Biogenic Silver for Disinfection of Water Contaminated with Viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag⁰. The antiviral action of biogenic Ag⁰ was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter⁻¹ biogenic Ag⁰ caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg⁰) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter⁻¹ ionic Ag⁺ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag⁰ were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag⁰ was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag⁰ m⁻² on the filter (135 mg biogenic Ag⁰ kg⁻¹ filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag⁰ and its promising opportunities for continuous water disinfection.At least 1 billion people do not have access to safe drinking water, according to the WHO (41). Contamination of drinking water and the subsequent outbreak of waterborne diseases are the leading cause of death in many developing nations. Moreover, the spectrum and incidence of some infectious diseases are increasing worldwide (40). Among them, the transmission of waterborne human noroviruses is considered to be the major cause of acute nonbacterial gastroenteritis (22). Numerous outbreaks of norovirus-associated gastroenteritis have been linked with ingestion of contaminated drinking water, in developed countries also (6; M. Kukkula, L. Maunula, E. Silvennoinen, and C. H. von Bonsdorff, presented at the International Workshop on Human Caliciviruses, Atlanta, GA, 29 to 31 March 1999). Therefore, the development of innovative drinking water quality control strategies is of the utmost importance in this decade.Recent interest has arisen in the use of nanotechnology for water disinfection (20). In particular the formation of by-products by conventional disinfection techniques (e.g., chlorination), has encouraged researchers to explore the antimicrobial activity of several nanomaterials, such as silver (18, 31). Silver-containing nanoparticles have previously been demonstrated to be effective against bacteria and viral particles (10, 28, 34). Several mechanisms of the antiviral activity have been ascribed to (chemically produced) zerovalent silver nanoparticles (nAg⁰) but still remain not fully understood. On the one hand, nAg⁰ can release Ag⁺ ions, which interact with thiol groups in proteins and interfere with DNA replication (11, 21, 24). On the other hand, the adhesion of nAg⁰ as such is responsible for the inactivation of HIV-1 virions (10).Previous studies showed that chemically produced nAg⁰ were unstable in solution and would easily aggregate with average particle sizes of <40 nm or at high concentrations (23). As a consequence, the specific surface of the nanomaterial decreases. Moreover, there is a need for environmentally friendly approaches to production of nanoparticles. To cope with these demands, biological processes have been developed using microorganisms. Microbial approaches to obtain nanoscale Ag⁰ have been demonstrated for the bacterium Pseudomonas stutzeri AG259 (17) and for fungi, e.g., Verticillium sp. (26), Phoma sp. (5), Fusarium sp. (2, 16), and Aspergillus sp. (12, 29, 39). However, these enzymatic reduction processes are slow and yield low concentrations of silver. Moreover, if the nanoparticles are produced intracellularly, specific treatments (e.g., heat treatment at 600°C for 6 h) are necessary to make the nanoparticles accessible for antibacterial or antiviral applications (39).Recently, lactic acid bacteria have been used as reducing agents for the fast, nonenzymatic, and extracellular production of nanoscale-sized Ag⁰ particles (33). The bacterial cell wall hereby serves as a microscale carrier matrix for the nanoparticles. The unique association of the nanoparticles with the (dead) bacterial carrier matrix, called biogenic Ag⁰, prevents them from aggregating and makes the association promising for disinfection technologies. In the case of virus inactivation, smaller nanoparticles are known to be more efficient due to a more effective binding to the glycoproteins of the viral envelope (10, 28). For biogenic Ag⁰ production using lactic acid bacteria, it was demonstrated that different particle sizes could be obtained, depending on the species used (33). Production by Lactobacillus fermentum resulted in the smallest average diameter and a narrow size distribution, potentially favorable for antimicrobial applications (33).The objective of the present study was to examine the inactivation of a bacteriophage (UZ1), isolated from hospital sewage, by biogenic Ag⁰. This DNA phage, a T7-like coliphage of the genus Podovirida (order Caudovirales) (38), is infective for Enterobacter aerogenes BE1, a species belonging to the normal digestive microbiota (30). The virucidal action of biogenic Ag⁰ was evaluated in drinking water and compared with the use of ionic Ag⁺ and chemically produced nAg⁰. To test the antiviral activity of biogenic Ag⁰ against noroviruses as well, the murine norovirus 1 (MNV-1) was used as a surrogate organism for human noroviruses (43). Finally, continuous disinfection by the biogenic nanoparticles was evaluated in a flowthrough system with a coated cartridge filter. To our knowledge, this is the first report to demonstrate the antiviral effect of extracellular biogenic Ag⁰.