Antiviral Natural Products, Their Mechanisms of Action and Potential Applications as Sanitizers and Disinfectants.

Plant extracts, natural products and plant oils contain natural virucidal actives that can be used to replace active ingredients in commercial sanitizers and disinfectants. This review focuses on the virucidal mechanisms of natural substances that may exhibit potential for indoor air and fomite disinfection. Review of scientific studies indicates: (1) most natural product studies use crude extracts and do not isolate or identify exact active antiviral substances; (2) many natural product studies contain unclear explanations of virucidal mechanisms of action; (3) natural product evaluations of virucidal activity should include methods that validate efficacy under standardized disinfectant testing procedures (e.g., carrier tests on applicable surfaces or activity against aerosolized viruses, etc.). The development of natural product disinfectants requires a better understanding of the mechanisms of action (MOA), chemical profiles, compound specificities, activity spectra, and the chemical formulations required for maximum activity. Combinations of natural antiviral substances and possibly the addition of synthetic compounds might be needed to increase inactivation of a broader spectrum of viruses, thereby providing the required efficacy for surface and air disinfection.

Evaluation of E. coli in sediment for assessing irrigation water quality using machine learning.

Fresh produce irrigated with contaminated water poses a substantial risk to human health. This study evaluated the impact of incorporating sediment information on improving the performance of machine learning models to quantify E. coli level in irrigation water. Field samples were collected from irrigation canals in the Southwest U.S., for which meteorological, chemical, and physical water quality variables as well as three additional flow and sediment properties: the concentration of E. coli in sediment, sediment median size, and bed shear stress. Water quality was classified based on E. coli concentration exceeding two standard levels: 1 E. coli and 126 E. coli colony forming units (CFU) per 100 ml of irrigation water. Two series of features, including (FIS) and excluding (FES) sediment features, were selected using multi-variant filter feature selection. The correlation analysis revealed the inclusion of sediment features improves the correlation with the target standards for E. coli compared to the models excluding these features. Support vector machine, logistic regression, and ridge classifier were tested in this study. The support vector machine model performed the best for both targeted standards. Besides, incorporating sediment features improved all models' performance. Therefore, the concentration of E. coli in sediment and bed shear stress are major factors influencing E. coli concentration in irrigation water.

Biocides and Novel Antimicrobial Agents for the Mitigation of Coronaviruses.

In December, 2019, a highly infectious and rapidly spreading new pneumonia of unknown cause was reported to the Chinese WHO Country Office. A cluster of these cases had appeared in Wuhan, a city in the Hubei Province of China. These infections were found to be caused by a new coronavirus which was given the name "2019 novel coronavirus" (2019-nCoV). It was later renamed "severe acute respiratory syndrome coronavirus 2," or SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11, 2020. It was named SARS-CoV-2 due to its close genetic similarity to the coronavirus which caused the SARS outbreak in 2002 (SARS-CoV-1). The aim of this review is to provide information, primarily to the food industry, regarding a range of biocides effective in eliminating or reducing the presence of coronaviruses from fomites, skin, oral/nasal mucosa, air, and food contact surfaces. As several EPA approved sanitizers against SARS-CoV-2 are commonly used by food processors, these compounds are primarily discussed as much of the industry already has them on site and is familiar with their application and use. Specifically, we focused on the effects of alcohols, povidone iodine, quaternary ammonium compounds, hydrogen peroxide, sodium hypochlorite (NaOCl), peroxyacetic acid (PAA), chlorine dioxide, ozone, ultraviolet light, metals, and plant-based antimicrobials. This review highlights the differences in the resistance or susceptibility of different strains of coronaviruses, or similar viruses, to these antimicrobial agents.

Review of water quality criteria for water reuse and risk-based implications for irrigated produce under the FDA Food Safety Modernization Act, produce safety rule.

