A comparison study of efficacy of food sanitizers with fresh leafy vegetables with different surface properties and analysis of the rotavirus replication cycle after inactivation by food sanitizers

Foodborne illness is a serious concern in the United States. Fresh produce is recognized as a vehicle of food borne illness pathogens including viruses. To prevent causes of foodborne illness, the use of sanitizers is essential for fresh produce. However, it is considered that the current sanitizers used in food industry may not be effective on virus inactivation on fresh produce, and little is known how the performance of sanitizers varies on virus inactivation on different fresh produce surface properties. In this study, we evaluated the efficacy of two kinds of food sanitizers Tsunami® 100 and malic acid + TDS on rotaviruses attached onto three cultivars’ surfaces with different surface properties. Moreover, we investigated the inhibitory effects of the sanitizers on the rotavirus replication cycle at a molecular level. As a result, while differences of sanitation efficacy on rotaviruses on the three cultivars were observed by Tsunami® 100, malic acid + TDS had similar disinfection efficacies on rotaviruses attached onto the produce. Moreover, the entry and replication step of the rotavirus replication cycle was significantly inhibited by the sanitizer treatments with rotaviruses, while the sanitizers did not inhibit the binding of rotaviruses onto cells. These observations suggest that the surface properties of fresh produce may affect the efficacy of sanitation on viruses, implying that food sanitizers should be carefully selected for different surface characteristics of fresh produce, and to identify how the food sanitizers function on rotaviruses, further studies on identification of virus damage caused by the sanitizers’ effect are needed

Effect Of Leaf Surface Chemical Properties On Efficacy Of Sanitizer For Rotavirus Inactivation

The use of sanitizers is essential for produce safety. However, little is known about how sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU were examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax contents and one cultivar of endive with a low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced by the 1-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3 log10) that were independent of leaf wax content. A statistical difference was observed with the disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. Significant reductions in the entry and replication of PRV were observed after treatment with either disinfectant. Moreover, the oxidant-based-sanitizer-treated PRV showed sialic acid-specific binding to the host cells, whereas the surfactant-based sanitizer increased the nonspecific binding of PRV to the host cells. These findings suggest that the surface properties of fresh produce may affect the efficacy of virus disinfection, implying that food sanitizers should be carefully selected for the different surface characteristics of fresh produce

Disinfection of fresh produce contaminated by enteric viruses and their inactivation mechanisms

