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Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles
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Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 282 – 286 www.nanomedjournal.com
Original Article: Engineering Nanomedicine, Antimicrobial Nanoparticles
Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles I. Mohammed Sadiq, MSc, Basudev Chowdhury, BTech, Natarajan Chandrasekaran, PhD, Amitava Mukherjee, PhD⁎ Nanobio-medicine Research Group, School of Biotechnology, Chemical & Biomedical Engineering, VIT-University, Vellore-632014, India Received 3 November 2008; accepted 5 January 2009
Abstract Metal oxide nanoparticles (NPs) are known to possess strong antimicrobial properties. Aluminum oxide NPs have wide-range applications in industrial as well as personal care products. In the absence of prior reports on the antimicrobial properties of alumina NPs for a wide concentration range, the principal objective of the present work was to study the growth-inhibitory effect of alumina NPs over a wide concentration range (10–1000 μg/mL) on an environmentally relevant gram-negative model microorganism, Escherichia coli. The mean diameter of the NPs was determined to be 179 nm in aqueous dispersion used for this study, and surface area was determined to be 21.23 m2/g. The concentration of 1000 μg/mL was found to be moderately inhibitory for bacteria. Almost negligible dependence of growth rate on the concentration of the NPs was observed. The extracellular protein content was found to be slightly lower in case of cells interacting with 1000 μg/mL alumina than the uninteracted control cells. Fourier transform–infrared studies established differences in structure between interacted and uninteracted cells. Alumina NPs showed a mild growth-inhibitory effect, only at very high concentrations, which might be due to surface charge interactions between the particles and cells. Free-radical scavenging properties of the particles might have prevented cell wall disruption and drastic antimicrobial action. This laboratory-scale study suggests that alumina NPs may only exhibit mild toxicity toward microorganisms in the environment. From the Clinical Editor: Metal oxide NPs, inluding aluminum oxide NPs, are known to possess strong antimicrobial properties. The study demonstrated a mild to moderate growth-inhibitory effect of alumina NPs over a wide concentration range (10-1000 μg/mL) on Escherichia coli. Almost negligible dependence on the concentration was observed. © 2009 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Alumina; Antimicrobial activity; Growth rate; Cell surface interactions
When the dimensions of a material are reduced to the atomic level it attains a range of unique properties that can be manipulated suitably for the desired applications.1 Because most of the biological processes also take place on the nanoscale, synergistic application of nanotechnology and biology can possibly address several important biomedical problems.2 Currently nanosized organic and inorganic nanoparticles (NPs) are finding increasing applications in medical devices3 as a result of their amenability to biological functionalization. Antimicrobial agents are highly relevant for a host of industrial applications in environmental, food, synthetic No conflict of interest was reported by the authors of this article. ⁎Corresponding author. Biomedical Engineering Division, School of Biotechnology, Chemical & Biomedical Engineering, VIT University, Old Madras Road, Vellore, Tamil Nadu-632014, India. E-mail address: [email protected] (A. Mukherjee).
textiles, packaging, health care, and medical care products. They can be grouped broadly into two types: organic and inorganic. The inorganic materials have gained importance over the past decade because of their ability to withstand adverse processing conditions.4,5 The antibacterial activity is known to be a function of the surface area in contact with the microorganisms; a larger surface area (as in the case of NPs) ensures a broad range of probable reactions with bioorganics present on the cell surface, as well as environmental inorganic and organic species.6 Published studies on the ecotoxicity of metal oxide NPs to bacterial species are limited, even though their bactericidal properties have been reported in the biomedical literature.4,7 One might therefore expect some of these materials to be toxic to microbes in the environment. Zinc oxide (ZnO) NPs seem to disrupt the gram-negative cell membrane structure in Escherichia coli,8 and it is proposed that NPs with a positive charge such as cerium oxide could bind the gram-negative
1549-9634/$ - see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.01.002 Please cite this article as: I.M. Sadiq, B. Chowdhury, N. Chandrasekaran, A. Mukherjee, Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles. Nanomedicine: NBM 2009;5:282-286, doi:10.1016/j.nano.2009.01.002
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cell membrane by electrostatic attraction. Clearly, the intimate relationship between the physicochemistry of the medium and membrane biology of the microbe is emerging as a key factor in NP toxicity to microorganisms. Aluminum oxide (Al2O3) NPs have important applications in the ceramics industry10 and can be used as an abrasive material, in heterogeneous catalysis, as an absorbent, as a biomaterial, and as reinforcements of metal-matrix composites.11,12 Alumina NPs are currently one of the two US market leaders for nanosized materials according to a recent report.13 Yamamoto et al investigated cytototoxic effects of metal oxide particles (Al2O3, titanium dioxide [TiO2], and zirconium oxide [ZrO2]) on murine fibroblasts and murine monocyte macrophages. They studied cytotoxicity as a function of shape, size, and surface area of the particles and also compared toxicity behavior of bulk and nanomaterials.14 Hanawa et al studied the toxicity of a range of metal oxide NPs, including Al2O3, having 500-nm to 3000-nm diameter on human fibroblast cells.15 Several recent studies with alumina NPs showed considerable cytotoxic effect on the mammalian cells.16-18 There have been very few studies available in the literature on the interaction of the alumina NPs with microbes. One past study found no detrimental effect of an alumina slurry between 62.5 and 250 mg/L concentration range on E. coli.19 Considering the paucity of prior literature reports on the effect of alumina NPs on bacterial species, the objective of the present work was to investigate the possible growth-inhibitory effect of alumina NPs over a wide concentration range (10–1000 μg/mL) on an environmentally relevant gram-negative model microorganism, E. coli. The implications of this type of growth-inhibitory study are mainly twofold: A. For biomedical applications the bactericidal properties of ceramic oxides must be studied. B. The growth inhibition effects of the oxides may reflect on toxicity behavior of metal oxides on bacterial species in the environment.
