INTRODUCTION
Bagged salads or Ready-to-eat (RTE) salads can contain a variety of food stuffs, ranging from leafy greens to seeds. According to the European Food Safety Authority (EFSA) RTE salads fall under the category ‘composite foods, multi-ingredient foods and other foods,’ and specifically in the sub-category, ‘mixed foods.’ This essay focuses on bagged salads containing minimally processed RTE salads of non-animal origin.
The rise in popularity of bagged salads in the 1990s, initiated by Fresh Express, Inc. in the United States (US), has led to the largest market size in North America, with rapid growth in the Asia Pacific region 1. Bagged salads, known for their convenience and freshness, undergo a complex supply chain involving diverse ingredients sourced from various fields, each presenting different microbial hazards. This complexity raises challenges in contaminant identification and traceability, potentially contributing to outbreak cases. The absence of a kill step, such as thermal treatment, further emphasizes the importance of microbial safety in ensuring freshness and nutritional value.
Bagged salads have been associated with outbreaks, particularly in the US. Notable cases include a 2006 outbreak of Escherichia coli O157:H7 in spinach and a recent 2022 outbreak of Salmonella Senftenberg linked to cherry-like tomatoes 2,3. Most outbreaks due to Salmonella, E. coli O157:H7 and Listeria monocytogenes gain higher attention because of the severity of the illness and cases of deaths. This essay focuses on these three microbiological hazards, even though fresh produce in bagged salads can host a range of other pathogenic bacteria (e.g. Listeria monocytogenes), virus (e.g. Norovirus), and parasites (e.g. Cryptosporidium).
MICROBIOLOGICAL HAZARDS
Enteric bacteria face a lot of hurdles to survive on the plant leafy surface. Factors like geography, ultraviolet (UV) light exposure, temperature, relative humidity, availability of moisture, availability of nutrients, cuticle damage, leaf age, epiphytic microbes, phytopathogenic microbes, plant species, etc. influence the survival and growth of enteric bacteria on the plant leaf surface. Moreover, these enteric bacteria have a vast array of cell surface moieties (exocellular polysaccharide, cell surface charge, presence/absence of fimbriae, and hydrophobicity) that influence the interaction or attachment to the plant tissue 2, 3.
E. coli outbreaks linked to fresh produce are more prevalent in the US than in the EU. Shiga toxin-producing E. coli (STEC), particularly the O157 serogroup, accounted for 92% of STEC cases in foodborne outbreaks between 1998 and 2013. Other implicated serogroups during this period were O26 (4%), O145 (3%), and O121 (1%) 4. A notable 2011 outbreak in Germany, associated with sprouts, involved E. coli O104:H4, resulting in 3816 cases and 54 fatalities 5. Sprouts and lettuce are commonly identified as vehicles for E. coli contamination. Although cattle are considered the primary reservoir of E. coli O157:H7, its presence extends to the faeces of various domestic and wild animals and birds. The widespread global contamination of leafy greens with E. coli O157:H7 lacks specific gene attributions. Studies suggest that curli fibres may not play a role in the attachment of E. coli O157:H7 to lettuce 2. But, use of curli, a downward shift in metabolism, and the suppression of biofilm formation are proposed causes for the interaction of E. coli O157:H7 on undamaged lettuce 6.
Salmonella enterica, has over 2500 serovars. The ability of each serovar to colonize specific plant species varies, with S. Tennessee showing a stronger adherence to lettuce than S. Negev. At the cultivar level, S. Typhimurium exhibits better adhesion to the lettuce cultivar "Nelly" compared to "Cancan" 4. Salmonella may enter plants through cuts in tissues, uptake by root systems, and surface contamination of flowering plants, subsequently becoming trapped during fruit or vegetable embryogenesis. Salmonella can adhere to and be internalized by plant surfaces. The contamination by Salmonella and L. monocytogenes via roots is significantly greater than direct infection on leaves. Once present in soil, Salmonella and Listeria can remain viable for extended periods. Analysis of manure-fertilized soil and contaminated water revealed Salmonella spp. presence 161 days after cultivating lettuce and 231 days after salsa cultivation 7.
Listeria monocytogenes, with the highest fatality rate among vulnerable populations, is associated with fewer outbreaks compared to E. coli and Salmonella. Serotype 4b is a major contributor to human listeriosis cases, responsible for outbreaks like the 32 cases reported in Switzerland from 2013 to 2014 4. Pre-harvest, temperature affects L. monocytogenes' attachment moieties 8. Post-harvest, L. monocytogenes adapts to slow growth under refrigeration, with refrigeration stress, virulence gene expression is potentially induced. Pioneer bacterial cells, like Pseudomonas fluorescens, form biofilms in plants, creating a favourable environment for L. monocytogenes proliferation. Also, it can penetrate plant tissues and be present within stomata, potentially reaching deeper intracellular spaces in leaf tissue 7.
