Faculty members from the Kansas State University College of Veterinary Medicine have developed a faster, more efficient method of detecting “Shiga toxin-producing E. coli,” or STEC, in ground beef, which often causes recalls of ground beef and vegetables.
“The traditional gold standard STEC detection, which requires bacterial isolation and characterization, is not amenable to high-throughput settings and often requires a week to obtain a definitive result,” said Jianfa Bai, section head of molecular research and development in the Kansas State Veterinary Diagnostic Laboratory.
The new method developed by Bai and colleagues requires only a day to obtain confirmatory results using a Kansas State University-patented method with the partition-based multichannel digital polymerase chain reaction system.
“We believe the new digital polymerase chain reaction detection method developed in this study will be widely used in food safety and inspection services for the rapid detection and confirmation of STEC and other foodborne pathogens,” said Jamie Henningson, director of the Kansas State Veterinary Diagnostic Laboratory.
When ingested through foods such as ground beef and vegetables, STEC can cause illnesses with symptoms including abdominal pain and diarrhea. Some illnesses caused by STEC may lead to kidney failure and can be life-threatening.
“Some E. coli strains do not produce Shiga toxins and thus do not affect human health as much,” said Xuming Liu, research assistant professor. “Because cattle feces and ground beef can contain harmless or less pathogenic E. coli along with STEC, the most commonly used polymerase chain reaction cannot identify pathogenic E. coli strains in a complex sample matrix.”
The new digital polymerase chain reaction test was developed for research and food safety inspections that require shorter turnaround and high throughput, without sacrificing detection accuracy.
“While the current, commonly used testing method is considered to be the gold standard, it is tedious and requires many days to obtain results that adequately differentiate the bacteria,” said Gary Anderson, director of the International Animal Health and Food Safety Institute at the K-State Olathe campus.
The study, “Single cell-based digital PCR detection and association of Shiga toxin-producing E. coli serogroups and major virulence genes,” which describes the test design and results, was published in the Journal of Clinical Microbiology.
Bacterial persistence is a form of phenotypic heterogeneity in which a subpopulation, persisters, has high tolerance to antibiotics and other stresses. Persisters of enteric pathogens may represent the subpopulations capable of surviving harsh environments and causing human infections. Here we examined the persister populations of several shiga toxin-producing Escherichia coli (STEC) outbreak strains under conditions relevant to leafy greens production.
The persister fraction of STEC in exponential-phase of culture varied greatly among the strains examined, ranging from 0.00003% to 0.0002% for O157:H7 strains to 0.06% and 0.08% for STEC O104:H4 strains. A much larger persister fraction (0.1–11.2%) was observed in STEC stationary cells grown in rich medium, which was comparable to the persister fractions in stationary cells grown in spinach lysates (0.6–3.6%). The highest persister fraction was measured in populations of cells incubated in field water (9.9–23.2%), in which no growth was detected for any of the STEC strains examined. Considering the high tolerance of persister cells to antimicrobial treatments and their ability to revert to normal cells, the presence of STEC persister cells in leafy greens production environments may pose a significant challenge in the development of effective control strategies to ensure the microbial safety of fresh vegetables.
Enhanced formation of shiga toxin-producing Escherichia coli persister variants in environments relevant to leafy greens production
Chris Koger of The Packer reported in late Dec. 2019 that Sprouts Unlimited, Marion, Iowa, is recalling clover sprouts, which have been linked to a cluster of E. coli cases under investigation in Iowa.
The Iowa Department of Inspections and Appeals is investigating the link between the outbreak and the product from Sprouts Unlimited, according to a Dec. 27 recall notice from the company.
The sprouts were shipped to Hy-Vee and Fareway Foods stores, and Jimmy John’s restaurants.
The retail packs in the recall are in pint containers with a blue label on the lid, according to Sprouts Unlimited. The Universal Product Code is 7 32684 00013 6 is on the bottom right side of the label.
The Iowa Department of Inspections and Appeals told Sprouts Unlimited the sprouts are epidemiologically linked to the outbreak. More tests are being conducted to determine the source, according to the recall notice.
