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Research Paper Bacterial tolerance to silver nanoparticles (SNPs): Aeromonas punctata isolated from sewage environment S. Sudheer Khan, E. Bharath Kumar, Amitava Mukherjee and N. Chandrasekaran Nanobio-Medicine Laboratory, School of Bio-Sciences and Technology, VIT University, Vellore, India
Use of silver nanoparticles (SNPs) is increasing in a large number of consumer products. Thus, the possible build-up of the nanoparticles in the environment is becoming a major concern. Aeromonas punctata isolated from sewage showed tolerance to 200 μg/ml SNPs. The growth kinetics data for A. punctata treated with nanoparticles were similar to those in the absence of nanoparticles. There was a reduction in the amount of exopolysaccharides (EPS) in bacterial culture supernatant after nanoparticle-supernatant interaction. EPS capping of the nanoparticles was confirmed by UV-visible, XRD and comparative FTIR analysis. The EPS-capped SNPs showed less toxicity to Escherichia coli, Staphylococcus aureus and Micrococcus luteus compared to the uncapped ones. The study suggests capping of nanoparticles by bacterially produced EPS as a probable physiological defense mechanism. Keywords: SNPs / Aeromonas punctata / EPS / Encapsulation / Bacterial tolerance / Toxicity reduction Received: February 19, 2010; accepted: July 08, 2010 DOI 10.1002/jobm.201000067
Introduction* Silver nanoparticles (SNPs) are potential candidates of strong antimicrobial activity and are used in significant amounts in consumer products, in the food industry for storage, packaging, and processing [1], in textiles [2], in medical applications for wound care products and therapeutic devices [3], and in diagnostics and drug delivery [4, 5]. But increasing concentrations of SNPs with varied physical and surface properties could pose a threat to human and environmental health [6]. Impellitteri et al. 2009 [7] revealed that the SNPs impregnated in clothes and washing systems can easily leak into wastewater during washing, thus potentially disrupting helpful bacteria used in wastewater treatment facilities or endangering aquatic organisms in lakes and streams. In vitro and in vivo toxicity studies in mammalian species proved that SNPs have the capability to enter cells and cause cellular damage [8]. They have the potential to cause chromosomal aberrations and DNA Correspondence: Prof. Dr. N. Chandrasekaran, Nano Bio-Medicine Laboratory, School of Bio-Sciences and Technology, VIT University, Vellore-632014, India E-mail:
[email protected];
[email protected] Phone: +91 416 2202624 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
damage and are capable of inducing proliferation arrest in cell lines [9]. Indiscriminate use of nano-sized silver materials in commercial products leads to their release into the environment and ultimately harms microbial communities in the ecosystem [10]. The release of SNPs entering sewage treatment plants was estimated to be 270 t/ year [11]. Due to this, sensitive bacteria get disturbed, besides harming some beneficial forms as well. Ultimately, the released SNPs destabilize the functioning of ecosystems [11]. On the other hand, some resistant bacteria exist due to their adaptive nature [12]. The hypothesis of the paper was to study the principle underlying bacterial tolerance to SNPs and its possible mechanism.