Questions related to the safety of alternative water sources, such as recycled water or reclaimed water (including grey water, produced water, return flows, and recycled wastewater), for produce production have been largely un-explored at the detail warranted for protection of public health. Additionally, recent outbreaks of Escherichia coli (E. coli) in fresh produce, in which agricultural water was suspected as the source, coupled with heightened media coverage, have elevated fruit and vegetable safety into the forefront of public attention. Exacerbating these concerns, new Federal regulations released by the U.S. Food and Drug Administration (FDA) as part of implementation of the FDA Food Safety Modernization Act (FSMA), require testing of agricultural water quality for generic E. coli. Here, we present a review of water quality criteria - including surface water, groundwater recreational water, and water reuse - in an attempt to better understand implications of new FDA regulations on irrigated produce. In addition, a Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate risks from pathogen contamination of food crops eaten fresh under the context of FDA regulations to provide perspective on current water reuse regulations across the country. Results indicate that irrigation water containing 126 CFU/100 mL of E. coli correspond to a risk of GI illness (diarrhea) of 9 cases in 100,000,000 persons (a 0.000009% risk) for subsurface irrigation, 1.1 cases in 100,000 persons (a 0.0011% risk) for furrow irrigation, and 1.1 cases in 1000 persons (a 0.11% risk) for sprinkler irrigation of lettuce. In comparison to metrics in states that currently regulate the use of recycled water for irrigation of food crops eaten fresh, the FDA FSMA water quality metrics are less stringent and therefore the use of recycled water presents a reduced risk to consumers than the FDA regulations. These findings, while limited to a one-time exposure event of lettuce irrigated with water meeting FSMA water quality regulations, highlight the need for additional assessments to determine if the scientific-basis of the regulation is protective of public health.

Antimicrobial efficacy of plant essential oils and extracts against Escherichia coli.

The efficacies of 11 plant-derived antimicrobials were evaluated against Escherichia coli in vitro in solution at room temperature. These included lemongrass, cinnamon, and oregano essential oils and their active components (citral, cinnamaldehyde, and carvacrol, respectively). Allspice and clove bud oils and olive, green tea, and grape seed extracts were also studied. The efficacies of the antimicrobials were both concentration- and exposure time-dependent. The essential oils and their active components demonstrated statistically significant >5.0-log10 reductions within 1-10 min. The plant extracts were less effective; green tea and grape seed extracts required 24 h before significant reductions were observed (1.93-log10 and 5.05-log10, respectively). Nevertheless, olive extract exhibited a reduction of ∼5-log10 within 30 min. Most of these plant-derived compounds exhibited strong bactericidal activity and can potentially be applied as alternatives to chemicals for foods/food contact surfaces since they are generally recognized as safe (GRAS) for human consumption. They may also be useful in applications in which other antimicrobials have reduced efficacy (e.g., in the presence of organics) or used with sensitive populations that are unable to tolerate exposure to harsher chemicals (e.g., elderly care facilities). These compounds could be used alone, in combination, or with fast-acting antimicrobials to provide a long-lasting residual.

Comparative Assessment of BGM and PLC/PRF/5 Cell Lines for Enteric Virus Detection in Biosolids.

The buffalo green monkey (BGM) cell line is required for the detection of enteric viruses in biosolids through a total culturable viral assay (TCVA) by the United States Environmental Protection Agency. In the present study, BGM and PLC/PRF/5 cell lines were evaluated for TCVA and for their use in determining the incidence of adenoviruses and enteroviruses in raw sludge and Class B biosolids. Six raw sludge and 17 Class B biosolid samples were collected from 13 wastewater treatment plants from seven U.S. states. Samples were processed via organic flocculation and concentrate volumes equivalent to 4 g total solids were assayed on BGM and PLC/PRF/5 cells. Cell monolayers were observed for cytopathic effect (CPE) after two 14-days passages. Cell lysates were tested for the presence of adenoviruses and enteroviruses by PCR or RT-PCR. The PLC/PRF/5 cells detected more culturable viruses than the BGM cells by CPE (73.9% vs. 56.5%, respectively). 52% of the samples were positive for CPE using both cell lines. No viruses were detected in either cell line by PCR in flasks in which CPE was not observed. No adenoviruses were detected in 13 CPE-positive samples from BGM lysates. In contrast, of the 17 samples exhibiting CPE on PLC/PRF/5 cells, 14 were positive for adenoviruses (82.4%). In conclusion, PLC/PRF/5 cells were superior for the detection of adenoviruses in both raw sludge and Class B biosolids. Thus, the use of BGM cells alone for TCVA may underestimate the viral concentration in sludge/biosolid samples.

Efficacy of copper and silver as residual disinfectants in drinking water.

Contamination events and biofilms can decrease the amount of free chlorine available in drinking water systems. The efficacy of 100 µg/L silver and 400 µg/L copper, individually and combined, were evaluated as secondary, longer-lasting residual disinfectants against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Mycobacterium fortuitum at 24 °C and 4 °C. A >5.0-log10 reduction was observed in E. coli and L. monocytogenes after three hours and S. Typhimurium following seven hours of exposure to silver. M. fortuitum was the most resistant species to silver (1.11-log10 after seven hours). Copper did not significantly reduce S. Typhimurium and E. coli at 24 °C; ≥2.80-log10 reductions were observed in the Gram-positive L. monocytogenes and M. fortuitum. Longer exposure times were required at 4 °C to achieve significant reductions in all species. A synergistic effect was observed when silver and copper were combined at 24 °C. In addition, silver was not affected by the presence of organic matter at concentrations that completely inhibited 0.2 mg/L chlorine. The results of this study suggest that combinations of silver and copper show promise as secondary residual disinfectants. They may also be used in conjunction with low chlorine levels or other disinfectants to provide additional, long-lasting residuals in distribution systems.

