Fresh fruit and vegetables as vehicles for the transmission of human pathogens

Much research into food‐borne human pathogens has focused on transmission from foods of animal origin. However, recent investigations have identified fruits and vegetables are the source of many disease outbreaks. Now believed to be a much larger contributor to produce‐associated outbreaks than previously reported, norovirus outbreaks are commonly caused by contamination of foods from hands of infected workers. Although infections with Shiga toxin‐producing E. coli O157 have been linked to beef more often than to any other food product, severe outbreaks have been traced to consumption of contaminated radish sprouts and pre‐packaged spinach. Similarly, while infections with Salmonella have mainly been linked to consumption of foods of animal origin, many outbreaks have been traced to contaminated fresh produce. E. coli O157 binds to lettuce leaves by alternative mechanisms involving the filamentous type III secretions system, flagella and the pilus curli. Association of Salmonella with fresh produce appears to be serovar‐specific involving flagella, curli, cellulose, and O antigen capsule. A better understanding of plant, microbiological, environmental, processing and food handling factors that facilitate contamination will allow development of evidence‐based policies, procedures and technologies aimed at reducing the risk of contamination of fresh produce.     

Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review

Consumption of fruit and vegetable products is commonly viewed as a potential risk factor for infection with enteropathogens such as Salmonella and Escherichia coli O157, with recent outbreaks linked to lettuce, spinach and tomatoes. Routes of contamination are varied and include application of organic wastes to agricultural land as fertilizer, contamination of waters used for irrigation with faecal material, direct contamination by livestock, wild animals and birds and postharvest issues such as worker hygiene. The ability of pathogens to survive in the field environment has been well studied, leading to the implementation of guidelines such as the Safe Sludge Matrix, which aim to limit the likelihood of viable pathogens remaining at point-of-sale. The behaviour of enteropathogens in the phyllosphere is a growing field of research, and it is suggested that inclusion in phyllosphere biofilms or internalization within the plant augments the survival. Improved knowledge of plant-microbe interactions and the interaction between epiphytic and immigrant micro-organisms on the leaf surface will lead to novel methods to limit enteropathogen survival in the phyllosphere.     

Untangling metabolic and communication networks: interactions of enterics with phytobacteria and their implications in produce safety

Recent outbreaks of vegetable-borne gastrointestinal illnesses across the globe demonstrate that human enteric pathogens can contaminate produce at any stage of production. Interactions of enterics with native plant-associated microbiota influence the microbiological safety of produce by affecting the attachment, persistence and proliferation of human pathogens on plants. Supermarket surveys have revealed that bacteria, but not fungi or mechanical damage, promote the growth of Salmonella enterica on produce. Field and laboratory studies have indicated that some plant pathogenic bacteria and fungi facilitate the entry and internalization of human pathogens in plants. Conversely, some phytobacteria, including those involved in biocontrol of plant diseases, significantly inhibit attachment and plant colonization by non-typhoidal Salmonella and enterovirulent Escherichia coli by producing antibiotics or competing for nutrients in the phyllosphere. In this review, we attempt to elucidate the mechanisms of interactions between human enteric pathogens and plant-associated microbiota, and describe how these interactions affect produce safety.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g. small farms), hospitals, nursing homes, cruise ships, and hotels.     

Do leafy green vegetables and their ready-to-eat [RTE] salads carry a risk of foodborne pathogens?

Over the past 10 years, there is an increasing demand for leafy green vegetables and their ready-to-eat (RTE) salads since people changed their eating habits because of healthier lifestyle interest. Nevertheless fresh leafy green vegetables and their RTE salads are recognized as a source of food poisoning outbreaks in many parts of the world. However, this increased proportion of outbreaks cannot be completely explained by increased consumption and enhanced surveillance of them. Both in Europe and in the USA, recent foodborne illness outbreaks have revealed links between some pathogens and some leafy green vegetables such as mostly lettuces and spinaches and their RTE salads since fresh leafy green vegetables carry the potential risk of microbiological contamination due to the usage of untreated irrigation water, inappropriate organic fertilizers, wildlife or other sources that can occur anywhere from the farm to the fork such as failure during harvesting, handling, processing and packaging. Among a wide range of pathogens causing foodborne illnesses, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes are the most common pathogens that contaminate leafy green vegetables. Children, the elderly, pregnant women and immunocompromised people are the most at risk for developing complications from foodborne illness as a result of eating contaminated leafy greens or their RTE salads. These outbreaks are mostly restaurant associated or they sometimes spread across several countries by international trade routes. This review summarizes current observations concerning the contaminated leafy green vegetables and their RTE salads as important vehicles for the transmission of some foodborne pathogens to humans.     

