Yup, they get into produce seeds, especially sprouts: Salmonella and E. coli internalization

Vegetable seeds contaminated with bacterial pathogens have been linked to fresh-produce-associated outbreaks of gastrointestinal infections. This study was undertaken to observe the physiological behavior of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) cells artificially internalized into vegetable seeds during the germination process.

Surface-decontaminated seeds of alfalfa, fenugreek, lettuce, and tomato were vacuum-infiltrated with four individual strains of Salmonella or EHEC. Contaminated seeds were germinated at 25°C for 9 days, and different sprout/seedling tissues were microbiologically analyzed every other day. The internalization of Salmonella and EHEC cells into vegetable seeds was confirmed by the absence of pathogens in seed-rinsing water and the presence of pathogens in seed homogenates after post-internalization seed surface decontamination.

Results show that 317 (62%) and 343 (67%) of the 512 collected sprout/seedling tissue samples were positive for Salmonella and EHEC, respectively. The average Salmonella populations were significantly larger (P < 0.05) than the EHEC populations. Significantly larger Salmonella populations were recovered from the cotyledon and seed coat tissues, followed by the root tissues, but the mean EHEC populations from all sampled tissue sections were statistically similar, except in pre-germinated seeds. Three Salmonella and two EHEC strains had significantly larger cell populations on sprout/seedling tissues than other strains used in the study.

Salmonella and EHEC populations from fenugreek and alfalfa tissues were significantly larger than those from tomato and lettuce tissues. The study showed the fate of internalized human pathogens on germinating vegetable seeds and sprout/seedling tissues and emphasized the importance of using pathogen-free seeds for sprout production.

Fate of Salmonella enterica and Enterohemorrhagic Escherichia coli cells artificially internalized into vegetable seeds during germination

Appl. Environ. Microbiol. January 2018 84:e01888-17; Accepted manuscript posted online 27 October 2017, doi:10.1128/AEM.01888-17

Da Liu, Yue Cui, Ronald Walcott and Jinru Chen




Produce sanitation: Gas is better than liquid, man

Produce safety has received much recent attention, with the emphasis being largely on discovery of how microbes invade produce.

comparison-of-aqueous-ozone-and-chlorine-as-sanitizers-in-food-processing110_900_691_aHowever, the sanitization operation deserves more attention than it has received. The ability of a sanitizer to reach the site of pathogens is a fundamental prerequisite for efficacy. This work addresses the transport processes of ozone (gaseous and liquid) sanitizer for decontamination of leafy greens.

The liquid sanitizer was ineffective against Escherichia coli K-12 in situations where air bubbles may be trapped within cavities. A model was developed for diffusion of sanitizer into the interior of produce.

The reaction rate of ozone with the surface of a lettuce leaf was determined experimentally and was used in a numerical simulation to evaluate ozone concentrations within the produce and to determine the time required to reach different locations. For aqueous ozone, the penetration depth was limited to several millimeters by ozone self-decomposition due to the significant time required for diffusion. In contrast, gaseous sanitizer was able to reach a depth of 100 mm in several minutes without depletion in the absence of reaction with surfaces. However, when the ozone gas reacted with the produce surface, gas concentration was significantly affected.

Simulation data were validated experimentally by measuring ozone concentrations at the bottom of a cylinder made of lettuce leaf. The microbiological test confirmed the relationship between ozone transport, its self-decomposition, reaction with surrounding materials, and the degree of inactivation of E. coli K-12.

Our study shows that decontamination of fresh produce, through direct contact with the sanitizer, is more feasible with gaseous than with aqueous sanitizers. Therefore, sanitization during a high-speed washing process is effective only for decontaminating the wash water.

