Spinach can be persistently infected with Salmonella

Posted on June 18, 2020 by Douglas Powell

When I was attempting a Masters degree (I never finished after almost three years, became a newspaper editor, then went back and got a PhD in Food Science; thanks Mansel for taking me on) looking at susceptible and resistant strains of tomato plants to Verticillium wilt, I had to inoculate these plants and walk up to the lab at all hours of the day and night to nurture and harvest the plants at prescribed times and look at their cells under a microscope – sometimes even electronic – and see what the fungus was doing.

That was 1985.

There have been improvements in technology.

The effects of using contaminated seed and water on the persistence and internalization of Salmonella Newport in organic spinach cultivars- Lazio, Space, Emilia and Waitiki were studied.

Seeds were contaminated by either immersing in a suspension of Salmonella and then sprouted or were sprouted in Salmonella contaminated water in the dark at 25 °C. After 5 days, germinated sprouts were analyzed for S. Newport population and internalization. Germinated sprouts were potted in soil and grown in a plant incubator for 4 weeks. Leaves, stems and roots were sampled for Salmonella population by plating on CHROMagar™. Plants surface-sterilized with chlorine were analyzed for internalized pathogen. Potting soil and water runoff were sampled for Salmonella after 4 weeks of plant growth.

Contaminated seeds and irrigation water had S. Newport populations of 7.64±0.43 log CFU/g and 7.12±0.04 log CFU/ml, respectively. Sprouts germinated using contaminated water or seeds had S. Newport populations of 8.09±0.04 and 8.08±0.03 log CFU/g, respectively and had a Salmonella population that was significantly higher than other spinach tissues (P<0.05). Populations of S. Newport in leaves, stem and roots of spinach plants were as follows: contaminated seed- 2.82±1.69, 1.69±0.86, and 4.41±0.62 log CFU/ml; contaminated water- 3.56±0.90, 3.04±0.31, and 4.03±0.42 log CFU/ml of macerated tissue suspension, respectively. Internalization was observed in plants developing from contaminated seeds and in sprouts germinated using contaminated water. S. Newport populations of 2.82±0.70 log CFU/g and 1.76±0.46 log CFU/ml were recovered from soil and water runoff, respectively.

The results indicate that contamination of spinach during germination can result in persistence, internalization and environmental reintroduction of Salmonella.

Contamination of spinach at germination: A route to persistence and environmental reintroduction by salmonella, 02 August 2020

International Journal of Food Microbiology

Govindaraj Dev Kumar, Jitendra Patel, Sadhana Ravishankar

https://arizona.pure.elsevier.com/en/publications/contamination-of-spinach-at-germination-a-route-to-persistence-an

Scientists in India figure out how Salmonella infect plants

Posted on January 11, 2019 by Douglas Powell

Sunderarajan Padmanabhan of Biotech Times writes that E. coli and Salmonella bacteria are the most common causes of food poisoning. Although most Salmonella outbreaks are linked to contamination during handling and transportation of the vegetables, there are also cases where the infectious bacterium had entered the plant when it was still in the farmland.

A new study by researchers at the Indian Institute of Science (IISc) and the University of Agricultural Sciences (UAS), Bengaluru, has solved the mystery.

They have found that unlike other disease-causing bacteria that enter the root, fruit or leaf by producing enzymes to break down the plant’s cell wall, Salmonella sneaks in through a tiny gap created when a lateral root branches out from the plant’s primary root.

The researchers were studying how different types of bacteria colonize the roots of tomato plants. While other bacteria were spread across the root, Salmonella clustered almost exclusively around areas where lateral roots emerge. When a lateral root pierces open the wall of the primary root to spread across the soil, it leaves behind a tiny opening. They figured out that it was entering through the gap with the help of fluorescent tagging and imaging.

They also noticed that under same conditions a plant with a greater number of lateral roots harbored a greater concentration of Salmonella than one with fewer lateral roots. Similarly, when plants were artificially induced to produce more lateral roots, Salmonella concentration increased.

Tomatoes plucked from these plants also tested positive for Salmonella infection, revealing its ability to travel all the way up to the fruit. “It is just like a systemic infection in humans,” said senior author Dipshikha Chakravortty, Professor, Department of Microbiology and Cell Biology, IISc. The researchers have published a paper on their work in the journal, BMC Plant Biology.

Kapudeep Karmakar, Ph.D. student in the Department of Microbiology and Cell Biology, IISc, and first author of the paper, noted that there are several possible sources from where Salmonella can reach the soil, such as manure containing animal faeces or contaminated irrigation water.

“Various studies show that irrigation water gets contaminated by sewage water. When that irrigation water is applied in the field, the soil becomes the portal for Salmonella to enter,” he said.

Norovirus inside leafy greens

Posted on June 3, 2018 by Douglas Powell

Lettuce has been implicated in human norovirus (HuNoV) outbreaks. The virus is stable on the leaf surface for at least 2 weeks; however, the dynamics of virus internalization have not been fully investigated. The purpose of this study was to assess the internalization and distribution of HuNoV and two surrogate viruses, porcine sapovirus (SaV) and Tulane virus (TV), in lettuce and spinach.

Viral inoculations through the roots of seedlings and the petiole of leaves from mature plants were performed, and the viruses were tracked on days 1 and 6 post-root inoculation and at 16 h and 72 h post-petiole inoculation. Confocal microscopy was used to visualize root-internalized HuNoV.

In both lettuce and spinach, (i) HuNoV was internalized into the roots and leaves at similar RNA titers, whereas surrogate viruses were more restricted to the roots, (ii) all three viruses were stable inside the roots and leaves for at least 6 days, and (iii) HuNoV disseminated similarly inside the central veins and leaf lamina, whereas surrogate viruses were more restricted to the central veins. Infectious TV, but not SaV, was detectable in all tissues, suggesting that TV has greater stability than SaV. HuNoV was visualized inside the roots’ vascular bundle and the leaf mesophyll of both plants.

In conclusion, using surrogate viruses may underestimate the level of HuNoV internalization into edible leaves. The internalization of HuNoV through roots and cut leaves and the dissemination into various spinach and lettuce tissues raise concerns of internal contamination through irrigation and/or wash water.

IMPORTANCE Human noroviruses are the leading cause of foodborne outbreaks, with lettuce being implicated in the majority of outbreaks. The virus causes acute gastroenteritis in all age groups, with more severe symptoms in children, the elderly, and immunocompromised patients, contributing to over 200,000 deaths worldwide annually. The majority of deaths due to HuNoV occur in the developing world, where limited sanitation exists along with poor wastewater treatment facilities, resulting in the contamination of water resources that are often used for irrigation.

Our study confirms the ability of lettuce and spinach to internalize HuNoV from contaminated water through the roots into the edible leaves. Since these leafy greens are consumed with minimal processing that targets only surface pathogens, the internalized HuNoV presents an added risk to consumers. Thus, preventive measures should be in place to limit the contamination of irrigation water. In addition, better processing technologies are needed to inactivate internalized viral pathogens.

Tissue distribution and visualization of internalized norovirus in leafy greens

April 2018

Applied Environmental Microbiology, vol.84 no.12

Malak A. Esseili ^a, Tea Meulia ^b, Linda J. Saif ^a and Qiuhong Wang ^a

doi:10.1128/AEM.00292-18

http://aem.asm.org/content/84/12/e00292-18.abstract?etoc

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

Posted on January 5, 2018 by Douglas Powell

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

http://aem.asm.org/content/84/1/e01888-17.abstract?etoc

Produce sanitation: Gas is better than liquid, man

Posted on December 8, 2015 by Douglas Powell

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

However, 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

http://www.ingentaconnect.com/content/iafp/jfp/2015/00000078/00000012/art00001

E. coli and Salmonella in tomatoes

Posted on November 8, 2015 by Douglas Powell

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.

Contamination 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.

http://www.mdpi.com/2076-2607/3/4/809

Can norovirus get into plants? Apparently, yes

Posted on March 29, 2015 by Douglas Powell

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.

The 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.

Collectively, 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

http://aem.asm.org/content/81/8/2727.abstract?etoc

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

Posted on March 2, 2015 by Douglas Powell

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.

In 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

01.mar.15

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

http://www.ingentaconnect.com/content/iafp/jfp/2015/00000078/00000003/art00006

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

Posted on May 25, 2014 by Douglas Powell

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 groups 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 min 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.

http://www.ingentaconnect.com/content/iafp/jfp/2014/00000077/00000006/art00007

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

Posted on May 24, 2014 by Douglas Powell

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.

Using 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.

http://www.ingentaconnect.com/content/iafp/jfp/2014/00000077/00000006/art00001

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