Lettuce cultivar mediates both phyllosphere and rhizosphere activity of Escherichia coli O157:H7

Plant roots and leaves can be colonized by human pathogenic bacteria, and accordingly some of the largest outbreaks of foodborne illness have been associated with salad leaves contaminated by E. coli O157. Integrated disease management strategies often exploit cultivar resistance to provide a level of protection from economically important plant pathogens; however, there is limited evidence of whether the genotype of the plant can also influence the extent of E. coli O157 colonization. To determine cultivar-specific effects on colonization by E. coli O157, we used 12 different cultivars of lettuce inoculated with a chromosomally lux-marked strain of E. coli O157:H7. Lettuce seedlings grown gnotobiotically in vitro did exhibit a differential cultivar-specific response to E. coli O157 colonization, although importantly there was no relationship between metabolic activity (measured as bioluminescence) and cell numbers. Metabolic activity was highest and lowest on the cultivars Vaila-winter gem and Dazzle respectively, and much higher in endophytic and tightly bound cells than in epiphytic and loosely bound cells. The cultivar effect was also evident in the rhizosphere of plants grown in compost, which suggests that cultivar-specific root exudate influences E. coli O157 activity. However, the influence of cultivar in the rhizosphere was the opposite to that in the phyllosphere, and the higher number and activity of E. coli O157 cells in the rhizosphere may be a consequence of them not being able to gain entry to the plant as effectively. If metabolic activity in the phyllosphere corresponds to a more prepared state of infectivity during human consumption, leaf internalization of E. coli O157 may pose more of a public health risk than leaf surface contamination alone.     

Same-day detection of Escherichia coli O157:H7 from spinach by using electrochemiluminescent and cytometric bead array biosensors

Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.     

Spatial and Temporal Factors Associated with an Increased Prevalence of Listeria monocytogenes in Spinach Fields in New York State

While rain and irrigation events have been associated with an increased prevalence of foodborne pathogens in produce production environments, quantitative data are needed to determine the effects of various spatial and temporal factors on the risk of produce contamination following these events. This study was performed to quantify these effects and to determine the impact of rain and irrigation events on the detection frequency and diversity of Listeria species (including L. monocytogenes) and L. monocytogenes in produce fields. Two spinach fields, with high and low predicted risks of L. monocytogenes isolation, were sampled 24, 48, 72, and 144 to 192 h following irrigation and rain events. Predicted risk was a function of the field''s proximity to water and roads. Factors were evaluated for their association with Listeria species and L. monocytogenes isolation by using generalized linear mixed models (GLMMs). In total, 1,492 (1,092 soil, 334 leaf, 14 fecal, and 52 water) samples were collected. According to the GLMM, the likelihood of Listeria species and L. monocytogenes isolation from soil samples was highest during the 24 h immediately following an event (odds ratios [ORs] of 7.7 and 25, respectively). Additionally, Listeria species and L. monocytogenes isolates associated with irrigation events showed significantly lower sigB allele type diversity than did isolates associated with precipitation events (P = <0.001), suggesting that irrigation water may be a point source of L. monocytogenes contamination. Small changes in management practices (e.g., not irrigating fields before harvest) may therefore reduce the risk of L. monocytogenes contamination of fresh produce.     

Avirulence proteins from haustoria-forming pathogens

A major insight that has emerged in the study of haustoria-forming plant pathogens over the last few years is that these eukaryotic biotrophs deliver suites of secreted proteins into host cells during infection. This insight has largely derived from successful efforts to identify avirulence (Avr) genes and their products from these pathogens. These Avr genes, identified from a rust and a powdery mildew fungus and three oomycete species, encode small proteins that are recognized by resistance proteins in the host plant cytoplasm, suggesting that they are transported inside plant cells during infection. These Avr proteins probably represent examples of fungal and oomycete effector proteins with important roles in subverting host cell biology during infection. In this respect, they represent a new opportunity to understand the basis of disease caused by these biotrophic pathogens. Elucidating how these pathogen proteins gain entry into plant cells and their biological function will be key questions for future research.     

A General Framework For Prioritizing Research To Reduce Injuries And Diseases in Mining

A strategy for prioritizing mining health and safety research by evaluating the potential for risk reduction through interventions is proposed. Mining has one of the highest incidence rates of injury and disease found in major industries. The main premise of this paper is that often the best opportunities to reduce these rates are not revealed by retrospective analysis of injury and illness data. Instead, a proactive approach is needed that accounts for risks to specific hazards that can be abated by engineering or behavioral interventions. The process proposed here begins with development of prospective interventions. The degree of reduction in risk to be expected from an intervention then is determined from statistics on the mining worker population, the expected degree of success of the intervention, and the expected change in the severity of injuries resulting from the intervention. Three disparate mining health and safety concerns are presented to demonstrate common problems in assessing risks of injury and illness and describe additional data needs. Information on events preceding injuries and illnesses and more detailed demographic data on the mining work force are needed to analyze injury and illness data more precisely. Detailed information on exposure to specific hazards is necessary to evaluate the potential for an intervention to reduce risk of injury or illness.     

