Antibacterial activities of gold and silver nanoparticles against Escherichia coli and bacillus Calmette-Guérin
© Zhou et al.; licensee BioMed Central Ltd. 2012
Received: 12 January 2012
Accepted: 16 April 2012
Published: 6 May 2012
Diseases such as tuberculosis (TB) have always had a large impact on human health. Bacillus Calmette-Guérin (BCG) is used as a surrogate for TB during the development of anti-TB drugs. Nanoparticles (NPs) have attracted great interest in drug development. The purpose of this study was to examine the potential of NPs as anti-TB compounds by studying the interacting mechanisms between NPs and bacteria.
We investigated effects of gold and silver NPs on BCG and Escherichia coli. Experimentally, particle size and shape were characterized using transmission electron microscopy (TEM). Different concentrations of NPs were applied in bacterial culture. The growth of E. coli was monitored through colony forming units (CFU). The mechanism of interaction between NPs and bacteria was analyzed through bacterial thin sections followed by TEM and scanning electron microscopy. Antibacterial effects on BCG were observed by recording fluorescent protein expression levels.
The results suggest NPs have potential applications as anti-TB compounds. The antibacterial effects and mechanism of action for NPs were dependent upon composition and surface modifications.
KeywordsAntibacterial effect Gold Silver Nanoparticle BCG
Diseases such as tuberculosis (TB) have always had a large impact on human health. According to a recent World Health Organization (WHO) report, there were an estimated 11.1 million prevalent cases and 9.4 million incident cases of TB in 2008. There are 1.3 million TB related deaths each year . As a widely used TB vaccine, bacillus Calmette-Guérin (BCG) has been prepared from a strain of attenuated Mycobacterium bovis, which causes bovine tuberculosis. The virulence of BCG was lost after being cultured in potato medium for decades. In biosafety level 2 labs, it has been used as a surrogate for TB during the development of anti-TB drugs. Combination drug therapies are normally used against TB, since monotherapy fails to clear infections and leads to rapid development of resistance. There are drawbacks in current therapies, including drug-induced disease and the increasing prevalence of multiple-drug-resistant tuberculosis (MDR-TB). Nanoparticles (NPs) have attracted great interest in their development as potential antibacterial drugs [2, 3]. It has been reported that biophysical interactions occur between NPs and bacteria including biosorption, NPs breakdown or aggregation, and cellular uptake, with effects including membrane damage and toxicity [4, 5]. The mechanisms of NPs inhibiting bacterial growth remain less well understood. It has been reported that the size and surface modifications of NPs could affect their antibacterial levels [5, 6]. Comprehensive understanding of antibacterial mechanisms is needed to improve the effectiveness of NPs in disease treatment.
Colloidal silver has been used as an antibacterial agent since ancient Greece . Unlike antibiotic drugs, bacteria cannot easily develop resistance because silver targets multiple components in the bacterial cell. As a result, silver is used in medical equipment coatings  and dental resin components . It is also reported that the mechanism behind its antibacterial activity is by weakening DNA replication and inactivating proteins . On the contrary, gold has low toxicity to biological systems, whether bacteria, animal, or human, due to its elemental properties .
To date, comprehensive studies on nanoparticles have rarely been carried out in bacteria. To understand their interactions, we investigated the antibacterial effects of NPs with different compositions and surface modifications. Model bacteria Escherichia coli were tested with different NPs. Gold and silver NPs were chosen to have similar sizes and shapes. The growth of E. coli was evaluated by colony forming units (CFU). In order to investigate the mechanisms involved, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) analyses were carried out. This mechanistic study provided information on how NPs interact with bacteria dynamically. The level of fluorescence expression was found to correlate with the numbers of viable BCG cells, as a result, we were able to monitor antibacterial activity of NPs on BCG cells. In terms of anti-TB drug development, this study suggests that NPs may represent useful candidates for therapeutics.
