ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2009, p. 3538–3540 0066-4804/09/$08.00ϩ0 doi:10.1128/AAC.01106-08Copyright 2009, American Society for Microbiology. All Rights Reserved.
Synergistic Interaction between Silver Nanoparticles and Membrane-Permeabilizing Antimicrobial Peptidesᰔ Serge Ruden,1 Kai Hilpert,2 Marina Berditsch,1 Parvesh Wadhwani,2 and Anne S. Ulrich1,2* KIT, IOC, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany,1 and KIT, IBG-2, POB 3640, 76021 Karlsruhe, Germany2 Received 18 August 2008/Returned for modification 13 November 2008/Accepted 2 June 2009 Silver nanoparticles, as well as antimicrobial peptides (AMPs), can be used to fight infectious diseases. Since
AMPs are known to permeabilize bacterial membranes and might therefore help silver nanoparticles to access
internal target sites, we investigated their combined activities and showed synergistic effects between polymyxin
B and silver nanoparticles for gram-negative bacteria.

The worldwide escalation of bacterial resistance to conven- methicin are hydrophobic and act against gram-positive bacte- tional medical antibiotics is a serious concern for modern med- ria by forming ion-selective channels (3) or barrel-stave pores icine (4). In view of this development, the aim of our study was (11), respectively. Gramicidin S is a cyclic ␤-sheet peptide (8), to investigate the antimicrobial and hemolytic properties of while PGLa and magainin 2 are ␣-helical (12). They are all silver(I) ions and silver nanoparticles in combination with dif- cationic and amphipathic and have similar activities against ferent antimicrobial peptides (AMPs). The synergistic action gram-negative and gram-positive bacteria, forming transmem- of antimicrobial agents can reduce the need for high dosages brane pores (12, 14). In combination with these diverse AMPs, and minimize side effects (5, 9, 18, 19). Examples include we tested silver nitrate (VWR, Darmstadt, Germany) and sil- PGLa plus magainin 2 from Xenopus laevis (18) and ␤-lactam ver nanoparticles (mean diameter, 25 nm) stabilized by a non- amoxicillin with silver nanoparticles (9), but a combination of biocidal carbon matrix (data not shown) (NovaCentrix, Austin, silver nanoparticles or silver ions with AMPs has not been TX). Electron microscopy demonstrated that these nanopar- tested. We therefore examined commercial polymyxin B, ticles accumulate at both the outer and inner membranes of gramicidin A and alamethicin (from Sigma-Aldrich, Buchs, bacteria (13). The exact mode of action of silver ions and silver Switzerland), gramicidin S extracted from Aneurinibacillus nanoparticles is unknown, but they seem to significantly reduce migulanus (2), and PGLa and magainin 2, which was synthe- cellular chemiosmotic potential (6, 10). Furthermore, it has sized in our laboratory. All noncommercial peptides were pu- been reported that within a period of 24 h, less than 5 ␮M of rified on a reverse-phase-high-performance liquid chromatog- free silver(I) ions are released into solution (13). Therefore, the antimicrobial effect of silver nanoparticles cannot be at- The cyclic polycationic lipopeptide polymyxin B is effective tributed to the release of silver(I) ions from the nanoparticles, against gram-negative bacteria, interacting with lipid A and yet sensitivity to silver may differ for various bacterial strains or disrupting their outer membranes (16). Gramicidin A and ala- TABLE 1. MIC values for silver and peptidesa MIC for AgNP MIC for PGLa MIC for Mag2 MIC for GS MIC for PMB MIC for Alam MIC for GA a AgNO , silver nitrate; AgNP, silver nanoparticles; PGLa, GMASKAGAIAGKIAKVAL-KAL-NH ; Mag2, magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS); GS, gramicidin S (cyclo͓VOLDFP͔ , where D shows the stereocenter of the amino acid); PMB, polymyxin B {(S)-6-methyloctanoyl-BTB-cyclo͓BBDFLBBT͔; B, diami- nobutyric acid}; Alam, alamethicin (Ac-XPXAXAQXVXGL-XPVXXEQ-Fol; X, ␣-aminoisobutyric acid); GA, gramicidin A (HCO-VGADLADVVVDWDLWDLW b E. coli, Escherichia coli; A. calcoaceticus, Acinetobacter calcoaceticus; E. helveticus, Enterobacter helveticus; A. bestiarum, Aeromonas bestiarum; P. myxofaciens, Proteus myxofaciens; P. fluorescens, Pseudomonas fluorescens; B. subtilis, Bacillus subtilis; K. rhizophila, Kocuria rhizophila; M. luteus, Micrococcus luteus.