Methods Characterization of alumina nanoparticles Dry alumina NPs were procured from Aldrich (St. Louis, Missouri; CAS Number 1344-28-1). The supplier's data can be summarized as follows: gamma phase alumina nanopowder, particle size b50 nm (BET surface area analyzer; Smart Instruments Co. Pvt. Ltd., Mumbai, India), surface area 35–43 m2/g, melting point 2040°C (from the literature). The aqueous dispersion of the particles was subjected to particle size analysis using 90Plus Particle Size Analyzer (Brookhaven Instruments Corp., Holtsville, New York). The morphological features and particle size of the procured NPs were characterized by scanning electron microscopy
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(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.). Preparation of nanoparticle dispersion The alumina NPs obtained from the suppliers were used as is to produce suspensions in deionized water. The suspensions at four different concentrations at 10, 50, 100, and 1000 μg/L were produced by means of ultrasonic vibration (100 W, 30 kHz) for 30 minutes. Bacterial strains The bactericidal experiments were carried out with gramnegative bacteria E. coli strain (NCIM No. 2666), which was procured from the National Chemical Laboratory (Pune, India) in Luria Bertani (LB) medium (Himedia Laboratories Ltd., Mumbai, India). Throughout this study the same nutrient medium was used. Growth inhibition study The minimum inhibitory concentration, defined as the lowest concentration of material that inhibits the growth of an organism, was determined based on batch cultures containing varying concentrations of alumina NPs in suspension (10, 50, 100, and 1000 μg/L). Sterile side-arm Erlenmeyer flasks (250 mL) containing 50 mL LB medium were sonicated for 10 minutes after adding the NPs to prevent aggregation of the NPs. Subsequently, the flasks were inoculated with 1 mL of the freshly prepared bacterial suspension to maintain initial bacterial concentration 108 colony-forming units per milliliter, and then incubated in an orbital shaker at 200 rpm and 30°C. The high rotary shaking speed was selected to minimize aggregation and settlement of the NPs over the incubation period. A lower rpm setting during incubation might cause underestimation of the antimicrobial activity of the NPs. Bacterial growth was measured as increase in absorbance at 600 nm determined using a spectrophotometer (CL-157 colorimeter; ELICO Company, Hyderabad, India). The experiments also included a positive control (flask containing NPs and nutrient medium, devoid of inoculum) and a negative control (flask containing inoculum and nutrient medium, devoid of NPs). The negative controls indicated the microbial growth profile in the absence of NPs. The absorbance values for positive controls were subtracted from the experimental values (flasks containing nutrient medium, inoculum, and NPs).20 All the experiments were carried out in triplicate and the mean value reported. Growth reversibility study To investigate whether the antibacterial effect of the NPs was due to the mere presence of these particles in the liquid broth, or due to their specific interactions with bacterial cellular components, the cells were cultured for 24 hours in the presence of alumina NPs, followed by sedimentation and reculturing them in fresh growth medium free of NPs. The
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Figure 1. Particle size distribution analysis of alumina NP dispersion. Figure 2. Scanning electron micrograph showing procured alumina NPs.
