Silver nanoparticles (Ag-NPs) can be considered as a cost-effective alternative to antibiotics. In the presence of Fe(III)-citrate and Ag+, Klebsiella oxytoca DSM 29614 produces biogenic Ag-NPs embedded in its peculiar exopolysaccharide (EPS). K. oxytoca DSM 29614 was cultivated in a defined growth medium–containing citrate (as sole carbon source) and supplemented with Ag+ and either low or high Fe(III) concentration. As inferred from elemental analysis, transmission and scanning electron microscopy, Fourier transform infrared spectrometry and dynamic light scattering, Ag-EPS NPs were produced in both conditions and contained also Fe. The production yield of high-Fe/Ag-EPS NPs was 12 times higher than the production yield of low-Fe/Ag-EPS NPs, confirming the stimulatory effect of iron. However, relative Ag content and Ag+ ion release were higher in low-Fe/Ag-EPS NPs than in high-Fe/Ag-EPS NPs, as revealed by emission-excitation spectra by luminescent spectrometry using a novel ad hoc established phycoerythrin fluorescence–based assay. Interestingly, high and low-Fe/Ag-EPS NPs showed different and growth medium–dependent minimal inhibitory concentrations against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. In addition, low-Fe/Ag-EPS NPs exert inhibition of staphylococcal and pseudomonal biofilm formation, while high-Fe/Ag-EPS NPs inhibits staphylococcal biofilm formation only. Altogether, these results, highlighting the different capability of Ag+ release, support the idea that Fe/Ag-EPS NPs produced by K. oxytoca DSM 29614 can be considered as promising candidates in the development of specific antibacterial and anti-biofilm agents.
• Klebsiella oxytoca DSM 29614 produces bimetal nanoparticles containing Fe and Ag.
• Fe concentration in growth medium affects nanoparticle yield and composition.
• Phycoerythrin fluorescence–based assay was developed to determine Ag+ release.
• Antimicrobial efficacy of bimetal nanoparticle parallels Ag+ ions release.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
All the data are available in the manuscript and in Supplementary Material.
Agnihotri S, Mukherji S, Mukherji S (2014) Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial efficacy. RSC Adv 4:3974–3983
Ahmed NT, Mazid MA (2018) Synthesis and functional evaluation of nanoparticle conjugated antibiotics. New Biotechnol 44:S95–S96. https://doi.org/10.1016/j.nbt.2018.05.960
Ahmed S, Ahmad M, Swami BL, Ikram S (2016) A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise. J Adv Res 7:17–28. https://doi.org/10.1016/j.jare.2015.02.007
Al-Asfar A, Zaheer Z, Aazam ES (2018) Eco-friendly green synthesis of Ag@Fe bimetallic nanoparticles: antioxidant, antimicrobial and photocatalytic degradation of bromothymol blue. J Photochem Photobiol B 185:143–152. https://doi.org/10.1016/j.jphotobiol.2018.05.028
Arčon I, Piccolo O, Paganelli S, Baldi F (2012) XAS analysis of a nanostructured iron polysaccharide produced anaerobically by a strain of Klebsiella oxytoca. Biometals 25:875–881
Arčon I, Paganelli S, Piccolo O, Gallo M, Vogel-Mikuš K, Baldi F (2015) XAS analysis of iron and palladium bonded to a polysaccharide produced anaerobically by a strain of Klebsiella oxytoca. J Synchrotron Radiat 22:1215–1226
Baldi F, Olson GJ (1987) Effects of cinnabar on pyrite oxidation by Thiobacillus ferrooxidans and cinnabar mobilization by a mercury-resistant strain. Appl Environ Microbiol 53:772–776
