E. coli O157 gets a boost from downy mildew in lettuce

Escherichia coli O157:H7 is more likely to contaminate lettuce when downy mildew is already present, according to U.S. Department of Agriculture (USDA) scientists.

downeymildew150723Downy mildew, a lettuce disease caused by the fungus-like water mold Bremia lactucae, is one of the biggest problems that lettuce growers must deal with.

But microbiologist Maria Brandl, with the USDA Agricultural Research Service’s (ARS) Produce Safety and Microbiology Research Unit in Albany, California, has been investigating why so many E. coli O157:H7 outbreaks can be traced back to lettuce fields when E. coli O157:H7 sources are as diverse as undercooked beef, sprouts, raw dairy, shelled walnuts, fruits and vegetables. ARS is USDA’s chief in-house research agency.

Lettuce leaves are actually a harsh place for microbes to survive. But the epidemiological evidence is indisputable about how often lettuce is the source of E. coli O157:H7 contamination.

In earlier research, Brandl found that E. coli O157:H7 preferred cut, injured and younger leaves to undamaged and older ones. Then, she collaborated with ARS geneticist and lettuce breeder Ivan Simko from the Crop Improvement and Protection Research Unit in Salinas, California.

They found that under warm temperature and on wet leaves, E. coli O157:H7 multiplied 1,000-fold more in downy mildew lesions than on healthy lettuce leaf tissue. Even on dry lettuce leaves, where most bacteria struggle to survive, E. coli O157:H7 persisted in greater numbers when downy mildew disease was present.

The researchers also found that E. coli O157:H7 did not grow as well in downy mildew lesions on the lettuce line RH08-0464, bred by Simko and a colleague to be less susceptible to the lettuce disease, as the bacteria did on Triple Threat, a commercial variety that is highly susceptible to downy mildew.

The exact factors that caused less growth of E. coli O157:H7 in the more resistant line still need to be carefully explored. But if a genetic hurdle to E. coli O157:H7 colonization could be bred into commercial lettuce varieties along with downy mildew resistance, it would add a new defensive line to contamination of lettuce, helping farmers to improve the microbial safety of their crop as well as control their number-one plant disease problem.

Salmonella sticks to older lettuce better

Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previously studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar.

lettuceIn this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the 1st and 5–6th true-leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.

 Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves.

Microbiology Letters [ahead of print]

Paul J. Hunter , Robert K. Shaw , Cedric N. Berger , Gad Frankel , David Pink , Paul Hand


Microbiological risk? Lettuce from British Columbia

As I sit and overlook Vancouver Harbour, it’s timely that hockey buddy Kevin Allen had a paper published about lettuce: because who doesn’t think of locally grown lettuce in Jan.

lettuceIncreased consumer demand for fresh leafy produce has been paralleled by an increase in outbreaks and illness associated with these foods.

Presently, data on the microbiological quality and safety of produce harvested in the Lower Mainland of British Columbia is lacking. Therefore, fresh green, red, and romaine lettuce samples (n = 68) were obtained from five regional farmers’ markets in late summer of 2012 and subsequently analyzed to determine total numbers of aerobic bacteria, coliforms, and Escherichia coli. Additionally, enrichment procedures were used to detect low concentrations of E. coli.

Obtained E. coli isolates were subjected to multiplex PCRs to determine phylogenetic groupings and the presence of virulence genes (eaeA, hlyA, stx 1, and stx 2). All E. coli were tested for resistance to 15 antibiotics using a disk diffusion assay.

Lettuce samples yielded mean aerobic colony counts of 6.3 log CFU/g. Coliforms were detected in 72% of samples, with a median concentration of 1.9 log CFU/g. Of samples, 13% were found to harbor E. coli, with a median level of 0.7 log CFU/g. Antibiogram typing of all E. coli (n = 33) revealed that 97% possessed resistance to one or more antimicrobials, with resistance to amikacin (58%), trimethoprim (48%), and trimethoprim-sulfamethoxazole (45%) being the most common.

