Intercellular Production of H2O2 in Tomatoes as a Prevention to Salmonella Proliferation

Introduction: Due to increases in produce associated gastroenteritis, it is becoming clearer that we know little about the ecology of human pathogens in vegetables. Previous research has shown that Salmonella (14028) can grow in numbers up to 10⁵ compared against the initial inoculum. It was also found that Piccolo (cherry) tomatoes were generally less conductive to Salmonella than any of the larger cultivars (up to 2 logs less). 

Purpose: We hypothesise that cherry tomatoes naturally produce higher levels of H₂O₂ in their intercellular fluid than larger tomatoes.

Methods: Around 100 cells of Salmonella were inoculated in wounds under the pericarp in both the cherry and Alicante (larger) tomatoes. Salmonella proliferation was measured after a week of incubation by plating on Xylose lysine deoxycholate agar (XLD agar). To test H₂O₂ levels a novel method was used. Small pieces of the pericarp (from both cultivars) totalling 2g were subjected to a pressured vacuum to infiltrate distilled water into the intercellular space. The pieces were then spun in a centrifuge to extract all fluid from the intercellular space. Samples were de-proteinised using 10kD spin columns. A fluorometric assay was then used to test the concentration of H₂O₂. Four biological and two technical replicas were carried out. 

Results: The increase of Salmonella from one week of initial incubation was up to 10⁴ CFU/tomato in Alicante cultivar, and 10² CFU/tomato in the cherry cultivar. The difference in Salmonella proliferation was significant (α=0.05). The cherry tomatoes showed an average of 0.76nmol/ml of H₂O₂ whereas the Alicante cultivar showed an average of 0.46nmol/ml. The difference in H₂O₂ concentration between the tomato cultivars was significant. 

Significance: These findings open opportunities into genetic research of the tomato. The oOxyR gene in Salmonella activates the regulon of H₂O₂ inducible genes. Such pathways can be involved in Salmonella proliferation in tomatoes.