Artificial phylloplanes: A novel tool to study parameters linked to bacterial and viral contamination of fresh produce

Every year, one-in-six Americans suffer from a food-related illness caused by bacteria, viruses, or parasites. Since 2010, fresh produce has been linked to seven foodborne outbreaks caused by Escherichia coli species alone. Produce surface properties, such as surface hydrophobicity and surface roughness play a key role determining the attachment of bacteria and viruses to and their removal from produce. A few previous studies have investigated the effect of surface roughness and surface hydrophobicity on the attachment and removal of bacteria and viruses from food and food contact surfaces. However, since produce surface properties undergo constant changes as a function of time and environmental factors, reports have shown inconsistent results for the same produce type with regard to bacteria attachment and removal. Researchers have realized the need to construct artificial plant surfaces to retain the surface characteristics of natural plant surfaces during sanitation tests. A few attempts have reported the use of polymers, stainless steel, zinc substrates, or alumina to fabricate surrogate surfaces that resemble food or food contact surfaces, with varying degrees of success. Nevertheless, even the most successful one among the pervious surrogate surfaces can only replicate the topographical characteristics of natural fresh produce surfaces, but not the chemical properties of the plant surfaces. Furthermore, most of the previous surrogate surfaces lack reusability due to the nature of the fabrication material. In a microbial attachment or removal study, the surrogate surface will be subjected to mechanical forces because they need to be placed in a stomacher to do the emulation; thus, the surrogate surfaces made from soft material will be damaged. The overall goal of this study is to develop a new method for the fabrication of reusable and reproducible artificial phylloplanes that replicate the three-dimensional topological features of natural produce leaves, thus having surface hydrophobicity, roughness values, and epicuticular composition resembling those of two selected fresh produce varieties. To achieve the goal, three inter-related studies were performed. In the first study, the effects of physiochemical characteristics, including produce leaf surface roughness, epicuticular wax composition, and produce and bacteria surface hydrophobicity on attachment/removal of E. coli K12 to/from plant surfaces was investigated. The attachment and removal of E. coli K12 was affected by multiple factors including produce genotype, produce surface roughness, and wax composition. Rougher surfaces resulted in higher attachment of E. coli and less removal. In addition, the removal of E. coli K12 was positively correlated with alkanes, ketones, and total wax content on the leaf surfaces. In study two, a method to create polydimethylsiloxane (PDMS)-based artificial phylloplane surface to resemble the topographical, chemical, and epicuticular characteristics of ‘Outredgeous’ romaine lettuce and ‘Carmel’ spinach to a high fidelity was developed. The artificial produce leaf surfaces were utilized to study the effect of surface hydrophobicity on the attachment of E. coli O157:H7 and Listeria innocua. The PDMS- artificial phylloplanes are reusable, economical, and recyclable. They can thus be used as a platform to investigate the interactions between bacteria and produce, and to develop new or enhanced fresh produce decontamination strategies. In study three, the newly developed artificial phylloplane surfaces were utilized to study the effect of produce leaf physiochemical characteristics on the attachment and removal of porcine rotavirus (PRV), strain OSU, and tulane virus (TV), a surrogate of human norovirus. In addition, the artificial phylloplanes were used to screen commercially available and new sanitizers and to study the use of ultrasonication as an enhancer of viral detachment in the washing step. No significant differences in attachment of PRV and TV inoculated to fresh leaves of ‘Outredgeous’ romaine lettuce and ‘Carmel’ spinach and their artificial phylloplanes were observed. In sanitation tests, the removal of virus attached to natural and artificial surfaces was virus type, sanitizer type, and produce cultivar dependent. In summary, the newly developed artificial phylloplanes establish a platform with constant surface properties for studying the interactions between bacteria and produce leaf surfaces. The new surfaces overcome the biological variations of produce surfaces originated from changes during preharvest, transportation, and post-harvest processing/storage, which oftentimes result in inconsistent sanitation results. The newly developed artificial phylloplanes provide a faithful replication of the surface characteristics of fresh produce in that they 1) resemble the 3D topological features of natural produce leaf surfaces, 2) have a similar surface hydrophobicity, 3) have similar epicuticular chemical composition, mainly epicuticular wax composition, 4) produce a similar bacterial attachment pattern, and 5) are reproducible and reusable, including autoclave-able and compatible with stomacher

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

Influence of Epicuticular Physicochemical Properties on Porcine Rotavirus Adsorption to 24 Leafy Green Vegetables and Tomatoes.

Foodborne diseases are a persistent problem in the United States and worldwide. Fresh produce, especially those used as raw foods like salad vegetables, can be contaminated, causing illness. In this study, we determined the number of rotaviruses adsorbed on produce surfaces using group A porcine rotaviruses and 24 cultivars of leafy vegetables and tomato fruits. We also characterized the physicochemical properties of each produce's outermost surface layer, known as the epicuticle. The number of rotaviruses found on produce surfaces varied among cultivars. Three-dimensional crystalline wax structures on the epicuticular surfaces were found to significantly contribute to the inhibition of viral adsorption to the produce surfaces (p = 0.01). We found significant negative correlations between the number of rotaviruses adsorbed on the epicuticular surfaces and the concentrations of alkanes, fatty acids, and total waxes on the epicuticular surfaces. Partial least square model fitting results suggest that alkanes, ketones, fatty acids, alcohols, contact angle and surface roughness together can explain 60% of the variation in viral adsorption. The results suggest that various fresh produce surface properties need to be collectively considered for efficient sanitation treatments. Up to 10.8% of the originally applied rotaviruses were found on the produce surfaces after three washing treatments, suggesting a potential public health concern regarding rotavirus contamination

Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.