Materials and methods Materials All chemicals and media were obtained from Himedia Laboratories Ltd., Mumbai, India. The bacterium was isolated from sewage (Vellore, India). Manufactured SNPs were obtained from Sigma Aldrich, USA. The nanoparticles were dispersed using an ultrasonic processor at a frequency of 132 kHz (Crest, USA). www.jbm-journal.com
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Characterization of SNPs The SNPs were characterized using UV-visible spectroscopy and a high-resolution transmission electron microscope (TEM; Tecnai G-20) after dispersion in LB medium. The samples were prepared by placing a drop of homogeneous suspension on a copper grid with a lacey carbon film and air-drying. The mean particle size was analyzed from the digitized images with Image Tool software. The morphological features of the manufactured SNPs were characterized by scanning electron microscopy (SEM; FEI Sirion, Eindhoven, The Netherlands). The surface area was measured using a Smart Sorb 93 single-point BET surface area analyzer (Smart Instruments Co. Pvt. Ltd., Mumbai, India). The received particles were also subjected to X-ray diffraction (XRD) analysis using a JEOL-JDX 8030 diffractometer. The target was Cu Kα (λ = 1.54 Å). The generator was operated at 45 kV and with a 30 mA current. The scanning range (2θ) was selected from 10 to 100°. Isolation and identification of microorganisms The sample collected from the sewage environment (1 ml) was grown on LB broth supplemented with 100 µg/ml SNPs and incubated at 30 °C for 24 h with agitation (200 rpm). The bacterial culture isolated from the above medium was identified using the Sherlock microbial identification system (MIS) following the method of Wintzingerode et al. [13] and 16S rRNA analysis. The 16S rRNA gene sequencing analysis was done by using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [14]. Nearly complete 16S rRNA nucleotides were aligned and bacterial identities were deduced by BLAST search to ascertain their closest relatives. A phylogenetic tree was constructed by using ClustalW. pls check Disc diffusion and agar well diffusion method Bacterial sensitivity to SNPs was tested by the disc diffusion method (25, 50 and 100 µg per disc) [15] and the well diffusion method (10, 25, 50, 100 and 200 μg/well) [16]. The average diameter of the inhibition zone surrounding the disc/well was measured. Six replicates were employed for all concentrations. The bacteria Escherichia coli (ATCC 13534 and ATCC 25922), Staphylococcus aureus (ATCC 25923) and Micrococcus luteus (clinical isolate) were used as positive control. The agar well diffusion method was carried out with silver nitrate solution at concentrations of 10, 25, 50, 100, 250 and 500 µg/ml. Dilution plate count method The different concentrations of SNPs (10, 25, 50, 100, and 200 µg) were applied uniformly on the surface of © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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LB agar plates and the colony-forming units (CFU) were determined at each concentration. Nanoparticle-free plates incubated under the same conditions were used as control. The counts on six plates corresponding to a particular concentration were averaged. Silver ion concentration measurement SNPs dispersed in LB broth at 100 and 200 µg/ml were centrifuged at 15,000 × g for 30 min. The clear supernatant was carefully collected and filtered through a 0.1 µm sterile filter. The ion concentrations were measured by an AAS after acidification by 1% nitric acid [17]. Growth kinetics study To examine the bacterial growth profile in the presence of SNPs at increasing concentrations (10 – 200 µg/ml), LB broth was sonicated for 30 min after adding the SNPs to prevent the aggregation of nanoparticles. Subsequently, the flasks were inoculated with 1 loop full of freshly prepared bacterial culture and incubated in an orbital shaker at 200 rpm and room temperature (30 °C). Nanoparticle-free media were used as control. The bacterial growth was measured every 2 h by using a colorimeter (Elico CL 157) at 600 nm. A positive control (flask containing nanoparticles and nutrient media, devoid of inoculum) and a negative control (flask containing inoculum and nutrient media, devoid of nanoparticles) were included in this experiment. The absorbance value for the positive control was subtracted from the experimental values (flasks containing nutrient media, inoculum and nanoparticles). The growth profiles of positive control strains were recorded at a concentration of 100 µg/ml SNPs. The growth studies of all the bacteria were performed with corresponding amounts of silver ions that were released during the time of dispersion of SNPs in the medium. FTIR analysis A loop full of culture of A. punctata was inoculated into 50 ml of LB broth and grown for 48 h at room temperature, under shaking at 180 rpm. The exopolysaccharides (EPS) were extracted [18] and quantified [19]. The purified EPS [18] were subjected to FTIR analysis (Perkin-Elmer spectrometer – one instrument in diffuse reflectance mode at a resolution of 4 cm–1 in KBr pellets). At the same time, another part of the A. punctata culture supernatant (10 ml) was collected and interacted with 100 µg/ml SNPs for 4 h, in a rotary shaker at 200 rpm to maintain a proper interaction with nanoparticles. After the interaction, the mixture was centriwww.jbm-journal.com
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fuged and the nanoparticle pellets were separated, lyophilized and subjected to FTIR analysis. The supernatant was collected, and the EPS left in the culture supernatant after interaction with SNPs were quantified. The EPS extraction and FTIR analysis were also done with positive control strains. UV-visible spectral analysis and XRD analysis The purified EPS were redissolved in deionized (DI) water and interacted with different concentrations of SNPs (10 – 100 µg/ml), in a rotary shaker at 200 rpm to maintain proper interaction with the nanoparticles. The absorbance of SNPs was measured after 4 h of interaction, by using a UV-visible spectrophotometer (Shimadzu UV – 1700, Japan) at 200 – 700 nm. After the interaction, the SNPs were separated, lyophilized and analyzed by XRD (PANalytical XPert Pro, Eindhoven, The Netherlands). Toxicity test for EPS-coated SNPs To examine the effect of EPS-coated nanoparticles, the coated and uncoated SNPs were interacted with positive control strains at a concentration of 100 µg/ml as per the method described earlier.