control cells were treated in a similar fashion but without any exposure to the NPs. Protein assay The extracellular protein was extracted using the polyethylene glycol method and was estimated using Lowry's method. The study was carried out in triplicate, and the mean value was reported. Standard error was within ±5%. Fourier transform–infrared study Fully grown culture after 24 hours incubation in the presence of 1000 μg/mL alumina NPs was harvested using centrifugation at 10,000 rpm for 15 minutes. The cell pellet was dispersed in deionized water, washed three times, and thereafter lyophilized for 4 hours, until the powder form was obtained. The powder was subjected to Fourier transform– infrared (FT-IR) by potassium bromide technique in a Nicolet 6700 FT-IR Spectrometer (Thermo Scientific Instruments Groups, Madison, Wisconsin). For controls the same protocol was followed in the absence of NPs. Results Characterization of as-procured nanoparticles The particle size distribution analysis (Figure 1) showed an effective diameter of 179.2 nm, with a polydispersity index of 0.191. The surface area was determined to be 21.23 m2/g. The high-resolution SEM image of procured alumina NPs is shown in Figure 2. Nearly spherical to spheroidal NPs were observed. The particles seemed to be agglomerated. The difference between the supplier's data and the experimentally obtained data may be due to agglomeration of the particles while present in aqueous suspension. Antimicrobial activity of alumina nanoparticles against E. coli In further experiments, strains of the gram-negative bacterium E. coli were inoculated in liquid LB medium
supplemented with increasing dosages of alumina NPs. Increasing concentration of NPs progressively retarded the growth of E. coli (Figure 3, A). The concentration of 1000 μg/mL was found to be mildly inhibitory for bacteria, in that about 6–8 hours passed before any noticeable growth was initiated. The steepness of the growth curve in the logarithmic phase and the final cell concentration were also noticeably lower at 500 and 1000 μg/mL concentrations, as compared with the lower ones used in this study. Prediction of bacterial growth in the presence of varying concentrations of alumina nanoparticles In the logarithmic phase the dynamic growth rate of a bacterial species is represented by the following equation: In N = lnN0 + lt
ð1Þ
where N is the bacterial cell count at time t, N0 is the initial cell count, and μ is the growth rate constant for the bacteria. From the growth curve (Figure 3, A) of the bacteria at different concentrations of alumina NPs the logarithmic phase was identified between 6 and 14 hours. The growth data in this time interval were plotted in the logarithmic scale (Figure 3, B) to derive the values of μ corresponding to various doses of the nanoparticles (x) studied. A linear relationship between x and μ (Figure 3, C) was derived: l = ax + b
ð2Þ
From Figure 3, C the equation obtained was l = 2 105 x + 0:0909
ð2aÞ
A very low value of a signifies almost negligible dependence of growth rate on the concentration of the NPs. Importantly, here μ positively correlated with concentration of the particles.
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Figure 4. Growth curve of cells recultured in particle-free fresh medium after interacting with the particles for 24 hours; uninteracted inoculum was used as control here.
extracellular proteins extracted from fully grown culture in the presence of 1000 μg/L alumina NP concentrations were found to be only 5.9 μg/mL. So the interaction with the particles had a diminishing effect on extracellular protein content in E. coli. Fourier transform–infrared study FT-IR spectra (not shown) of the uninteracted bacterial samples revealed the following bands: 3726.6 and 3626.8 cm–1 band predominantly due to OH stretching vibrations; 2150 and 2002.7 cm–1 may be overtones due to combination of NH3+ torsion and NH3+ antisymmetric deformation for amino acids; 1542.1 cm–1 due to amide II. When the cells were interacted with 1000 μg/L alumina NPs, their FT-IR spectra revealed 2383.3, 2162.4, and 1986.8 cm–1 bands signifying probably overtones due to combination of NH3+ torsion and NH3+ antisymmetric deformation for amino acids. The bands for OH stretching and amide II were conspicuously absent in the spectra. Discussion
Figure 3. (A) Effect of alumina NP concentrations on growth of E. coli. (B) Cell growth of E. coli in the logarithmic phase as a function of alumina NP concentrations. (C) Dynamic cell growth rate (μ) of E. coli as a function of alumina NP concentrations. CFU, colony-forming units; OD, optical density.