Preparation and characterization of nanoparticles
Antibacterial effect of NPs on E. coli
Electron microscopic analysis
Antibacterial effect of NPs on BCG
Our results suggest a different antibacterial mechanism for PAH Au NPs from citrate Au NPs. A previous report shows that PAH Au NPs self-assemble into 4−5 micron long chains, which is an indication of strong interactions between NPs and PAH . As a result, PAH NPs did not further aggregate in culture media and dose-dependent relations seen in Figure 3B. Furthermore, PAH organized chain-like aggregates were disturbed once they entered the cell, and more scattered Au NPs were found within pools of released cytoplasm (Figure 4F−4I). The hypothesis for this mechanism is shown in Figure 7B. PAH facilitated the delivery of a large number of Au NPs that strongly bond to PAH on the bacterial cell surface. Bacteria cell wall tends to attract positive charged PAH due to the total charge of cell wall being negative . As a result, cell walls encounter high stress as they accumulate PAH and Au NPs. Since the MIC of PAH is well above the concentrations likely to be present in the NPs, as shown in Figure 3D, the PAH is not responsible for the toxic effects of NPs for bacteria. Once Au NPs penetrate the cell wall and enter the cytoplasm, PAH is more likely to have direct contact with the cell membrane through damaged cell wall. Then, PAH could play a role in accelerating cell wall breakdown and cytoplasm release. It has been reported that cationic coated Au NPs are more toxic than anionic coated ones , and this supports the concept that PAH Au NPs caused immediate cell lysis while citrate Au NPs did not. Furthermore, the mechanism of interaction between Ag NPs and E. coli is described in Figure 7C. The high antibacterial activity resulted from the nature of the silver element, and possibly the neutral TX-100, which could participate in the interaction. Based on FE-SEM images, several Au NPs were very close to each other tending to aggregate (arrows in Figure 5B). NPs tend to bind to the long end of the cell, it is likely that the ends providing a better site for attachment.
In antibacterial tests against BCG, NPs successfully reduced BCG’s fluorescence. Since the level of fluorescence correlates with numbers of viable BCG as tested by CFU ( 6A), this assay provides a fast way to monitor the growth of slow-glowing mycobacteria. From the reduction of fluorescence, we can conclude that BCG growth is inhibited by NPs, which could be ideal candidates for drug development.
In conclusion, we have demonstrated that gold and silver nanoparticles display excellent antibacterial potential for the Gram negative bacteria E. coli and the Gram positive bacteria BCG. These NPs display their best performance when aggregation is not observed at high levels. By changing surface modifications agents, gold NPs with the same shape and size exhibited different inhibitory effects. Our mechanistic analyses indicated that PAH capped gold NPs caused cell lysis, while citrate capped gold NPs did not. Strong antibacterial activities were observed for silver NPs due to their inherent elemental properties. In terms of anti-TB drug development, this study suggests that NPs may represent useful candidates, but will require significant development to ensure optimal bactericidal activity and low host toxicity.
Citrate stabilized gold (Au) NPs were prepared following previously reported methods . Citrate ions produce negative charges on the surface of nanoparticles. By controlling citrate and hydrogen tetrachloroaurate tri-hydrate (HAuCl4) concentrations, 20−30 nm diameter Au NPs were synthesized. Poly-allylamine hydrochloride (PAH) stabilized Au NPs with 22 nm mean size were synthesized in an aqueous solution of HAuCl4 and PAH and in the presence of gold seed particles . A positive charge was demonstrated on Au NPs’ surface because of the presence of PAH. Silver (Ag) NPs were produced by photoirradiating AgNO3 in Triton X-100 (TX-100) solution for 60 min, the average size was around 30 nm . Citrate Au NPs and Ag NPs solutions were centrifuged at 8000 rpm for 15 min to remove extra citrate and TX-100. The precipitated NPs were suspended in DI water. This process was repeated 3−4 times. PAH Au NPs were used immediately after they were made, since precipitated NPs were unable to dissolve in water.
Bacterial Growth and Exposure
pBlueScriptKSII + plasmid (Agilent Technologies, Santa Clara, CA) was transformed into Escherichia coli DH5α (Invitrogen, Carlsbad, CA). E. coli was grown in liquid Luria-Bertani (LB) medium at 37°C and 250 rpm. Bacillus Calmette-Guérin (Pasteur) cultures expressing tdTomato fluorescent protein were grown in M-OADC-TW broth with Middlebrook (M) 7 H9 broth (Difco) supplemented with 10% (v/v) oleic acid albumin dextrose complex, 0.5% (v/v) glycerol, and 0.05% (v/v) Tween 80. Medium was supplemented with 25 μg/ml kanamycin. NPs in solution were added into bacteria cultures to reach specific concentrations.
