente FE, Erviti JP, Kubiczek D, Otero-González A, Rodríguez A, Ständker L, Rosenau F (2021) Antimicrobial peptides Pom-1 and Pom-2 from Pomacea poeyana are active against Candida auris, C. parapsilosis and C. albicans biofilms. Pathogens 10(4):496. https://doi.org/10.3390/pathogens10040496
Rather IA, Sabir JSM, Asseri AH, Ali S (2022) Antifungal activity of human cathelicidin LL-37, a membrane disrupting peptide, by triggering oxidative stress and cell cycle arrest in Candida auris. J Fungi (basel) 8(2):204. https://doi.org/10.3390/jof8020204
Rautenbach M, Troskie AM, Vosloo JA (2016) Antifungal peptides: to be or not to be membrane active. Biochimie 130:132–145. https://doi.org/10.1016/j.biochi.2016.05.013
Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H (2009) Candida auris sp nov a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol 53(1):41–44. https://doi.org/10.1111/j.1348-0421.2008.00083
Schneider JJ, Unholzer A, Schaller M, Schäfer-Korting M, Korting HC (2005) Human defensins. J Mol Med 83(8):587–595. https://doi.org/10.1007/s00109-005-0657-1
Scorzoni L, de Paula E, Silva AC, Marcos CM, Assato PA, de Melo WC, de Oliveira HC, Costa-Orlandi CB, Mendes-Giannini MJ, Fusco-Almeida AM (2017) Antifungal therapy: new advances in the understanding and treatment of mycosis. Front Microbiol 8:36. https://doi.org/10.3389/fmicb.2017.00036
Singh K, Shekhar S, Yadav Y, Xess I, Dey S (2017) DS6: anticandidal, antibiofilm peptide against Candida tropicalis and exhibit synergy with commercial drug. J Pept Sci 23(3):228–235. https://doi.org/10.1002/psc.2973
Spitzer M, Robbins N, Wright GD (2017) Combinatorial strategies for combating invasive fungal infections. Virulence 8(2):169–185. https://doi.org/10.1080/21505594.2016.1196300
Struyfs C, Cammue BPA, Thevissen K (2021) Membrane-interacting antifungal peptides. Front Cell Dev Biol 9:649875. https://doi.org/10.3389/fcell.2021.649875
Suchodolski J, Feder-Kubis J, Krasowska A (2017) Antifungal activity of ionic liquids based on (-)-menthol: a mechanism study. Microbiol Res 197:56–64. https://doi.org/10.1016/j.micres.2016.12.008
van Eijk M, Boerefijn S, Cen L, Rosa M, Morren MJH, van der Ent CK, Kraak B, Dijksterhuis J, Valdes ID, Haagsman HP, de Cock H (2020) Cathelicidin-inspired antimicrobial peptides as novel antifungal compounds. Med Mycol 58(8):1073–1084. https://doi.org/10.1093/mmy/myaa014
Vicente FEM, González-Garcia M, Diaz Pico E, Moreno-Castillo E, Garay HE, Rosi PE, Jimenez AM, Campos-Delgado JA, Rivera DG, Chinea G, Pietro RCLR, Stenger S, Spellerberg B, Kubiczek D, Bodenberger N, Dietz S, Rosenau F, Paixão MW, Ständker L, Otero-González AJ (2019) Design of a helical-stabilized, cyclic, and nontoxic analogue of the peptide Cm-p5 with improved antifungal activity. ACS Omega 4(21):19081–19095. https://doi.org/10.1021/acsomega.9b02201
Vylkova S, Li XS, Berner JC, Edgerton M (2006) Distinct antifungal mechanisms: β-defensins require Candida albicans Ssa1 protein, while Trk1p mediates activity of cysteine-free cationic peptides. Antimicrob Agents Chemother 50(1):324–331. https://doi.org/10.1128/AAC.50.1.324-331.2006
Wei GX, Bobek LA (2004) In vitro synergic antifungal effect of MUC7 12-mer with histatin-5 12-mer or miconazole. J Antimicrob Chemother 53(5):750–758. https://doi.org/10.1093/jac/dkh181
Zhu Y, O’Brien B, Leach L, Clarke A, Bates M, Adams E, Ostrowsky B, Quinn M, Dufort E, Southwick K, Erazo R, Haley VB, Bucher C, Chaturvedi V, Limberger RJ, Blog D, Lutterloh E, Chaturvedi S (2020) Laboratory analysis of an outbreak of Candida auris in New York from 2016 to 2018: impact and lessons learned. J Clin Microbiol 58(4):e01503-e1519. https://doi.org/10.1128/JCM.01503-19
Zimmermann GR, Lehár J, Keith CT (2007) Multi-target therapeutics: when the whole is greater than the sum of the parts. Drug Discov Today 12(1–2):34–42. https://doi.org/10.1016/j.drudis.2006.11.008
Funding
A.A. has received a research funding from National Health Laboratory Service (RA21-013).
Author information
Authors and Affiliations
Contributions
Aijaz Ahmad and Mrudula Patel contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Siham Shaban. The first draft of the manuscript was written by Siham Shaban, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval
Ethical approval for this study was obtained from the Human Research Committee of University of Witwatersrand, Johannesburg, South Africa (Ref no: W-CBP-210428–01).
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
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.
About this article
Cite this article
Shaban, S., Patel, M. & Ahmad, A. Fungicidal activity of human antimicrobial peptides and their synergistic interaction with common antifungals against multidrug-resistant Candida auris. Int Microbiol 26, 165–177 (2023). https://doi.org/10.1007/s10123-022-00290-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10123-022-00290-5