Growth reversibility study In reversibility studies a significant retardation in growth of recultured gram-negative bacteria E. coli that had had prior exposure to the NPs was observed in the fresh medium (Figure 4). Control cells did not display any deviation from normal growth characteristics. These observations were consistent with sustained interaction between NPs and cellular components of gram-negative bacteria, in that otherwise the growth curves of recultured bacteria would have been comparable to that of the control cells. Protein assay of the interacted microorganisms The extracellular protein content from the uninteracted control cells of E. coli was measured to be 7.75 μg/mL. The
The hypothesis in this study was that alumina NPs at the tested concentrations inhibit growth of the test microorganism, E. coli, under the experimental conditions. The important observations from the present work with gramnegative E. coli can be summarized as mild growthinhibitory effect of alumina NPs in the concentration range 10–1000 μg/mL. The intimate relationship between the physicochemistry of the oxide NPs, medium, and membrane biology of the microbe is emerging as a key factor in the sensitivity of microorganisms to the particles in suspension. ZnO NPs appear to disrupt the gram-negative cell membrane structure in E. coli,8 and it is proposed that NPs with a positive charge such as cerium oxide could bind the gram-negative cell membrane by electrostatic attraction.9 The decrease in cell viability as observed in the present study may be due to bacterial adhesion to the particle surfaces. Due to positive surface charges on the alumina NPs at near-neutral pH, an electrostatic interaction is possible between negatively charged E. coli cells and the particles, leading to bacterial adhesion onto NP surfaces. As the adhesion increased with increase in concentration of the particles in the suspension, a
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negative effect on growth was observed with respect to concentration. This electrostatic interaction between bacteria and particle surface, along with hydrophobic interactions and polymer bridging, may be responsible for the phenomenon of bacterial adhesion onto the particles.21 The strong bactericidal effect as observed with other metal oxides such as ZnO2, TiO2, etc. was not observed in the case of alumina. The disruption of cell wall due to reactive oxygen species (ROS) generation is one of the important mechanisms behind cell death leading to the strong antimicrobial property of these metal oxides.8 But alumina NPs may act as freeradical scavengers.22 Alumina is thermodynamically stable over a wide temperature range and has a corundum-like structure, with oxygen atoms adopting hexagonal closepacking and Al3+ ions filling two thirds of the octahedral sites in the lattice. These NPs are able to rescue cells from oxidative stress-induced cell death in a manner that appears to be dependent upon the structure of the particle but independent of its size within the range of 6–1000 nm. According to another explanation, they may act as direct antioxidants, blocking ROS production, thus inhibiting the apoptotic pathway, and they may directly inhibit ROS production, which rapidly induces a ROS defense system before the glutamate-induced cell death program is complete.22 It is evident from this study that alumina NPs do not possess strong antimicrobial properties; minor growth inhibition was noticed at high NP concentrations up to 1000 μg/mL. These observations are pertinent regarding ecotoxicity of alumina NPs with respect to microorganisms. E. coli is one important environmentally relevant gram-negative microorganism. This laboratory-scale study suggests that alumina may have only mild toxicity toward microorganisms in the environment. However, the environmental behavior may be more complex in nature— depending upon physicochemical interactions of the particles or presence of microbial consortia, among other factors. In conclusion, alumina NPs showed a mild growthinhibitory effect on E. coli, only at very high concentrations. A concentration-dependent negative effect was observed on extracellular protein content of gram-negative bacteria E. coli. Almost negligible dependence of growth rate was observed on the concentration of the NPs. References 1. Gleiter H. Nanostructured materials, basic concepts and microstructure. Acta Mater 2000;48:1-12. 2. Curtis A, Wilkinson C. Nanotechniques and approaches in biotechnology. Trends Biotechnol 2001;19:97-101. 3. Waren CW, Nie S. Quantum dot bioconjugates for ultra sensitive nonisotopic detection. Science 1998;281:2016-8.
4. Fu G, Vary PS, Lin CT. Anatase TiO2 nanocomposites for antimicrobial coating. J Phys Chem B 2005;109:8889-98. 5. Makhluf S, Dror R, Nitzan Y, Abramovich Y, Jelinek R, Gedanken A. Microwave-assisted synthesis of nanocrystalline MgO and its use as bactericide. Adv Funct Mater 2005;15:1708-15. 6. Holister P, Weener JW, Romas Vas C, Harper T. Nanoparticles: technology white papers 3. London: Cientific Ltd; 2003. p. 2-11. 7. Duran N, Marcato PD, De Souza GIH, Alves OL, Esposito E. Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J Biomed Nanotech 2007;3: 203-8. 8. Brayner R, Ferrari-Iliou R, Brivois N, Djediat S, Benedetti MF, Fievet F. Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Lett 2006;6: 866-70. 9. Thill A, Zeyons O, Spalla O, Chauvat F, Rose J, Auffan M, et al. Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physicochemical insight of the cytotoxicity mechanism. Environ Sci Technol 2006;40:6151-6. 