Baldi F, Bralia A, Riccobono F, Sabatini G (1991) Bioleaching of cobalt and zinc from pyrite ore in relation to calcitic gangue content. World J Microbiol Biotechnol 7:298–308
Baldi F, Minacci A, Pepi M, Scozzafava A (2001) Gel sequestration of heavy metals by Klebsiella oxytoca isolated from iron mat. FEMS Microbiol Ecol 36:169–174
Baldi F, Marchetto D, Battistel D, Daniele S, Faleri C, De Castro C, Lanzetta R (2009) Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage. J Appl Microbiol 107:1241–1250. https://doi.org/10.1111/j.1365-2672.2009.04302.x
Baldi F, Marchetto D, Zanchettin D, Sartorato E, Paganelli S, Piccolo O (2010) A bio-generated Fe(III)-binding exopolysaccharide used as new catalyst for phenol hydroxylation. Green Chem 12:1405–1409. https://doi.org/10.1039/C004967K
Baldi F, Daniele S, Gallo M, Paganelli S, Battistel D, Piccolo O, Faleri C, Puglia AM, Gallo G (2016a) Polysaccharide-based silver nanoparticles synthesized by Klebsiella oxytoca DSM 29614 cause DNA fragmentation in E. coli cells. BioMetals 29. https://doi.org/10.1007/s10534-016-9918-4
Baldi F, Gallo M, Paganelli S, Tassini R, Sperni L, Piccolo O, Zambon S, Piazza R, Natile MM, Armelao L (2016b) Hydrodechlorination of aroclor 1260 in aqueous two-phase mixture catalyzed by biogenerated bimetallic catalysts. Int Res J Pure Appl Chem 11:1–9
Banerjee A, Halder U, Bandopadhyay R (2017) Preparations and applications of polysaccharide based green synthesized metal nanoparticles: a state-of-the-art. J Clust Sci 28:1803–1813. https://doi.org/10.1007/s10876-017-1219-8
Battistel D, Baldi F, Gallo M, Faleri C, Daniele S (2015) Characterisation of biosynthesised silver nanoparticles by scanning electrochemical microscopy (SECM) and voltammetry. Talanta 132:294–300. https://doi.org/10.1016/j.talanta.2014.09.023
Bekasova OD, Brekhovskikh AA, Revina AA, Dubinchuk VT (2008) Preparation and optical properties of silver nanoparticles in R-phycoerythrin, a protein matrix. Inorg Mater 44:835–841. https://doi.org/10.1134/S0020168508080098
Bekasova OD, Ryvkina NG, Chmutin IA, Shtein-Margolina VA, Kurganov BI (2017) Electrical properties of R-phycoerythrin containing Ag0 nanoparticles in its channels. Inorg Mater 53:1249–1253. https://doi.org/10.1134/S0020168517120032
Bose JL, Lehman MK, Fey PD, Bayles KW (2012) Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation. PLoS One 7:e42244. https://doi.org/10.1371/journal.pone.0042244
Buttacavoli M, Albanese NN, Di Cara G, Alduina R, Faleri C, Gallo M, Pizzolanti G, Gallo G, Feo S, Baldi F, Cancemi P (2018) Anticancer activity of biogenerated silver nanoparticles: an integrated proteomic investigation. Oncotarget 9:9–9705. https://doi.org/10.18632/oncotarget.23859
Escárcega-González CE, Garza-Cervantes JA, Vazquez-Rodríguez A, Montelongo-Peralta LZ, Treviño-Gonzalez MT, Díaz Barriga Castro E, Saucedo-Salazar EM, Chávez Morales RM, Regalado-Soto DI, Treviño-González FM, Carrazco Rosales JL, Villalobos Cruz R, Morones-Ramirez JR (2018) In vivo antimicrobial activity of silver nanoparticles produced via a green chemistry synthesis using Acacia rigidula as a reducing and capping agent. Int J Nanomed 13:2349–2363. https://doi.org/10.2147/IJN.S160605
