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