- Email: [email protected]
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman* Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel *Correspondence: [email protected] A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recently developed in our lab. We hypothesized that this hybrid carries potential as new antibacterial agent to combat biofilms: by hybrid penetration into the biofilm and scavenging of glucose traces, hydrogen peroxide will be formed by the enzyme, subsequently releasing silver ions from the hybrid’s silver “shell” by local chemical oxidation. These in situ released silver ions are expected effectively to kill bacterial cells located within their immediate vicinity. We designed and established a working flow system for in vitro biofilm growth and comparison of the efficacy of the antibacterial activity of several forms of silver and the hybrid on E. coli biofilms. Results obtained demonstrated the feasibility of the working hypothesis, thus paving the way for subsequent in vivo studies. Key words: Bacterial biofilm – Antibacterial activity – Silver – Glucose oxidase – Enzyme-metal hybrids – Nanoparticles.
Infection of medical devices and chronic wounds is often associated with the formation of bacterial biofilms [1,2]. Such biofilms are formed by the proliferation of a single microorganism, attached to the surface of a wound or implant, into micro-colonies embedded within a complex structure [3]. The bacterial cells within this structure are attached to each other by means of proteins and an interwoven exopolysaccharide matrix [4,5]. Several types of inter-cellular communications take place in these biofilms [6]. As the biofilm matures, single microorganisms are detached into the medium, migrate and re-colonize a new site, expanding the size of the infected site [7, 8]. Silver has been used for a long time as an antimicrobial agent in many medical preparations. Silver ions, either soluble or complexed, colloidal silver preparations and silver nanoparticles have been found effective for the treatment of bacterial infections of chronic wounds or biomaterials [9-11]. The antibacterial activity of silver is primarily attributed to intracellular interaction of these ions with enzymatic activities and DNA [12-15]. The migration of these ions is usually very limited by the interaction of silver ions with anions such as chloride, resulting in water-insoluble salts. Effective supply or release of silver ions has therefore to be provided within the close vicinity of the targeted bacterial cells. A novel methodology for the preparation of enzyme-silver hybrids was recently developed in our lab [16]. The hybrids were prepared by a new approach directing the process of electroless deposition of silver to the surface of single, soluble enzyme molecules. The first silver-enzyme hybrid prepared using this method was silver-glucose oxidase hybrid which retained its glucose oxidation biocatalytic activity. Furthermore, it was demonstrated that the crystalline silver coating of the enzyme’s surface (Figure 1) was effective for the “nanowiring” of this hybrid to electrodes for glucose determination in the absence of oxygen [16]. In the presence of oxygen, glucose oxidase will oxidize glucose to gluconolactone, a process accompanied by the formation of hydrogen peroxide as by-product [17]. The hydrogen peroxide thus obtained may in turn oxidize some of the metallic silver coating of the enzyme to release silver ions [18]. In this paper we propose and demonstrate in vitro feasibility of a new mechanism of enzymatically attenuated in situ release of silver ions from the silver-glucose oxidase hybrid, to combat infections based on bacterial biofilm (Figure 2). This working hypothesis makes use of the solubility and the nanometric size of the silver-enzyme hybrid,
Ag
Figure 1 - High resolution transmission electron microscope (Tecnai F20, Philips) micrographs, obtained without staining for silver-glucose oxidase hybrid.The chemical identity of the silver deposits was confirmed by EDX (inserts) [16]. Bar: 5 nm.
allowing its diffusion into the targeted biofilm followed by scavenging residual glucose molecules present within the biofilm’s pores. The resulting in situ release of silver ions within the close proximity of the targeted cells is designed to result in effective antibacterial activity.
I. MATERIALS AND METHODS 1. Bacterial strain
E. coli MG1655 K12 was used as model strain for in vitro biofilm growth. LB standard growth medium was used for cell growth [19]. Silver-based antibacterial treatments were carried out in 50 mmol imidazole isotonic buffer pH 7.5, containing 150 mmol sodium nitrate. This buffer is compatible with silver ions without precipitation. 25
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
A
H B
+ O2 + H2O2
C D F G
E
H2O2 + 2Ag0 + 2H+ 2Ag+ + 2H2O
PGA (polyglutaraldehyde)
Figure 3 - Details of culture well chamber: (A) Inlet solution hose; (B) chamber seal cover; (C) culture well chamber; (D) liquid solution; (E) glass disc; (F) the biofilm; (G) closer look at the biofilm on the glass disc; outlet solution hose (H). Thick arrows indicate the direction of flow.