Fresh produce has been identified as the major cause of foodborne viral illnesses in the United States. Viruses from contaminated irrigation water could be carried to crops, resulting in foodborne infection risks, especially for fresh produce eaten raw. Upon harvest, vegetables are commonly washed by a sanitizer to inactivate viruses that may cause foodborne infections. Despite this sanitation step, foodborne viral outbreaks associated with fresh vegetables still occurred. Therefore, to improve the vegetable sanitation efficacy, it is important to understand how viruses are inactivated by vegetable sanitizers. However, inactivation mechanisms of viruses by vegetable sanitizers are not fully understood. To fill this knowledge gap, the objective of this study is to understand inactivation mechanisms of viruses exposed to vegetable sanitizers. First, to investigate the effect of post-harvest disinfection efficacy on foodborne disease risks associated with consumption of viral-contaminated fresh produce, a quantitative microbial risk assessment (QMRA) model was developed. Using this model, I estimated the health risks associated with consumption of rotavirus (RV)-contaminated fresh produce (‘Totem’ Belgian endive and ‘Red Russian’ kale), disinfected with either peracetic acid (PAA) or a surfactant-based sanitizer, assuming that vegetables were irrigated with RV-contaminated water. As the disinfection efficacy of RV attached to the produce surface varied depending on the produce type and sanitizer, the health risks of RV illness were greatly influenced by the combination of sanitizer and vegetable type. Global sensitivity analyses of this QMRA model demonstrated that RV concentration in irrigation water and post-harvest disinfection efficacy were the two most influential factors in infection risks. For this reason, these two factors should be well controlled for vegetable production and sanitation so that the foodborne illness risk is below the WHO threshold for RV. To understand whether free chlorine, the most commonly used sanitizer in the vegetable industry, is effective to inactivate virus attached to vegetable surfaces, free chlorine was exposed to virus-contaminated vegetable surfaces of ‘Red Russian’ kale and southern giant curly mustard. Disinfection efficacy of RV attached to vegetable surfaces depended on the vegetable type (higher disinfection efficacy of RV attached to mustard than kale). In contrast, disinfection efficacy of TV, a Human Norovirus surrogate, was not influenced by the vegetable type. However, resistance of suspended TV was significantly more resistant to free chlorine than suspended RV. Free chlorine inactivation mechanisms on viruses were also investigated to reveal that damage on both genome and binding proteins caused by free chlorine led virus inactivation. RV lost its ability to bind to host receptors after exposure to free chlorine at 1.7 ppm for 1 min. For the same level of binding reduction of TV, free chlorine at 16 ppm for 1 min was needed. This study results indicate that free chlorine efficacy depends on the virus type and findings here will potentially enhance free chlorine sanitation strategies for fresh vegetables. Hydroponics for growing vegetables has been gaining popularity. However, hydroponically grown vegetables can be internally contaminated through the hydroponics feed water which may contain viruses. Sanitation of post-harvest vegetable with internalized viruses may not be effective because sanitizers might not be able to reach viruses internalized inside the vegetable tissues. To test this hypothesis, the effect of where viruses are localized in fresh vegetables (vegetable surfaces or inside the vegetable tissues) on the disinfection efficacy was determined by exposing PAA to viral-contaminated arugula microgreens. Disinfection efficacy of RV was higher when RV was on the arugula surface, compared to RV internalized inside the arugula interior. However, TV showed similar disinfection efficacy, independent on the virus location in arugula. The inactivation efficacy of viruses attached to the arugula microgreen surfaces was greater for RV compared to TV, while the inactivation efficacy of viruses internalized into the arugula microgreens was equal for RV and TV. For both arugula-internalized TV and RV, disinfection efficacies were less than 2-log10. These findings indicate that the type of virus and where the virus is localized in fresh vegetables may influence the virus disinfection of post-harvest vegetables. Virus inactivation mechanism of PAA was also investigated. Suspended TV and RV were exposed to PAA. TV showed greatly high resistance to PAA than RV. RV exposed to PAA at 1 mg/L for 3 min had 2-log10 inactivation. In contrast for TV, to achieve the same level of infectivity reduction as RV, 10 mg/L PAA over 30 min was needed. The resistance of TV to PAA can be due to aggregation of TV in PAA that was observed in this study. This resistance of TV caused by TV aggregation suggests that the use of these sanitizers, which are often used for vegetable washing, might facilitate aggregation of some viruses, resulting in less inactivation efficacy. Moreover, it was found that both binding protein damage and genome damage due to PAA exposure to viruses led inactivation of viruses. To summarize, this study revealed that disinfection efficacy of vegetable sanitizers depend on several factors, including virus properties (ability to propagate, resistance to low pH, resistance to free chlorine and PAA, RNase-permeability, and free chlorine- and PAA-permeability), the kind of sanitizer (free chlorine and PAA), and type of fresh vegetables (hydrophobicity and hydrophilicity due to epicuticular wax concentration on the leaf surfaces), the route of virus contamination (surface contamination and internal contamination in vegetable). Thus, knowledge obtained in this comprehensive study about virus inactivation mechanisms will facilitate to optimize vegetable sanitation practice to reduce foodborne viral infection risks.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

Disinfection of fresh produce contaminated by enteric viruses and their inactivation mechanisms