Physics of fresh produce safety: Role of diffusion and tissue reaction in sanitization of leafy green vegetables with liquid and gaseous ozone-based sanitizers

Journal of Food Protection, Number 12, December 2015

Mykola V. Shynkaryk, Taras Pyatkovskyy, Hussein M. Mohamed, Ahmed E. Yousef, and Sudhir K. Sastry


E. coli and Salmonella in tomatoes

Salmonella serovars have been associated with the majority of foodborne illness outbreaks involving tomatoes, and E. coli O157:H7 has caused outbreaks involving other fresh produce.

tomatoContamination by both pathogens has been thought to originate from all points of the growing and distribution process. To determine if Salmonella serovar Typhimurium and E. coli O157:H7 could move to the mature tomato fruit of different tomato cultivars following contamination, three different contamination scenarios (seed, leaf, and soil) were examined. Following contamination, each cultivar appeared to respond differently to the presence of the pathogens, with most producing few fruit and having overall poor health.

The Micro-Tom cultivar, however, produced relatively more fruit and E. coli O157:H7 was detected in the ripe tomatoes for both the seed- and leaf- contaminated plants, but not following soil contamination. The Roma cultivar produced fewer fruit, but was the only cultivar in which E. coli O157:H7 was detected via all three routes of contamination. Only two of the five cultivars produced tomatoes following seed-, leaf-, and soil- contamination with Salmonella Typhimurium, and no Salmonella was found in any of the tomatoes. Together these results show that different tomato cultivars respond differently to the presence of a human pathogen, and for E. coli O157:H7, in particular, tomato plants that are either contaminated as seeds or have a natural opening or a wound, that allows bacteria to enter the leaves can result in plants that have the potential to produce tomatoes that harbor internalized pathogenic bacteria.

 Movement of Salmonella serovar Typhimurium and E. coli O157:H7 to ripe tomato fruit following various routes of contamination

Microorganisms 2015, 3(4), 809-825

Deering, A.J.; Jack, D.R.; Pruitt, R.E.; Mauer, L.J.


Can norovirus get into plants? Apparently, yes

Human norovirus (NoV) is the leading cause of foodborne disease in the United States, and epidemiological studies have shown that fresh produce is one of the major vehicles for the transmission of human NoV. However, the mechanisms of norovirus contamination and persistence in fresh produce are poorly understood.

sorenne.strawberry.13The objective of this study is to determine whether human NoV surrogates, murine norovirus (MNV-1) and Tulane virus (TV), can attach and become internalized and disseminated in strawberries grown in soil.

The soil of growing strawberry plants was inoculated with MNV-1 and TV at a level of 108 PFU/plant. Leaves and berries were harvested over a 14-day period, and the viral titer was determined by plaque assay. Over the course of the study, 31.6% of the strawberries contained internalized MNV-1, with an average titer of 0.81 ± 0.33 log10 PFU/g. In comparison, 37.5% of strawberries were positive for infectious TV, with an average titer of 1.83 ± 0.22 log10 PFU/g. A higher percentage (78.7%) of strawberries were positive for TV RNA, with an average titer of 3.15 ± 0.51 log10 RNA copies/g as determined by real-time reverse transcriptase quantitative PCR (RT-qPCR).

In contrast, no or little virus internalization and dissemination were detected when TV was inoculated into bell peppers grown in soil.

strawberryCollectively, these data demonstrate (i) virally contaminated soils can lead to the internalization of virus via plant roots and subsequent dissemination to the leaf and fruit portions of growing strawberry plants and (ii) the magnitude of internalization is dependent on the type of virus and plant.