β-alanine Ag atomic Ag ion
0-0.17 mg/mL (0-1 mmol) were prepared in 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5. Suspensions of silver nanoparticles were prepared in two concentrations as follows: a 0.17 mg/mL silver nanoparticle suspension was prepared by mixing 1.25 mL of 2 mmol poly-glutaraldehyde (PGA) in DDW with 1 mL of a 50 mmol b-alanine in DDW, followed by the addition of 0.5 mL of a 8.494 mg/mL AgNO3 in DDW. The resulting solution was diluted with DDW to a final volume of 25 mL and incubated overnight at 30°C. The suspension of silver nanoparticles thus obtained (15-33 nm in size) was purified from silver ions by centrifugation (10 min, 6000 g) and the pellet was resuspended in 25 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5. Suspension of 0.017 mg/mL silver nanoparticles was similarly prepared by using one-tenth of the mixed solution input. A combined 2 mg/mL untreated glucose oxidase and 0.017 mg/mL silver nanoparticle suspension was prepared by resuspension of the nanoparticles pellet in 20 mL of a 2.5 mg/mL glucose oxidase (type VII from Aspergillus niger, Sigma) dissolved in 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 and further dilution with 5 mL of the same isotonic buffer. Treatment solutions were pumped in the dark into each of the four cells for 4 h at a flow rate of 0.025 mL/min.
Figure 2 - Proposed mechanism for attenuated in situ release of silver ions from the silver-glucose oxidase hybrid In the presence of glucose and oxygen. Enzymatic reaction is depicted in black while silver oxidation in grey.
2. Establishment of working system
A working system allowing controlled and reproducible growth and treatment of model E. coli biofilm was developed as a set of simultaneously operated four-flow cells, equipped with 13 mm diameter sterile glass discs, serving as the substrate for biofilm development. Growth or treatment solutions were pumped into each flow cell at a flow rate of 0.29 mL/min (see Figure 3 for in-well settings) under sterile conditions by means of a multi-channeled peristaltic pump (Minipuls 3, Gilson).
3. Biofilm growth
The model biofilm was constructed by a two step procedure: attachment and growth. Cell attachment was effected onto each glass disc by the addition of E. coli cell suspension (OD620 = 0.17) in 2 mL of standard LB growth medium into each well of a set of four wells in a Costar Cat No 3524 multiwell plate, each fitted with glass disc. Following static incubation for one hour at 30°C for cell attachment the glass discs were removed from the hosting wells, washed with 2 mL sterile PBS and transferred under sterile conditions – with their treated side upwards – into a new set of four wells. Each well was then sealed with an application unit connected to the multi-channel peristaltic pump and fresh LB medium was pumped through the well for 48 h at a flow rate of 0.29 mL/min to effect biofilm growth (see Figure 3 for in-well settings).
5. Silver-glucose oxidase hybrid solution
Silver-glucose oxidase hybrid was prepared after Dagan-Moscovich et al. [16]. Silver-glucose oxidase hybrid treatment solutions were produced by diluting the product obtained after [16] with 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 to final concentrations of 0.1-2 mg/mL. Hybrid solutions containing glucose were prepared using the same procedure with 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 containing 0.2% (w/v) glucose. High resolution transmission electron microscopy (Tecnai F20 TEM, Philips) micrographs of the hybrid were obtained without staining by absorbing 10 µL solution on TEM grids (Carbon coated 200 mesh
4. Silver ion and silver nanoparticle solutions
Silver ion solutions (AgNO3) within the concentration range of 26
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
grids, SPI), and drying at room temperature for 24 h. The chemical identity of the silver deposits was confirmed by energy dispersive X-ray spectroscopy (EDX). Treatment solutions were pumped in the dark into each cell for 4 h at a flow rate of 0.025 ml/min.
6. Biofilm post-treatment residual viability
The residual post-treatment viability of treated biofilms was determined by standard viable count assay [20]. The cells embedded within the treated biofilms were recovered as freely suspended cell population by sonication carried out following washing of treated biofilms with 6 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 (3 mL applied on each side of the glass disc to remove any washable cells). The remaining bacterial biofilm core was detached from the surface of the glass disc by sonication for 5 min in Sonicator bath (SC-52H, Sonicor) in 2 mL of 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 followed by vortex mixing (5 min). The number of colony forming units (CFU) surviving the antibacterial treatments was determined by dilution and growth on LB agar plates [21]. Data obtained was presented as relative residual CFU for comparing the efficacy of each treatment.
7. Characterization of the biofilms by scanning confocal laser microscopy (SCLM)
Characterization of untreated biofilms by SCLM was carried out by staining with 0.2 mL of a 0.5 mg/mL Safranine-O (Sigma) for 5 min [22-24]. The stained biofilms were rinsed with 6 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 by rinsing with 3 mL of each side of the glass disc. Double staining was carried out with 0.2 mL of a 2.5 µmol Sytox green (Molecular probes) for 10 min [25]. The double-stained glass discs were rinsed again with the treatment buffer. Visualization of live or dead cells embedded within the stained biofilm was carried out by scanning confocal laser microscope (LSM 510, Zeiss). The SCLM argon gas excited the Safranine-O and the Sytox green stains at 488 nm and emitted light was detected at 586 and 523 nm, respectively.