Saltelli Global Sensitivity Analysis and Simulation Modelling to Identify Intervention Strategies to Reduce the Prevalence of Escherichia coli O157 Contaminated Beef Carcasses

Introduction

Strains of Shiga-toxin producing Escherichia coli O157 (STEC O157) are important foodborne pathogens in humans, and outbreaks of illness have been associated with consumption of undercooked beef. Here, we determine the most effective intervention strategies to reduce the prevalence of STEC O157 contaminated beef carcasses using a modelling approach.

Method

A computational model simulated events and processes in the beef harvest chain. Information from empirical studies was used to parameterise the model. Variance-based global sensitivity analysis (GSA) using the Saltelli method identified variables with the greatest influence on the prevalence of STEC O157 contaminated carcasses. Following a baseline scenario (no interventions), a series of simulations systematically introduced and tested interventions based on influential variables identified by repeated Saltelli GSA, to determine the most effective intervention strategy.

Results

Transfer of STEC O157 from hide or gastro-intestinal tract to carcass (improved abattoir hygiene) had the greatest influence on the prevalence of contaminated carcases. Due to interactions between inputs (identified by Saltelli GSA), combinations of interventions based on improved abattoir hygiene achieved a greater reduction in maximum prevalence than would be expected from an additive effect of single interventions. The most effective combination was improved abattoir hygiene with vaccination, which achieved a greater than ten-fold decrease in maximum prevalence compared to the baseline scenario.

Conclusion

Study results suggest that effective interventions to reduce the prevalence of STEC O157 contaminated carcasses should initially be based on improved abattoir hygiene. However, the effect of improved abattoir hygiene on the distribution of STEC O157 concentration on carcasses is an important information gap—further empirical research is required to determine whether reduced prevalence of contaminated carcasses is likely to result in reduced incidence of STEC O157 associated illness in humans. This is the first use of variance-based GSA to assess the drivers of STEC O157 contamination of beef carcasses.     

Incidence and tracking of Escherichia coli O157:H7 in a major produce production region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.     

Detection and surveillance of waterborne protozoan parasites

The majority of the world's population still live without access to healthy water and the contamination of drinking water with protozoan pathogens poses a serious threat to millions of people in the developing world. Even in the developed world periodic outbreaks of diarrhoeal diseases are caused by the protozoan parasites Cryptosporidium sp., Giardia duodenalis and Entamoeba histolytica. Thus, surveillance of drinking water is imperative to minimize such contaminations and ensure continuous supplies of healthy water world-wide. This article reviews the progress in technology for detection and surveillance of these important waterborne parasites.     

Foodborne viruses

Foodborne and waterborne viral infections are increasingly recognized as causes of illness in humans. This increase is partly explained by changes in food processing and consumption patterns that lead to the worldwide availability of high-risk food. As a result, vast outbreaks may occur due to contamination of food by a single foodhandler or at a single source. Although there are numerous fecal-orally transmitted viruses, most reports of foodborne transmission describe infections with Norwalk-like caliciviruses (NLV) and hepatitis A virus (HAV), suggesting that these viruses are associated with the greatest risk of foodborne transmission. NLV and HAV can be transmitted from person to person, or indirectly via food, water, or fomites contaminated with virus-containing feces or vomit. People can be infected without showing symptoms. The high frequency of secondary cases of NLV illness and - to a lesser extent - of hepatitis A following a foodborne outbreak results in amplification of the problem. The burden of illness is highest in the elderly, and therefore is likely to increase due to the aging population. For HAV, the burden of illness may increase following hygienic control measures, due to a decreasing population of naturally immune individuals and a concurrent increase in the population at risk. Recent advances in the research of NLV and HAV have led to the development of molecular methods which can be used for molecular tracing of virus strains. These methods can be and have been used for the detection of common source outbreaks. While traditionally certain foods have been implicated in virus outbreaks, it is clear that almost any food item can be involved, provided it has been handled by an infected person. There are no established methods for detection of viruses in foods other than shellfish. Little information is available on disinfection and preventive measures specifically for these viruses. Studies addressing this issue are hampered by the lack of culture systems. As currently available routine monitoring systems exclusively focus on bacterial pathogens, efforts should be made to combine epidemiological and virological information for a combined laboratory-based rapid detection system for foodborne viruses. With better surveillance, including typing information, outbreaks of foodborne infections could be reported faster to prevent further spread.     

