Assessments of Total and Viable Escherichia coli O157:H7 on Field and Laboratory Grown Lettuce

Abstract

Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 106 CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 104 greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable.

Factors affecting cell population density during enrichment and subsequent molecular detection of Salmonella enterica and Escherichia coli O157:H7 on lettuce contaminated during field production

“…Using the same Salmonella strain as in this study to inoculate melon surfaces during field production, a significant reduction of the population was observed during the first few days (Lopez-Velasco, . These and other studies, provide evidence that there is an initial loss of viability or recovery after enteric bacteria are inoculated on plant surfaces, however surviving bacteria may establish and persist at very low cell numbers that require concentration or enrichment for their detection (Moyne et al, 2013;Poza-Carrion, Suslow, & Lindow, 2013). How these data-based observations reflect the post-arrival fate of natural sources of contamination and the factors that influence this potential, other than atypical practices such as non-treated wastewater used for irrigation (Pachepsky, Shelton, Mclain, Patel, & Mandrell, 2011;Suslow, 2010), are largely unknown.…”

“…However independent studies have demonstrated that the accuracy of PCR based methods, in general, might be challenged when testing naturally contaminated samples as their sensitivity can be compromised by intrinsic or extrinsic factors (D'Aoust et al, 2007;Hoorfar, 2011). These challenges involve the inability of differentiating viable from dead cells, without significant additional sample processing, or those cells that can be in a viable but not culturable physiological condition (VBNC) (Moyne, Harris, & Marco, 2013). Furthermore, there may be substantial interference, difficult to predict in a nonvalidated matrix, of plant or food components that can cause inhibition of the PCR reaction (Harris & Griffiths, 1992;Jacobson, Gill, Irvin, Wang, & Hammack, 2012;Kim et al, 2012;Taskila, Toumola, & Ojamo, 2012).…”

Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

“…While the occurrence of the pathogen in leafy greens on that same sample date was relatively low, subsequent dissemination of high levels of E. coli O157:H7 from the feedlot surface to the plots may have increased the contamination of leafy greens that were sampled on the later dates. Numerous studies have evaluated the risks associated with preharvest contamination of leafy vegetables and have found that E. coli O157:H7 can persist on spinach and lettuce for days up to weeks (31)(32)(33)(34)(35)(36)(37)(38). Both growth chamber and field studies indicate that after inoculation onto leafy greens to mimic a contamination event, populations of E. coli O157:H7 initially decrease rapidly, followed by a more gradual rate of decline of remaining cells.…”

Multifactorial Effects of Ambient Temperature, Precipitation, Farm Management, and Environmental Factors Determine the Level of Generic Escherichia coli Contamination on Preharvested Spinach

“…The culture-based detection, as applied in the present study, is unable to detect viable but nonculturable (VBNC) cells, and this may vary across seasons and time since planting (55). However, Moyne et al reported that under field conditions, the loss in culturability of E. coli O157:H7 was most likely due to cell death rather than an inability to form colonies on standard media (55). The resurrection of VBNC has been shown possible under optimal laboratory conditions (e.g., with temperature upshift or nutrient availability) (56).…”