Figure 4 - Images of a mature untreated biofilm from a scanning confocal laser microscopy (SCLM). (A) A 2D X-Z plane view and (B) a 2D X-Y plane view (upper left box - Safranine-O stain, upper right box - Sytox green stain, lower left box - transmitted light image and lower right box - all 3 boxes together). Bar : 20 µm.
II. RESULTS 1. Establishment of working system
Infectious biofilms usually thrive on nutrients supplied by a continuous stream of moving aqueous phase [26]. The working system adopted for this study was accordingly established to mimic biofilm growth in nature. This working system provided continuous flow of solutions providing nutrients for biofilm growth, antibacterial treatment and washing, mediated by means of a multi-channel peristaltic pump into a set of four culture wells. Continuous drainage was simultaneously effected (see Figure 3 for in-well setting).
3. Antibacterial activity of silver ions and silver nanoparticles
The impact of continuous flow of a series of input concentration of silver ions and silver nanoparticles on biofilm viability was investigated. The antibacterial activity of a series of silver ion input concentrations is shown in Figure 5. Relative residual CFU values resulting from the impact of streaming 0-0.1 mg/mL (0-0.06 mmol) silver nitrate in treatment medium for 4 h clearly indicated a threshold effect for silver nitrate (silver ionic form) input at 0.06 mg/mL (0.036 mmol or higher) required for effecting total loss of bacterial biofilm viability. The impact of shifting from silver ion form to suspension of silver metallic nanoparticles was compared for same low and high silver inputs (0.017 and 0.17 mg/mL, respectively, Figure 6). Results obtained indicate that for the same low input of silver, the silver ionic form was much more effective, exhibiting a higher specific activity (residual CFU of ~6 vs ~19% recorded for the nanoparticles).At 0.17 mg/mL input of silver nanoparticles total loss of bacterial biofilm viability was readily achieved.
2. In vitro biofilm growth and its characterization
The established working system allowed a continuous stream of fresh growth medium into the culture-well chamber providing a steady-state environment to support the growth of biofilm on the surface of inoculated glass discs. Growth phase was maintained for 48 h. The biofilms thus obtained exhibited a three-dimensional structure (see Figure 4 for images obtained by confocal microscopy). The confocal microscopy images indicated that the biofilm grew on the glass disc as sporadic spots ~120-300 µm in diameter and 15-40 µm in height. Double staining assay confirmed the presence of live bacteria within the biofilm (Figure 4) and that the biofilms obtained were mature [27, 28]. The reproducibility and viability of the biofilms thus obtained were confirmed to be 1.43 · 107 colony forming units (CFU) ± 18% per disc, by repetitive growth experiments and determination of CFU obtained from biofilms disintegrated by sonication.
4. Antibacterial activity of silver-glucose oxidase hybrid
The effect of a series of input concentrations of silver-glucose oxidase inputs on biofilm viability was investigated in the presence or absence of added glucose (Figure 7). 27
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
Relative residual CFU values resulting from the impact of streaming an input of 0-2 mg/mL hybrid in 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 for 4 hrs clearly indicated a threshold effect observed for hybrid concentrations of 1 mg/mL and higher in the presence of 0.2% (W/V) glucose and 0.2 mg/mL in the absence of glucose. The presence of glucose effected a decrease in the antimicrobial activity of the hybrid, reflected in the distinctively lower threshold recorded in the absence of glucose. The combination of untreated glucose oxidase with suspension of 0.017 mg/mL silver nanoparticles exhibited antimicrobial activity lower than the activity recorded for the same silver nanoparticles input in the absence of the enzyme.
Silver ion concentration impact on biofilm viability 14
Relative residual CFU (%)
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0 0
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0.02
0.03
0.04
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5. Comparison of antibacterial performance of ionic, particulate and enzyme-hybrid silver formulations
Silver ions concentration (mg/ml)
Figure 5 - The impact of silver ion input on biofilm viability.
The antibacterial effectiveness of the silver-glucose oxidase hybrid in the presence and absence of glucose were compared with that of the same input of silver in the form of ions, nanoparticles and nanoparticles combined with native glucose oxidase (Figure 8). Results clearly indicated that for the same silver input tested (0.017 mg/mL) the silver-glucose oxidase hybrid exhibited the most effective antibacterial activity.