<p>Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.</p

Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts.

Salmonella enterica subspecies I (ssp 1) is the leading cause of hospitalizations and deaths due to known bacterial foodborne pathogens in the United States and is frequently implicated in foodborne disease outbreaks associated with spices and nuts. However, the underlying mechanisms of this association have not been fully elucidated. In this study, we evaluated the influence of storage temperature (4 or 25°C), relative humidity (20 or 60%), and food surface characteristics on the attachment and survival of five individual strains representing S. enterica ssp 1 serovars Typhimurium, Montevideo, Braenderup, Mbandaka, and Enteritidis on raw in-shell black peppercorns, almonds, and hazelnuts. We observed a direct correlation between the food surface roughness and S. enterica ssp 1 attachment, and detected significant inter-strain difference in survival on the shell surface under various storage conditions. A combination of low relative humidity (20%) and ambient storage temperature (25°C) resulted in the most significant reduction of S. enterica on shell surfaces (p &lt; 0.05). To identify genes potentially associated with S. enterica attachment and survival on shell surfaces, we inoculated a library of 120,000 random transposon insertion mutants of an S. Enteritidis strain on almond shells, and screened for mutant survival after 1, 3, 7, and 14 days of storage at 20% relative humidity and 25°C. Mutants in 155 S. Enteritidis genes which are involved in carbohydrate metabolic pathways, aerobic and anaerobic respiration, inner membrane transport, and glutamine synthesis displayed significant selection on almond shells (p &lt; 0.05). Findings of this study suggest that various food attributes, environmental factors, and an unexpectedly complex metabolic and regulatory network in S. enterica ssp 1 collectively contribute to the bacterial attachment and survival on low moisture shell surface, providing new data for the future development of knowledge-based intervention strategies

Comparison of physiochemical epicuticular properties between cultivars with 2-D or 3-D wax crystals on leaf surfaces.

<p>Absence or presence of 3-D wax crystals was determined by SEM. LSD test was used to indicate significant difference for the variables between cultivars with and without wax crystals on leaf surface. Tomato cultivars were excluded because of different tissue type.</p><p>Comparison of physiochemical epicuticular properties between cultivars with 2-D or 3-D wax crystals on leaf surfaces.</p

Chemical composition of epicuticular waxes from 24 vegetable leaves and tomato fruits and the genome copies from adsorbed rotaviruses on these produce surfaces.

<p>^a The percentage was calculated using number of adsorbed rotaviruses divided by OSU rotavirus genome copies in the initial viral solution (7.17 ± 0.05 log₁₀ genome copies/ml). Wax components were quantified by GC-FID and the total amount of wax was calculated as the sum of single components. LSD value was calculated by Student’s T-test at <i>P</i> = 0.05.</p><p>Chemical composition of epicuticular waxes from 24 vegetable leaves and tomato fruits and the genome copies from adsorbed rotaviruses on these produce surfaces.</p

Physical properties of epicuticular layers of 24 vegetable leaves and tomato fruits.

<p>(Contact angle is presented in °, and roughness is in μm.). Ad and ab indicate adaxial and abaxial leaf, respectively. Stoma lengths were measured on adaxial leaf surfaces.</p><p>Physical properties of epicuticular layers of 24 vegetable leaves and tomato fruits.</p

Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.

<p>Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.</p

Epicuticular images from various vegetable leaves and tomato fruits.

<p>All SEM images were generated at 500 × resolution. Scale bar in the image is 50 μm. Inset images were taken at higher resolutions. White arrows indicate stomata. Alphabetical order matches sample list from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132841#pone.0132841.t001" target="_blank">Table 1</a>. A: Tokyo bekana; B: ‘Perseo’ radicchio; C: ‘Rhodos’ endive; D: ‘Southern Giant Curled’ mustard; E: Mizuna; F: ‘Tyee’ spinach; G: ‘Racoon’ spinach; H: ‘Carmel’ spinach; I: Tatsoi; J: ‘Top Bunch’ collard; K: ‘Starbor’ kale; L: ‘Red Russian’ kale; M: Arugula; N: ‘Totem’ Belgian Endive; O: ‘Two Star’ lettuce; P: ‘Tropicana’ lettuce; Q: ‘Outredgeous’ romaine lettuce; R: ‘Super Red’ cabbage; S: ‘Gonzales’ cabbage; T: ‘Ruby Perfection’ cabbage; U: ‘Alcosa’ cabbage; V: ‘Sun Gold’ cherry tomato; W: ‘Indigo Rose’ tomato; X: ‘Rose’ tomato.</p