Wednesday, November 30, 2016

Salad Leaf Juice Enhances the Growth, Attachment, and Virulence of Salmonella

A study recently published in Applied and Environmental Microbiology shows that juices from leafy greens can enhance the growth, colonization, and virulence of Salmonella.  When chopping salad leaves, juices will be released from the cut surface.  The metabolites in these juices will enhance the growth of Salmonella as well as enhance its ability to form biofilms attaching to the leaf and bag surfaces.  There was even increased virulence.

While no one should avoid eating leafy greens, this study reinforces the need for good practices when growing, harvesting, processing and storing bagged chopped leafy greens.  Contamination on leafy greens is generally low, however, if improperly processed or handled, even a few inconsequential organisms can increase to the point where it becomes a problem.  For storage, bagged chopped salad should always be stored at refrigeration temperatures.

Health News | Wed Nov 30, 2016 | 7:24am EST
Bagged salads may encourage Salmonella growth
By Carolyn Crist
(Reuters Health) – - Crushed leaves in bagged lettuces at the supermarket may leak juice that fosters the right environment for Salmonella growth, according to a new study from the UK.

Salad juices increased the growth of Salmonella bacteria by 110 percent over normal levels, researchers found.

“Salad leaves pose a particular infection risk because they are usually minimally processed after harvesting and consumed raw,” said senior study author Primrose Freestone, a clinical microbiology lecturer at the University of Leicester.

Researchers are paying more attention to salad produce contamination after 100 people in the United States contracted Salmonella infections from bean sprouts in 2014. Salmonella causes 1.4 million cases of foodborne illness and 400 deaths annually in the United States, according to the Centers for Disease Control and Prevention.

“Our project does not indicate any increased risk for eating leafy salads, but it does provide a better understanding of the factors contributing to food poisoning risks,” Freestone told Reuters Health by email. “It also highlights the need for continued good practice in salad leaf production and preparation.”

Freestone and colleagues measured growth of Salmonella enterica, the strain commonly found in foodborne outbreaks in recent years. They crushed several salad leaf types - such as spinach, red chard and red romaine lettuce - to obtain leaf juice.

During a five-day refrigeration period, which is typical storage time for bagged salad, 100 Salmonella bacteria multiplied to more than 100,000. Salad leaf juice also enhanced the bacteria’s ability to attach to the sides of the plastic bags and containers, as well as to the leaves themselves.

“Most concerning was that we found exposure to the juices released from the salad leaves appeared to enhance the Salmonella’s capacity to establish an infection in the consumer,” Freestone said. “Salad leaves are an important part of a healthy diet but have been associated in recent years with a growing risk of food poisoning.”

Leafy salads carry a 3 percent risk for food poisoning due to pathogens such as Salmonella, the study authors write in the Journal of Applied and Environmental Microbiology. The European Food Safety Authority has classified leafy green salads as one of the top sources of foodborne infections, with salmonellosis accounting for more than 30 percent of outbreaks.

“Consumers seem to be more preoccupied with nutritional facts, but they should not forget that foodborne pathogens can be deadly,” said Kimon Karatzas, an assistant professor of food microbiology at the University of Reading in the UK who was not involved with the study. “Avoiding fresh produce is not a solution, but if possible, consumers should buy fresh uncut produce over chopped.”

Future studies should investigate how Salmonella survives in different kinds of fresh produce, Karatzas told Reuters Health by email. His research team is developing disinfectants that eliminate microorganisms from fresh produce.“The fact that bacteria growth is enhanced by the presence of nutrients from a food is not very surprising,” said Martin Adams, a food microbiology professor at the University of Surrey in the UK who was not involved with the study. “What did concern me was that the particular strain of Salmonella was able to grow at 4 degrees Celsius (39.2 degrees F), or refrigeration temperature.”

Salmonella strains typically don’t grow below 7 degrees Celsius, Adams said, and the accepted absolute minimum growth temperature is 5 degrees Celsius (41 degrees F).

“It is very important that salad vegetables are washed thoroughly before consumption, which is good advice that goes back many years,” Adams told Reuters Health by email. “Although prepared bagged salads have already been washed, another washing before use would give an added level of reassurance.”
SOURCE: Journal of Applied and Environmental Microbiology, online November 18, 2016.

Applied and Environmental Microbiology
Salad leaf juices enhance Salmonella growth, fresh produce colonisation and virulence

Giannis Koukkidis1,
Richard Haigh2,
Natalie Allcock3,
Suzanne Jordan4* and
Primrose Freestone1*

+ Author Affiliations
1Department of Infection, Immunity and Inflammation, Maurice Shock Medical Sciences Building. 2Department of Genetics, 3Core Biotechnology Services, University of Leicester, University Road, Leicester LE1 9HN UK
4Campden BRI, Station Road, Chipping Campden, Gloucestershire GL55 6LD UK


We show in this report that traces of juices released from salad leaves as they became damaged can significantly enhance Salmonella enterica salad leaf colonisation. Salad juices in water increased Salmonella growth by 110% over the un-supplemented control, and in host-like serum based media by more than 2400-fold over controls. In serum based media salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonisation and retention were enhanced, such as motility and biofilm formation, which increased over controls by >220% and 250% respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked advantage from fluids released from salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days refrigeration (a typical storage time for bagged salad leaves) even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonisation, which could be an unappreciated factor in pathogen fresh produce retention. Collectively, this study shows that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves, and strongly emphasizes the importance of ensuring the microbiological safety of fresh produce.

Importance Salad leaves are an important part of a healthy diet, but in recent years have been associated with a growing risk of food poisoning from bacterial pathogens such as Salmonella enterica. Although this is considered a significant public health problem, very little is known about what happens to the behaviour of the Salmonella when in the actual salad bag. We show that juices released from the cut-ends of the salad leaves enabled the Salmonella to grow in water, even when it was refrigerated. Salad juice exposure also helped the Salmonella cells to attach to the salad leaves so strongly that washing could not remove them. Collectively, this study shows that exposure to even traces of salad leaf juice may contribute to the persistence of Salmonella on salad leaves as well as priming it for establishing an infection in the consumer.


*Corresponding authors: Dr. Primrose Freestone, Email:, Tel: +44 (0)116 252 5656, Fax: +44 (0)116 252 5030
*Dr. Suzanne Jordan, Email:, Telephone: +44(0)1386 842013
Copyright © 2016 Koukkidis et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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