25 Silver ions Silver nanoparticles
Relative residual CFU (%)
20
15
10
III. DISCUSSION
A working system providing simple methodology and monitoring for the growth and antibacterial treatment of E. coli biofilms was developed. This system provided a convenient tool for a comparative study of the alternative modes of applying silver formulations as antibacterial treatments to combat biofilms under continuous flow as well as demonstration of the feasibility of the working hypothesis. E. coli biofilms were routinely readily established within the continuous flow cell systems developed. Ionic silver was found to be an effective agent for combating the model biofilm at 36 µmol and higher concentrations. This result is substantially higher than results reported for the concentration required to kill bacterial cells in wounds (100 ppm) [29], a result reflecting the limited diffusion of free silver ions within the biofilm. The introduction of silver in the form of nanoparticles resulted in lower antibacterial efficacy either in comparison with the abovementioned performance of ionic silver or with the value of 0.06 mg/mL required for total growth inhibition in agar plates [11]. A combination of silver nanoparticles suspension with native glucose oxidase was less effective than the same nanoparticles input. As the antibacterial treatment of wounds with the ionic form of silver involves side effects such as precipitation, coloration and irritation, we proposed the mechanism and use of enzymatically attenuated in situ release of silver ions from the new silver-glucose oxidase hybrid as an alternative mode of silver application. The working hypothesis was that this nano-sized soluble and enzymatically active hybrid may diffuse into the pores of bacterial biofilms. Upon scavenging residual glucose traces remaining inside the biofilm, hydrogen peroxide will be locally produced by the enzyme, subsequently releasing by chemical oxidation silver ions from its adjunct hybrid’s silver “shell”. As these ions are released into the immediate vicinity of the targeted cells they are expected to be a highly effective antibacterial agent. The results described above from our feasibility study indicate the feasibility of this working hypothesis: application of the silver-glucose oxidase hybrid at different concentrations exhibited a threshold effect of 0.1 mg/mL hybrid concentration (estimated silver input of 0.017 mg/mL), indicating high specific antibacterial activity exhibited under continuous flow conditions providing the highest antibacterial activity of all silver forms tested for this input. Furthermore, it clearly appeared that glucose addition into the medium resulted in lower specific antibacterial activity. This observation may be explained by silver ions released from the hybrid into the bulk medium instead of
5
0 0.017
0.17
Silver concentration (mg/ml)
Figure 6 - Comparison of the impact of silver ions and silver nanoparticles on biofilm viability. 60
In the absence of glucose In the presence of glucose
Relative residual CFU (%)
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Figure 7 - The impact of silver-glucose oxidase hybrid on biofilm viability in presence and absence of glucose. 70
0.017 mg/ml silver ions 0.017 mg/ml silver nanoparticles
Relative residual CFU (%)
60
50
2 mg/ml glucose oxidase + 0.017 mg/ml silver nanoparticles 0.1 mg/ml silver-glucose oxidase hybrid (0.017 mg/ml silver) + 0.2% glucose 0.1 mg/ml silver-glucose oxidase hybrid (0.017 mg/ml silver)
40
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0
Antimicrobial treatment
Figure 8 - Comparison of the impact of same input of silver in the forms of ions, nanoparticles and silver-glucose oxidase hybrid on biofilm viability.
28
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
inside the targeted biofilm, resulting in silver ion release into the bulk medium with dilution and low efficacy. Moreover, results obtained from mixing of silver nanoparticles with untreated enzyme also exhibited poor antibacterial activity, indicating the essential role of integration of the glucose oxidase enzymatic activity and the metallic silver into a hybrid form required for achieving highly effective antibacterial activity. All the above mentioned results indicate that, in accordance with the literature, the final silver form which has the killing effect is the silver ionic form [30]. The data obtained from this study now pave the way for the design and investigation of the antibacterial efficacy of the new hybrid in vivo in preclinical trials.
16.
17. 18. 19. 20.