Phylogroup typing showed that 79% of these isolates belonged to group B1, with the remaining assigned to groups A (9%) or D (12%); no virulence genes were detected. Considering that phylogroup indicators suggestive of fecal contamination (groups A and D E. coli) were recovered in lettuce samples presented at retail, further work is required to explore at what point along the food chain contamination occurs.

Also, this study shows the presence of multidrug-resistant E. coli in fresh vegetables. Summed, these data provide important information on the microbiological quality of leafy vegetables grown in British Columbia through the detection and characterization of frequently used indicator organisms.


Microbiological survey of locally grown lettuce sold at farmers’ markets in Vancouver, British Columbia


Journal of Food Protection®, Number 1, January 2015, pp. 4-234, pp. 203-208(6)

Wood, Jayde L.; Chen, Jessica C.; Friesen, Elsie; Delaquis, Pascal; Allen, Kevin J.


Biggest PR screw-up in NZ for 2014? Bad lettuce

The handling of a food poisoning scare involving carrots and lettuce has been deemed the biggest public relations challenge this year by a Wellington PR firm.

lettuce.skull.e.coli.O145The handling of the Yersinia pseudotuberculosis issue by the Ministry for Primary Industries beat the closure of regional flight routes by Air New Zealand and Roger Sutton’s resignation by the State Services Commission to make the top of the list.

“In a year of dirty politics, what really concerned New Zealanders most was dirty lettuce and carrots,” BlacklandPR director Mark Blackham said.

“Everyone had these vegetables in our fridges, yet no one in authority could say for some time whether they were a health threat.

Millions of people were affected and little information is a recipe for fear, rumours and anger.”

Epidemiology, just trying to do this jigsaw puzzle: Outbreak of E. coli O157:H7 associated with lettuce served at fast food chains in the Maritimes and Ontario, Canada, Dec 2012

Background: Identification and control of multi-jurisdictional foodborne illness outbreaks can be complex because of their multidisciplinary nature and the number of investigative partners involved.

spongebob.oil.colbert.may3.10Objective: To describe the multi-jurisdictional outbreak response to an E. coli O157:H7 outbreak in Canada that highlights the importance of early notification and collaboration and the value of centralized interviewing.

Methods: Investigators from local, provincial and federal jurisdictions, using a national outbreak response protocol to clarify roles and responsibilities and facilitate collaboration, conducted a rapid investigation that included centralized re-interview of cases, descriptive methods, binomial probability, and traceback findings to identify the source of the outbreak.

Results: There were 31 laboratory confirmed cases identified in New Brunswick, Nova Scotia, and Ontario. Thirteen cases (42%) were hospitalized and one case (3%) developed hemolytic uremic syndrome; there were no deaths. Due to early notification a coordinated investigation was initiated before laboratory subtyping was available. Re-interview of cases identified 10 cases who had not initially reported exposure to the source of the outbreak. Less than one week after the Outbreak Investigation Coordinating Committee was formed, consumption of shredded lettuce from a fast food chain was identified as the likely source of the illnesses and the implicated importer/processor initiated a precautionary recall the same day.

Conclusion: This outbreak investigation highlights the importance of early notification, prompt re-interviewing and collaboration to rapidly identify the source of an outbreak.

Canada Communicable Disease Report CCDR

Tataryn J, Morton V, Cutler J, McDonald L, Whitfield Y, Billard B, Gad RR and Hexemer A


Tracking an Escherichia coli O157:H7–contaminated batch of leafy greens through a pilot-scale fresh-cut processing line

Cross-contamination of fresh-cut leafy greens with residual Escherichia coli O157:H7–contaminated product during commercial processing was likely a contributing factor in several recent multistate outbreaks.

lettuceConsequently, radicchio was used as a visual marker to track the spread of the contaminated product to iceberg lettuce in a pilot-scale processing line that included a commercial shredder, step conveyor, flume tank, shaker table, and centrifugal dryer. Uninoculated iceberg lettuce (45 kg) was processed, followed by 9.1 kg of radicchio (dip inoculated to contain a four-strain, green fluorescent protein–labeled nontoxigenic E. coli O157:H7 cocktail at 106 CFU/g) and 907 kg (2,000 lb) of uninoculated iceberg lettuce. After collecting the lettuce and radicchio in about 40 bags (∼22.7 kg per bag) along with water and equipment surface samples, all visible shreds of radicchio were retrieved from the bags of shredded product, the equipment, and the floor. E. coli O157:H7 populations were quantified in the lettuce, water, and equipment samples by direct plating with or without prior membrane filtration on Trypticase soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Based on triplicate experiments, the weight of radicchio in the shredded lettuce averaged 614.9 g (93.6%), 6.9 g (1.3%), 5.0 g (0.8%), and 2.8 g (0.5%) for bags 1 to 10, 11 to 20, 21 to 30, and 31 to 40, respectively, with mean E. coli O157:H7 populations of 1.7, 1.2, 1.1, and 1.1 log CFU/g in radicchio-free lettuce. After processing, more radicchio remained on the conveyor (9.8 g; P < 0.05), compared with the shredder (8.3 g), flume tank (3.5 g), and shaker table (0.1 g), with similar E. coli O157:H7 populations (P > 0.05) recovered from all equipment surfaces after processing.

These findings clearly demonstrate both the potential for the continuous spread of contaminated lettuce to multiple batches of product during processing and the need for improved equipment designs that minimize the buildup of residual product during processing.

Journal of Food Protection®, Number 9, September 2014, pp. 1448-1648, pp. 1487-1494(8)

Buchholz, Annemarie L.1; Davidson, Gordon R.1; Marks, Bradley P.2; Todd, Ewen C.D.3; Ryser, Elliot T.


Barriers to trace-back in a salad-associated EHEC outbreak, Sweden, June 2013

In June-July 2013, six counties notified the Swedish Institute for Communicable Disease Control of enterohaemorrhagic E.coli (EHEC) infections among attendees at a hotel in Dalarna, Sweden. An outbreak control team investigated to identify the source and implement control measures.

lettuce.skull.e.coli.O145We included individuals who attended the hotel between June 19th-25th in a cohort. We asked them about animal contact, swimming, and consumption of food items during this time using a questionnaire. A confirmed case was an EHEC O157:H7 outbreak strain positive individual who developed abdominal pain or diarrhoea between June 20th-July 2nd. We described the outbreak in time, place and person, calculated risk ratios (RR) and 95% confidence intervals (CI). We investigated the kitchen, tested and traced back implicated food items.

172 individuals responded. We identified 19 confirmed cases (Median age: 17 years, 64% female) with symptom onset between June 22nd-27th. Eating green salad on June 20th was associated with illness (RR:3.7;CI:1.3–11). The kitchen mixed green salads without records and destroyed leftovers immediately. Hence we could not conduct trace-back or obtain microbiological confirmation.

Green salad contaminated before entering the kitchen was the likely outbreak source. We recommended early collaboration with food agencies and better restaurant records to facilitate future investigations.

PLOS Currents Outbreaks

Michael Edelstein, Camilla Sundborger, Maria-Pia Hergens, Sofie Ivarsson, Rikard Dryselius, Mona Insulander, Cecilia Jernberg, Yvan Hutin, Anders Wallensten


Leafy green cone of silence: lawsuits dismissed against Tanimura & Antle

With different interpretations offered by opposing legal teams, two E. coli-related lawsuits filed by Seattle lawyer Bill Marler against Salinas-based Tanimura & Antle have been dismissed.

lettuce.skull.noroMarler told The Packer both cases had merit and were dismissed for reasons not related to the legitimacy of the cases.

Wesley Van Camp, vice president legal and general counsel for Tanimura & Antle, said the cases illustrated the importance of putting up a rigorous legal fight if there is no clear-cut connection between foodborne illnesses and specific fresh produce. She said the government must investigate to find a link between foodborne illness and specific produce items.’

In Pueblo, Colo., Tanimura & Antle had been named as a third-party defendant in an E. coli food contamination lawsuit, Liebnow vs. Boston Enterprises. The trial, which had been set for June 3, ended when the plaintiff dismissed her lawsuit against Tanimura & Antle, according to a release from the company.