NPs samples for TEM imaging were prepared by slow evaporation of freshly made NPs in solution on a carbon-coated copper grid at room temperature. Bacterial samples were fixed in 2% (v/v) glutaraldehyde for 0.5 h at 37°C by adding fixative into culture medium. The following process was performed with cold microwave technology in the BioWave. The microwave was set for a 6 minute cycle (2 min power on, 2 min power off, 2 min power on) at 200 W for primary fixation. Alternating vacuum cycles of 30 seconds were used during 6 minutes of fixation. The microwave temperature was set at 20°C for all steps. Bacteria were spun down and washed 3 times with 0.1 M HEPES for 1 min at 200 W with consistent vacuum. Subsequently, 1% (v/v) osmium tetroxide in HEPES buffer was used for overnight post fixation. Samples were microwaved for 6 min at 100 W at the same power and vacuum cycles as used in primary fixation. Methyl alcohol was used for dehydration in 10% (v/v) steps from 10% to 100%. Microwave was set at 100 W with 1 min per step. Quetol 651 epoxy resin was used for specimen embedding. Thin sections were picked up on 200 mesh copper grids and imaged with a JEOL 1200EX TEM with 100 keV acceleration voltage . FE-SEM sample preparation followed the same fixation and dehydration procedure described above. Images were taken on an FEI Quanta 600 FE-SEM at an acceleration voltage of 10 keV. ImageJ software was used for measuring nanoparticle size distribution.
E. coli cells were grown in LB liquid medium at 37°C for 12 hours before they were diluted in fresh LB liquid medium to reach OD600 = 0.003 (optical density). Gradient concentrations of NPs were then added to the culture medium. Bacteria/NP mixed cultures were put into a 37°C incubator. At different time points of 80, 120, 160, 200, and 260 min the medium was withdrawn from each sample. Dilutions were then made and cultured on LB agar plates. Plates were incubated overnight at 37°C and CFU was determined. NP-free E. coli was used as a negative control and 20 μg/ml hygromycin was used as a positive control for bactericidal activity. The toxicity of PAH was tested by determining its minimum inhibitory concentration (MIC). Overnight cultured E. coli suspensions were adjusted in fresh LB liquid medium to reach an OD600 = 0.1 and diluted by a factor of 1:100. PAH was dissolved in LB media. After mixing 50 μL E. coli dilution and 50 μL PAH solution, 10 μL of the sample was immediately drawn out and a series of dilutions made. Samples were plated on LB agar plates, and colonies were counted the next day .
BCG::tdTomato with OD600 = 0.05 was cultured in 96 well plates with different NPs at various concentrations. Five days after mixing, fluorescence was measured using a Perkin Elmer Envision spectrophotometer with an excitation wavelength of 554 nm and emission wavelength of 581 nm. Wells with NP-free BCG::tdTomato were used as negative controls, and the wells with 20 μg/ml hygromycin in NP-free 7 H9 medium were used as positive controls for comparison. The t test (student version) was used to determine the significance of results.
BCG = Bacillus Calmette-Guérin
CFU = Colony forming units
FE-SEM = Field emission scanning electron microscopy
Hyg = Hygromycin
LB = Luria-Bertani
MIC = Minimum inhibitory concentration
NP = Nanoparticle
OD600 = Optical density at 600 nm
PAH = Poly-allylamine hydrochloride
TB = Tuberculosis
TEM = Transmission electron microscope
TX-100 = Triton X-100.
Assistance in TEM by Ann Ellis at the Microscopy Imaging Center (MIC), Texas A&M University, was greatly appreciated. Funding was provided by National Science Foundation 0506082. This work was in part supported by the Bill and Melinda Gates Foundation grant no. 48523. The authors declare no conflicts of interest.
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