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reductase in a marine Vibrio alginolyticus. - Biochim. Biophys. Acta, 1099, 145-151, 1992. Dagan-Moscovich H., Cohen-Hadar N., Porat C., Rishpon J., Shacham-Diamand Y., Freeman A. - Nanowiring of the catalytic site of novel molecular enzyme-metal hybrids to electrodes. - J. Phys. Chem. C, 111, 5766-5769, 2007. Wilson R., Turner A.P.F. - Glucose oxidase: an ideal enzyme. Biosens. Bioelectron., 7, 165-185, 1992. Al-Salehi S.K., Hatton P.V., McLeod C.W., Cox A.G. - The effect of hydrogen peroxide concentration on metal ion release from dental amalgam. - J. Dent., 35, 172-176, 2007. Domka J., Lee J., Bansal T., Wood T.K. - Temporal gene-expression in Escherichia coli K-12 biofilms. - Environ. Microbiol., 9, 332-346, 2007. Whiteley M., Brown E., McLean R.J.C. - An inexpensive chemostat apparatus for the study of microbial biofilms. - J. Microbiol. Methods, 30, 125-132, 1997. Adams J.L., McLean R.J.C. - Impact of rpoS deletion on Escherichia coli biofilms. - Appl. Environ. Microbiol., 65, 4285-4287, 1999. Beveridge T.J. - Use of the gram stain in microbiology. - Biotech. Histochem., 76, 111-118, 2001. Prior D.A.M., Oparka K.J., Roberts I.M. - En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope. - J. Microsc., 193, 20-27, 1999. Akiyama H., Hamada T., Huh W-K., Yamasaki O., Oono T., Fujimoto W., Iwatsuki K. - Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of bullous impetigo, atopic dermatitis and pemphigus foliaceus. - Br. J. Dermatol., 148, 526-532, 2003. Burnett S.L., Beuchat L.R. - Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers. - Int. J. Food Microbiol., 74, 37-45, 2002. McLean R.J., Bates C.L., Barnes M.B., McGowin C.L., Aron G.M. - Methods of studying biofilms. - In: Microbial Biofilms, M. Ghannoum, G.A. O'Toole Eds., ASM Press, Washington DC, 2004, pp. 379-413. Tolker-Nielsen T., Molin S. - Spatial organization of microbial biofilm communities. - Microb. Ecol., 40, 75-84, 2000. Banin E., Vasil M.L., Greenberg E.P. - Iron and Pseudomonas aeruginosa biofilm formation. - Proc. Natl. Acad. Sci. USA, 102, 11076-11081, 2005. Melaiye A, Youngs WJ. - Silver and its application as an antimicrobial agent - Expert Opinion Ther. Patents, 15, 125-130, 2005. Lok C-N., Ho C-M., Chen R., He Q-Y., Yu W-Y., Sun H., Tam P.K-H., Chiu J-F., Che C-M. - Proteomic analysis of the mode of antibacterial action of silver nanoparticles. - J. Proteome Res., 5, 916-924, 2006.
Acknowledgments This study was supported in part by the Seroussi Chair for Protein NanoBiotechnology.
Manuscript Received 5 June 2007, accepted for publication 23 August 2007.
29
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman* Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel *Correspondence: [email protected] A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recently developed in our lab. We hypothesized that this hybrid carries potential as new antibacterial agent to combat biofilms: by hybrid penetration into the biofilm and scavenging of glucose traces, hydrogen peroxide will be formed by the enzyme, subsequently releasing silver ions from the hybrid’s silver “shell” by local chemical oxidation. These in situ released silver ions are expected effectively to kill bacterial cells located within their immediate vicinity. We designed and established a working flow system for in vitro biofilm growth and comparison of the efficacy of the antibacterial activity of several forms of silver and the hybrid on E. coli biofilms. Results obtained demonstrated the feasibility of the working hypothesis, thus paving the way for subsequent in vivo studies. Key words: Bacterial biofilm – Antibacterial activity – Silver – Glucose oxidase – Enzyme-metal hybrids – Nanoparticles.
Infection of medical devices and chronic wounds is often associated with the formation of bacterial biofilms [1,2]. Such biofilms are formed by the proliferation of a single microorganism, attached to the surface of a wound or implant, into micro-colonies embedded within a complex structure [3]. The bacterial cells within this structure are attached to each other by means of proteins and an interwoven exopolysaccharide matrix [4,5]. Several types of inter-cellular communications take place in these biofilms [6]. As the biofilm matures, single microorganisms are detached into the medium, migrate and re-colonize a new site, expanding the size of the infected site [7, 8]. Silver has been used for a long time as an antimicrobial agent in many medical preparations. Silver ions, either soluble or complexed, colloidal silver preparations and silver nanoparticles have been found effective for the treatment of bacterial infections of chronic wounds or biomaterials [9-11]. The antibacterial activity of silver is primarily attributed to intracellular interaction of these ions with enzymatic activities and DNA [12-15]. The migration of these ions is usually very limited by the interaction of silver ions with anions such as chloride, resulting in water-insoluble salts. Effective supply or release of silver ions has therefore to be provided within the close vicinity of the targeted bacterial cells. A novel methodology for the preparation of enzyme-silver hybrids was recently developed in our lab [16]. The hybrids were prepared by a new approach directing the process of electroless deposition of silver to the surface of single, soluble enzyme molecules. The first silver-enzyme hybrid prepared using this method was silver-glucose oxidase hybrid which retained its glucose oxidation biocatalytic activity. Furthermore, it was demonstrated that the crystalline silver coating of the enzyme’s surface (Figure 1) was effective for the “nanowiring” of this hybrid to electrodes for glucose determination in the absence of oxygen [16]. In the presence of oxygen, glucose oxidase will oxidize glucose to gluconolactone, a process accompanied by the formation of hydrogen peroxide as by-product [17]. The hydrogen peroxide thus obtained may in turn oxidize some of the metallic silver coating of the enzyme to release silver ions [18]. In this paper we propose and demonstrate in vitro feasibility of a new mechanism of enzymatically attenuated in situ release of silver ions from the silver-glucose oxidase hybrid, to combat infections based on bacterial biofilm (Figure 2). This working hypothesis makes use of the solubility and the nanometric size of the silver-enzyme hybrid,
Ag
Figure 1 - High resolution transmission electron microscope (Tecnai F20, Philips) micrographs, obtained without staining for silver-glucose oxidase hybrid.The chemical identity of the silver deposits was confirmed by EDX (inserts) [16]. Bar: 5 nm.
allowing its diffusion into the targeted biofilm followed by scavenging residual glucose molecules present within the biofilm’s pores. The resulting in situ release of silver ions within the close proximity of the targeted cells is designed to result in effective antibacterial activity.