Fresh produce has been identified as the major cause of foodborne viral illnesses in the United States. Viruses from contaminated irrigation water could be carried to crops, resulting in foodborne infection risks, especially for fresh produce eaten raw. Upon harvest, vegetables are commonly washed by a sanitizer to inactivate viruses that may cause foodborne infections. Despite this sanitation step, foodborne viral outbreaks associated with fresh vegetables still occurred. Therefore, to improve the vegetable sanitation efficacy, it is important to understand how viruses are inactivated by vegetable sanitizers. However, inactivation mechanisms of viruses by vegetable sanitizers are not fully understood. To fill this knowledge gap, the objective of this study is to understand inactivation mechanisms of viruses exposed to vegetable sanitizers. First, to investigate the effect of post-harvest disinfection efficacy on foodborne disease risks associated with consumption of viral-contaminated fresh produce, a quantitative microbial risk assessment (QMRA) model was developed. Using this model, I estimated the health risks associated with consumption of rotavirus (RV)-contaminated fresh produce (‘Totem’ Belgian endive and ‘Red Russian’ kale), disinfected with either peracetic acid (PAA) or a surfactant-based sanitizer, assuming that vegetables were irrigated with RV-contaminated water. As the disinfection efficacy of RV attached to the produce surface varied depending on the produce type and sanitizer, the health risks of RV illness were greatly influenced by the combination of sanitizer and vegetable type. Global sensitivity analyses of this QMRA model demonstrated that RV concentration in irrigation water and post-harvest disinfection efficacy were the two most influential factors in infection risks. For this reason, these two factors should be well controlled for vegetable production and sanitation so that the foodborne illness risk is below the WHO threshold for RV. To understand whether free chlorine, the most commonly used sanitizer in the vegetable industry, is effective to inactivate virus attached to vegetable surfaces, free chlorine was exposed to virus-contaminated vegetable surfaces of ‘Red Russian’ kale and southern giant curly mustard. Disinfection efficacy of RV attached to vegetable surfaces depended on the vegetable type (higher disinfection efficacy of RV attached to mustard than kale). In contrast, disinfection efficacy of TV, a Human Norovirus surrogate, was not influenced by the vegetable type. However, resistance of suspended TV was significantly more resistant to free chlorine than suspended RV. Free chlorine inactivation mechanisms on viruses were also investigated to reveal that damage on both genome and binding proteins caused by free chlorine led virus inactivation. RV lost its ability to bind to host receptors after exposure to free chlorine at 1.7 ppm for 1 min. For the same level of binding reduction of TV, free chlorine at 16 ppm for 1 min was needed. This study results indicate that free chlorine efficacy depends on the virus type and findings here will potentially enhance free chlorine sanitation strategies for fresh vegetables. Hydroponics for growing vegetables has been gaining popularity. However, hydroponically grown vegetables can be internally contaminated through the hydroponics feed water which may contain viruses. Sanitation of post-harvest vegetable with internalized viruses may not be effective because sanitizers might not be able to reach viruses internalized inside the vegetable tissues. To test this hypothesis, the effect of where viruses are localized in fresh vegetables (vegetable surfaces or inside the vegetable tissues) on the disinfection efficacy was determined by exposing PAA to viral-contaminated arugula microgreens. Disinfection efficacy of RV was higher when RV was on the arugula surface, compared to RV internalized inside the arugula interior. However, TV showed similar disinfection efficacy, independent on the virus location in arugula. The inactivation efficacy of viruses attached to the arugula microgreen surfaces was greater for RV compared to TV, while the inactivation efficacy of viruses internalized into the arugula microgreens was equal for RV and TV. For both arugula-internalized TV and RV, disinfection efficacies were less than 2-log10. These findings indicate that the type of virus and where the virus is localized in fresh vegetables may influence the virus disinfection of post-harvest vegetables. Virus inactivation mechanism of PAA was also investigated. Suspended TV and RV were exposed to PAA. TV showed greatly high resistance to PAA than RV. RV exposed to PAA at 1 mg/L for 3 min had 2-log10 inactivation. In contrast for TV, to achieve the same level of infectivity reduction as RV, 10 mg/L PAA over 30 min was needed. The resistance of TV to PAA can be due to aggregation of TV in PAA that was observed in this study. This resistance of TV caused by TV aggregation suggests that the use of these sanitizers, which are often used for vegetable washing, might facilitate aggregation of some viruses, resulting in less inactivation efficacy. Moreover, it was found that both binding protein damage and genome damage due to PAA exposure to viruses led inactivation of viruses. To summarize, this study revealed that disinfection efficacy of vegetable sanitizers depend on several factors, including virus properties (ability to propagate, resistance to low pH, resistance to free chlorine and PAA, RNase-permeability, and free chlorine- and PAA-permeability), the kind of sanitizer (free chlorine and PAA), and type of fresh vegetables (hydrophobicity and hydrophilicity due to epicuticular wax concentration on the leaf surfaces), the route of virus contamination (surface contamination and internal contamination in vegetable). Thus, knowledge obtained in this comprehensive study about virus inactivation mechanisms will facilitate to optimize vegetable sanitation practice to reduce foodborne viral infection risks.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated