Purpose: The aim of our study was to elucidate the risk of internalization of surface inoculated E. coli O157:H7 upon vacuum cooling of lettuce as a function of microbial inoculation (high: 6 log CFU per leaf disk) or low: 3 log CFU per disk), surface moisture, and on both sides (abaxial and adaxial side) of lettuce leaves. To measure internalization of microbes in intact leafy greens, multiphoton 3-D microscopy was used. This novel microscopy approach addresses the challenges of autofluorescence and limited depth penetration to enable 3D microscopy of intact leaves.
Methods: Commercial lettuce purchased from a grocery store was washed, and the leaf surface was sprayed evenly with a solution of E. coli O157:H7 GFP to inoculate the surface of lettuce. After vacuum cooling treatment, the lettuce leaves were imaged with multiphoton microscopy. For quantitative and statistical analysis, the number of microbes associated with stomata and infiltrated into the leaf was quantified.
Results: Results based on imaging measurements demonstrated that the vacuum cooling process does not significantly increase the risk of internalization (P > 0.5) of surface inoculated E. coli into an intact lettuce leaf. The imaging results also show that the vacuum cooling process increased the number of bacteria associated with stomata (P < 0.5). These results were observed for both high moisture and low moisture conditions for samples inoculated on abaxial surface of lettuce leaves.
Significance: The imaging measurements highlight that the vacuum cooling process does not significantly increase the risk of internalization of microbes.