I. MATERIALS AND METHODS 1. Bacterial strain
E. coli MG1655 K12 was used as model strain for in vitro biofilm growth. LB standard growth medium was used for cell growth [19]. Silver-based antibacterial treatments were carried out in 50 mmol imidazole isotonic buffer pH 7.5, containing 150 mmol sodium nitrate. This buffer is compatible with silver ions without precipitation. 25
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
A
H B
+ O2 + H2O2
C D F G
E
H2O2 + 2Ag0 + 2H+ 2Ag+ + 2H2O
PGA (polyglutaraldehyde)
Figure 3 - Details of culture well chamber: (A) Inlet solution hose; (B) chamber seal cover; (C) culture well chamber; (D) liquid solution; (E) glass disc; (F) the biofilm; (G) closer look at the biofilm on the glass disc; outlet solution hose (H). Thick arrows indicate the direction of flow.
β-alanine Ag atomic Ag ion
0-0.17 mg/mL (0-1 mmol) were prepared in 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5. Suspensions of silver nanoparticles were prepared in two concentrations as follows: a 0.17 mg/mL silver nanoparticle suspension was prepared by mixing 1.25 mL of 2 mmol poly-glutaraldehyde (PGA) in DDW with 1 mL of a 50 mmol b-alanine in DDW, followed by the addition of 0.5 mL of a 8.494 mg/mL AgNO3 in DDW. The resulting solution was diluted with DDW to a final volume of 25 mL and incubated overnight at 30°C. The suspension of silver nanoparticles thus obtained (15-33 nm in size) was purified from silver ions by centrifugation (10 min, 6000 g) and the pellet was resuspended in 25 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5. Suspension of 0.017 mg/mL silver nanoparticles was similarly prepared by using one-tenth of the mixed solution input. A combined 2 mg/mL untreated glucose oxidase and 0.017 mg/mL silver nanoparticle suspension was prepared by resuspension of the nanoparticles pellet in 20 mL of a 2.5 mg/mL glucose oxidase (type VII from Aspergillus niger, Sigma) dissolved in 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 and further dilution with 5 mL of the same isotonic buffer. Treatment solutions were pumped in the dark into each of the four cells for 4 h at a flow rate of 0.025 mL/min.
Figure 2 - Proposed mechanism for attenuated in situ release of silver ions from the silver-glucose oxidase hybrid In the presence of glucose and oxygen. Enzymatic reaction is depicted in black while silver oxidation in grey.
2. Establishment of working system
A working system allowing controlled and reproducible growth and treatment of model E. coli biofilm was developed as a set of simultaneously operated four-flow cells, equipped with 13 mm diameter sterile glass discs, serving as the substrate for biofilm development. Growth or treatment solutions were pumped into each flow cell at a flow rate of 0.29 mL/min (see Figure 3 for in-well settings) under sterile conditions by means of a multi-channeled peristaltic pump (Minipuls 3, Gilson).
3. Biofilm growth
The model biofilm was constructed by a two step procedure: attachment and growth. Cell attachment was effected onto each glass disc by the addition of E. coli cell suspension (OD620 = 0.17) in 2 mL of standard LB growth medium into each well of a set of four wells in a Costar Cat No 3524 multiwell plate, each fitted with glass disc. Following static incubation for one hour at 30°C for cell attachment the glass discs were removed from the hosting wells, washed with 2 mL sterile PBS and transferred under sterile conditions – with their treated side upwards – into a new set of four wells. Each well was then sealed with an application unit connected to the multi-channel peristaltic pump and fresh LB medium was pumped through the well for 48 h at a flow rate of 0.29 mL/min to effect biofilm growth (see Figure 3 for in-well settings).
5. Silver-glucose oxidase hybrid solution
Silver-glucose oxidase hybrid was prepared after Dagan-Moscovich et al. [16]. Silver-glucose oxidase hybrid treatment solutions were produced by diluting the product obtained after [16] with 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 to final concentrations of 0.1-2 mg/mL. Hybrid solutions containing glucose were prepared using the same procedure with 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 containing 0.2% (w/v) glucose. High resolution transmission electron microscopy (Tecnai F20 TEM, Philips) micrographs of the hybrid were obtained without staining by absorbing 10 µL solution on TEM grids (Carbon coated 200 mesh
4. Silver ion and silver nanoparticle solutions
Silver ion solutions (AgNO3) within the concentration range of 26
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
grids, SPI), and drying at room temperature for 24 h. The chemical identity of the silver deposits was confirmed by energy dispersive X-ray spectroscopy (EDX). Treatment solutions were pumped in the dark into each cell for 4 h at a flow rate of 0.025 ml/min.
