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Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of GramNegative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa B Ramalingam, Thanusu Parandhaman, and Sujoy K. Das ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00161 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 3, 2016
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Table of Content Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa Baskaran Ramalingam,†,≠ Thanusu Parandhaman,†,‡,≠ and Sujoy K. Das*,†,‡ †
Bioproducts Laboratory, Council of Scientific and Industrial Research (CSIR)Central leather Research Institute (CLRI), Chennai600020, India, and ‡Academy of Scientific and Innovative Research (AcSIR), New Delhi110001, India. ≠ Both have equal contribution
1 µm
*
Address for communication: Dr. Sujoy K. Das Email:
[email protected];
[email protected] Tel: +914424437133, Fax: +914424911589
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Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa Baskaran Ramalingam,†,≠ Thanusu Parandhaman,†,‡,≠ and Sujoy K. Das*,†,‡ †
Bioproducts Laboratory, Council of Scientific and Industrial Research (CSIR)Central leather Research Institute (CLRI), Chennai600020, India, and ‡Academy of Scientific and Innovative Research (AcSIR), New Delhi110001, India. ≠ Both have equal contribution
ABSTRACT: Understanding the interactions of silver nanoparticles (AgNPs) with the cell surface is crucial for the evaluation of bactericidal activity and for advanced biomedical and environmental applications. Biosynthesis of AgNPs was carried out through in situ reduction of silver nitrate (AgNO3) by cell free protein of Rhizopus oryzae and the synthesized AgNPs was characterized by UV-vis spectroscopy, high resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), Zeta potential analysis and FTIR spectroscopy. The HRTEM measurement confirmed the formation of 7.1 ± 1.2 nm AgNPs, whereas DLS study demonstrated average hydrodynamic size of AgNPs as 9.1 ± 1.6 nm. The antibacterial activity of the biosynthesized AgNPs ( = 17.1 ± 1.2 mV) was evaluated against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. The results showed that AgNPs exhibited concentration dependent antibacterial activity and 100% killing of E. coli and P. aeruginosa achieved when the cells were treated with 4.5 and 2.7 µg/mL AgNPs, respectively for 4 h. Furthermore, the intracellular reactive oxygen species (ROS) production suppressed the antioxidant defense and exerted mechanical damage to the membrane. AgNPs also induced surface charge neutralization and altered of the cell membrane permeability causing non-viability of the cells. Atomic force microscopic (AFM) studies depicted alteration of ultrastructural and nanomechanical properties of the cell surface following interaction with AgNPs, while FTIR spectroscopic analysis demonstrated that cell membrane of the treated cells underwent an orderto-disorder transition during the killing process and chemical composition of the cell membrane including fatty acids, proteins, and carbohydrates was decomposed following interaction with AgNPs. KEYWORDS: Silver nanoparticles, Antibacterial activity, Reactive oxygen species, Force spectroscopy, Nanomechanical properties
*Address for communication: Dr. Sujoy K. Das Email:
[email protected];
[email protected]; Tel: +914424437133, Fax: +914424911589 2 ACS Paragon Plus Environment
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1. INTRODUCTION Bacterial infections remain a major cause of death, disability, and socioeconomic loss for millions of people worldwide and rapid emergences of antibiotic-resistant microbes have made the situation more complicated.1 It is estimated that 50% of the hospitalized patients suffer from nosocomial infection with drug-resistant bacteria annually worldwide.2 Development of drug resistance microorganisms against common antibiotics urges to new drug and/ or material to combat pathogenic microorganisms.3 Recently, the nanoparticle based disinfection and therapeutic system has been considered as a promising alternative for improving the diagnostic system due to their unique physical and chemical properties of nanoparticles.4-8 In particular, silver nanoparticles (AgNPs) possess broad spectrum antimicrobial activity and thus increasingly use in medicine, consumer products and water treatment to prevent microbial growth.5-11 Inspite of that, the applications of AgNPs unavoidably suffer from certain limitations. The AgNPs are very prone to oxidation, which causes aggregation of AgNPs and thus, lose antibacterial activity.12,13 Moreover, leaching of Ag+ ions often creates health and environmental issues. In addition, the synthesis and functionalization processes often raise health issues due to use of toxic chemicals.14,15 To solve these issues, green synthesis of metal nanoparticles using proteins or other biomolecules has gained significant importance.15-19 In order to enhance the biomedical applications of biosynthesized AgNPs, it is essential to understand the interaction of AgNPs with the microorganisms and their subsequent cellular responses. However, the fundamental molecular mechanism of antibacterial activity of AgNPs did not understand very well; there is still debate on the mechanism of action. Various mechanisms have been proposed for AgNPs mediated cell death including disruption of the cell envelope, oxidation of the cell components, inactivation of the respiratory chain enzymes, production of the