 Evidence of the Internalization of Animal Caliciviruses via the Roots of Growing Strawberry Plants and Dissemination to the Fruit

Applied and Environmental Microbiology, April 2015, Volume 81, Number 8, doi:10.1128/AEM.03867-14

DiCaprio E, Culbertson D, Li J


Careful with that cookie dough: E. coli O157:H7 can survive in wheat (at least in the lab)

Escherichia coli O157:H7 is a human pathogen that can cause bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. E. coli O157:H7 illnesses are mainly associated with undercooked beef; however, in recent years, outbreaks have been linked to fresh produce, such as spinach, lettuce, and sprouts.

nestle.toll.house.cookie.doughIn 2009, flour was implicated as the contamination source in an outbreak involving consumption of raw cookie dough that resulted in 77 illnesses. The objectives of this research were to determine (i) whether E. coli O157:H7 could be translocated into the internal tissues of wheat (Triticum aestivum) seedlings from contaminated seed, soil, or irrigation water and (ii) whether the bacterium could survive on flowering wheat heads. The levels of contamination of kanamycin-resistant E. coli O157:H7 strains in seed, soil, and irrigation water were 6.88 log CFU/g, 6.60 log CFU/g, and 6.76 log CFU/ml, respectively.

One hundred plants per treatment were sown in pot trays with 50 g of autoclaved soil or purposely contaminated soil, watered every day with 5 ml of water, and harvested 9 days postinoculation. In a fourth experiment, flowering wheat heads were spray inoculated with water containing 4.19 log CFU/ml E. coli O157:H7 and analyzed for survival after 15 days, near the harvest period. To detect low levels of internalization, enrichment procedures were performed and Biotecon real-time PCR detection assays were used to determine the presence of E. coli O157:H7 in the wheat, using a Roche Applied Science LightCycler 2.0 instrument.

The results showed that internalization was possible using contaminated seed, soil, and irrigation water in wheat seedlings, with internalization rates of 2, 5, and 10%, respectively. Even though the rates were low, to our knowledge this is the first study to demonstrate the ability of this strain to reach the phylloplane in wheat. In the head contamination experiment, all samples tested positive, showing the ability of E. coli O157:H7 to survive on the wheat head.


Transmission of Escherichia coli O157:H7 to internal tissues and its survival on flowering heads of wheat


Journal of Food Protection®, Number 3, March 2015, pp. 484-627, pp. 518-524(7)

Martinez, Bismarck; Stratton, Jayne; Bianchini, Andréia; Wegulo, Stephen; Weaver, Glen


How long until chefs catch up? Turn steaks every two minutes for safety

Keeping with the internalization theme, do beef steaks that are needle or blade tenderized need to be cooked to a higher temperature to ensure food safety? Or can other procedures like regular turning ensure safety. (I’ve done this for a long time because it led to a better product, but chefs are stuck with the one-turn principle.)

Colin Gill and colleagues write:

Beef steaks (2 cm thick) were each inoculated at three sites in the central plane with Escherichia coli O157:H7 at 5.9 ± 0.3 log CFU per site. Temperatures at steak centers were monitored during cooking on a hot plate or the grill of a gas barbeque. Steaks were cooked in hank.hill.bbqgroups of five using the same procedures and cooking each steak to the same temperature, and surviving E. coli O157:H7 at each site was enumerated. When steaks cooked on the hot plate were turned over every 2 or 4 min during cooking to between 56 and 62°C, no E. coli O157:H7 was recovered from steaks cooked to ≥58 or 62°C, respectively. When steaks were cooked to ≤71°C and turned over once during cooking, E. coli O157:H7 was recovered from steaks in groups turned over after ≤8 min but not from steaks turned over after 10 or 12 min.

E. coli O157:H7 was recovered in similar numbers from steaks that were not held or were held for 3 min after cooking when steaks were turned over once after 4 or 6 min during cooking. When steaks were cooked on the grill with the barbeque lid open and turned over every 2 or 4 min during cooking to 63 or 56°C, E. coli O157:H7 was recovered from only those steaks turned over at 4-min intervals and cooked to 56°C. E. coli O157:H7 was recovered from some steaks turned over once during cooking on the grill and held or not held after cooking to 63°C. E. coli O157:H7 was not recovered from steaks turned over after 4 min during cooking to 60°C on the grill with the barbeque lid closed or when the lid was closed after 6 min.