6. Biofilm post-treatment residual viability
The residual post-treatment viability of treated biofilms was determined by standard viable count assay [20]. The cells embedded within the treated biofilms were recovered as freely suspended cell population by sonication carried out following washing of treated biofilms with 6 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 (3 mL applied on each side of the glass disc to remove any washable cells). The remaining bacterial biofilm core was detached from the surface of the glass disc by sonication for 5 min in Sonicator bath (SC-52H, Sonicor) in 2 mL of 50 mmol imidazole150 mmol sodium nitrate isotonic buffer pH 7.5 followed by vortex mixing (5 min). The number of colony forming units (CFU) surviving the antibacterial treatments was determined by dilution and growth on LB agar plates [21]. Data obtained was presented as relative residual CFU for comparing the efficacy of each treatment.
7. Characterization of the biofilms by scanning confocal laser microscopy (SCLM)
Characterization of untreated biofilms by SCLM was carried out by staining with 0.2 mL of a 0.5 mg/mL Safranine-O (Sigma) for 5 min [22-24]. The stained biofilms were rinsed with 6 mL of 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 by rinsing with 3 mL of each side of the glass disc. Double staining was carried out with 0.2 mL of a 2.5 µmol Sytox green (Molecular probes) for 10 min [25]. The double-stained glass discs were rinsed again with the treatment buffer. Visualization of live or dead cells embedded within the stained biofilm was carried out by scanning confocal laser microscope (LSM 510, Zeiss). The SCLM argon gas excited the Safranine-O and the Sytox green stains at 488 nm and emitted light was detected at 586 and 523 nm, respectively.
Figure 4 - Images of a mature untreated biofilm from a scanning confocal laser microscopy (SCLM). (A) A 2D X-Z plane view and (B) a 2D X-Y plane view (upper left box - Safranine-O stain, upper right box - Sytox green stain, lower left box - transmitted light image and lower right box - all 3 boxes together). Bar : 20 µm.
II. RESULTS 1. Establishment of working system
Infectious biofilms usually thrive on nutrients supplied by a continuous stream of moving aqueous phase [26]. The working system adopted for this study was accordingly established to mimic biofilm growth in nature. This working system provided continuous flow of solutions providing nutrients for biofilm growth, antibacterial treatment and washing, mediated by means of a multi-channel peristaltic pump into a set of four culture wells. Continuous drainage was simultaneously effected (see Figure 3 for in-well setting).
3. Antibacterial activity of silver ions and silver nanoparticles
The impact of continuous flow of a series of input concentration of silver ions and silver nanoparticles on biofilm viability was investigated. The antibacterial activity of a series of silver ion input concentrations is shown in Figure 5. Relative residual CFU values resulting from the impact of streaming 0-0.1 mg/mL (0-0.06 mmol) silver nitrate in treatment medium for 4 h clearly indicated a threshold effect for silver nitrate (silver ionic form) input at 0.06 mg/mL (0.036 mmol or higher) required for effecting total loss of bacterial biofilm viability. The impact of shifting from silver ion form to suspension of silver metallic nanoparticles was compared for same low and high silver inputs (0.017 and 0.17 mg/mL, respectively, Figure 6). Results obtained indicate that for the same low input of silver, the silver ionic form was much more effective, exhibiting a higher specific activity (residual CFU of ~6 vs ~19% recorded for the nanoparticles).At 0.17 mg/mL input of silver nanoparticles total loss of bacterial biofilm viability was readily achieved.
2. In vitro biofilm growth and its characterization
The established working system allowed a continuous stream of fresh growth medium into the culture-well chamber providing a steady-state environment to support the growth of biofilm on the surface of inoculated glass discs. Growth phase was maintained for 48 h. The biofilms thus obtained exhibited a three-dimensional structure (see Figure 4 for images obtained by confocal microscopy). The confocal microscopy images indicated that the biofilm grew on the glass disc as sporadic spots ~120-300 µm in diameter and 15-40 µm in height. Double staining assay confirmed the presence of live bacteria within the biofilm (Figure 4) and that the biofilms obtained were mature [27, 28]. The reproducibility and viability of the biofilms thus obtained were confirmed to be 1.43 · 107 colony forming units (CFU) ± 18% per disc, by repetitive growth experiments and determination of CFU obtained from biofilms disintegrated by sonication.