Nutritional and perceived health benefits have contributed to the increasing popularity of raw sprouted seed products. In the past two decades, sprouted seeds have been a recurring food safety concern, with at least 55 documented foodborne outbreaks affecting more than 15,000 people. A compilation of selected publications was used to yield an analysis of the evolving safety and risk communication related to raw sprouts, including microbiological safety, efforts to improve production practices, and effectiveness of communication prior to, during, and after sprout-related outbreaks. Scientific investigation and media coverage of sprout-related outbreaks has led to improved production guidelines and public health enforcement actions, yet continued outbreaks call into question the effectiveness of risk management strategies and producer compliance. Raw sprouts remain a high-risk product and avoidance or thorough cooking are the only ways that consumers can reduce risk; even thorough cooking messages fail to acknowledge the risk of cross-contamination. Risk communication messages have been inconsistent over time with Canadian and U.S. governments finally aligning their messages in the past five years, telling consumers to avoid sprouts. Yet consumer and industry awareness of risk remains low. To minimize health risks linked to the consumption of sprout products, local and national public health agencies, restaurants, retailers and producers need validated, consistent and repeated risk messaging through a variety of sources.
Several types of mettwurst, manufactured by a South Australian Company, have been recalled after it was discovered the products may be contaminated with harmful bacteria.
Wintulichs, based in Gawler, recalled their Metwurst Garlic 300g, 375g, 500g, 700g, Mettwurst Plain 700g and Mettwurst Pepperoni 375g products.
Food Standards Australia New Zealand say the products have been sold at Woolworths, IGA and independent stores across SA.
The recall is due to incorrect pH and water activity levels, which may lead to microbial contamination and could cause illness if consumed.
Customers should return the products to the place of purchase for a full refund.
In Australia and around the world, the incidence of reported foodborne illness is on the increase. Regularly cited estimates suggest that Australia is plagued with over two million cases of foodborne illness each year, costing the community in excess of $1 billion annually.
Based on the case studies cited here and a thorough examination of a variety of documents disseminated for public consumption, government and industry in Australia are well aware of the challenges posed by greater public awareness of foodborne illness. They are also well aware of risk communication basics and seem eager to enter the public fray on contentious issues. The primary challenge for government and industry will be to provide evidence that approaches to managing microbial foodborne risks are indeed mitigating and reducing levels of risk; that actions are matching words.
There is a further challenge in impressing upon all producers and processors the importance of food safety vigilance, as well as the need for a comprehensive crisis management plan for critical food safety issues.
On Feb. 1, 1995, the first report of a food poisoning outbreak in Australia involving the death of a child from hemolytic uremic syndrome (HUS) after eating contaminated mettwurst reached the national press. The next day, the causative organism was identified in news stories as E. coli 0111, a Shiga-toxin E. coli (STEC) which was previously thought to be destroyed by the acidity in fermented sausage products like mettwurst, an uncooked, semi-dry fermented sausage. By Feb. 3, 1995, the child was identified as a four-year-old girl and the number sickened in the outbreak was estimated at 21.
The manager of the company that allegedly produced the contaminated mettwurst had to hire security guards to protect his family home as threats continued to be made on his life, and the social actors began jockeying for position in the public discourse. The company, Garibaldi, blamed a slaughterhouse for providing the contaminated product, while the State’s chief meat hygiene officer insisted that meat inspections and slaughtering techniques in Australian abattoirs were “top class and only getting better.”
On Feb. 4, just three days after the initial, national report, the South Australian state government announced it was implementing new food regulations effective March 1, 1995. The federal government followed suit the next day, announcing intentions to bolster food processing standards and launching a full inquiry. Even the coroner investigating the death of the girl said on Feb. 9 that investigations relating to inquests usually took about three months to complete, but he would start the hearing the next day if possible.
By Feb. 6, 1995, Garibaldi Smallgoods declared bankruptcy. Sales of smallgoods like mettwurst were down anywhere from 50 to 100 per cent according to the National Smallgoods Council.
The outbreak of E. coli O111 and the reverberations fundamentally changed the public discussion of foodborne illness in Australia, much as similar outbreaks of STEC in Japan, the U.K. and the U.S. subsequently altered public perception, regulatory efforts and industry pronouncements in those countries. The pattern of public reporting and response followed a similar pattern of reporting on the medical implications of the illness, attempts to determine causation and finger pointing. Such patterns of reporting are valid; when people are sick and in some cases dying from the food they consume, people want to know why. The results altered both the scientific and public landscapes regarding microbial foodborne illness, and can inform future risk communication and management efforts.