reactive oxygen species (ROS), decomposition of the cellular components, etc.20-27 It is reported that AgNPs kill the bacterial cells through contact inhibition mode.23,24 The membrane permeability increases following incorporation of AgNPs into the cell membrane, which subsequently forms permeable pits. This leads to an osmotic collapse in the cells and releases the intracellular materials. The other possible mechanism includes initial binding of AgNPs with the cell surface proteins or carbohydrate moieties damages the cell membrane, which causes denaturation of the enzymes and interruption of the electron transport pathway leading to the cell death.25,26 Oxidative stress induced reactive oxygen species (ROS) generation is also proposed as another important mechanism for antibacterial activity of AgNPs.21,22,27 The inhibition of respiratory enzymes by AgNPs leads to the formation of ROS such as hydroxyl radicals OH•, superoxide ions O2•, H2O2 and hydroperoxyl radicals, which
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triggers cells damage through oxidative decomposition of the cellular components. Among all these mechanistic pathways, the interaction of AgNPs with the bacterial cells disrupts the cell membrane and kills the bacteria. However, effect of AgNPs on physico-chemical properties, such as surface charge, roughness, chemical composition, cell wall rigidity, and adhesion property, of the bacterial cell have yet to be investigated. Atomic force microscopy (AFM) has provided an opportunity to probe nanomechanical properties including morphology of the microbial cells at physiological condition.28,29 Recently only few studies described the effect of AgNPs on alteration of cellular morphology using AFM.30-32 In this manuscript we have demonstrated the physico-chemical interactions of the biosynthesized AgNPs with bacterial cells using Zeta potential, fluorescence and atomic force microscopy along with Fourier transform infrared spectroscopy (FTIR) analysis. Specifically, we have synthesized AgNPs by cell free protein of Rhizopus oryzae and investigated the antibacterial activity of the synthesized AgNPs against Gram-negative bacteria viz. Escherichia coli and Pseudomonas aeruginosa as model bacteria. The alteration of morphological and physico-chemical properties of the bacterial cell surface has been monitored through Zeta potential, fluorescence microscopic, AFM and FTIR studies.
2. EXPERIMENTAL SECTION Materials. Silver nitrate (AgNO3), potassium bromide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and all other chemicals were purchased from Merck, India. All microbiological media and ingredients were purchased from HiMedia, India.
Microorganisms. Escherichia coli (MTCC 062), Pseudomonas aeruginosa (MTCC 424) and Rhizopus oryzae (MTCC 262) were obtained from Institute of Microbial Technology, India. The fungal and bacterial strains were maintained on potato dextrose (20% potato extract, 2% dextrose and 1.5% agar) and nutrient (0.3% beef extract, 0.5% peptone and 1.5% agar) agar slants, respectively. The organisms were stored at 4 C and sub-cultured at regular intervals of 30 days to maintain the cell viability.
Preparation of R. oryzae protein extract. R. oryzae mycelia were grown in 50 mL of potato dextrose (20% potato extract and 2% dextrose) broth for 72 h at 30 C were harvested and washed several times using phosphate buffer (50 mM, pH 7.0). The mycelia were then crushed with sea sand using mortar and pestle at low temperature (1.35 µg/mL) the bacterial growth was inhibited. At low concentration of AgNPs, the initial lag phase was extended compared to the control cells and the AgNPs at a concentration of 1.35 ± 0.1 µg/mL inhibited 50% of the growth of both bacteria. With further increase in concentration of AgNPs to 3.6 ± 0.23 and 2.25 ± 0.2 µg/mL caused complete inhibition (P1.35 µg/mL AgNPs, the intracellular ROS production was induced and with further increase in AgNPs the concentration of ROS increased significantly indicating ROS mediated cell death. Moreover, in comparison to positive control (1 mM H2O2), ROS production
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level in E. coli and P. aeruginosa varied from 70-80% when treated with 4.5 and 2.7 µg/mL AgNPs, respectively. This implied that despite ROS production other factor like physical damage of the cell membrane might also be responsible for bacterial killing by AgNPs. The intracellular ROS production was further verified by measuring glutathione (GSH) concentration. In most of the Gram-negative bacteria GSH, a thiol-containing tripeptide, is predominantly present in reduced form at high levels (0.110 mM) to maintain the cellular redox environment and protect the cells against oxidative stress by scavenging ROS.47 Preserving the GSH mediated antioxidant defense is therefore, very crucial for cell survival. However, GSH spontaneously oxidize to disulfide (GSSG) upon exposure to the molecular oxygen (O2 + 2R-SH RSSR + H2O2; G0= 96 kJ/mol).48 Significant oxidation of GSH into GSSG is leading to the cell death. The intracellular GSH concentration was therefore, measured to understand the cellular oxidative stress upon treatment with AgNPs. The results (Figure 3C) demonstrated that GSH concentration depleted in the treated cells with subsequent increase in AgNPs concentration. The GSH concentration was 1.14 ± 0.04 and 1.09 ± 0.03 mM respectively in untreated E. coli and P aeruginosa and reduced significantly to 0.51 ± 0.004 and 0.44 ± 0.009 mM (P value is
