Kelly R. Bright

Silver as a Disinfectant

The antimicrobial effects of silver (Ag) have been recognized for thousands of years. In ancient times, it was used in water containers (Grier 1983) and to prevent putrefaction of liquids and foods. In ancient times in Mexico, water and milk were kept in silver containers (Davis and Etris 1997). Silver was also mentioned in the Roman pharmacopoeia of 69 b.c. (Davis and Etris 1997).

New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water.

ABSTRACT The methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years, with the most common technique being the use of 1MDS Virozorb filters followed by organic flocculation for secondary concentration. Recently, a few studies have investigated alternatives; however, many of these methods are impractical for use in the field or share some of the limitations of this traditional method. In the present study, the NanoCeram virus sampler, an electropositive pleated microporous filter composed of microglass filaments coated with nanoalumina fibers, was evaluated. Test viruses were first concentrated by passage of 20 liters of seeded water through the filter (average filter retention efficiency was ≥99.8%), and then the viruses were recovered using various salt-based or proteinaceous eluting solutions. A 1.0% sodium polyphosphate solution with 0.05 M glycine was determined to be the most effective. The recovered viruses were then further concentrated using Centricon Plus-70 centrifugal ultrafilters to a final volume of 3.3 (±0.3 [standard deviation]) ml; this volume compares quite favorably to that of previously described methods, such as organic flocculation (∼15 to 40 ml). The overall virus recovery efficiencies were 66% for poliovirus 1, 83% for echovirus 1, 77% for coxsackievirus B5, 14% for adenovirus 2, and 56% for MS2 coliphage. In addition, this method appears to be compatible with both cell culture and PCR assays. This new approach for the recovery of viruses from water is therefore a viable alternative to currently used methods when small volumes of final concentrate are an advantage.

Silver as a residual disinfectant to prevent biofilm formation in water distribution systems.

ABSTRACT Biofilms can have deleterious effects on drinking water quality and may harbor pathogens. Experiments were conducted using 100 μg/liter silver to prevent biofilm formation in modified Robbins devices with polyvinyl chloride and stainless steel surfaces. No significant difference was observed on either surface between the silver treatment and the control.

Rapid reduction of Legionella pneumophila on stainless steel with zeolite coatings containing silver and zinc ions

Aims: To determine the rate of reduction of Legionella pneumophila by stainless steel surfaces with zeolite ceramic coatings containing 2·5% (w/w) silver (Ag) and 14% zinc (Zn) ions.

Occurrence of Bacteria and Viruses on Elementary Classroom Surfaces and the Potential Role of Classroom Hygiene in the Spread of Infectious Diseases.

The presence of microorganisms on common classroom contact surfaces (fomites) was determined to identify the areas most likely to become contaminated. Six elementary classrooms were divided into control and intervention groups (cleaned daily with a quaternary ammonium wipe) and tested for heterotrophic bacteria. Three classrooms were also tested for norovirus and influenza A virus. Frequently used fomites were the most contaminated; water fountain toggles, pencil sharpeners, keyboards, and faucet handles were the most bacterially contaminated; desktops, faucet handles, and paper towel dispensers were the most contaminated with viruses. Influenza A virus was detected on up to 50% and norovirus on up to 22% of surfaces throughout the day. Children in the control classrooms were 2.32 times more likely to report absenteeism due to illness than children in the intervention classrooms and were absent longer (on average). Improved classroom hygiene may reduce the incidence of infection and thus student absenteeism.

Antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus

To investigate the antiviral efficacy of oregano oil and its primary active component, carvacrol, against the nonenveloped murine norovirus (MNV), a human norovirus surrogate.