In all, 173 people were stricken by foodborne illness linked to consumption of mettwurst manufactured by Garibaldi smallgoods. Twenty-three people, mainly children, developed HUS, and one died. Although sporadic cases of HUS had been previously reported, this was the first outbreak of this condition recognized in Australia.
Once public attention focused on Garibaldi as the source of the offending foodstuff, the company quickly deflected criticism, blaming an unnamed Victorian-based company of supplying contaminated raw meat, and citing historical precedent as proof of safety. Garibaldi’s administration manager Neville Mead was quoted as saying that he was confident hygiene and processing at the plant were up to standard, adding, “We stand by our processing. We’ve done this process now for 24 years and it’s proved successful.” Such blind faith in tradition, even in the face of changing science-based recommendations, even in the face of tragedy, is often a hallmark of outbreaks of foodborne illness, reflecting the deep cultural and social mythologies that are associated with food.
However, given the uncertainties at the time, a spokesman with the Australian Meat and Livestock Association appropriately rejected such allegations, saying, “I believe it is irresponsible of them (Garibaldi) to make that statement when there is absolutely no evidence of that at all.” Likewise, Victorian Meat Authority chairman John Watson said his officers were investigating Garibaldi’s claims, but that even if the raw meat had come from
Victoria, the supplier may not necessarily be the source of the disease, but rather it could be based in Garibaldi’s processing techniques.
Similarly, when Garibaldi accused the watchdog South Australian Health Commission of dragging its feet with investigations, Health Minister, Dr. Michael Armitage responded by publicly stating that, “They indicated to us that they wanted their lawyers first to be involved before they provided us with information (concerning the mettwurst). It was only (after) earlier this week, under the Food Act, we issued a demand for that information, that we got it. So indeed, I would put it to Garibaldi that the boot is completely on the other foot.”
Likewise, South Australia’s chief meat hygiene officer, Robin Van de Graaff rejected such claims, saying that, “These organisms are part of a large family of bugs that are normal inhabitants of the gut of farm animals … If a tragedy like this occurs it is usually because, and it no doubt is in this case, not because of a small amount of contamination at the point of slaughter but because of the method of handling and processing after that.” The statements of government regulators would be subsequently validated.
Escherichia coli O157:H7 (EcO157) infections have been recurrently associated with produce. The physiological state of EcO157 cells surviving the many stresses encountered on plants is poorly understood. EcO157 populations on plants in the field generally follow a biphasic decay in which small subpopulations survive over longer periods of time. We hypothesized that these subpopulations include persister cells, known as cells in a transient dormant state that arise through phenotypic variation in a clonal population.
Using three experimental regimes (with growing, stationary at carrying capacity, and decaying populations), we measured the persister cell fractions in culturable EcO157 populations after inoculation onto lettuce plants in the laboratory. The greatest average persister cell fractions on the leaves within each regime were 0.015, 0.095, and 0.221%, respectively. The declining EcO157 populations on plants incubated under dry conditions showed the largest increase in the persister fraction (46.9-fold). Differential equation models were built to describe the average temporal dynamics of EcO157 normal and persister cell populations after inoculation onto plants maintained under low relative humidity, resulting in switch rates from a normal cell to a persister cell of 7.7 × 10−6 to 2.8 × 10−5 h−1. Applying our model equations from the decay regime, we estimated model parameters for four published field trials of EcO157 survival on lettuce and obtained switch rates similar to those obtained in our study. Hence, our model has relevance to the survival of this human pathogen on lettuce plants in the field. Given the low metabolic state of persister cells, which may protect them from sanitization treatments, these cells are important to consider in the microbial decontamination of produce.
IMPORTANCE Despite causing outbreaks of foodborne illness linked to lettuce consumption, E. coli O157:H7 (EcO157) declines rapidly when applied onto plants in the field, and few cells survive over prolonged periods of time. We hypothesized that these cells are persisters, which are in a dormant state and which arise naturally in bacterial populations. When lettuce plants were inoculated with EcO157 in the laboratory, the greatest persister fraction in the population was observed during population decline on dry leaf surfaces. Using mathematical modeling, we calculated the switch rate from an EcO157 normal to persister cell on dry lettuce plants based on our laboratory data. The model was applied to published studies in which lettuce was inoculated with EcO157 in the field, and switch rates similar to those obtained in our study were obtained. Our results contribute important new knowledge about the physiology of this virulent pathogen on plants to be considered to enhance produce safety.
