Survival and virulence of Escherichia coli O157:H7 on romaine lettuce and its packaging film when kept under extended refrigerated storage

Foodborne disease outbreaks due to fresh produce contaminated with Escherichia coli O157:H7 have been a growing concern despite the continuous advancements in inactivation methods. Leafy vegetables like lettuce and spinach are susceptible to contamination through multiple agents (water, manure, soil, processing, and storage practices). Post-harvest pathogen inactivation/sanitation is the last defense to ensure food safety, but the increasing number of foodborne outbreaks indicates the ineffectiveness of current sanitation methods. In this study, the survival of E. coli O157:H7 was evaluated post-harvest on romaine lettuce leaves and packaging film at 4 °C over a 15-day period. Besides the conventional plate counting method, PMA-qPCR and LIVE/DEAD BacLight assay were performed. The aim was to detect and quantify the total viable cells during the 15-day refrigerated storage. At the 12th and 15th days of refrigerated storage, no viable E. coli cells were recovered on non-selective agar media; however, the LIVE/DEAD BacLight and PMA-qPCR method revealed the presence of viable cells. The expression of selected virulence (stx1a, stx2a, fliC, and eaeA), stress (rpoS, cspA, sodB, and uspB), Toxin-antitoxin (TA) system (chpB and hipA) genes were quantified by reverse transcription real-time qPCR assay. Expressions of all virulence genes (stx1a, stx2a, fliC, and eaeA), including the Shiga-toxin producing genes, were upregulated throughout the refrigerated storage at 4 °C. The trend of upregulation over the 15-day period was assessed, and it suggested an increase in upregulation with the increase in the days under refrigerated storage on the romaine lettuce as well as its packaging film. An exception was seen in the flagellin encoding gene, fliC which showed a decreasing trend of upregulation on the packaging film with a 2.4 log fold decrease (P < 0.05) on day 12 in the refrigerated storage. The expression of the key member of cold-shock regulon (cspA) was mostly constant with an average of 3.6 log fold increase (P < 0.05) on all days during the 15-day refrigerated period. There was no significant change in rpoS on the romaine lettuce, and its packaging film. However, the expression was variable with more frequent upregulation readings during the initial refrigerated storage period. The universal stress protein (uspB) expression showed upregulation on most days with an average of 0.6 log fold increase on romaine lettuce and 0.2 log fold increase on packaging film during the storage period. The overall trend was the increase in upregulation on romaine lettuce but a decrease in upregulation on packaging film. The TA system genes illustrated an increasing trend of gene upregulation for chpB but a decreasing trend for hipA. Average log fold-change of mRNA expression levels on romaine lettuce and packaging film was 1.8 log fold increase and 0.12 log fold increase for chpB and hipA, respectively. This expression trend was similar on the romaine lettuce as well as packaging film. In conclusion, the upregulation of virulence gene expression suggests that E. coli O157:H7 may become more virulent during storage at refrigerated temperatures. Our study indicates that E. coli can survive in the supply chain during prolonged refrigerated storage.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Agarwal, Saumya

Contributors dc:contributor

• Banerjee, Pratik
• Miller , Michael
• Stasiewicz, Matthew John
• Feng, Hao

• Foodborne Illness
• E. Coli O157:h7
• Romaine Lettuce
• Refrigerated

Statement dc:rights

• © 2023 Saumya Agarwal

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/120505

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F120505