Results and discussion Characterization of SNPs UV-visible spectroscopy is one of the techniques used for the structural characterization of SNPs. UV-visible absorption spectra for the manufactured SNPs showed an absorption band at 425 nm (Fig. 1). The surface area of the manufactured SNPs was determined to be
Figure 1. UV-visible absorption spectra of dispersed SNPs in LB broth (a) before dilution, (b) after 10 times dilution. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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0.26 m2/g. The TEM images showed that the SNPs were spherical in shape and polydispersed with diameters in the range of 5 to 40 nm. The SEM images showed SNPs whose size was below 40 nm. The XRD pattern of the dispersed SNPs was characterized by a major peak at 38.115 and minor peaks at 44.299, 64.443, 77.397, 81.541 and 98.241. This confirms the stability of the nanoparticles in the LB medium. Microbial identification MIS studies and 16S rRNA analysis showed that our bacterial isolate from sewage was A. punctata (data not shown). Fig. 2 represents the phylogenetic tree with boot strap values. The BLAST search shows 99% similarity to the A. punctata strain. The 16S rRNA sequence was submitted to Genbank (Accession number GQ401237) and the organism was named A. punctata VITSCA01. Antimicrobial tests The antimicrobial properties of SNPs against the isolate A. punctata were investigated by disc diffusion test, agar well diffusion method, dilution plate count method and growth kinetics studies. The disc impregnated with SNPs in the A. punctata culture plate gave no zone of inhibition. There was also no zone of inhibition observed in the agar well diffusion method (Fig. 3). In the disc diffusion test, E. coli 13534, E. coli 25922 and M. luteus (clinical isolate) showed a small inhibition at 50 µg/disc. However, S. aureus 25923 gave an inhibition zone at 100 µg/disc. In the agar well diffusion method, the zone of inhibition was observed in all control organisms at 100 and 200 µg (Fig. 3). Three different agar media (Muller Hinton agar, Nutrient agar and LB agar) were used in both experiments. There was no difference in the activity of SNPs observed in all the media tested. The study of Ruparelia et al. [15] has shown that a disc impregnated with spherical SNPs showed a large inhibition zone for E. coli, S. aureus and Bacillus subtilis. The study with spherical SNPs in the size range from 10 to 100 nm diameter exhibited excellent antibacterial activity against the bacteria S. aureus, B. cereus, E. coli and Pseudomonas aeruginosa, with the agar well diffusion method, and clearing zones around the holes with bacteria growth were observed [16]. A. punctata showed tolerance to silver nitrate at 100, 250, and 500 µg/ml concentration. A narrow zone at 500 µg/ml that measured approximately 1 mm in diameter was observed (not shown). This indicates that A. punctata was resistant to 100 and 250 µg/ml silver and showed the lowest resistance at 500 µg/ml. The studies proved the mechanism of resistance to be plasmid mediated [20 – 22]. www.jbm-journal.com
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Figure 2. Phylogenetic tree based on 16S rRNA gene sequence comparison showing the position of the silver nanoparticle-tolerant bacterial strain isolated from sewage.