Tanimura & Antle’s legal counsel was present at a mediation of the Colorado case and insisted upon dismissal of all claims against the company and defendants with prejudice, Van Camp said. The company was not a party to the settlement and no confidentiality provisions applied to Tanimura & Antle, she said.

Tanimura & Antle said in the release that a report filed by David Acheson, former associate commissioner for foods at the Food and Drug Administration, was pivotal in the case. Though a state epidemiologist had earlier named Tanimura & Antle as the lettuce supplier in the case, Acheson said in his lengthy deposition that public health officials did not conduct a full traceback of romaine lettuce and therefore could not rule out other sources of exposure. Acheson said FDA officials indicated the agency did not believe there was a compelling case to undertake the traceback on romaine lettuce.

Marler said May 3 that the Colorado case was ultimately dismissed by agreement of both parties through mediation. He said the law firm presented expert testimony that refuted Acheson’s conclusions. Timothy Jones, medical doctor and expert witness for Marler’s plaintiff, said in a report that the 2009 E. coli outbreak that caused ten cases of illness — including then 10-year old Emily Liebnow of Colorado Pueblo, Colo. Victims of the outbreak were identified in six states, including Colorado, Connecticut, Iowa, Minnesota, Missouri and North Carolina, he said. Jones said that evidence makes it “extremely unlikely” that anything other than a widely distributed food product was the source of multiple cases of illnesses caused by the E. coli O157:H7 strain.

spongebob.oil_.colbert.may3_.10-300x234“It is far more likely than not that Emily Liebnow’s infection resulted from consumption of contaminated Romaine lettuce from Tanimura and Antle, served at Giacomo’s restaurant on Sept. 6, 2009,” Jones said in this report.

In California, Marler also filed a motion to dismiss the lawsuit brought by Oroville Bernacki on behalf of his deceased wife Gail Bernacki in the U.S. District Court in Northern California against Tanimura & Antle. Tanimura & Antle’s attorney Gregory Rockwell said the plaintiff would not be able to prove that the E. coli infection resulted from the consumption of the defendant’s product.

Disputing that point, Marler said that the evidence presented in the lawsuit showed a genetic match between the woman’s illness and Tanimura & Antle product in the same time frame. Marler said the case had merit but was dismissed because the elderly plaintiff, a Canadian resident, was in declining health and was not mentally or physically up to litigation.

Fresh herbs and bagged salads can pose food safety risks

While the Canadian Food Inspection Agency was playing statistical silliness and gushing that 98 per cent of fresh leafy herbs sampled in 2009/2010 were not contaminated with bacterial pathogens or generic E. coli, researchers in Tennessee and the U.S. Centers for Disease Control published a more rigorous analysis of a multistate outbreak of Escherichia coli O157:H7 associated with bagged salad.

lettuce-skullI know; salad is not herbs. But they both have the potential to be contaminated.

Shiga toxin–producing Escherichia coli O157:H7 (STEC O157) is the most commonly identified serotype of STEC in the United States. An estimated 63,000 STEC O157 infections occur annually. Infection typically results in diarrhea, bloody stool, abdominal cramps, and, in some cases, hemolytic uremic syndrome. Recent outbreaks of STEC O157 have increasingly been associated with consumption of leafy greens such as lettuce and spinach. We investigated an outbreak of STEC O157 associated with the consumption of bagged salad with cases clustered in various institutional settings. A case–control study was conducted among cases from selected schools with controls matched by school and grade. Seventeen cases from three U.S. states were identified. The median age of cases was 23 years (range: 3–88) and 13 (76%) were female. Six cases were hospitalized and two died. Onset dates ranged from April 29 to May 12, 2012.

The matched case–control analysis identified a single significant food service exposure: consumption of lettuce provided by a school cafeteria (median odds ratio=9.4, 95% confidence interval: 1.4–∞, p=0.0469). The implicated bagged salad product was traced back to a single production facility. Implicated growing areas were scheduled for heightened inspection for the upcoming growing season. A combination of analytical epidemiologic studies among subclusters of cases, surveillance, and traceback implicated bagged salad in this outbreak investigation.

Foodborne Pathogens and Disease

Marder Ellyn P., Garman Katie N., Ingram Lily Amanda, and Dunn John R.

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