Potential of Human Norovirus Surrogates and Salmonella enterica Contamination of Pre-harvest Basil (Ocimum basilicum) via Leaf Surface and Plant Substrate

Abstract

Fresh produce has been identified as an important vehicle for foodborne pathogen transmission and fresh culinary herbs have occasionally been associated with human pathogens and illness. In this study, the fate of human NoV surrogates [murine norovirus 1 (MNV-1) and Tulane virus (TV)] and three strains of Salmonella enterica on pre-harvest basil (Ocimum basilicum) was investigated. The persistence after contamination via either leaf surface or plant substrate was tested respectively. After 3 days, both MNV-1 and TV on pre-harvest leaves were at non-detectable levels (>5.5-log reduction for MNV-1 and >3.3-log reduction for TV). The three Salmonella strains showed consistent reductions of 3- to 4-log. At day 6 and 9, all the tested samples showed low levels of infectivity which were close or below the detection limits (1.7-log PFU/sample leaf for MNV-1 and TV, 0.7-log CFU/sample leaf for Salmonella) except for S. Thompson FMFP 899, one out of three samples showed to maintain present at exceptional high levels (day 6: 5.5-log CFU/sample leaf; day 9: 6.7-log CFU/sample leaf). Possibilities of microbial internalization into the edible parts of basil via the roots was demonstrated with both MNV-1 and S. enterica Thompson FMFP 899. The infectivity of internalized MNV-1 and S. enterica both decreased to non-detectable levels within 9 days after inoculation. Moreover, it should be noticed that very high microbial inoculation was used in the experimental set-up (8.46-log PFU/ml of MNV-1, 8.60-log CFU/ml of S. enterica), which is abnormal in the real-life expected contamination scenario. Within the tested scenarios in this study, S. enterica contaminated on the adaxial leaf surface of basil plants while in growth, and remained/reached a high population of over 6-log CFU/sample leaf after 9 days in one out of three samples, thus showed the highest potential for causing foodborne infection.

Salmonella Establishment in Agricultural Soil and Colonization of Crop Plants Depend on Soil Type and Plant Species

“…Typhimurium S1 into the root of different plants, including lettuce, was demonstrated recently using a sterile system and subsequent FISH-CLSM analysis (Kljujev et al, 2018). In the case of basil, internalization of S. Thompson strain FMFP 899 via roots was demonstrated using Salmonella -soaked germination discs using high cell counts of Salmonella for inoculation (Li and Uyttendaele, 2018). In the present study, we could observe all tested Salmonella strains in lettuce and corn salad leaves, indicating that the bacteria were able to internalize into the roots and spread within the plant, probably through the vascular system.…”

Risk of Human Pathogen Internalization in Leafy Vegetables During Lab-Scale Hydroponic Cultivation

“…Sharma et al [32] tested three strains of genetically engineered GFP-expressing E. coli O157:H7 detected by immunofluorescence. Additionally, not all investigators performed a leaf surface sterilization prior to microbial detection to rule out epiphytic bacteria [46,47,52]. However, the natural contamination of bacteria at significant levels is unlikely due to the high inoculation levels of the specific strains used in the study combined with the aseptic environment of lab-scale systems.…”

“…In some lab-based systems, plants were cultivated using an agar-solidified hydroponic nutrient solution rather than a fluid solution. Two studies have utilized a NFT or NFT-like system [50,51], while one study utilized a continuous drip system, but inoculated the solid phase as opposed to the nutrient solution [52]. Research addressing the internalization of pathogens in leafy vegetables across a variety of hydroponic systems has been summarized in Table 2.…”

“…An additional study investigated the internalization of S. Thompson and MNV into the edible parts of basil via the roots [52]. Here, the authors used a four-pot hydroponic drip system filled with clay pebbles.…”

“…Basil seeds were germinated in peat pellets and then transplanted to the drip system. At six weeks old, basil plants in the peat pellets were removed from the pots and soaked in an inoculum of either MNV or S. Thompson for 1 h. Li and Uyttendaele [52] reported varying levels of MNV internalization on days 1 and 3 post-inoculation and positive S. Thompson on days 3 and 6 following sample enrichment. This study presents unique differences from the previously discussed research utilizing DWC-like systems.…”