4. Antibacterial activity of silver-glucose oxidase hybrid
The effect of a series of input concentrations of silver-glucose oxidase inputs on biofilm viability was investigated in the presence or absence of added glucose (Figure 7). 27
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
Relative residual CFU values resulting from the impact of streaming an input of 0-2 mg/mL hybrid in 50 mmol imidazole-150 mmol sodium nitrate isotonic buffer pH 7.5 for 4 hrs clearly indicated a threshold effect observed for hybrid concentrations of 1 mg/mL and higher in the presence of 0.2% (W/V) glucose and 0.2 mg/mL in the absence of glucose. The presence of glucose effected a decrease in the antimicrobial activity of the hybrid, reflected in the distinctively lower threshold recorded in the absence of glucose. The combination of untreated glucose oxidase with suspension of 0.017 mg/mL silver nanoparticles exhibited antimicrobial activity lower than the activity recorded for the same silver nanoparticles input in the absence of the enzyme.
Silver ion concentration impact on biofilm viability 14
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Figure 5 - The impact of silver ion input on biofilm viability.
The antibacterial effectiveness of the silver-glucose oxidase hybrid in the presence and absence of glucose were compared with that of the same input of silver in the form of ions, nanoparticles and nanoparticles combined with native glucose oxidase (Figure 8). Results clearly indicated that for the same silver input tested (0.017 mg/mL) the silver-glucose oxidase hybrid exhibited the most effective antibacterial activity.
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III. DISCUSSION
A working system providing simple methodology and monitoring for the growth and antibacterial treatment of E. coli biofilms was developed. This system provided a convenient tool for a comparative study of the alternative modes of applying silver formulations as antibacterial treatments to combat biofilms under continuous flow as well as demonstration of the feasibility of the working hypothesis. E. coli biofilms were routinely readily established within the continuous flow cell systems developed. Ionic silver was found to be an effective agent for combating the model biofilm at 36 µmol and higher concentrations. This result is substantially higher than results reported for the concentration required to kill bacterial cells in wounds (100 ppm) [29], a result reflecting the limited diffusion of free silver ions within the biofilm. The introduction of silver in the form of nanoparticles resulted in lower antibacterial efficacy either in comparison with the abovementioned performance of ionic silver or with the value of 0.06 mg/mL required for total growth inhibition in agar plates [11]. A combination of silver nanoparticles suspension with native glucose oxidase was less effective than the same nanoparticles input. As the antibacterial treatment of wounds with the ionic form of silver involves side effects such as precipitation, coloration and irritation, we proposed the mechanism and use of enzymatically attenuated in situ release of silver ions from the new silver-glucose oxidase hybrid as an alternative mode of silver application. The working hypothesis was that this nano-sized soluble and enzymatically active hybrid may diffuse into the pores of bacterial biofilms. Upon scavenging residual glucose traces remaining inside the biofilm, hydrogen peroxide will be locally produced by the enzyme, subsequently releasing by chemical oxidation silver ions from its adjunct hybrid’s silver “shell”. As these ions are released into the immediate vicinity of the targeted cells they are expected to be a highly effective antibacterial agent. The results described above from our feasibility study indicate the feasibility of this working hypothesis: application of the silver-glucose oxidase hybrid at different concentrations exhibited a threshold effect of 0.1 mg/mL hybrid concentration (estimated silver input of 0.017 mg/mL), indicating high specific antibacterial activity exhibited under continuous flow conditions providing the highest antibacterial activity of all silver forms tested for this input. Furthermore, it clearly appeared that glucose addition into the medium resulted in lower specific antibacterial activity. This observation may be explained by silver ions released from the hybrid into the bulk medium instead of
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Figure 6 - Comparison of the impact of silver ions and silver nanoparticles on biofilm viability. 60
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Figure 7 - The impact of silver-glucose oxidase hybrid on biofilm viability in presence and absence of glucose. 70
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Figure 8 - Comparison of the impact of same input of silver in the forms of ions, nanoparticles and silver-glucose oxidase hybrid on biofilm viability.
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Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study H. Ben-Yoav, A. Freeman
J. DRUG DEL. SCI. TECH., 18 (1) 25-29 2008
inside the targeted biofilm, resulting in silver ion release into the bulk medium with dilution and low efficacy. Moreover, results obtained from mixing of silver nanoparticles with untreated enzyme also exhibited poor antibacterial activity, indicating the essential role of integration of the glucose oxidase enzymatic activity and the metallic silver into a hybrid form required for achieving highly effective antibacterial activity. All the above mentioned results indicate that, in accordance with the literature, the final silver form which has the killing effect is the silver ionic form [30]. The data obtained from this study now pave the way for the design and investigation of the antibacterial efficacy of the new hybrid in vivo in preclinical trials.
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Acknowledgments This study was supported in part by the Seroussi Chair for Protein NanoBiotechnology.
Manuscript Received 5 June 2007, accepted for publication 23 August 2007.
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