Cultivation Conditions of Spinach and Rocket Influence Epiphytic Growth of Listeria monocytogenes

Abstract

Leafy vegetables are associated with Listeriosis outbreaks due to contamination with Listeria monocytogenes. To date, contradictory findings were reported on spinach, rocket, and kale, where some studies reported growth of L. monocytogenes, while others did not. Thus, the current study investigated the reason for conflicting findings by producing leafy vegetables, where cultivation factors were known for growth potential studies. Of all polytunnel produce, kale Nero di Toscana demonstrated the highest growth potential (2.56 log cfu g−1), followed by spinach F1 Cello (1.84 log cfu g−1), rocket Buzz (1.41 log cfu g−1), spinach F1 Trumpet (1.37 log cfu g−1), and finally rocket Esmee (1.23 log cfu g−1). Thus, plant species and variety influenced L. monocytogenes growth potentials. Moreover, significantly lower growth potentials of 0.3 log cfu g−1 were identified when rocket Buzz was cultivated in open fields (1.11 log cfu g−1) instead of a polytunnel. The opposite effect was observed for spinach F1 Trumpet, where growth potentials increased significantly by 0.84 log cfu g−1 when cultivated in open fields (2.21 log cfu g−1). Furthermore, a significant seasonality effect between batches was found (p < 0.05). This study revealed that spinach and rocket cultivation conditions are at least co-factors in the reporting of differing growth potentials of L. monocytogenes across literature and should be considered when conducting future growth potential studies.

Bacterial community structure analysis onListeria monocytogenesinoculated spinach leaves is affected by PCR based methods to exclude chloroplast co-amplification

“…Presence of the family Listeriaceae, which contain the genus Listeria was not detected in the majority of samples, even at the utilized sequence depth. A sequence depth of over 16,000 reads after rarefication should be able to identify at least 0.1 % relative abundant taxa, which would be the case for L. monocytogenes , when cultivation based abundances are taken into account (selective for L. monocytogenes as well as total heterotrophic counts (Culliney and Schmalenberger, 2022)). Consequently, a direct relationship of relative sequence abundances of certain phylogenetic groups over time alongside L. monocytogenes could not be analysed.…”

“…All three groups also displayed similar trends of increasing Lactobacillales content from day 0 (0.23 to 0.32 %) to 5 (0.76 to 1.19 %) followed by decreases until day 9 near absence. The largest decrease in Lactobacillales content from day 7 (0.49 to 0.64%) to 9 (0.03 to 0.04 %) was associated with the greatest increase in L. monocytogenes i.e., 1.10 log 10 cfu g -1 (Culliney and Schmalenberger, 2022). Thus, the reduction of Lactobacillales (which are associated with decreasing L. monocytogenes growth) content to near absence in combination with an increase in Pseudomonodaceae content (+ 1.69 to 2.34 increase across three methods from day 7 to 9) may have been responsible for the greater increase in L. monocytogenes growth observed alongside the higher storage temperate from day6-9.…”

“…The same authors revealed that bacterial densities were low at approximately 2 to 3 logs cfu g -1 . Thus, suggesting PNA clamps may not be an appropriate method for other leafy vegetables expecting lower reads due to lower bacterial densities such as kale which started at approximately 2 to 3 logs compared to 7 to 8 logs for spinach (Culliney and Schmalenberger, 2022). However, similar to the observations of Kim and colleagues (2020), utilisation of PNA clamps did increase recovery of usable bacterial sequences by 5,395 reads belonging to the apple flower phytobiome (Steven et al, 2018).…”

“…Additionally, the change in the structure of the spinach phyllosphere over time is important to fully understand the behaviour of L. monocytogenes on leafy vegetables. From day 7 to 9, chloroplast content decreased to 2.64 % while the associated L. monocytogenes’ populations increased drastically (Culliney and Schmalenberger, 2022). Thus, while blocking methods were effective, chloroplast content also naturally reduced throughout storage to low contents by day 9.…”

“…DNA extractions were conducted on 20 samples from L. monocytogenes growth potential experiments on open-field spinach (F1 Trumpet) that was stored for 0, 2, 5,7 and 9 days at 7°C for days 0-6 and at 12°C for days 7-9, where L. monocytogenes and total heterotrophic bacteria were enumerated on cultivation media (Culliney and Schmalenberger, 2022). Growth experiments were executed according to the EU guidance document’s guidelines for conducting growth potential studies (EURL Lm, 2019).…”