Gallo G, Presta L, Perrin E, Gallo M, Marchetto D, Puglia AM, Fani R, Baldi F (2018) Genomic traits of Klebsiella oxytoca DSM 29614, an uncommon metal-nanoparticle producer strain isolated from acid mine drainages. BMC Microbiol 18:198. https://doi.org/10.1186/s12866-018-1330-5
Green NJB, Pimblott SM, Tachiya M (1993) Generalizations of the Stern-Volmer relation. J Phys Chem 97:196–202. https://doi.org/10.1021/j100103a034
Guibaud G, Comte S, Bordas F, Dupuy S, Baudu M (2005) Comparison of the complexation potential of extracellular polymeric substances (EPS), extracted from activated sludges and produced by pure bacteria strains, for cadmium, lead and nickel. Chemosphere 59:629–638. https://doi.org/10.1016/j.chemosphere.2004.10.028
Harbarth S, Balkhy HH, Goossens H, Jarlier V, Kluytmans J, Laxminarayan R, Saam M, Van Belkum A, Pittet D (2015) Antimicrobial resistance: one world, one fight! Antimicrob Resist Infect Control 4:49
Javaid A, Oloketuyi SF, Khan MM, Khan F (2018) Diversity of bacterial synthesis of silver nanoparticles. Bionanoscience 8:43–59. https://doi.org/10.1007/s12668-017-0496-x
Kathiraven T, Sundaramanickam A, Shanmugam N, Balasubramanian T (2015) Green synthesis of silver nanoparticles using marine algae Caulerpa racemosa and their antibacterial activity against some human pathogens. Appl Nanosci 5:499–504. https://doi.org/10.1007/s13204-014-0341-2
Khan F, Khan MM, Kim Y-M (2018) Recent progress and future perspectives of antibiofilm drugs immobilized on nanomaterials. Curr Pharm Biotechnol 19:631–643. https://doi.org/10.2174/1389201019666180828090052
Khan F, Lee J-W, Manivasagan P, Pham DTN, Oh J, Kim Y-M (2019a) Synthesis and characterization of chitosan oligosaccharide-capped gold nanoparticles as an effective antibiofilm drug against the Pseudomonas aeruginosa PAO1. Microb Pathog 135:103623. https://doi.org/10.1016/j.micpath.2019.103623
Khan F, Manivasagan P, Lee J-W, Pham D, Oh J, Kim Y-M (2019b) Fucoidan-stabilized gold nanoparticle-mediated biofilm inhibition, attenuation of virulence and motility properties in Pseudomonas aeruginosa PAO1. Mar Drugs 17:208. https://doi.org/10.3390/md17040208
Khan F, Manivasagan P, Pham DTN, Oh J, Kim S-K, Kim Y-M (2019c) Antibiofilm and antivirulence properties of chitosan-polypyrrole nanocomposites to Pseudomonas aeruginosa. Microb Pathog 128:363–373. https://doi.org/10.1016/j.micpath.2019.01.033
Kim Y, Oh S, Kim SH (2009) Released exopolysaccharide (r-EPS) produced from probiotic bacteria reduce biofilm formation of enterohemorrhagic Escherichia coli O157:H7. Biochem Biophys Res Commun 379:324–329. https://doi.org/10.1016/j.bbrc.2008.12.053
Kim SW, Baek Y-W, An Y-J (2011) Assay-dependent effect of silver nanoparticles to Escherichia coli and Bacillus subtilis. Appl Microbiol Biotechnol 92:1045–1052. https://doi.org/10.1007/s00253-011-3611-x
Le Ouay B, Stellacci F (2015) Antibacterial activity of silver nanoparticles: a surface science insight. Nano Today 10:339–354. https://doi.org/10.1016/j.nantod.2015.04.002
Leone S, De Castro C, Parrilli M, Baldi F, Lanzetta R (2007) Structure of the iron-binding exopolysaccharide produced anaerobically by the Gram-negative bacterium Klebsiella oxytoca BAS-10. Eur J Org Chem 2007:5183–5189
Losasso C, Belluco S, Cibin V, Zavagnin P, Mičetić I, Gallocchio F, Zanella M, Bregoli L, Biancotto G, Ricci A (2014) Antibacterial activity of silver nanoparticles: sensitivity of different Salmonella serovars. Front Microbiol 5:227. https://doi.org/10.3389/fmicb.2014.00227