The occurrence of Naegleria fowleri in recreational waters in Arizona.

Naegleria fowleri is a free-living amoeba found in waters in warmer regions that causes primary amoebic meningoencephalitis, a rare but almost universally fatal disease. The goal of this project was to assess the occurrence of N. fowleri and other thermophilic amoebae in 33 recreational surface waters across Arizona to determine if their presence could be correlated with seasonal or other environmental factors. First, 1-L grab samples were collected over two years and analyzed using polymerase chain reaction and amoebae viability. Seasonality was observed, with N. fowleri and thermophilic amoebae (20% and 30%, respectively) being detected more often in the winter and spring combined than in the summer and fall combined (7.9% and 9.5%, respectively). The spring and fall both had an average temperature of 18°C, yet had different occurrence data (18.2% versus 5.9% for N. fowleri, respectively; 27.3% versus 0% for viable amoebae, respectively). These results are in stark contrast to previous studies in which N. fowleri has been found almost exclusively during warmer months. Over the two-year study, N. fowleri was detected in six and thermophilic amoebae in eight of the 33 recreational water bodies. Five of these were lakes near Phoenix that tested positive for N. fowleri and thermophilic amoebae over multiple seasons. These lakes differed significantly (P ≤ 0.05) from the other 28 surface waters, with a lower average temperature in the spring, a higher temperature in the fall, a higher pH and turbidity in the summer, and a lower electro-conductivity in the spring. They also had lower Escherichia coli and heterotrophic bacteria levels during colder months. Future N. fowleri monitoring in Arizona should focus on these five lakes to further elucidate the factors that contribute to the low occurrence of this amoeba in the summer or which might explain why these lakes appear to be reservoirs for the organism.

Mechanisms of antiviral action of plant antimicrobials against murine norovirus.

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.

Survival of Respiratory Viruses on Fresh Produce.

In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory coronaviruses and influenza viruses may potentially be transmitted via contaminated foods. The goal of this study was to determine the recovery efficiencies and the survival of two respiratory viruses, namely, adenovirus 2 (Ad2) and coronavirus 229E (CoV229E), on fresh produce in comparison to the enteric poliovirus 1 (PV1). Adenovirus was recovered with efficiencies of 56.5, 31.8, and 34.8 % from lettuce, strawberries, and raspberries, respectively. Coronavirus was recovered from lettuce with an efficiency of 19.6 % yet could not be recovered from strawberries. Poliovirus was recovered with efficiencies of 76.7 % from lettuce, but only 0.06 % from strawberries. For comparison purposes, the survival of Ad2, CoV229E, and PV1 was determined for periods up to 10 days on produce. The enteric PV1 survived better than both respiratory viruses on lettuce and strawberries, with only ≤1.03 log10 reductions after 10 days of storage at 4 °C compared to CoV229E not being recovered after 4 days on lettuce and reductions of 1.97 log10 and 2.38 log10 of Ad2 on lettuce and strawberries, respectively, after 10 days. Nevertheless, these respiratory viruses were able to survive for at least several days on produce. There is therefore the potential for transfer to the hands and subsequently to the mucosa via rubbing the eyes or nose. In addition, some respiratory coronaviruses (e.g., severe acute respiratory syndrome coronavirus) and adenoviruses are also capable of replication in the gut and there is thus some potential for acquisition through the consumption of contaminated produce.

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.

Effectiveness of poliovirus concentration and recovery from treated wastewater by two electropositive filter methods.

