A Simple Assay to Assess Salmonella enterica Persistence in Lettuce Leaves After Low Inoculation Dose

Abstract

Salmonella enterica is an enterobacterium associated with numerous foodborne illnesses worldwide. Leafy greens have been a common vehicle for disease outbreaks caused by S. enterica. This human pathogen can be introduced into crop fields and potentially contaminate fresh produce. Several studies have shown that S. enterica can survive for long periods in the plant tissues. Often, S. enterica population does not reach high titers in leaves; however, it is still relevant for food safety due to the low infective dose of the pathogen. Thus, laboratory procedures to study the survival of S. enterica in fresh vegetables should be adjusted accordingly. Here, we describe a protocol to assess the population dynamics of S. enterica serovar Typhimurium 14028s in the leaf apoplast of three cultivars of lettuce (Lactuca sativa L.). By comparing a range of inoculum concentrations, we showed that vacuum infiltration of a bacterium inoculum level in the range of 3.4 Log CFU ml −1 (with a recovery of approximately 170 cells per gram of fresh leaves 2 h post inoculation) allows for a robust assessment of bacterial persistence in three lettuce cultivars using serial dilution plating and qPCR methods. We anticipate that this method can be applied to other leaf-human pathogen combinations in an attempt to standardize the procedure for future efforts to screen for plant phenotypic variability, which is useful for breeding programs.

Intraspecies competition among Salmonella enterica isolates in the lettuce leaf apoplast

“…Taking advantage of the lettuce genotypic variability, we used two contrasting lettuce cultivars, Red Tide (RT) and Lollo Rossa (LR) and observed that the S. enterica isolates used in this study overall tend to survive better on RT than LR, individually ( Figure 1 ) or combined in pools ( Supplementary Figure S2 ). This finding is in agreement with the previously described increased fitness of E. coli O157:H7 and S. enterica serovar Typhimurium 14028s on RT ( Jacob and Melotto, 2020 ; Oblessuc and Melotto, 2020 ). It is possible that these bacterial pathogens of humans interact with the plant as commensals, occupying the phyllosphere while not benefiting or harming the host.…”

“…LSLB medium was supplemented with either 15 μg/mL tetracycline or 60 μg/mL kanamycin, as appropriate. Upon reaching an OD 600 of 0.7 to 0.9, bacterial cells were collected by centrifugation and a two-step dilution process was used to prepare the inoculum as described by Oblessuc and Melotto (2020) for a final concentration of 1 x 10 3 CFU/mL. To make inocula with pools of multiple S. enterica isolates ( Table 2 ), equal amounts of each isolate were added.…”

“…Leaves were vacuum-infiltrated with inoculum and the apoplastic bacterial population size (CFU/g foliar tissue) was estimated through enumeration using serial-dilution plating as previously described ( Oblessuc and Melotto, 2020 ; Supplementary Figure S1 ). The bacterial population size was estimated at 0 and 10 days post-inoculation (DPI).…”