10. Zielínski PA, Schulz R, Kaliaguine S, Van Neste A. Structural transformations of alumina by high energy ball milling. J Mater Res 1993;8:2985-92. 11. Ganguly P, Poole WJ. In situ measurement of reinforcement stress in an aluminium–alumina metal matrix composite under compressive loading. Mater Sci Eng A 2003;352:46-54. 12. Martínez Flores E, Negrete J, Torres Villaseñor G. Structure and properties of Zn-Al-Cu alloy reinforced with alumina particles. Mater Des 2003;24:281-6. 13. Abraham T. Healthy prospects for nanoceramic powders. Ceramic Industry. http://www.ceramicindustry.com/Articles/Feature_Article/284026788a9c7010VgnVCM100000f932a8c0. 2000 Accessed on 5 September 2008. 14. Yamamoto A, Honma R, Sumita M, Hanawa T. Cytotoxicity evaluation of ceramic particles of different sizes and shapes. J Biomed Mater Res 2004;68A:244-56. 15. Hanawa T, Kaga M, Itoh Y, Echizenya T, Oguchi H, Ota M. Cytotoxicities of oxides, phosphates and sulphides of metals. Biomaterials 1992;13:20-4. 16. Dey S, Bakthavatchalu V, Tseng MT, Wu P, Florence RL, Grulke EA, et al. Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells. Carcinogenesis 2008;29:1920-9. 17. Oesterling E, Chopra N, Gavalas V, Arzuaga X, Lim EJ, Sultana R, et al. Alumina nanoparticles induce expression of endothelial cell adhesion molecules. Toxicol Lett 2008;178:160-66. 18. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro 2005;19:975-83. 19. Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. Evaluation of growth inhibitory effect of ceramics powder slurry on bacteria by conductance method. J Chem Eng Jpn 1995;28:288-93. 20. Williams DN, Ehrman SH, Holoman TRP. Evaluation of the microbial growth response to inorganic nanoparticles. J Nanobiotechnol 2006. Published online February 28, 2006. doi:10.1186/1477-3155-4-3. 21. Li B, Logan BE. Bacterial adhesion to glass and metal oxide surfaces. Colloids Surf B 2004;36:81-90. 22. Mohammad G, Mishra VK, Pandey HP. Antioxidant properties of some nanoparticles may enhance wound healing in T2DM patient. Digest J Nanomater Biostruct 2008;3:159-62.
Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 282 – 286 www.nanomedjournal.com
Original Article: Engineering Nanomedicine, Antimicrobial Nanoparticles
Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles I. Mohammed Sadiq, MSc, Basudev Chowdhury, BTech, Natarajan Chandrasekaran, PhD, Amitava Mukherjee, PhD⁎ Nanobio-medicine Research Group, School of Biotechnology, Chemical & Biomedical Engineering, VIT-University, Vellore-632014, India Received 3 November 2008; accepted 5 January 2009
Abstract Metal oxide nanoparticles (NPs) are known to possess strong antimicrobial properties. Aluminum oxide NPs have wide-range applications in industrial as well as personal care products. In the absence of prior reports on the antimicrobial properties of alumina NPs for a wide concentration range, the principal objective of the present work was to study the growth-inhibitory effect of alumina NPs over a wide concentration range (10–1000 μg/mL) on an environmentally relevant gram-negative model microorganism, Escherichia coli. The mean diameter of the NPs was determined to be 179 nm in aqueous dispersion used for this study, and surface area was determined to be 21.23 m2/g. The concentration of 1000 μg/mL was found to be moderately inhibitory for bacteria. Almost negligible dependence of growth rate on the concentration of the NPs was observed. The extracellular protein content was found to be slightly lower in case of cells interacting with 1000 μg/mL alumina than the uninteracted control cells. Fourier transform–infrared studies established differences in structure between interacted and uninteracted cells. Alumina NPs showed a mild growth-inhibitory effect, only at very high concentrations, which might be due to surface charge interactions between the particles and cells. Free-radical scavenging properties of the particles might have prevented cell wall disruption and drastic antimicrobial action. This laboratory-scale study suggests that alumina NPs may only exhibit mild toxicity toward microorganisms in the environment. From the Clinical Editor: Metal oxide NPs, inluding aluminum oxide NPs, are known to possess strong antimicrobial properties. The study demonstrated a mild to moderate growth-inhibitory effect of alumina NPs over a wide concentration range (10-1000 μg/mL) on Escherichia coli. Almost negligible dependence on the concentration was observed. © 2009 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Alumina; Antimicrobial activity; Growth rate; Cell surface interactions
When the dimensions of a material are reduced to the atomic level it attains a range of unique properties that can be manipulated suitably for the desired applications.1 Because most of the biological processes also take place on the nanoscale, synergistic application of nanotechnology and biology can possibly address several important biomedical problems.2 Currently nanosized organic and inorganic nanoparticles (NPs) are finding increasing applications in medical devices3 as a result of their amenability to biological functionalization. Antimicrobial agents are highly relevant for a host of industrial applications in environmental, food, synthetic No conflict of interest was reported by the authors of this article. ⁎Corresponding author. Biomedical Engineering Division, School of Biotechnology, Chemical & Biomedical Engineering, VIT University, Old Madras Road, Vellore, Tamil Nadu-632014, India. E-mail address: [email protected] (A. Mukherjee).