Early events in the elicitation of plant defence

Plants successfully use inducible defence mechanisms to combat potential pathogens. Elicitors are signaling compounds that stimulate any of such defence responses. Recent progress in the isolation of pure elicitors has made possible investigations on elicitor-binding proteins which might function as receptors in signal transduction pathways that ultimately activate the defences. The elicitor-binding sites studied so far show a high degree of ligand specificity, as do the candidate binding proteins identified for some of the ligands. Following elicitor perception, a number of rapid reactions are detectable in plant cells, including enhanced ion fluxes across the plasma membrane, formation of reactive oxygen intermediates, changes in protein phosphorylation, and lipid oxidation. Intriguing questions arising from these observations are whether the elicitor-binding proteins constitute receptors in plant defence signaling and whether any of the rapid events participate in signal transduction during defence activation. Received: 17 November 1997 / Accepted: 22 April 1998     

Assessing the mycotoxigenic threat of necrotrophic pathogens of wheat

Pathogenic fungi are the causal agents of many significant plant diseases around the world. These diseases often result in significant yield reductions, leading to lower food production rates and economic losses. Several of these pathogenic fungi also produce mycotoxins during infection, which are harmful to human and animal health. Whilst some of these toxins and the fungi that produce them have been studied intensively, the mycotoxigenic potential of many of these pathogens remains largely unknown. Included within these fungi are the necrotrophic pathogens of wheat, Stagonospora nodorum, Pyrenophora tritici-repentis and Alternaria alternata. Recent studies have demonstrated that each of these pathogens is capable of synthesizing an array of mycotoxic compounds during disease development, questioning their status as non-mycotoxin producers. This review summarises recent mycotoxin findings in these necrotrophic wheat pathogens by briefly discussing the mycotoxins identified, their toxicity and their synthesis. Future and emerging threats are also considered.     

National outbreak of Salmonella serotype saintpaul infections: importance of Texas restaurant investigations in implicating jalapeño peppers

Background

In May 2008, PulseNet detected a multistate outbreak of Salmonella enterica serotype Saintpaul infections. Initial investigations identified an epidemiologic association between illness and consumption of raw tomatoes, yet cases continued. In mid-June, we investigated two clusters of outbreak strain infections in Texas among patrons of Restaurant A and two establishments of Restaurant Chain B to determine the outbreak''s source.

Methodology/Principal Findings

We conducted independent case-control studies of Restaurant A and B patrons. Patients were matched to well controls by meal date. We conducted restaurant environmental investigations and traced the origin of implicated products. Forty-seven case-patients and 40 controls were enrolled in the Restaurant A study. Thirty case-patients and 31 controls were enrolled in the Restaurant Chain B study. In both studies, illness was independently associated with only one menu item, fresh salsa (Restaurant A: matched odds ratio [mOR], 37; 95% confidence interval [CI], 7.2–386; Restaurant B: mOR, 13; 95% CI 1.3–infinity). The only ingredient in common between the two salsas was raw jalapeño peppers. Cultures of jalapeño peppers collected from an importer that supplied Restaurant Chain B and serrano peppers and irrigation water from a Mexican farm that supplied that importer with jalapeño and serrano peppers grew the outbreak strain.

Conclusions/Significance

Jalapeño peppers, contaminated before arrival at the restaurants and served in uncooked fresh salsas, were the source of these infections. Our investigations, critical in understanding the broader multistate outbreak, exemplify an effective approach to investigating large foodborne outbreaks. Additional measures are needed to reduce produce contamination.     

Surveillance for outbreaks of respiratory tract infections in nursing homes

BACKGROUND: Outbreaks of respiratory tract infections are common in long-term care facilities for older people. The objective of our study was to determine both the frequency of such outbreaks and their clinical and epidemiological features. METHODS: Prospective surveillance for outbreaks of respiratory tract infections and a retrospective audit of surveillance records were conducted in 5 nursing homes in metropolitan Toronto over 3 years. The clinical manifestations of infected residents were identified and microbiological investigations for causal agents were conducted. RESULTS: Sixteen outbreaks, involving 480 of 1313 residents, were identified prospectively during 1 144 208 resident-days of surveillance, for an overall rate of 0.42 infections per 1000 resident-days. Another 30 outbreaks, involving 388 residents, were identified retrospectively. Outbreaks occurred year-round, with no seasonal pattern. Pathogens included influenza virus, parainfluenza virus, respiratory syncytial virus, Legionella sainthelensi and Chlamydia pneumoniae. Multiple pathogens were detected in 38% (6/16) of the prospectively identified outbreaks. Of the 480 residents in the prospectively identified outbreaks 398 (83%) had a cough, 194 (40%) had fever and 215 (45%) had coryza. Clinical findings were nonspecific and could not be used to distinguish between causal agents. Pneumonia developed in 72 (15%) of the 480 residents, and 58 (12%) required transfer to hospital. The case-fatality rate was 8% (37/480). INTERPRETATION: Our findings emphasize the importance of adequate surveillance for outbreaks of respiratory tract infections in nursing homes and of early diagnosis so that appropriate interventions can be promptly instituted.