Manivasagan P, Khan F, Hoang G, Mondal S, Kim H, Hoang Minh Doan V, Kim Y-M, Oh J (2019) Thiol chitosan-wrapped gold nanoshells for near-infrared laser-induced photothermal destruction of antibiotic-resistant bacteria. Carbohydr Polym 225:115228. https://doi.org/10.1016/j.carbpol.2019.115228
Markowska K, Grudniak AM, Wolska KI (2013) Silver nanoparticles as an alternative strategy against bacterial biofilms. Acta Biochim Pol 60:523–530
Mecozzi M, Acquistucci R, Di Noto V, Pietrantonio E, Amici M, Cardarilli D (2001) Characterization of mucilage aggregates in Adriatic and Tyrrhenian Sea: structure similarities between mucilage samples and the insoluble fractions of marine humic substance. Chemosphere 44:709–720
Mijnendonckx K, Leys N, Mahillon J, Silver S, Van Houdt R (2013) Antimicrobial silver: uses, toxicity and potential for resistance. Biometals 26:609–621
Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR (2019) Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: a review. Front Microbiol 10:539. https://doi.org/10.3389/fmicb.2019.00539
Paganelli S, Piccolo O, Baldi F, Tassini R, Gallo M, La Sorella G (2013) Aqueous biphasic hydrogenations catalyzed by new biogenerated Pd-polysaccharide species. Appl Catal A Gen 451:144–152. https://doi.org/10.1016/j.apcata.2012.10.040
Paganelli S, Piccolo O, Baldi F, Gallo M, Tassini R, Rancan M, Armelao L (2015a) A new biogenerated Rh-based catalyst for aqueous biphasic hydroformylation. Catal Commun 71:32–36. https://doi.org/10.1016/j.catcom.2015.08.001
Paganelli S, Tassini R, La Sorella G, Piccolo O, Baldi F, Rathod VD (2015b) Aqueous biphasic treatment of some nitrocompounds with hydrogen in the presence of a biogenerated Pd-polysaccharide. New Biotechnol 32:313–317. https://doi.org/10.1016/j.nbt.2015.01.010
Panáček A, Kvitek L, Prucek R, Kolář M, Večeřová R, Pizúrová N, Sharma VK, Jana NT, Zbořil R (2006) Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. J Phys Chem B 110:16248–16253
Pham DTN, Khan F, Phan TTV, Park S-K, Manivasagan P, Oh J, Kim Y-M (2019) Biofilm inhibition, modulation of virulence and motility properties by FeOOH nanoparticle in Pseudomonas aeruginosa. Braz. J Microbiol 50:791–805. https://doi.org/10.1007/s42770-019-00108-z
Picceri GG, Leonardi P, Iotti M, Gallo M, Baldi F, Zambonelli A, Amicucci A, Vallorani L, Piccoli G, Ciccimarra G, Arshakyan M, Burattini S, Falcieri E, Chiarantini L (2018) Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii). Appl Microbiol Biotechnol 102:1429–1441. https://doi.org/10.1007/s00253-017-8615-8
Raimondi MV, Maggio B, Raffa D, Plescia F, Cascioferro S, Cancemi G, Schillaci D, Cusimano MG, Vitale M, Daidone G (2012) Synthesis and anti-staphylococcal activity of new 4-diazopyrazole derivatives. Eur J Med Chem 58:64–71. https://doi.org/10.1016/j.ejmech.2012.09.041
Raimondi MV, Listro R, Cusimano MG, La Franca M, Faddetta T, Gallo G, Schillaci D, Collina S, Leonchiks A, Barone G (2019) Pyrrolomycins as antimicrobial agents. Microwave-assisted organic synthesis and insights into their antimicrobial mechanism of action. Bioorg Med Chem. https://doi.org/10.1016/j.bmc.2019.01.010
Rendueles O, Kaplan JB, Ghigo J-M (2013) Antibiofilm polysaccharides. Environ Microbiol 15:334–346. https://doi.org/10.1111/j.1462-2920.2012.02810.x