textiles, packaging, health care, and medical care products. They can be grouped broadly into two types: organic and inorganic. The inorganic materials have gained importance over the past decade because of their ability to withstand adverse processing conditions.4,5 The antibacterial activity is known to be a function of the surface area in contact with the microorganisms; a larger surface area (as in the case of NPs) ensures a broad range of probable reactions with bioorganics present on the cell surface, as well as environmental inorganic and organic species.6 Published studies on the ecotoxicity of metal oxide NPs to bacterial species are limited, even though their bactericidal properties have been reported in the biomedical literature.4,7 One might therefore expect some of these materials to be toxic to microbes in the environment. Zinc oxide (ZnO) NPs seem to disrupt the gram-negative cell membrane structure in Escherichia coli,8 and it is proposed that NPs with a positive charge such as cerium oxide could bind the gram-negative
1549-9634/$ - see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.01.002 Please cite this article as: I.M. Sadiq, B. Chowdhury, N. Chandrasekaran, A. Mukherjee, Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles. Nanomedicine: NBM 2009;5:282-286, doi:10.1016/j.nano.2009.01.002
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cell membrane by electrostatic attraction. Clearly, the intimate relationship between the physicochemistry of the medium and membrane biology of the microbe is emerging as a key factor in NP toxicity to microorganisms. Aluminum oxide (Al2O3) NPs have important applications in the ceramics industry10 and can be used as an abrasive material, in heterogeneous catalysis, as an absorbent, as a biomaterial, and as reinforcements of metal-matrix composites.11,12 Alumina NPs are currently one of the two US market leaders for nanosized materials according to a recent report.13 Yamamoto et al investigated cytototoxic effects of metal oxide particles (Al2O3, titanium dioxide [TiO2], and zirconium oxide [ZrO2]) on murine fibroblasts and murine monocyte macrophages. They studied cytotoxicity as a function of shape, size, and surface area of the particles and also compared toxicity behavior of bulk and nanomaterials.14 Hanawa et al studied the toxicity of a range of metal oxide NPs, including Al2O3, having 500-nm to 3000-nm diameter on human fibroblast cells.15 Several recent studies with alumina NPs showed considerable cytotoxic effect on the mammalian cells.16-18 There have been very few studies available in the literature on the interaction of the alumina NPs with microbes. One past study found no detrimental effect of an alumina slurry between 62.5 and 250 mg/L concentration range on E. coli.19 Considering the paucity of prior literature reports on the effect of alumina NPs on bacterial species, the objective of the present work was to investigate the possible growth-inhibitory effect of alumina NPs over a wide concentration range (10–1000 μg/mL) on an environmentally relevant gram-negative model microorganism, E. coli. The implications of this type of growth-inhibitory study are mainly twofold: A. For biomedical applications the bactericidal properties of ceramic oxides must be studied. B. The growth inhibition effects of the oxides may reflect on toxicity behavior of metal oxides on bacterial species in the environment.
Methods Characterization of alumina nanoparticles Dry alumina NPs were procured from Aldrich (St. Louis, Missouri; CAS Number 1344-28-1). The supplier's data can be summarized as follows: gamma phase alumina nanopowder, particle size b50 nm (BET surface area analyzer; Smart Instruments Co. Pvt. Ltd., Mumbai, India), surface area 35–43 m2/g, melting point 2040°C (from the literature). The aqueous dispersion of the particles was subjected to particle size analysis using 90Plus Particle Size Analyzer (Brookhaven Instruments Corp., Holtsville, New York). The morphological features and particle size of the procured NPs were characterized by scanning electron microscopy
283
(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.). Preparation of nanoparticle dispersion The alumina NPs obtained from the suppliers were used as is to produce suspensions in deionized water. The suspensions at four different concentrations at 10, 50, 100, and 1000 μg/L were produced by means of ultrasonic vibration (100 W, 30 kHz) for 30 minutes. Bacterial strains The bactericidal experiments were carried out with gramnegative bacteria E. coli strain (NCIM No. 2666), which was procured from the National Chemical Laboratory (Pune, India) in Luria Bertani (LB) medium (Himedia Laboratories Ltd., Mumbai, India). Throughout this study the same nutrient medium was used. Growth inhibition study The minimum inhibitory concentration, defined as the lowest concentration of material that inhibits the growth of an organism, was determined based on batch cultures containing varying concentrations of alumina NPs in suspension (10, 50, 100, and 1000 μg/L). Sterile side-arm Erlenmeyer flasks (250 mL) containing 50 mL LB medium were sonicated for 10 minutes after adding the NPs to prevent aggregation of the NPs. Subsequently, the flasks were inoculated with 1 mL of the freshly prepared bacterial suspension to maintain initial bacterial concentration 108 colony-forming units per milliliter, and then incubated in an orbital shaker at 200 rpm and 30°C. The high rotary shaking speed was selected to minimize aggregation and settlement of the NPs over the incubation period. A lower rpm setting during incubation might cause underestimation of the antimicrobial activity of the NPs. Bacterial growth was measured as increase in absorbance at 600 nm determined using a spectrophotometer (CL-157 colorimeter; ELICO Company, Hyderabad, India). The experiments also included a positive control (flask containing NPs and nutrient medium, devoid of inoculum) and a negative control (flask containing inoculum and nutrient medium, devoid of NPs). The negative controls indicated the microbial growth profile in the absence of NPs. The absorbance values for positive controls were subtracted from the experimental values (flasks containing nutrient medium, inoculum, and NPs).20 All the experiments were carried out in triplicate and the mean value reported. Growth reversibility study To investigate whether the antibacterial effect of the NPs was due to the mere presence of these particles in the liquid broth, or due to their specific interactions with bacterial cellular components, the cells were cultured for 24 hours in the presence of alumina NPs, followed by sedimentation and reculturing them in fresh growth medium free of NPs. The
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Figure 1. Particle size distribution analysis of alumina NP dispersion. Figure 2. Scanning electron micrograph showing procured alumina NPs.