Apparently, the microbiological safety of mechanically tenderized steaks can be assured by turning steaks over at intervals of about 2 200297777-001min during cooking to ≥60°C in an open skillet or on a barbecue grill. When steaks are turned over only once during cooking to ≥60°C, microbiological safety may be assured by covering the skillet or grill with a lid during at least the final minutes of cooking.


Effects of selected cooking procedures on the survival of Escherichia coli O157:H7 in inoculated steaks cooked on a hot plate or gas barbecue grill

Journal of Food Protection, Number 6, June 2014, pp. 872-1042, pp. 919-926(8)

Gill, C. O., Devos, J., Youssef, M. K., Yang, X.


Biotic and abiotic variables affecting internalization and fate of Escherichia coli O157:H7 isolates in leafy green roots

In the on-going discussion of whether dangerous pathogens can be internalized in leafy greens, Erickson, et al. write:

Preharvest internalization of Escherichia coli O157:H7 into the roots of leafy greens is a food safety risk because the pathogen may be systemically transported to edible portions of the plant. In this study, both abiotic (degree of soil moisture) and biotic (E. coli O157:H7 exposure, presence of Shiga toxin genes, and type of leafy green) factors were examined to determine their potential effects on pathogen internalization into roots of leafy greens.

lettuceUsing field soil that should have an active indigenous microbial community, internalized populations in lettuce roots were 0.8 to 1.6 log CFU/g after exposure to soil containing E. coli O157:H7 at 5.6 to 6.1 log CFU/g. Internalization of E. coli O157:H7 into leafy green plant roots was higher when E. coli O157:H7 populations in soil were increased to 7 or 8 log CFU/g or when the soil was saturated with water. No differences were noted in the extent to which internalization of E. coli O157:H7 occurred in spinach, lettuce, or parsley roots; however, in saturated soil, maximum levels in parsley occurred later than did those in spinach or lettuce. Translocation of E. coli O157:H7 from roots to leaves was rare; therefore, decreases observed in root populations over time were likely the result of inactivation within the plant tissue.

Shiga toxin–negative (nontoxigenic) E. coli O157:H7 isolates were more stable than were virulent isolates in soil, but the degree of internalization of E. coli O157:H7 into roots did not differ between isolate type. Therefore, these nontoxigenic isolates could be used as surrogates for virulent isolates in field trials involving internalization.

Journal of Food Protection, Number 6, June 2014, pp. 872-1042, pp. 872-879(8)

Erickson, Marilyn C., Webb, Cathy C., Davey, Lindsey E., Payton, Alison S., Flitcroft, Ian D., Doyle, Michael P.


Internalization and fate of Escherichia coli O157:H7 in leafy green phyllosphere tissue using various spray conditions

In the past decade, leafy greens have been implicated in several outbreaks of foodborne illness, and research has focused on contamination during preharvest operations. Concerns have been raised that internalization of pathogens into the edible tissue occurs where postharvest chemical interventions would be ineffective. This study was initiated to measure the lettucedegree and fate of Escherichia coli O157:H7 internalized in the phyllosphere tissue of leafy greens when spray conditions, inoculum level, and type of leafy green were varied. Two spraying treatments were applied: (i) spraying individual spinach or lettuce leaves on plants once with a high dose (7 to 8 log CFU/ml) of E. coli O157:H7 and (ii) spraying spinach, lettuce, or parsley plants repeatedly (once per minute) with a low dose (2.7 to 4.2 log CFU/ml) of E. coli O157:H7 over a 10- to 20-min period. With the high-dose spray protocol, no significant differences in the prevalence of internalization occurred between Shiga toxin–negative E. coli O157:H7 isolates and virulent isolates (P > 0.05), implying that the Shiga toxin virulence factors did not influence internalization or the subsequent fate of those populations under these test conditions. Significantly greater internalization of E. coli O157:H7 occurred in spinach leaves compared with lettuce leaves when leaves were sprayed once with the high-dose inoculum (P < 0.05), whereas internalization was not observed in lettuce leaves but continued to be observed in spinach and parsley leaves following repeated spraying of the low-dose inoculum. Based on these results, it is surmised that a moisture film was generated when spraying was repeated and this film assisted in the mobilization of pathogen cells to plant apertures, such as stomata. E. coli O157:H7 cells that were internalized into spinach tissue using a low-dose repeat-spray protocol were temporary residents because they were not detected 2 days later, suggesting that plant-microbe interactions may be responsible.