Figure 3. Representative images of agar plates containing SNPimpregnated discs and wells. (A) A. punctata, (B) E. coli (ATCC 13534), (C) E. coli (ATCC 25922), and (D) S. aureus (ATCC 25923); (a) 25, (b) 50, (c) 100, and (d) 200 μg SNPs. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
When nanoparticles were present on the surface of the nutrient agar plates, they could more completely inhibit bacterial growth as compared to liquid broth. The growth of A. punctata in agar plates supplemented with different concentrations of SNPs was not found to be different compared to the control plate. The observed bacterial counts (mean ± standard error) in the agar plates were 84.6 ± 0.8, 84.8 ± 0.9, 83.3 ± 0.8, 84.8 ± 0.6, 84.3 ± 0.9, and 83.5 ± 0.8 for control, 10, 25, 50, 100, and 200 µg SNPs, respectively (not shown). Fig. 4 shows the bacterial growth curves for different concentrations of SNPs. In comparison with the control, no growth inhibition was observed at all the concentrations of SNPs tested. The present study with positive control strains showed a great reduction in the growth of the bacteria (Fig. 5), similar to the studies of Sondi and Salopek-Sondi [23]. Fig. 6 shows the interaction of A. punctata with SNPs. Most of the studies reported bacterial growth inhibition by nanoparticles at several optimal concentrations [15, 23 – 25]. www.jbm-journal.com
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Figure 4. Representative batch growth profile of A. punctata in LB broth dosed with 10, 25, 50, 100 and 200 μg/ml SNPs and control broth (without nanoparticles) at 30 °C.
Overuse of nanoparticles in consumer products and washing of these into sewage systems might induce resistance to environmental strains [26]. On dispersion, a substantial amount of silver ions was quickly released from SNPs into the LB medium. At 100 µg/ml SNPs, 1.4 ± 0.002 µg/ml silver ions were released, and 200 µg/ml of SNPs released 2.9 ± 0.01 µg/ml silver ions into the LB medium. To evaluate the toxic effect of silver ions released during the dispersion of SNPs, the LB medium was centrifuged at 15,000 × g for 30 min after dispersion. The SNPs settle down and the silver ions remain in the medium. The growth kinetics studies of A. punctata did not show any growth reduction
Figure 5. Toxicity studies of EPS-coated and uncoated SNPs with E. coli 13534. The same pattern was observed with three other control species. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. SEM image of A. punctata interacted with SNPs. The production of EPS can be observed in the figure.
in the above medium. A. punctata may not be affected by these silver ions due to its above-mentioned resistant nature. The growth kinetics studies with E. coli (ATCC 13534, ATCC 25922), S. aureus (ATCC 25923) and M. luteus (clinical isolate) showed only a negligible growth reduction. This suggests that there might be some other mechanism behind the action of SNPs. The release of silver ions from SNPs may vary depending on the medium used for dispersion. Morones et al. [25] reported that the bactericidal mechanisms of SNPs and silver ions are distinctly different. For treatment with silver nitrate, a low-molecular-weight central region was formed within the cells as a defense mechanism, whereas for treatment with nanoparticles no such phenomenon was observed, although the nanoparticles were found to penetrate the cell wall [23]. Role of EPS in defense mechanism The results of EPS quantification showed that the amount of EPS present in the supernatant after interaction with SNPs was lower (82.3 ± 0.9 µg/ml) compared to the amount of EPS extracted before interaction (113.7 ± 0.6 µg/ml). A reduction in the amount of EPS was observed in the supernatant after SNPs-supernatant interaction. This result suggests that the EPS might have coated the SNPs during interaction. The FTIR spectral profile obtained in the 1000 – 1200 cm–1 region mainly reflected the absorption of sugars present in the EPS. The studies showed that FTIR www.jbm-journal.com
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spectroscopy affords a rapid and easy means for indicating the nature of major components of A. punctata EPS (Fig. 7a). The three peaks near 1000 – 1200 cm–1 indicate that the polysaccharide was α-pyranose. After interaction of the SNPs with A. punctata culture supernatant, the FTIR spectra matched all the peak values of the extracted EPS (Fig. 7b). The coating with EPS during the