control cells were treated in a similar fashion but without any exposure to the NPs. Protein assay The extracellular protein was extracted using the polyethylene glycol method and was estimated using Lowry's method. The study was carried out in triplicate, and the mean value was reported. Standard error was within ±5%. Fourier transform–infrared study Fully grown culture after 24 hours incubation in the presence of 1000 μg/mL alumina NPs was harvested using centrifugation at 10,000 rpm for 15 minutes. The cell pellet was dispersed in deionized water, washed three times, and thereafter lyophilized for 4 hours, until the powder form was obtained. The powder was subjected to Fourier transform– infrared (FT-IR) by potassium bromide technique in a Nicolet 6700 FT-IR Spectrometer (Thermo Scientific Instruments Groups, Madison, Wisconsin). For controls the same protocol was followed in the absence of NPs. Results Characterization of as-procured nanoparticles The particle size distribution analysis (Figure 1) showed an effective diameter of 179.2 nm, with a polydispersity index of 0.191. The surface area was determined to be 21.23 m2/g. The high-resolution SEM image of procured alumina NPs is shown in Figure 2. Nearly spherical to spheroidal NPs were observed. The particles seemed to be agglomerated. The difference between the supplier's data and the experimentally obtained data may be due to agglomeration of the particles while present in aqueous suspension. Antimicrobial activity of alumina nanoparticles against E. coli In further experiments, strains of the gram-negative bacterium E. coli were inoculated in liquid LB medium
supplemented with increasing dosages of alumina NPs. Increasing concentration of NPs progressively retarded the growth of E. coli (Figure 3, A). The concentration of 1000 μg/mL was found to be mildly inhibitory for bacteria, in that about 6–8 hours passed before any noticeable growth was initiated. The steepness of the growth curve in the logarithmic phase and the final cell concentration were also noticeably lower at 500 and 1000 μg/mL concentrations, as compared with the lower ones used in this study. Prediction of bacterial growth in the presence of varying concentrations of alumina nanoparticles In the logarithmic phase the dynamic growth rate of a bacterial species is represented by the following equation: In N = lnN0 + lt
ð1Þ
where N is the bacterial cell count at time t, N0 is the initial cell count, and μ is the growth rate constant for the bacteria. From the growth curve (Figure 3, A) of the bacteria at different concentrations of alumina NPs the logarithmic phase was identified between 6 and 14 hours. The growth data in this time interval were plotted in the logarithmic scale (Figure 3, B) to derive the values of μ corresponding to various doses of the nanoparticles (x) studied. A linear relationship between x and μ (Figure 3, C) was derived: l = ax + b
ð2Þ
From Figure 3, C the equation obtained was l = 2 105 x + 0:0909
ð2aÞ
A very low value of a signifies almost negligible dependence of growth rate on the concentration of the NPs. Importantly, here μ positively correlated with concentration of the particles.