Enteric viruses are often present in low numbers in various water matrices. Virus sampling therefore involves multiple concentration steps to condense large samples down to small volumes for detection by cell culture or molecular assays. The NanoCeram® Virus Sampler has been demonstrated to be effective for the recovery of viruses from tap water, surface waters, and seawater. The goal of this study was to evaluate a new method using NanoCeram® filters for the recovery of poliovirus 1 (PV-1) from treated wastewater. Activated sludge effluent samples were spiked with PV-1 and concentrated in side-by-side tests by two methods: (1) NanoCeram® filtration, elution with sodium polyphosphate buffer, secondary concentration via centrifugal ultrafiltration; and (2) 1MDS filtration, elution with beef extract, secondary concentration via organic flocculation. The virus retention and elution efficiencies did not differ significantly between the two methods. In contrast, the secondary concentrate volume was smaller for the NanoCeram® method (8.4 vs. 30 mL) and the secondary concentration efficiencies were different between the two methods with 98 % for centrifugal ultrafiltration (NanoCeram® and 45 % for organic flocculation (1MDS). The overall method efficiencies were significantly different (P ≤ 0.05) with the NanoCeram® method yielding a 57 % and the 1MDS a 23 % virus recovery. In addition, there appeared to be less interference with viral detection via polymerase chain reaction with the NanoCeram® concentrates. This NanoCeram® method therefore is able to efficiently recover PV-1 from large volumes of wastewater and may serve as an inexpensive alternative to the standard 1MDS filter method for such applications.

Occurrence of household mold and efficacy of sodium hypochlorite disinfectant.

The occurrence and distribution of mold on household surfaces and the efficacy of bleach-based (sodium hypochlorite, NaOCl) disinfectants on mold viability and allergenicity was documented. Household microenvironments prone to increased moisture were specifically targeted. Using the sticky tape method, 1330 samples were collected from non-porous indoor surfaces of 160 homes across the United States, and analyzed for mold. Homes were randomly selected and recruited via phone interviews. Culture and immunoassays were used to measure the viability and reduction of allergenic properties of Aspergillus fumigatus following 2.4% NaOCl treatment. All homes and 72.9% of surfaces tested positive for mold. Windowsills were the most frequently contaminated site (87.5%) and Cladosporium the most commonly identified mold (31.0%). Five-minute exposures to 2.4% NaOCl resulted in a >3 to >6-log10 reduction of culturable mold counts in controlled laboratory studies. Organisms were nonculturable after 5- and 10-min contact times on non-porous and porous ceramic carriers, respectively, and A. fumigatus spore-eluted allergen levels were reduced by an average 95.8% in 30 sec, as indicated by immunoassay. All homes are contaminated with some level of mold, and regrowth is likely in moisture-prone microenvironments. The use of low concentrations (2.4%) of NaOCl for the reduction of culturable indoor mold and related allergens is effective and recommended.

Concentration and recovery of viruses from water: a comprehensive review.

Enteric viruses are a cause of waterborne disease worldwide, and low numbers in drinking water can present a significant risk of infection. Because the numbers are often quite low, large volumes (100-1,000 L) of water are usually processed. The VIRADEL method using microporous filters is most commonly used today for this purpose. Negatively charged filters require the addition of multivalent salts and acidification of the water sample to effect virus adsorption, which can make large-volume sampling difficult. Positively charged filters require no preconditioning of samples, and are able to concentrate viruses from water over a greater pH range than electronegative filters. The most widely used electropositive filter is the Virosorb 1MDS; however, the Environmental Protection Agency has added the positively charged NanoCeram filters to their proposed Method 1615. Ultrafilters concentrate viruses based on size exclusion rather than electrokinetics, but are impractical for field sampling or processing of turbid water. Elution (recovery) of viruses from filters following concentration is performed with organic (e.g., beef extract) or inorganic solutions (e.g., sodium polyphosphates). Eluates are then reconcentrated to decrease the sample volume to enhance detection methods (e.g., cell culture infectivity assays and molecular detection techniques). While the majority of available filters have demonstrated high virus retention efficiencies, the methods to elute and reconcentrate viruses have met with varying degrees of success due to the biological variability of viruses present in water.

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

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.

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.

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

Biofilms can have deleterious effects on drinking water quality and may harbor pathogens. Experiments were conducted using 100 microg/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.

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 microg/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 degrees 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.

Silver as a disinfectant.

Silver has been used as an antimicrobial for thousands of years. Over the past several decades, it has been introduced into numerous new venues such as in the treatment of water, in dietary supplements, in medical applications, and to produce antimicrobial coatings and products. Silver is often used as an alternative disinfectant in applications in which the use of traditional disinfectants such as chlorine may result in the formation of toxic by-products or cause corrosion of surfaces. Silver has also been demonstrated to produce a synergistic effect in combination with several other disinfectants. Many mechanisms of the antibacterial effect of silver have been described, but its antiviral and antiprotozoal mechanisms are not well understood. Both microbial tolerance and resistance to silver have been reported; however, the effect of silver has been observed against a wide variety of microorganisms over a period of years. Further research is needed to determine the antimicrobial efficacy of silver in these new applications and the effects of its long-term usage.