Rigo C, Zamengo L, Rampazzo G, Argese E (2009) Characterization of a former dump site in the Lagoon of Venice contaminated by municipal solid waste incinerator bottom ash, and estimation of possible environmental risk. Chemosphere 77:510–517. https://doi.org/10.1016/j.chemosphere.2009.07.046
Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017) ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinform 18:529. https://doi.org/10.1186/s12859-017-1934-z
Russo M, Armetta F, Riela S, Martino DC, Lo MP, Noto R (2015) Silver nanoparticles stabilized by a polyaminocyclodextrin as catalysts for the reduction of nitroaromatic compounds. J Mol Catal A Chem 408:250–261. https://doi.org/10.1016/j.molcata.2015.07.031
Russo M, Meli A, Sutera A, Gallo G, Chillura Martino D, Lo Meo P, Noto R (2016) Photosynthesized silver–polyaminocyclodextrin nanocomposites as promising antibacterial agents with improved activity. RSC Adv 6:40090–40099. https://doi.org/10.1039/C6RA00042H
Sahukhal GS, Batte JL, Elasri MO (2015) msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus. FEMS Microbiol Lett 362. https://doi.org/10.1093/femsle/fnv006
Saravanan C, Rajesh R, Kaviarasan T, Muthukumar K, Kavitake D, Shetty PH (2017) Synthesis of silver nanoparticles using bacterial exopolysaccharide and its application for degradation of azo-dyes. Biotechnol Rep 15:33–40. https://doi.org/10.1016/j.btre.2017.02.006
Schillaci D, Maggio B, Raffa D, Daidone G, Cascioferro S, Cusimano MG, Raimondi MV (2008) 4-Diazopyrazole derivatives as potential new antibiofilm agents. Chemotherapy 54:456–462. https://doi.org/10.1159/000159271
Sfriso AA, Gallo M, Baldi F (2018) Phycoerythrin productivity and diversity from five red macroalgae. J Appl Phycol 30:2523–2531. https://doi.org/10.1007/s10811-018-1440-3
Wang C, Gao X, Chen Z, Chen Y, Chen H (2017) Preparation, characterization and application of polysaccharide-based metallic nanoparticles: a review. Polymers (Basel) 9:689
Yuan Y-G, Peng Q-L, Gurunathan S (2017) Effects of silver nanoparticles on multiple drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa from mastitis-infected goats: an alternative approach for antimicrobial therapy. Int J Mol Sci 18:18. https://doi.org/10.3390/ijms18030569
Zhao X, Zhou L, Riaz Rajoka MS, Yan L, Jiang C, Shao D, Zhu J, Shi J, Huang Q, Yang H, Jin M (2018) Fungal silver nanoparticles: synthesis, application and challenges. Crit Rev Biotechnol 38:817–835. https://doi.org/10.1080/07388551.2017.1414141
TEM experimental data were provided by ATeN Center of University of Palermo. The FE-SEM microanalysis was performed by Davide Cristofori, Scientific Campus DSMN Cà Foscari University.
This work was supported by the University of Palermo and Italian MIUR to GG and by Ca’ Foscari University of Venice to FB.
Conflict of interest
The authors declare that they have no conflict of interest.
Consent to participate
Consent for publication
All the authors red and approved the manuscript for publication.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Cusimano, M.G., Ardizzone, F., Nasillo, G. et al. Biogenic iron-silver nanoparticles inhibit bacterial biofilm formation due to Ag+ release as determined by a novel phycoerythrin-based assay. Appl Microbiol Biotechnol 104, 6325–6336 (2020). https://doi.org/10.1007/s00253-020-10686-w
- Biogenic bimetal nanoparticles
- Bacterial exopolysaccharide
- Nanoparticle production yield
- Silver ion release
- Phycoerythrin fluorescence–based assay
- Biofilm formation inhibition