Minimal processing of iceberg lettuce has no substantial influence on the survival, attachment and internalization of E. coli O157 and Salmonella

• Cut edges are the main entrance for internalization of E. coli O157 and Salmonella.
• Atmospheric conditions and light storage did not affect pathogen attachment.
• Sanitizing wash did not reduce the attachment and internalization of the pathogens.

The influence of a selection of minimal processing techniques (sanitizing wash prior to packaging, modified atmosphere, storage conditions under light or in the dark) was investigated in relation to the survival of, attachment to and internalization of enteric pathogens in fresh produce. Cut Iceberg lettuce was chosen as a model for fresh produce, Escherichia coli O157:H7 (E. coli O157) and Salmonella enterica were chosen as pathogen models. Care was taken to simulate industrial post-harvest processing. A total of 50 ± 0.1 g of fresh-cut Iceberg lettuce was packed in bags under near ambient atmospheric air with approximately 21% O2 (NAA) conditions or equilibrium modified atmosphere with 3% O2 (EMAP). Two lettuce pieces inoculated with E. coli O157 BRMSID 188 or Salmonella Typhimurium labeled with green fluorescent protein (GFP) were added to each package. The bags with cut lettuce were stored under either dark or light conditions for 2 days at 7 °C. The pathogens' capacity to attach to the lettuce surface and cut edge was evaluated 2 days after inoculation using conventional plating technique and the internalization of the bacteria was investigated and quantified using confocal microscopy. The effect of a sanitizing wash step (40 mg/L NaClO or 40 mg/L peracetic acid + 1143 mg/L lactic acid) of the cut lettuce prior to packaging was evaluated as well. Our results indicate that both pathogens behaved similarly under the investigated conditions. Pathogen growth was not observed, nor was there any substantial influence of the investigated atmospheric conditions or light/dark storage conditions on their attachment/internalization. The pathogens attached to and internalized via cut edges and wounds, from which they were able to penetrate into the parenchyma. Internalization through the stomata into the parenchyma was not observed, although some bacteria were found in the substomatal cavity. Washing the cut edges with sanitizing agents to reduce enteric pathogen numbers was not more effective than a rinse with precooled tap water prior to packaging. Our results confirm that cut surfaces are the main risk for postharvest attachment and internalization of E. coli O157 and Salmonella during minimal processing and that storage and packaging conditions have no important effect.