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Figure 4. Growth curve of cells recultured in particle-free fresh medium after interacting with the particles for 24 hours; uninteracted inoculum was used as control here.
extracellular proteins extracted from fully grown culture in the presence of 1000 μg/L alumina NP concentrations were found to be only 5.9 μg/mL. So the interaction with the particles had a diminishing effect on extracellular protein content in E. coli. Fourier transform–infrared study FT-IR spectra (not shown) of the uninteracted bacterial samples revealed the following bands: 3726.6 and 3626.8 cm–1 band predominantly due to OH stretching vibrations; 2150 and 2002.7 cm–1 may be overtones due to combination of NH3+ torsion and NH3+ antisymmetric deformation for amino acids; 1542.1 cm–1 due to amide II. When the cells were interacted with 1000 μg/L alumina NPs, their FT-IR spectra revealed 2383.3, 2162.4, and 1986.8 cm–1 bands signifying probably overtones due to combination of NH3+ torsion and NH3+ antisymmetric deformation for amino acids. The bands for OH stretching and amide II were conspicuously absent in the spectra. Discussion
Figure 3. (A) Effect of alumina NP concentrations on growth of E. coli. (B) Cell growth of E. coli in the logarithmic phase as a function of alumina NP concentrations. (C) Dynamic cell growth rate (μ) of E. coli as a function of alumina NP concentrations. CFU, colony-forming units; OD, optical density.
Growth reversibility study In reversibility studies a significant retardation in growth of recultured gram-negative bacteria E. coli that had had prior exposure to the NPs was observed in the fresh medium (Figure 4). Control cells did not display any deviation from normal growth characteristics. These observations were consistent with sustained interaction between NPs and cellular components of gram-negative bacteria, in that otherwise the growth curves of recultured bacteria would have been comparable to that of the control cells. Protein assay of the interacted microorganisms The extracellular protein content from the uninteracted control cells of E. coli was measured to be 7.75 μg/mL. The
The hypothesis in this study was that alumina NPs at the tested concentrations inhibit growth of the test microorganism, E. coli, under the experimental conditions. The important observations from the present work with gramnegative E. coli can be summarized as mild growthinhibitory effect of alumina NPs in the concentration range 10–1000 μg/mL. The intimate relationship between the physicochemistry of the oxide NPs, medium, and membrane biology of the microbe is emerging as a key factor in the sensitivity of microorganisms to the particles in suspension. ZnO NPs appear to disrupt the gram-negative cell membrane structure in E. coli,8 and it is proposed that NPs with a positive charge such as cerium oxide could bind the gram-negative cell membrane by electrostatic attraction.9 The decrease in cell viability as observed in the present study may be due to bacterial adhesion to the particle surfaces. Due to positive surface charges on the alumina NPs at near-neutral pH, an electrostatic interaction is possible between negatively charged E. coli cells and the particles, leading to bacterial adhesion onto NP surfaces. As the adhesion increased with increase in concentration of the particles in the suspension, a
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negative effect on growth was observed with respect to concentration. This electrostatic interaction between bacteria and particle surface, along with hydrophobic interactions and polymer bridging, may be responsible for the phenomenon of bacterial adhesion onto the particles.21 The strong bactericidal effect as observed with other metal oxides such as ZnO2, TiO2, etc. was not observed in the case of alumina. The disruption of cell wall due to reactive oxygen species (ROS) generation is one of the important mechanisms behind cell death leading to the strong antimicrobial property of these metal oxides.8 But alumina NPs may act as freeradical scavengers.22 Alumina is thermodynamically stable over a wide temperature range and has a corundum-like structure, with oxygen atoms adopting hexagonal closepacking and Al3+ ions filling two thirds of the octahedral sites in the lattice. These NPs are able to rescue cells from oxidative stress-induced cell death in a manner that appears to be dependent upon the structure of the particle but independent of its size within the range of 6–1000 nm. According to another explanation, they may act as direct antioxidants, blocking ROS production, thus inhibiting the apoptotic pathway, and they may directly inhibit ROS production, which rapidly induces a ROS defense system before the glutamate-induced cell death program is complete.22 It is evident from this study that alumina NPs do not possess strong antimicrobial properties; minor growth inhibition was noticed at high NP concentrations up to 1000 μg/mL. These observations are pertinent regarding ecotoxicity of alumina NPs with respect to microorganisms. E. coli is one important environmentally relevant gram-negative microorganism. This laboratory-scale study suggests that alumina may have only mild toxicity toward microorganisms in the environment. However, the environmental behavior may be more complex in nature— depending upon physicochemical interactions of the particles or presence of microbial consortia, among other factors. In conclusion, alumina NPs showed a mild growthinhibitory effect on E. coli, only at very high concentrations. A concentration-dependent negative effect was observed on extracellular protein content of gram-negative bacteria E. coli. Almost negligible dependence of growth rate was observed on the concentration of the NPs. References 1. Gleiter H. Nanostructured materials, basic concepts and microstructure. Acta Mater 2000;48:1-12. 2. Curtis A, Wilkinson C. Nanotechniques and approaches in biotechnology. Trends Biotechnol 2001;19:97-101. 3. Waren CW, Nie S. Quantum dot bioconjugates for ultra sensitive nonisotopic detection. Science 1998;281:2016-8.
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