Journal of Food Protection®, Number 5, May 2014, pp. 696-863 , pp. 713-721(9)

Erickson, Marilyn C.1; Webb, Cathy C.2; Davey, Lindsey E.2; Payton, Alison S.2; Flitcroft, Ian D.3; Doyle, Michael P.2

Effects of post-harvest handling conditions on internalization and growth of Salmonella Enterica in tomatoes

Journal of Food Protection®, Number 3, March 2014, pp. 352-521 , pp. 365-370(6)

Zhou, Bin; Luo, Yaguang; Nou, Xiangwu; Yang, Yang; Wu, Yunpeng; Wang, Qin


Salmonella internalization in tomatoes during postharvest handling is a major food safety concern. This study was conducted to determine the effect of immersion time, immersion depth, and temperature differential between bacterial suspension and tomato pulp on the internalization of Salmonella enterica in tomato fruits. The effect of storage temperature and tomato.dump.tankduration on the survival and growth of internalized Salmonella cells was also evaluated. Overall, immersion time significantly affected the incidence and extent of S. enterica internalization (P < 0.0001), with a linear correlation between immersion time and Salmonella internalization. The depth of Salmonella internalization in tomato tissues also increased with increasing immersion time. Immersion time also significantly influenced the degree to which the temperature differential affected Salmonella internalization. With an immersion time of 2 min, the temperature differential had no significant effect on Salmonella internalization (P = 0.2536). However, with an immersion time of 15 min, a significantly larger Salmonella population became internalized in tomatoes immersed in solutions with a –30°F (–16.7°C) temperature differential. Internalized S. enterica cells persisted in the core tissues during 14 days of storage. Strain type and storage duration significantly affected (P < 0.05) both the frequency detected and the population of internalized Salmonella recovered, but storage temperatures of 55 to 70°F (12.8 to 21.1°C) did not (P > 0.05). These findings indicate the importance of preventing pathogen internalization during postharvest handling.


Pathogen internalization by root uptake into food crops

Can pathogens like E. coli or Salmonella be internalized by growing fresh produce like lettuce, spinach and tomatoes?


Researchers from the University of Delaware and the U.S. Department of Agriculture report in Foodborne Pathogens and Disease that enteric pathogens localized at subsurface sites on leafy green plant tissue prevent their removal during washing and inactivation by sanitizers. Root uptake of enteric pathogens and subsequent internalization has been a large area of research with results varying due to differences in experimental design, systems tested, and pathogens and crops used.

The potential for uptake of foodborne pathogen, both bacterial and viral, through roots into food crops is reviewed. Various factors shown to affect the ability of human pathogens to internalize include growth substrate (soil vs. hydroponic solution), plant developmental stage, pathogen genus and/or strain, inoculum level, and plant species and cultivar. Several mechanisms of internalization (“active” vs. “passive”) of bacteria to plant roots have also been hypothesized.

The authors do conclude:

• uptake through internalization is a plant–pathogen specific interaction;
• the plant growth substrate used plays a large role in the uptake of both
bacterial and viral pathogens in plants;
• intact, healthy, non-injured roots seem to discourage the uptake of bacteria cells and viruses into plants; and,
• generally, the presence of internalized pathogens in roots of plants does not directly correlate with internalized pathogens in the edible or foliar tissues of crops.

The authors also note that contaminated soil, for the most part, resulted in little to no observed internalization as compared to contaminated hydroponic solution.