Naegleria fowleri in Well Water

To the Editor: Naegleria fowleri, a protozoon found in hot springs and warm surface water, can cause primary amebic meningoencephalitis in humans. A survey of drinking water supply wells in Arizona determined that wells can be colonized and may be an unrecognized source of this organism that could present a human health risk. N. fowleri is a free-living ameboflagellate found in warm bodies of water such as ponds, irrigation ditches, lakes, coastal waters, and hot springs and can cause primary amebic meningoencephalitis. Humans come into contact with N. fowleri by swimming or bathing, particularly in surface waters. The ameba enters the nasal passages, penetrates the nasopharyngeal mucosa, and migrates to the olfactory nerves, eventually invading the brain through the cribriform plate (1). From 1995 to 2004, N. fowleri killed 23 persons in the United States (2), including 2 children in the Phoenix, Arizona, area in 2002, who had been exposed to well water but had not consumed it (3). There have been 6 documented deaths in 2007, all in warmer regions (Arizona, Texas, Florida) (4). Although N. fowleri’s presence in surface waters is well documented (5,6), no previous studies on its occurrence in wells have been conducted. We studied high-volume drinking water wells operated by municipal utilities or private water companies in the greater Phoenix and Tucson, Arizona, areas. Previous data from 500 wells in the region showed temperatures ranging from 13°C to 46°C. Typical well discharges ranged from hundreds to >3,780 L per minute. Well depths varied from 100 m to >300 m. Well water samples were collected by using 1-L sterile polyethylene bottles at or near the wellhead before disinfection by well owners or utilities (7). In phase 1, samples were collected after wells were flushed until the water was clear. During phase 2, samples were collected as water was turned on from spigots at or near wellheads (initial) and after a 3-borehole volume had flushed through the system (purged). Additional wells were sampled during this phase. Samples were tested for temperature, pH, turbidity, chlorine residual, conductance, coliforms, heterotrophic bacterial plate counts (HPC), and Escherichia coli following standard methods (7). To test for viable amebas, we spread aliquots on nonnutrient agar seeded with E. coli at 37°C (3,8). We placed scrapings from the advancing front of subsequent ameba plaques in distilled water to identify enflagellation (5); however, precise species identification was not possible. Live amebae were therefore harvested for PCR analysis to specifically identify N. fowleri. We chose PCR over the mouse pathogenicity test because other Naegleria species that are nonpathogenic in humans are lethal in mice (8). The genotype of isolates was not determined because all of the described genotypes found in the United States have been shown to be pathogenic in humans (9). To concentrate trophozoites/cysts, we gently agitated samples for 2 minutes and then centrifuged and filtered them through polyethylene filters (2-μm pore; Millipore, Bedford, MA, USA). A 10-μL volume of concentrate was used as a template for nested PCR (3,8) (triplicate tests were conducted immediately and after a 2-week 37°C incubation). Positive and negative PCR products were frozen at –80°C, coded to prevent bias, and shipped to Francine Marciano-Cabral at Virginia Commonwealth University for confirmation by cloning and sequencing (3). The general microbial quality of the wells was as follows: 73 (51%) had >500 HPC/mL; 8 (5.5%) were positive for coliforms; none were positive for E. coli. Oils used to lubricate well motors may result in the growth of HPC in well water (10). N. fowleri feeds on heterotrophic bacteria in water and could multiply in the well casing. This may explain N. fowleri’s colonization of wells. The recent association in Arizona between unchlorinated drinking water and the transmission of N. fowleri suggests that groundwater has been an unrecognized source of this organism. PCR detected N. fowleri DNA in 11 (7.7%) of 143 wells. Of 185 total samples, 30 (16.2%) tested positive for N. fowleri (Table). The organism was most often detected after the wells had been purged (17.9% purged vs. 10.0% initial samples), suggesting that N. fowleri was present in the aquifer or was released from the well casing or column during pumping. The wells testing positive for N. fowleri ranged in temperature from 21.9°C to 37.4°C (average 29.0°C; median 29.5°C). Table Naegleria fowleri in well water samples, Arizona The live trophozoite form was confirmed in only 1 well, though 11 of 143 wells tested positive according to PCR. This discrepancy may be due to the low occurrence of trophozoites in water or to differences in assay volumes for detection of live trophozoites (0.75 mL) versus PCR (30 mL equivalent unconcentrated volume). PCR is also more sensitive, capable of detecting 100 organisms/L in an unconcentrated sample (8); however, PCR did not determine if the amebas were infectious. Although PCR can determine the species by using primers for a specific gene sequence not found in other Naegleria species, it cannot determine the life stage (cyst/trophozoite). Trophozoites are believed to be the infectious form of the organism (1); nonetheless, cysts can be equally harmful because they may revert to trophozoites under optimal conditions (1). The surprisingly common occurrence of N. fowleri in drinking water wells suggests that groundwaters may be an unrecognized human health threat.

Inactivation of Pseudomonas aeruginosa and Aeromonas hydrophila by silver in tap water

This study was conducted to assess the efficacy of silver as a secondary disinfectant to replace or reduce the level of chlorine utilized in water distribution systems. Pseudomonas aeruginosa and Aeromonas hydrophila are opportunistic pathogens present in drinking water and have been associated with waterborne disease. After 8 hours of exposure to 100 μ g/L of silver, there was a > 6-log10 reduction in P. aeruginosa in tap water at room temperature at pH7 and a 5.55-log10 reduction in the presence of 3 mg/L humic acid. Similar reductions were observed at pH9. At 4°C, reductions greater than 4-log10 were observed after 24 hours. For A. hydrophila, a > 6-log10 reduction occurred at both pH7 and pH9 within nine hours. The World Health Organization has determined that this amount of silver could be used for water disinfection without health risks. Furthermore, silver shows promise as a secondary disinfectant, even in the presence of organic matter in concentrations that would reduce the effectiveness of free chlorine.

Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples.

The extraction and purification of nucleic acids is a critical step in the molecular detection of enteric viruses from environmental or fecal samples. In the present study, the performance of three commercially available kits was assessed: the MO BIO PowerViral Environmental DNA/RNA Isolation kit, the Qiagen QIAamp Viral RNA Mini kit, and the Zymo ZR Virus DNA/RNA Extraction kit. Viral particles of adenovirus 2 (AdV), murine norovirus (MNV), and poliovirus type 1 (PV1) were spiked in molecular grade water and three different types of sample matrices (i.e., biosolids, feces, and surface water concentrates), extracted with the kits, and the yields of the nucleic acids were determined by quantitative PCR (qPCR). The MO BIO kit performed the best with the biosolids, which were considered to contain the highest level of inhibitors and provided the most consistent detection of spiked virus from all of the samples. A qPCR inhibition test using an internal control plasmid DNA and a nucleic acid purity test using an absorbance at 230 nm for the nucleic acid extracts demonstrated that the MO BIO kit was able to remove qPCR inhibitors more effectively than the Qiagen and Zymo kits. These results suggest that the MO BIO kit is appropriate for the extraction and purification of viral nucleic acids from environmental and clinical samples that contain high levels of inhibitors.

Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

ABSTRACT Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log10 to 1.88 log10). After 24 h, the reductions were 2.74, 3.00, and 3.41 log10 for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.