SOURCE, SPREAD, AND CONTROL OF CONTAMINATION
Fresh herbs and salad leaves are susceptible to contamination from various sources throughout their lifecycle. These sources include irrigation or pesticide application with contaminated water, the use of sewage as fertilizer, washing crops with contaminated water post-harvest, contact with animals, contaminated equipment, distribution-related factors, and cross-contamination during food preparation, including handling by infected food handlers. The potential for contamination and microbial proliferation exists during cultivation, harvest, post-harvest handling, processing, storage, distribution, or consumer handling of leafy vegetables.
Pre-harvest control measures
Interventions to control microbiological hazards in soil are typically applied at the start of a production cycle. UV treatment or filtration-based systems with zero-valent iron sand have demonstrated up to 6 log reduction in bacterial pathogen populations in irrigation water. Within the first 4 weeks of the growing cycle electrostatic application of lactic acid bacteria has demonstrated nearly 3-log reduction in E. coli O157:H7. Applied one day prior to harvest, acetic acid spray treatment impacts the prevalence of pathogens like E. coli O157:H7 or Salmonella on leaf lettuce, spinach, or cabbage. Spinach plants on treatment with Bacillus spp. has shown a 1-log reduction in Salmonella. A widely practiced treatment for animal fertilizers, composting has been effective in reducing the transfer of E. coli from amended soil to growing lettuce plants. Physical barriers such as plastic mulch to prevent direct contact between soil and edible portions provides an additional layer of control 9. By amending the leaves with a carbon source that is metabolized by spinach epiphytic bacteria but not by E.coli O157:H7, it may be possible to decrease populations of E. coli O157:H7. However, some epiphytes have no effect and some even promote the persistence of enteric pathogens on the phyllosphere, likely because these did not compete for the same carbon sources as E. coli O157:H7 10.
Post-harvest control measures
Numerous Natural antimicrobial Compounds (NACs) are under consideration for interventions against foodborne pathogens during or after minimal processing of fruits and vegetables. Lab-scale studies show that NACs can inactivate enteric bacterial foodborne pathogens at various stages of processing and on various leafy vegetables. However, there is variability in efficacy reported in different studies and on diverse commodities. Challenges include a lack of data on antiviral or antiparasitic effects, as well as solubility issues, necessitating means to improve functionality. Realistic and practical data on their impact on product quality in commercial processes and distribution channels are lacking 9. The use of generally recognized as safe (GRAS) additives, such as organic acids, is considered an alternative to control the multiplication of pathogenic microorganisms during food processing 7.
Irradiation by gamma ray, electron beam, or X-ray consistently reduces bacterial foodborne pathogens by 5–6 log in leafy vegetables without compromising the quality. The FDA approves irradiation of leafy vegetables at doses up to 1 kGy 9. However, the continued use of UV-C radiation can cause changes in colour 7.
Modified Atmosphere Packaging (MAP) is widely used to improve the shelf-life, sensory, appearance, and quality of minimally processed leafy vegetables, like oxidative browning of lettuce. However, it may promote the proliferation of pathogenic, facultative anaerobic bacteria, such as L. monocytogenes and E. coli O157: H7. Salmonella also has shown to persist high CO2. Exposure to antimicrobials in the gas phase, such as 10 percent hydrogen peroxide treatment, has shown reductions in bacterial populations on lettuce 7,9. Sanitizers, such as chlorine, chlorine dioxide, ozone, and peracetic acid, are commonly added to processing tanks to reduce microbial loads. However, their efficacy against bacteria attached to vegetable surfaces is limited, achieving only a 9.99% reduction instead of the desired 99.999%. Despite the widespread use of chlorine in processing plants, its FDA- approved limits may not be sufficient to eliminate important pathogens like Salmonella.
Alternative sanitizers, such as chlorine dioxide and peracetic acid, are being explored. The persistence of Salmonella and L. monocytogenes after appropriate sanitizer use may beattributed to greater tolerance, resistance to sublethal methods, or enhanced adhesion to surfaces. Enteric pathogens, including E. coli and Salmonella, can survive on fruit or vegetable surfaces, particularly in micro-holes or damaged areas. Disinfection process struggles to reduce strongly adherent biofilms by more than one logarithmic cycle. In a study example, shredded lettuce samples inoculated with L. monocytogenes exhibited varied responses to different washing conditions, concluding that washing using heated chlorinated water (47°C) favoured microbial growth in the samples stored at 10°C unlike cold water (4°C) 7.
Combining different agents, such as peracetic acid, neutral electrolyzed water, and UV-C radiation, can be effective in extending the shelf-life of vegetables. Ultrasound techniques, when combined with organic acids, have shown synergistic effect in reducing microbial load in different agricultural products 7.
Programmes such as Good Manufacturing Practices (GMP) in domestic kitchens and a Hazard Analysis Critical Control Point (HACCP) plan in the industry monitors and minimizes the occurrence of foodborne disease outbreaks 7. Emerging tools, such as electrochemical sensors, offer real-time information on microbiological risks, potentially integrating into industrial production lines. The sensors demonstrated efficient detection of bacterial contaminations in leafy green salads, even at low concentrations 11.
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