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interaction of SNPs with the bacterial culture supernatant was confirmed by evaluating the FTIR peak value in both these cases. Hydroxyl, carboxyl, carbonyl and amine groups were found in the FTIR spectra of EPS. The FTIR spectra of EPS secreted by E. coli (ATCC 13534, ATCC 25922) and S. aureus were entirely different from that of the test
a)
b)
Figure 7. (a) FTIR spectra of EPS extracted from A. punctata culture supernatant. (b) FTIR spectra of lyophilized SNPs after 4 h of interaction with A. punctata culture supernatant. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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strain. Production of EPS was not found in the case of M. luteus. Noble metal SNPs exhibit unique and tunable optical properties, on account of their surface plasmon resonance (SPR) which depends on shape, size and size distribution of the nanoparticles. When SNPs were interacted with EPS extracted from the culture supernatant of A. punctata, UV-visible spectrophotometric analysis gave a peak at 394 nm for the SNPs. On the other hand, the λmax value for the uninteracted SNP dispersion was noted at 425 nm. A decrease in absorbance at 425 nm was observed when it was interacted with EPS. After interaction with EPS, a peak shift was observed (blue shift). This might be due to the repelling action of EPScapped SNPs. The breaking up of the agglomerates/flocs would result in a blue shift. The blue shift (towards lower wavelength) in the λmax value after interaction with EPS demonstrated that the shape of the nanoparticles did not suffer distinct modifications. The blue shift indicates that the decrease in size might be due to the coating with EPS that prevents the further agglomeration of SNPs. EPS coating was confirmed by XRD analysis. The XRD study corroborated the adsorption of EPS on the nanoparticle surface; the coverage was so strong that no characteristic band of silver could be revealed through XRD, whereas the manufactured SNPs gave six peaks in XRD analysis. Ravindran et al. [27] reported that, in XRD analysis, SNPs that were interacted with BSA did not give the characteristic peak of silver, due to the coating of SNPs with BSA. The toxicity test of EPS-coated and uncoated SNPs (100 µg/ml) with four different strains, E. coli 13534, E. coli 25922, S. aureus 25923 and M. luteus (clinical isolate), showed a great reduction in the growth rate of these bacteria when treated with uncoated nanoparticles compared to the control growth profile (without nanoparticles). But not much reduction was observed when the culture medium was supplemented with coated SNPs, indicating lower toxicity (Fig. 5). Nanosilver may compromise to control harmful bacteria. Besides that it may affect the beneficial bacteria present in the soil and sewage treatment plants. This study confirmed that the release of nanoparticles into the wastewater in a wastewater treatment plant would not affect all the beneficial microbes involved in the sewage treatment process. A. punctata might protect the survival of the beneficial microbes by providing an EPS capping to the nanoparticles. On the other hand, the EPS-capped SNPs can be used for commercial application including drug delivery and biosensing. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Conclusions A. punctata isolated from sewage showed tolerance to SNPs at the tested concentrations of up to 200 μg/ml. The findings from this study suggest that, when grown in the presence of increasing concentrations of SNPs, environmental isolates may acquire tolerance/resistance. One of the important implications of our findings is that future predictions on the environmental toxicity of nanoparticles, especially for microbes, need to take into account the possible adaptation of the environmental strains to high concentrations of nanoparticles. This physiological defense mechanism developed might be due to the presence of other toxic compounds in the sewage system. Although the toxicity effects in situ will definitely differ from those observed in a controlled laboratory environment, we strongly feel that, in the absence of prior reports on acquired nanoparticle tolerance in environmental isolates, our study gains importance. Finally, we stress here the need to take up future in situ studies involving different nanoparticles and further microbial strains for a clearer understanding of the environmental toxicity effects of nanoparticles.
Acknowledgement The authors thank the Management of VIT University for the provision of funding to carry out this research.
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