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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Research Project #441365

Research Project: Assessing Romaine Lettuce “Forward Processing” for Potential Impacts on EHEC Growth, Antimicrobial Susceptibility, and Infectivity

Location: Environmental Microbial & Food Safety Laboratory

Project Number: 8042-32420-009-025-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Jan 1, 2022
End Date: Dec 31, 2023

a. Comprehensive assessment of forward processing practice under routine operation conditions for product integrity and microbiological quality. b. Assessment of microbiome dynamics on Romaine lettuce from harvest to retail for products being forward and source processed. c. Determine the impacts of different practices on EHEC outbreak strains, on cell physiology that may affect their growth potential, susceptibility to antimicrobial treatment, or virulence. d. Improvement of forward processing management by applying findings above.

Assessment of forward processing. To compare forward and source processing, freshly harvested Romaine lettuce from a common commercial lot destined to both forward and source processing facilities is tracked from farm to processing floors by embedding data loggers in designated bins. The conditions for fresh-cut washing and packaging are also recorded. Whole lettuce heads from the farm, and from the tracked bins, cut lettuce before washing, and packaged lettuce from both forward and source facilities are collected for visual quality assessment and for microbiological analyses, including total bacterial counts, key indicator organism counts, and microbiome. Samples collected from lettuce farm and source processing facility are sent to ARS laboratories in Maryland by overnight express service in refrigerated packages and processed upon receipt. Samples from the forward processing facility (Local) are immediately brought to the laboratory for processing. Packaged lettuce samples from both facilities are also stored at 4 oC and 10 oC for up to 2 weeks to compare the impact of forward processing on products during storage at compliant and abuse temperatures. This process is repeated 5 times for each collaborating company, and the data are used to determine the impact of forward processing on lettuce quality and microbiology. Impacts of forward processing conditions on EHEC. The forward and source processing conditions are simulated at ARS labs in Maryland with EHEC outbreak strains inoculated at a low level that would not significantly alter the microbiome dynamics on lettuce. Lettuce samples collected at various stages of these simulated processes are subjected to microbiological analyses as above. In addition, the growth, survival, susceptibility to antimicrobial treatment, and virulence gene expression of the inoculated EHEC (Strains associated with recent outbreaks) are analyzed. Higher inoculation levels are used for assessing EHEC gene expression. Produce quality and microbiological analyses. Whole head Romaine lettuce from the farm and from the processing facilities are compared for occurrence of visible damages during the transportation. Packaged lettuce products from both facilities are assessed for quality changes before and after storage at compliant and abusive temperatures by trained evaluation panels. Total bacterial counts on various lettuce samples are determined by non-selective plating on TSA; key indicator organism counts are determined using 3M Petrifilm plates for coliform, E. coli, and Yeast/Mold. Inoculated EHEC on lettuce is quantitatively detected by IMS-plating and by selective enrichment or qPCR. Microbiome and EHEC gene expressing analyses. Total microorganism recovered from lettuce samples are treated with PMA to ensure DNA is extracted from viable bacterial cells. The microbiome on lettuce samples is then determined by 16S rRNA gene sequencing as previously described or by shotgun metagenomics. Metatranscriptomic analyses are used to assess gene expression at the microbiome level. Expression of selected EHEC genes involved in stress responses and virulence are determined using arrayed RT-PCR analyses.