Formation of Escherichia coli O157:H7 persister cells in the lettuce phyllosphere and application of differential equation models to predict their prevalence on lettuce plants in the field
08 November 2019
Applied and Environmental Microbiology
Daniel S. Munther, Michelle Q. Carter, Claude V. Aldric, Renata Ivanek, Maria T. Brandl
Don and Ben start the episode talking about kimchi fermentation and all the cabbage that needed to be washed and salted. The conversation went towards collaborations with fun people that might seem a bit unnatural to outsiders. The guys talk about a few outbreaks including two pathogenic E. coli ones linked to Romaine lettuce and Hep A in blackberries. They then do some listener feedback on foreign objects, bad cleaning and sanitizing machines and chitterlings. Also, bacteria is everywhere.
Food safety recalls are always either too early or too late. If you’re right, it’s always too late. If you’re wrong, it’s always too early.
– Dr. Paul Mead
Wheat flour has recently been recognised as an exposure vehicle for the foodborne pathogen Shiga toxin-producing Escherichia coli (STEC). Wheat flour milled on two sequential production days in October 2016, and implicated in a Canada wide outbreak of STEC O121:H19, was analysed for the presence of STEC in November 2018.
Stored in sealed containers at ambient temperature, the water activity of individual flour samples was below 0.5 at 6 months post-milling and remained static or decreased slightly in individual samples during 18 months of additional storage. STEC O121 was isolated, with the same genotype (stx2a, eae, hlyA) and core genome multilocus sequence type as previous flour and clinical isolates associated with the outbreak. The result of this analysis demonstrates the potential for STEC to persist in wheat flour at levels associated with outbreak infections for periods of up to two years. This has implications for the potential for STEC to survive in other foods with low water activity.
Shiga toxin-producing Escherichia coli survives storage in wheat flour for two years
This study was conducted to validate a simulated commercial baking process for plain muffins against E. coli O121 (isolated from the recent illness outbreak associated with flour), and compare the thermal inactivation parameters (D- and z-values) of cocktails of four isolates of E. coli O121 and three serovars of Salmonella (Newport, Typhimurium, and Senftenberg) in muffin batter.
Flour samples were spray inoculated with the E. coli O121 or Salmonella cocktails, dried back to the pre-inoculation weight to achieve ~7 log10 CFU/g, and used to prepare muffin batter. For the muffin baking validation study using E.coli O121, muffin batter was baked at 375 °F (190.6 °C) oven temperature for 21 min followed by 30 min of ambient cooling. The E. coli O121 population decreased by >7 log10 CFU/g in muffins by 17 min of baking, and was completely eradicated after 21 min of baking and ambient cooling. The D-values of E. coli O121 and Salmonella cocktails in muffin batter at 60, 65 and 70 °C were 42.0 and 38.4, 7.5 and 7.2, and 0.4 and 0.5 min, respectively; whereas the z-values of E. coli O121 and Salmonella were 5.0 and 5.2 °C, respectively.
Comparison of survival and heat resistance of Escherichia coli O121 and salmonella in muffins
Chris Koger of The Packer writes the U.S. Food and Drug Administration will be collecting romaine samples in California and Arizona for a year to test for salmonella and E. coli following several foodborne outbreaks linked to the lettuce.
The new program begins this month, according to the FDA, citing two E. coli outbreaks in 2018 linked to romaine, and another one in October that was suspected to be from the leafy green. In its notice on the surveillance program, the FDA also cited a 2012 Salmonella Newport outbreak from romaine.
“Consistent with the FDA’s mission to protect consumers, if one of the target pathogens is detected as a result of this assignment, the agency will perform whole genome sequencing of the microorganism’s DNA to determine its virulence and whether it is genetically related to isolates causing human illness,” according to the notice.
All samples will be tested before processing to allow the FDA to quickly find the point of origin, which has been problematic in recent outbreaks as public and federal health agencies traced lettuce through the supply chain. In part, traceability hurdles have led to the FDA’s New Era of Smarter Food Safety program, which tasks the industry with enhancing traceability methods and technology.
Trimmed and washed lettuce will be tested, but not fresh-cut lettuce, and no lettuce at the farm-level will be involved in the surveillance program.
Samples will be targeted at facilities and farms identified in the outbreaks starting in 2017, including wholesalers, foodservice distribution centers, and commercial cooling and cold storage facilities, according to the FDA notice.