* Corresponding author. Mailing address: KIT, IOC, Fritz-Haber- Weg 6, 76131 Karlsruhe, Germany. Phone and fax: 49-0721-6083912.
E-mail: [email protected].
ᰔ Published ahead of print on 15 June 2009.
The MIC is the lowest concentration of a (single) antibiotic that inhibits the visible growth of microorganisms (1). Assayswere carried out in salt-free Luria/Miller-bouillon growth me- dium for three different gram-positive and six gram-negative bacterial strains. To dissolve silver nanoparticles for the MIC and hemolytic assays, an 8 mM Tris/HCl buffer [pH 7.6] con- taining 150 mM NaCl was used. Typical MICs, summarized in Table 1, were around 16 ␮g/ml of PGLa (for both gram- positive and gram-negative bacteria) and 1 ␮g/ml of gramicidin A (for gram-positive bacteria only) and polymyxin B (for gram- negative bacteria only). Similar MICs of about 16 ␮g/ml of both silver nitrate and silver nanoparticles were obtained. It is remarkable that the metallic silver nanoparticles and the ionic solution exhibit similar activities at almost the same mass per volume, given that each nanoparticle contains about 650,000 silver atoms. This observation supports the suggestion that the toxicity of the nanoparticles cannot be attributed to the release of free silver ions (see above) (13).
To detect any synergism between the peptides and silver, a two-dimensional microdilution assay was used (7). Assays were carried out in salt-free Luria/Miller-bouillon growth medium.
The combined antibiotic effect of agents A and B (where A is either AgNO or silver nanoparticles, and B is one of six AMPs) was calculated as follows: the fractional inhibitory con- centration (FIC) index ϭ MIC (A in combination with B)/MIC (A alone) ϩ MIC (B in combination with A)/MIC (B alone).
FIC index values above 2.0 indicate antagonistic effects, values between 0.5 and 2.0 indicate additive effects, and values lowerthan 0.5 indicate synergistic effects (7). The synergistic pair of PGLa and magainin 2 was used as a positive control. The FIC indices in Table 2 show the following results: polymyxin B acts synergistically with silver nanoparticles against all gram-nega- tive bacteria, while with AgNO it shows synergy only against Escherichia coli, Enterobacter helveticus, and Proteus myxofa- ciens. In addition, gramicidin S shows synergy with silver nano- particles against E. helveticus, P. myxofaciens, and Pseudomo- nas fluorescens, and with AgNO only against E. helveticus. All the other combinations work only in an additive manner. ‘Acin- etobacter calcoaceticus’ was not sensitive to silver nanoparticles even at the highest concentration (1,024 ␮g/ml). We may nev- ertheless conclude that silver nanoparticles generally show more synergy than silver(I) ion against most strains.
Hemolytic activity was examined using a modified serial dilution assay carried out in 8 mM Tris/HCl buffer [pH 7.6] containing 150 mM NaCl (17). The ability of AMPs to release hemoglobin from human erythrocytes was measured photo- metrically at different peptide concentrations. Triton X-100(Roth, Karlsruhe, Germany) was used to define 100% hemo- lysis. For every experimental run, the value for autohemolysis was measured and subsequently subtracted from all hemolytic values. All peptides were tested alone and in combination with two different concentrations of AgNO and silver nanopar- ticles to obtain the AMP concentration (in ␮g/ml) required to induce 50% hemolysis (HC ). It was shown that increasing the concentration of AgNO in combination with any AMP en- hances its hemolytic activity (Table 3). However, the combina- tion of AMPs with silver nanoparticles does not increase he- their own could not be determined, as they had low levels of values for silver alone or in combination with peptides values are in ␮g/ml. Values in brackets are standard deviations for three independent measurements. For abbreviations of antibacterial agents, see Table 1, hemolytic activity even at very high concentrations (1,024 respiratory chain of Escherichia coli: an electrochemical and scanning elec- trochemical microscopy study of the antimicrobial mechanism of micromolar
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